Bioactive Lipid Mediators - (2015)
Bioactive Lipid Mediators - (2015)
Bioactive Lipid Mediators - (2015)
Editors
Bioactive
Lipid
Mediators
Current Reviews and Protocols
Bioactive Lipid Mediators
Takehiko Yokomizo Makoto Murakami
Editors
According to legend, Hippocrates advocated the use of willow bark, Salix alba, to
ameliorate the pain of childbirth and fever. Salicylates were later isolated as the
active component in willow bark and conjugated with an acetyl group to generate
aspirin. Today, aspirin is the most well-known nonsteroidal antiinflammatory drug
and has numerous recognized clinical benefits, ranging from reducing the risk of
heart attack and colon polyposis to its frequently exploited antiinflammatory and
pain-relief actions, by decreasing the production of prostaglandins and thrombox-
ane from arachidonic acid. The other arachidonate-derived lipid mediators leukotri-
ene B4 and cysteinyl leukotrienes have also attracted research interest and are now
well known, particularly in the immunology field, as a potent leukocyte attractant
and a bronchoconstrictor, respectively.
Recent advances in biochemistry and molecular biology have enabled us to
purify and cDNA-clone multiple biosynthetic enzymes and receptors responsible
for the production and recognition of individual lipid mediators. Numerous lines of
transgenic and knockout mice in relation to lipid mediators have been established
and analyzed to unveil their in vivo roles. Given that some lipid mediators were
shown to cause inflammatory, immune, or oncogenic disorders, some of these
enzymes and receptors are now recognized as targets for new drugs. Furthermore,
whole sequencing of the human genome led us to discover many orphan receptors,
some of which were later identified as receptors for lysophospholipids or their
derivatives, such as platelet-activating factor, lysophosphatidic acid, and sphingo-
sine 1-phosphate, and their physiological and pathophysiological roles are currently
being extensively analyzed. It has also become evident that omega-3 polyunsatu-
rated fatty acids, which have long been believed to be beneficial for our health, are
metabolized to another class of lipid mediators (the so-called pro-resolving lipid
mediators), which include resolvins and protectins. The molecular nature of lyso-
phospholipid acyltransferases, which are involved in the remodeling and asymme-
try formation of membrane phospholipids, has finally been identified. In addition to
progress in protein chemistry and molecular biology, the development of highly
sensitive mass spectrometry has greatly contributed to the direct identification of
novel lipid mediators that are lowly abundant in tissues. Recently, imaging mass
v
vi Preface
vii
viii Contents
1.1 Introduction
H. Shindou (*)
Department of Lipid Signaling, Research Institute, National Center for Global Health
and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
CREST, Japan Science and Technology Agency,
4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
e-mail: [email protected]
T. Harayama D. Hishikawa
Department of Lipid Signaling, Research Institute, National Center for Global Health
and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
Phospholpid biosynthesis
Phospholipids Lysophospholipids
TAG
PC LPC
G3P
GPATs
DAG PE LPLATs LPE
LPA
LPAATs PS LPS
PA
PI LPI
PLAs
CDP-DAG PG LPG
CL LCL
PAF biosynthesis
cytosolic PLA2 Lyso-PAFAT
1-alkyl-PC lyso-PAF PAF
Fig. 1.1 Phospholipid biosynthetic pathways. Acyltransferase steps are indicated in red, phospho-
lipase (PLA) steps to release fatty acids in blue, and phospholipids in yellow
Table 1.1 Summary of lysophospholipid acyltransferases (LPLATs) from the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) and membrane-bound
O-acyltransferases (MBOAT) families. Other names were registered in HomoloGene of the NCBI database
Name Other name Product Mouse Human Family
GPAT1 GPAM; GPAT; GPAT1; RP11-426E5.2 LPA NP_032175.2 NP_065969 AGPAT
GPAT2 xGPAT1; CT123 LPA NP_001074558.1 NP_997211.2 AGPAT
GPAT3 AGPAT8; AGPAT9; LPAAT; MAG1; HMFN0839 LPA NP_766303.1 NP_116106.2 AGPAT
GPAT4 AGPAT4; LPAAT; dJ473J16.2; RP3-473 J16.2 LPA NP_061213.2 NP_848934 AGPAT
Lysophospholipid Acyltransferases
Lysophosphatidic acid (LPA) is the substrate for LPAAT enzymes and is biosynthe-
sized as part of the acyltransferase reaction. Four mammalian GPATs, which syn-
thesize LPA from glycerophospholipids (GP), have been cloned from the AGPAT
family [2, 4]. GPAT1 and GPAT2 function in the outer mitochondrial membrane,
whereas GPAT3 and GPAT4 are localized to the endoplasmic reticulum (ER) [4].
GPAT4 is also found in lipid droplets [11]. The four GPATs preferentially use satu-
rated and monounsaturated fatty acyl-CoAs to produce LPA, which is then con-
verted to PA by LPAAT. To date, four LPAATs (LPAAT1, -2, -3, and -4) have been
cloned and characterized. The representative LPAAT-catalyzed reaction is shown in
Fig. 1.2.
Human LPAAT1 and LPAAT2 were cloned based on their homology with yeast,
Escherichia coli, and coconut AGPATs. LPAAT1 has a higher activity toward 14:0-,
16:0-, and 18:2-CoAs, whereas LPAAT2 exhibited higher activity toward 20:4-CoA
compared with 16:0- or 18:0-CoA. Both mRNAs are found in a broad range of tis-
sues [4]. LPAAT2 mutations have been linked to congenital generalized lipodystro-
phy (also known as BerardinelliSeip syndrome) [12], suggesting that LPAAT2
might be involved in triacylglycerol (TAG) synthesis and storage in adipocytes.
1.2.2 LPAAT3
A O O
P OH
O O -
O
HO H O
LPA(18:0) CoA
S
18:1-CoA
LPAAT
SH-CoA
O O
P OH
O O -
O
O H
O
PA(18:0/18:1)
B O O
P OH
O O -
O
HO H O
LPA(16:0) CoA
S
22:6-CoA
LPAAT
SH-CoA
O O
P OH
O O -
O
O H
O
PA(16:0/22:6)
1.2.3 LPAAT4
Recently, LPAAT4 was also reported to possess LPAAT activity with 22:6-CoA
[17]. LPAAT4 mRNA is expressed predominantly in the brain. The brain contains
an abundant amount of DHA-containing phospholipids. Therefore, LPAAT4 might
have important roles in brain function.
8 H. Shindou et al.
AGPAT5 (also called LPAAT) was reported to be a LPAAT and lyso-PE (LPE) AT,
but it has not yet been analyzed in detail [18, 19]. AGPAT5 also contains the AGPAT
motifs. Further studies are needed to identify the AGPAT5 roles.
1.3.1 LPCAT1
LPCAT1 was the first enzyme identified as having LPCAT activity [20, 21]. LPCAT1
preferentially uses 16:0-CoA to produce dipalmitoyl-PC (DPPC), which is the main
component of the pulmonary surfactant that prevents alveolar collapse, small air-
way closure, and alveolar flooding by decreasing surface tension. Alveolar type II
cells produce the pulmonary surfactant that is essential for respiration. Consistent
with this, LPCAT1 is expressed mainly in the lung, particularly in alveolar type II
cells, and its mRNA expression is upregulated during the perinatal period [21].
Pulmonary surfactant deficiency is an important contributing factor during the
pathogenesis of infant respiratory distress syndrome (IRDS), acute respiratory dis-
tress syndrome (ARDS), bronchial asthma, and bronchiolitis because pulmonary
surfactant plays a critical role in respiratory physiology [22]. Recently, several
groups reported the function of LPCAT1 in the lung. LPCAT1 gene trap mice exhib-
ited a decreased level of saturated PC and increased perinatal mortality because of
respiratory failure [23]. In addition, LPCAT1-knockout mice had low levels of
DPPC, as well as higher sensitivity for acute lung injury than control mice [24].
These reports suggest that the saturated PC generated by LPCAT1 is important for
lung surfactant production and function.
Retinal degeneration and visual dysfunction were also found in a mouse strain
with a LPCAT1 mutation (rd11) [25]. LPCAT1 mRNA levels decreased in the retina
and the brain in response to the onset of diabetes in Ins2(Akita) and db/db mice,
mouse models of type 1 and type 2 diabetes, respectively [26]. LPCAT1 expression
was reported in colorectal cancer and was also correlated with the progression of
prostate cancer [27, 28].
1 Lysophospholipid Acyltransferases 9
A O O
O O P O
H -O N+
HO O
LPC(16:0) CoA
S
16:0-CoA
LPCAT
SH-CoA
O O
O O P O
H -O N+
O
O
PC(16:0/16:0)
B O O
P O
O O - N+
O
HO H O
LPC(18:0)
CoA
S
20:4-CoA
LPCAT
SH-CoA
O O
P O
O O - N+
O
O H
O
PC(18:0/20:4)
Fig. 1.3 Representative LPCAT-catalyzed reaction: PC (16:0/16:0, DPPC) (a); PC (18:0/20:4) (b)
1.3.2 LPCAT2
1.3.3 LPCAT3
LPCAT3 mRNA is expressed ubiquitously, and the protein exhibits LPCAT, LPEAT,
and LPSAT activities with PUFA-CoAs such as 20:4-CoA and 18:2-CoA [31, 32].
LPCAT3 knockdown in HEK293 cells induced apoptosis and altered cellular mor-
phology [33]. Liver-specific LPCAT3 knockdown in mice showed increased levels of
LPC, which led to decreased hepatic triglyceride levels and increased triglyceride-rich
lipoprotein production and apolipoprotein-B secretion [34]. LPCAT3 knockdown in
mammalian cells also enhanced the palmitic acid-induced unfolded protein response
[35]. LPCAT3 is induced by agonists for peroxisome proliferator-activator receptor-
and liver X receptor, as well as during the differentiation of C3H10T1/2 cells into
adipocyte-like cells [32, 36, 37]. LPCAT1, LPCAT2, LPCAT3, and LPCAT4 were
induced in a model of nonalcoholic steatohepatitis [38]. In addition, hepatic LPCAT3
and LPCAT4 were induced by treatment with fibrates [39]. Interestingly, the
Drosophila orthologue of LPCAT3, nessy, is controlled by ultrabithorax (Ubx),
homeobox (Hox), and other Hox proteins during Drosophila embryogenesis [40].
1.3.4 LPCAT4
LPCAT4 has both LPCAT and LPEAT activities [31, 41]. LPCAT4 preferentially uses
18:1-CoA as a donor. Mouse LPCAT4 mRNA is highly expressed in the epididymis,
brain, testis, and ovary. However, biological roles of LPCAT4 remain unclear.
O
P O
O O - N+
O
HO H
LysoPAF O
CoA
S
2:0-CoA
LysoPAFAT
SH-CoA
O
P O
O O - N+
O
O H
PAF
O
1.5.1 LPEAT1
LPEAT1 exhibits both LPEAT and LPSAT activities and has a preference for 18:1-
CoA as a donor [31, 41]. LPEAT1 mRNA is highly expressed in the stomach, epi-
didymis, and colon. Furthermore, the human LPEAT1 gene, which is located on
chromosome 6, was disrupted in a brachydactyly-syndactyly syndrome patient [51],
suggesting that LPEAT1 contributes to the turnover of phospholipids during normal
development and organogenesis.
O O
P NH3+
O O -O
H O O
HO
LPE(18:0) CoA
S
22:6-CoA
LPEAT
SH-CoA
O O
PO NH3+
O O
O-
O H
O
PE(18:0/22:6)
1.5.2 LPEAT2
LPEAT2 was identified from the AGPAT family. Although LPEAT2 exhibits
LPEAT, LPGAT, LPSAT, and LPCAT activities using 18:1-CoA or 20:4-CoA as the
acyl donor in vitro, only LPEAT activity was decreased by its siRNA transfection in
HEK293T cells [52]. LPEAT2 is expressed at high levels in the brain, suggesting
that it might be important for the biogenesis of brain PE. However, the reported
biochemical activities of LPEAT2 are inconsistent with the brain PE composition
[53]. It has been reported that hepatic LPEAT2 expression increases on exposure to
lithocholic acid exposure [54]. Confusingly, LPEAT2 is also called LPCAT4 (see
Table 1.1).
LPCAT3 and LPCAT4 both exhibit LPEAT activity [31, 41]; however, their biologi-
cal roles as LPEAT are yet to be clarified. The regulation of these enzymes is
described earlier in the LPCAT enzymes section.
O
O O H O-
PO NH 3+
O O -
O
HO H O
LPS(16:0) CoA
S
22:6-CoA
LPSAT
SH-CoA
O
O O H O-
PO NH 3+
O O -
O
O H
O
PS(16:0/22:6)
LPCAT3 and LPCAT4 both exhibit LPSAT activity [31, 41]; however, their biologi-
cal roles as LPSAT are yet to be clarified. The regulation of these enzymes is
described earlier in the LPCAT enzymes section.
1.7.1 LPIAT1
LPIAT1 is the first reported LPIAT and was identified from the MBOAT family
[55]. LPIAT1 prefers 20:4-CoA and 20:5-CoA as donors. In LPIAT1-KO mice, PI
and PI phosphates containing arachidonic acid decreased, and the mice exhibited
abnormal brain morphology, delayed neural migration, and reduced neurite out-
growth. LPIAT1-KO mice were significantly smaller than their littermates and were
born at a frequency less than would be expected from the Mendelian ratio [56, 57].
Arachidonic acid containing PI produced by LPIAT1 is converted to phosphoinosit-
ides. They play an important role in brain development [56]. A Caenorhabditis
elegans LPIAT1 (mboa-7) mutant showed a bag of worms phenotype, whereby
the embryos hatched within the mother, leaving a cuticle sack containing multiple
wriggling larvae [55].
O O OH OH OH
P OH
O O -O OH
H O O
LPI(18:0)
HO
CoA
S
20:4-CoA
LPIAT
SH-CoA
O O OH OH OH
P OH
O O -O OH
O
O H
O
PI(18:0/20:4)
O O OH
P
O O -O OH
O
HO H O
LPG(16:0) CoA
S
18:1-CoA
LPGAT
SH-CoA
O O OH
PO OH
O O
O-
O H
O
PG(16:0/18:1)
LCLAT1, a member of the AGPAT family, has LPIAT activity and uses 18:1-CoA
as the donor [58]. LCLAT1 can also incorporate 18:0-CoA into the sn-1 position of
LPI [59], as is described in detail in the LCLAT enzyme section.
1.8.1 LPGAT1
PG is synthesized from LPG by LPGAT enzyme during the Lands cycle. LPGAT1
was cloned as an LPGAT enzyme from the AGPAT family [60]. Human LPGAT1
has a preference for 16:0-, 18:0-, and 18:1-CoAs as donors and is widely expressed
in several tissues. PG is a precursor for the synthesis of CL.
O O
P O-
O O
O
O H
HO H
O O O
O
O O P
H O-
HO
O
LCL(18:2/18:2/18:2) CoA
S
18:2-CoA
LCLAT
SH-CoA
O O
P O-
O O
O
O H
HO H
O O O
O
O O P
H O-
O
O
CL(18:2/18:2/18:2/18:2)
1.9.1 LCLAT1
1.10 Conclusion
The Kennedy pathway and the Lands cycle were first proposed in the 1950s. More
than ten LPLATs have been identified during the past decade, resulting in signifi-
cant advancement of the LPLAT field. However, the nomenclature should be stan-
dardized in the international conferences to bring about progress in phospholipid
research because most enzymes have several confusing names. It is possible that
additional LPLATs with preferences for different substrates might contribute to the
generation of membrane diversity and will be identified in future studies. The
redundant and pleiotropic substrate preferences of LPLATs might help regulate
membrane diversity in tissues, which could be changed in response to external stim-
uli (Fig. 1.10). Further in vivo studies are needed to elucidate the biological roles of
LPLATs and to understand the biological significance of membrane diversity and
asymmetry.
Acknowledgments We are grateful to Prof. Takao Shimizu and all members of Shimizus
laboratory (National Center for Global Health and Medicine, and The University of Tokyo) for
their valuable suggestions.
Note This work is supported by CREST, the Japan Science and Technology Agency (H.S.), a
grant-in-aid for Scientific Research (C) (H.S.), and a Grant-in-Aid for Young Scientists (B) (D.H.)
from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
References
1. Shimizu T (2009) Lipid mediators in health and disease: enzymes and receptors as therapeutic
targets for the regulation of immunity and inflammation. Annu Rev Pharmacol 49:123150
2. Shindou H, Shimizu T (2009) Acyl-CoA: lysophospholipid acyltransferases. J Biol Chem
284:15
3. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how
they behave. Nat Rev Mol Cell Biol 9:112124
4. Yamashita A, Hayashi Y, Nemoto-Sasaki Y, Ito M, Oka S, Tanikawa T, Waku K, Sugiura T
(2013) Acyltransferases and transacylases that determine the fatty acid composition of glyc-
erolipids and the metabolism of bioactive lipid mediators in mammalian cells and model
organisms. Prog Lipid Res 53C:1881
5. Kennedy EP, Weiss SB (1956) The function of cytidine coenzymes in the biosynthesis of phos-
pholipids. J Biol Chem 222:193214
6. Lands WE (1958) Metabolism of glycerolipides; a comparison of lecithin and triglyceride
synthesis. J Biol Chem 231:883888
7. Shindou H, Hishikawa D, Harayama T, Eto M, Shimizu T (2013) Generation of membrane
diversity by lysophospholipid acyltransferases. J Biochem 154:2128
8. Hishikawa D, Hashidate T, Shimizu T, Shindou H (2014) Diversity and function of membrane
glycerophospholipids generated by the remodeling pathway in mammalian cells. J Lipid Res
55:799807
9. Harayama T, Shindou H, Ogasawara R, Suwabe A, Shimizu T (2008) Identification of a novel
noninflammatory biosynthetic pathway of platelet-activating factor. J Biol Chem
283:1109711106
10. Shindou H, Eto M, Morimoto R, Shimizu T (2009) Identification of membrane O-acyltransferase
family motifs. Biochem Biophys Res Commun 383:320325
11. Wilfling F, Wang H, Haas JT, Krahmer N, Gould TJ, Uchida A, Cheng JX, Graham M,
Christiano R, Frohlich F, Liu X, Buhman KK, Coleman RA, Bewersdorf J, Farese RV Jr,
Walther TC (2013) Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocal-
izing from the ER to lipid droplets. Dev Cell 24:384399
12. Agarwal AK, Arioglu E, De Almeida S, Akkoc N, Taylor SI, Bowcock AM, Barnes RI, Garg A
(2002) AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome
9q34. Nat Genet 31:2123
13. Yuki K, Shindou H, Hishikawa D, Shimizu T (2009) Characterization of mouse lysophospha-
tidic acid acyltransferase 3: an enzyme with dual functions in the testis. J Lipid Res
50:860869
14. Koeberle A, Shindou H, Harayama T, Yuki K, Shimizu T (2012) Polyunsaturated fatty acids
are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransfer-
ase 3. FASEB J 26:169180
15. Koeberle A, Shindou H, Harayama T, Shimizu T (2010) Role of lysophosphatidic acid acyl-
transferase 3 for the supply of highly polyunsaturated fatty acids in TM4 Sertoli cells. FASEB
J 24:49294938
1 Lysophospholipid Acyltransferases 19
16. Schmidt J, Brown W (2009) Lysophosphatidic acid acyltransferase 3 regulates Golgi complex
structure and function. J Cell Biol 186:211218
17. Eto M, Shindou H, Shimizu T (2014) A novel lysophosphatidic acid acyltransferase enzyme
(LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophos-
pholipids. Biochem Biophys Res Commun 443:718724
18. Lu B, Jiang YJ, Zhou Y, Xu FY, Hatch GM, Choy PC (2005) Cloning and characterization of
murine 1-acyl-sn-glycerol 3-phosphate acyltransferases and their regulation by PPARalpha in
murine heart. Biochem J 385:469477
19. Prasad SS, Garg A, Agarwal AK (2011) Enzymatic activities of the human AGPAT isoform 3
and isoform 5: localization of AGPAT5 to mitochondria. J Lipid Res 52:451462
20. Nakanishi H, Shindou H, Hishikawa D, Harayama T, Ogasawara R, Suwabe A, Taguchi R,
Shimizu T (2006) Cloning and characterization of mouse lung-type acyl-
CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1): expression in alveolar type II cells
and possible involvement in surfactant production. J Biol Chem 281:2014020147
21. Chen X, Hyatt BA, Mucenski ML, Mason RJ, Shannon JM (2006) Identification and charac-
terization of a lysophosphatidylcholine acyltransferase in alveolar type II cells. Proc Natl Acad
Sci U S A 103:1172411729
22. Stevens TP, Sinkin RA (2007) Surfactant replacement therapy. Chest 131:15771582
23. Bridges JP, Ikegami M, Brilli LL, Chen X, Mason RJ, Shannon JM (2010) LPCAT1 regulates
surfactant phospholipid synthesis and is required for transitioning to air breathing in mice. J
Clin Invest 120:17361748
24. Harayama T, Eto M, Shindou H, Kita Y, Otsubo E, Hishikawa D, Ishii S, Sakimura K, Mishina
M, Shimizu T (2014) Lysophospholipid acyltransferases mediate phosphatidylcholine diversi-
fication to achieve the physical properties required in vivo. Cell Metab 20:295305
25. Friedman JS, Chang B, Krauth DS, Lopez I, Waseem NH, Hurd RE, Feathers KL, Branham
KE, Shaw M, Thomas GE, Brooks MJ, Liu C, Bakeri HA, Campos MM, Maubaret C, Webster
AR, Rodriguez IR, Thompson DA, Bhattacharya SS, Koenekoop RK, Heckenlively JR,
Swaroop A (2010) Loss of lysophosphatidylcholine acyltransferase 1 leads to photoreceptor
degeneration in rd11 mice. Proc Natl Acad Sci U S A 107:1552315528
26. Cheng L, Han X, Shi Y (2009) A regulatory role of LPCAT1 in the synthesis of inflammatory
lipids, PAF and LPC, in the retina of diabetic mice. Am J Physiol Endocrinol Metab
297:E1276E1282
27. Mansilla F, da Costa KA, Wang S, Kruhoffer M, Lewin TM, Orntoft TF, Coleman RA,
Birkenkamp-Demtroder K (2009) Lysophosphatidylcholine acyltransferase 1 (LPCAT1) over-
expression in human colorectal cancer. J Mol Med (Berl) 87:8597
28. Zhou X, Lawrence TJ, He Z, Pound CR, Mao J, Bigler SA (2012) The expression level of
lysophosphatidylcholine acyltransferase 1 (LPCAT1) correlates to the progression of prostate
cancer. Exp Mol Pathol 92:105110
29. Shindou H, Hishikawa D, Nakanishi H, Harayama T, Ishii S, Taguchi R, Shimizu T (2007) A
single enzyme catalyzes both platelet-activating factor production and membrane biogenesis
of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransfer-
ase. J Biol Chem 282:65326539
30. Moessinger C, Kuerschner L, Spandl J, Shevchenko A, Thiele C (2011) Human lysophospha-
tidylcholine acyltransferases 1 and 2 are located in lipid droplets where they catalyze the for-
mation of phosphatidylcholine. J Biol Chem 286:2133021339
31. Hishikawa D, Shindou H, Kobayashi S, Nakanishi H, Taguchi R, Shimizu T (2008) Discovery
of a lysophospholipid acyltransferase family essential for membrane asymmetry and diversity.
Proc Natl Acad Sci U S A 105:28302835
32. Zhao Y, Chen YQ, Bonacci TM, Bredt DS, Li S, Bensch WR, Moller DE, Kowala M, Konrad
RJ, Cao G (2008) Identification and characterization of a major liver lysophosphatidylcholine
acyltransferase. J Biol Chem 283:82588265
33. Jain S, Zhang X, Khandelwal PJ, Saunders AJ, Cummings BS, Oelkers P (2009) Characterization
of human lysophospholipid acyltransferase 3. J Lipid Res 50:15631570
20 H. Shindou et al.
34. Li Z, Ding T, Pan X, Li Y, Li R, Sanders PE, Kuo MS, Hussain MM, Cao G, Jiang XC (2012)
Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine
accumulation promotes very low density lipoprotein production by enhancing microsomal tri-
glyceride transfer protein expression. J Biol Chem 287:2012220131
35. Ariyama H, Kono N, Matsuda S, Inoue T, Arai H (2010) Decrease in membrane phospholipid
unsaturation induces unfolded protein response. J Biol Chem 285:2202722035
36. Demeure O, Lecerf F, Duby C, Desert C, Ducheix S, Guillou H, Lagarrigue S (2011) Regulation
of LPCAT3 by LXR. Gene (Amst) 470:711
37. Eto M, Shindou H, Koeberle A, Harayama T, Yanagida K, Shimizu T (2012)
Lysophosphatidylcholine acyltransferase 3 is the key enzyme for incorporating arachidonic
acid into glycerophospholipids during adipocyte differentiation. Int J Mol Sci
13:1626716280
38. Tanaka N, Matsubara T, Krausz KW, Patterson AD, Gonzalez FJ (2012) Disruption of phos-
pholipid and bile acid homeostasis in mice with nonalcoholic steatohepatitis. Hepatology
56:118129
39. Yamazaki T, Wakabayashi M, Ikeda E, Tanaka S, Sakamoto T, Mitsumoto A, Kudo N,
Kawashima Y (2012) Induction of 1-acylglycerophosphocholine acyltransferase genes by
fibrates in the liver of rats. Biol Pharm Bull 35:15091515
40. Maurel-Zaffran C, Chauvet S, Jullien N, Miassod R, Pradel J, Aragnol D (1999) nessy, an
evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.
Mech Dev 86:159163
41. Gijn M, Riekhof W, Zarini S, Murphy R, Voelker D (2008) Lysophospholipid acyltransfer-
ases and arachidonate recycling in human neutrophils. J Biol Chem 283:3023530245
42. Prescott SM, Zimmerman GA, McIntyre TM (1990) Platelet-activating factor. J Biol Chem
265:1738117384
43. Ishii S, Shimizu T (2000) Platelet-activating factor (PAF) receptor and genetically engineered
PAF receptor mutant mice. Prog Lipid Res 39:4182
44. Shindou H, Ishii S, Yamamoto M, Takeda K, Akira S, Shimizu T (2005) Priming effect of
lipopolysaccharide on acetyl-coenzyme A: lyso-platelet-activating factor acetyltransferase is
MyD88 and TRIF independent. J Immunol 175:11771183
45. Morimoto R, Shindou H, Tarui M, Shimizu T (2014) Rapid production of platelet-activating
factor is induced by protein kinase C alpha-mediated phosphorylation of lysophosphatidylcho-
line acyltransferase 2 protein. J Biol Chem 289:1556615576
46. Morimoto R, Shindou H, Oda Y, Shimizu T (2010) Phosphorylation of lysophosphatidylcho-
line acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-
stimulated macrophages. J Biol Chem 285:2985729862
47. Smith WL, Langenbach R (2001) Why there are two cyclooxygenase isozymes. J Clin Invest
107:14911495
48. Tarui M, Shindou H, Kumagai K, Morimoto R, Harayama T, Hashidate T, Kojima H, Okabe T,
Nagano T, Nagase T, Shimizu T (2014) Selective inhibitors of a PAF biosynthetic enzyme
lysophosphatidylcholine acyltransferase 2. J Lipid Res 55:13861396
49. Okubo M, Yamanaka H, Kobayashi K, Kanda H, Dai Y, Noguchi K (2012) Up-regulation of
platelet-activating factor synthases and its receptor in spinal cord contribute to development of
neuropathic pain following peripheral nerve injury. Mol Pain 8:8
50. Kihara Y, Yanagida K, Masago K, Kita Y, Hishikawa D, Shindou H, Ishii S, Shimizu T (2008)
Platelet-activating factor production in the spinal cord of experimental allergic encephalomy-
elitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis. J Immunol
181:50085014
51. Dauwerse JG, de Vries BB, Wouters CH, Bakker E, Rappold G, Mortier GR, Breuning MH,
Peters DJ (2007) A t(4;6)(q12;p23) translocation disrupts a membrane-associated O-acetyl
transferase gene (MBOAT1) in a patient with a novel brachydactyly-syndactyly syndrome. Eur
J Hum Genet 15:743751
1 Lysophospholipid Acyltransferases 21
52. Cao J, Shan D, Revett T, Li D, Wu L, Liu W, Tobin JF, Gimeno RE (2008) Molecular
identification of a novel mammalian brain isoform of acyl-CoA:lysophospholipid acyltransfer-
ase with prominent ethanolamine lysophospholipid acylating activity, LPEAT2. J Biol Chem
283:1904919057
53. Yabuuchi H, OBrien JS (1968) Positional distribution of fatty acids in glycerophosphatides of
bovine gray matter. J Lipid Res 9:6567
54. Matsubara T, Tanaka N, Sato M, Kang DW, Krausz KW, Flanders KC, Ikeda K, Luecke H,
Wakefield LM, Gonzalez FJ (2012) TGF-beta-SMAD3 signaling mediates hepatic bile acid
and phospholipid metabolism following lithocholic acid-induced liver injury. J Lipid Res
53:26982707
55. Lee HC, Inoue T, Imae R, Kono N, Shirae S, Matsuda S, Gengyo-Ando K, Mitani S, Arai H
(2008) Caenorhabditis elegans mboa-7, a member of the MBOAT family, is required for selec-
tive incorporation of polyunsaturated fatty acids into phosphatidylinositol. Mol Biol Cell
19:11741184
56. Lee HC, Inoue T, Sasaki J, Kubo T, Matsuda S, Nakasaki Y, Hattori M, Tanaka F, Udagawa O,
Kono N, Itoh T, Ogiso H, Taguchi R, Arita M, Sasaki T, Arai H (2012) LPIAT1 regulates ara-
chidonic acid content in phosphatidylinositol and is required for cortical lamination in mice.
Mol Biol Cell 23:46894700
57. Anderson KE, Kielkowska A, Durrant TN, Juvin V, Clark J, Stephens LR, Hawkins PT (2013)
Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) is required to maintain physiological
levels of PtdIns and PtdInsP2 in the mouse. PLoS One 8:e58425
58. Cao J, Liu Y, Lockwood J, Burn P, Shi Y (2004) A novel cardiolipin-remodeling pathway
revealed by a gene encoding an endoplasmic reticulum-associated acyl-CoA:lysocardiolipin
acyltransferase (ALCAT1) in mouse. J Biol Chem 279:3172731734
59. Imae R, Inoue T, Nakasaki Y, Uchida Y, Ohba Y, Kono N, Nakanishi H, Sasaki T, Mitani S,
Arai H (2012) LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty
acid composition of phosphatidylinositol in mice. J Lipid Res 53:335347
60. Yang Y, Cao J, Shi Y (2004) Identification and characterization of a gene encoding human
LPGAT1, an endoplasmic reticulum-associated lysophosphatidylglycerol acyltransferase. J
Biol Chem 279:5586655874
61. Zhao Y, Chen YQ, Li S, Konrad RJ, Cao G (2009) The microsomal cardiolipin remodeling
enzyme acyl-CoA lysocardiolipin acyltransferase is an acyltransferase of multiple anionic
lysophospholipids. J Lipid Res 50:945956
62. Li J, Romestaing C, Han X, Li Y, Hao X, Wu Y, Sun C, Liu X, Jefferson LS, Xiong J, Lanoue
KF, Chang Z, Lynch CJ, Wang H, Shi Y (2010) Cardiolipin remodeling by ALCAT1 links
oxidative stress and mitochondrial dysfunction to obesity. Cell Metab 12:154165
63. Liu X, Ye B, Miller S, Yuan H, Zhang H, Tian L, Nie J, Imae R, Arai H, Li Y, Cheng Z, Shi Y
(2012) Ablation of ALCAT1 mitigates hypertrophic cardiomyopathy through effects on oxida-
tive stress and mitophagy. Mol Cell Biol 32:44934504
Chapter 2
Phospholipase A2
Abstract Phospholipase A2s (PLA2s) are a group of enzymes that hydrolyze the
sn-2 position of phospholipids to generate fatty acids and lysophospholipids, which
serve as lipid mediators or their precursors. Mammalian genomes encode genes for
more than 30 PLA2s or related enzymes, which are subdivided into several groups
on the basis of their structures, enzymatic properties, and evolutional relationships.
Among them, the Ca2+-dependent cytosolic PLA2 (cPLA2), Ca2+-independent PLA2
(iPLA2), and secreted PLA2 (sPLA2) families are regarded as the big three. From
a general point of view, cPLA2 (the prototypic cPLA2) plays a major role in the
initiation of arachidonic acid (AA) metabolism, the iPLA2 family contributes to
membrane homeostasis or energy metabolism, and the sPLA2 family affects various
biological events by modulating extracellular phospholipid milieus in response to
given microenvironmental cues. In this chapter, we overview current understanding
of the biological functions of PLA2s as revealed by gene-manipulated mice and
human diseases.
2.1 Introduction
mammals, and these have been subdivided into several groups based on their struc-
tures, catalytic mechanisms, localizations. and evolutionary relationships. The
cPLA2 family contains 6 enzymes (cPLA2), which (except for cPLA2) contain
an N-terminal C2 domain for Ca2+-dependent association with the membrane. The
iPLA2 or patatin-like phospholipase domain-containing lipase (PNPLA) family
includes 9 enzymes, some of which act principally on phospholipids and others on
neutral lipids such as triglyceride (TG). The sPLA2 family, in which 10 catalytically
active enzymes have been identied, are low molecular weight, extracellular enzymes
that require Ca2+ of the mM order for optimal enzymatic activity. Because of this
diversity, PLA2 enzymes have been implicated in various biological processes such
as lipid mediator production, membrane remodeling, and energy metabolism.
During the past few decades, studies of various PLA2 transgenic and/or knockout
mice as well as human diseases with PLA2 gene mutations have provided new
insights into the emerging biological roles of individual PLA2s. The functions of
individual PLA2s may not simply reect changes in lipid mediator signaling, or
more particularly eicosanoid signaling, but may also be attributable to hydrolysis of
one or a combination of various target membrane lipids. Herein, we focus on the
pathophysiology of various PLA2s as revealed by information from transgenic or
knockout mice, as well as human diseases.
2.2.2 cPLA2
cPLA2, also known as group IVA PLA2, is localized in the cytosol of resting cells,
and in response to an increase in cytosolic Ca2+ levels after cell activation, it trans-
locates to the perinuclear or, more specically, the Golgi membranes to encounter
its preferred substrate, AA-containing phosphatidylcholine (PC). Ceramide-1-
phosphate or phosphoinositide-4,5-bisphosphate (PIP2) enhances the membrane
2 Phospholipase A2 25
Fig. 2.1 Activation of cytosolic PLA2 (cPLA2) in mast cells (MCs). In response to Ca2+ inux
following FcRI activation with IgE and cognate antigen, cPLA2 translocates from the cytosol to
the perinuclear membrane and is phosphorylated by mitogen-activated protein kinase (MAPK) for
optimal activation. The arachidonic acid (AA) released from membrane phospholipids by cPLA2
is then converted to PGD2 by the sequential action of cyclooxygenase (COX)-1 (or COX-2 when
the cells are primed by particular stimuli) and hematopoietic PGD2 synthase (H-PGDS) to PGD2
or by the sequential action of 5-lipoxygenase (5-LOX) incorporation with 5-LOX-activating pro-
tein (FLAP) and LTC4 synthase (LTC4S) to LTC4
cPLA2, , , and (group IVB, IVD, IVE, and IVF PLA2s) map to the same chro-
mosomal locus and are therefore evolutionally more related [16]. cPLA2 is a dual
PLA1/PLA2 enzyme, although cPLA2 has a robust PLA1 activity in preference to
PLA2 activity. cPLA2 (group IVC PLA2) is unique in that it lacks the C2 domain
and displays lysophospholipase and transacylase activities in addition to PLA2
activity [17]. The in vivo functions of these cPLA2 isoforms are entirely unknown
because knockout studies have yet to be performed. A recent study has shown that
cPLA2 may drive recycling through clathrin-independent endocytosis [18].
The human genome encodes nine iPLA2/PNPLA enzymes, which share a protein
motif known as the patatin domain with an unusual folding topology that differs
from classical lipases (Fig. 2.2). The cPLA2 and iPLA2 families seem to have
evolved from a common ancestral gene, as their catalytic domains are commonly
characterized by a three-layer // architecture employing a conserved Ser/Asp
catalytic dyad instead of the classical catalytic triad [19]. iPLA2/PNPLA enzymes
are found in virtually all eukaryotes including yeast, plants, invertebrates, and ver-
tebrates, suggesting that they possess fundamental roles in cellular lipid metabolism
conserved in the eukaryote kingdom. The designation PNPLA appears to be more
appropriate than iPLA2, as some of the isoforms have enzymatic activities appar-
ently distinct from bona fide PLA2 activity. For instance, iPLA2/PNPLA2 functions
as a major TG lipase in adipose and many other tissues, whereas iPLA2/PNPLA3
may act mainly as an acyltransferase or transacylase for accumulation of TG, par-
ticularly in the liver [20]. Here, we focus on two particular iPLA2s, iPLA2/PNPLA9
and iPLA2/PNPLA8, which have robust PLA2 activity.
2 Phospholipase A2 27
Fig. 2.2 Evolutional relationship between the cPLA2 and iPLA2 families. The iPLA2 family is
present in all eukaryotes, whereas the cPLA2 family emerged from the iPLA2 family at the stage of
divergence of vertebrates
2.3.2 iPLA2
iPLA2 (PNPLA9 or group VIA PLA2), the best characterized iPLA2, has long been
thought to be involved in homeostatic phospholipid remodeling through deacylation
of phospholipids in the Lands cycle. Indeed, the composition of phospholipids,
particularly those containing docosahexaenoic acid (DHA), is noticeably altered in
the brain (but not other tissues) of mice lacking iPLA2 (Pla2g6/) [21]. Notably,
human PLA2G6 mutations are associated with neurodegenerative diseases such as
infantile neuroaxonal dystrophy (INAD), neurodegeneration with brain iron accu-
mulation (NBIA), and Schindlers disease, which share the distinctive pathological
feature of axonal degeneration with spheroid bodies in the nervous system [22].
Similar neurodegenerative phenotypes are also evident in Pla2g6/ mice or Pla2g6
mutant mice (Pla2g6-inad, in which the Pla2g6 gene harbors a point mutation),
which show motor dysfunction caused by widespread degeneration of axons and
synapses, accompanied by the appearance of spheroids and vacuoles [23, 24].
iPLA2 has also been proposed to have more diverse signaling roles. These Pla2g6/
phenotypes include male infertility [25], defective opening of store-operated Ca2+
entry, probably caused by reduced production of lysophosphatidylcholine (LPC)
[26], impaired insulin secretion by pancreatic -cells [27], reduced apoptosis [28],
decreased eicosanoid generation in vascular cells [29], and protection from ovarian
cancer, possibly through reduction of lysophosphatidic acid (LPA) [30]. In most
cases, however, the iPLA2-driven lipid metabolic processes underlying these
events are poorly characterized.
28 M. Murakami and Y. Taketomi
2.3.3 iPLA2
More than one third of the PLA2 enzymes belong to the sPLA2 family, which con-
tains ten catalytically active isoforms (IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA).
Individual sPLA2s exhibit unique tissue and cellular localizations and enzymatic
properties, suggesting their distinct pathophysiological roles. Classical group I/
II/V/X sPLA2s are closely related, 14- to 19-kDa secreted enzymes with a highly
conserved Ca2+-binding loop and a His/Asp catalytic dyad as well as conserved
disulde bonds, whereas group III and XII sPLA2s are atypical and classied into
distinct classes. As sPLA2s are secreted, their target membranes should reside in the
extracellular spaces. Individual sPLA2s contribute to various biological events
through production of lipid mediators, promotion of membrane remodeling, modi-
cation of extracellular noncellular lipid components such as surfactant, micropar-
ticles, and lipoproteins, or degradation of foreign phospholipids such as those
originating from microbes and dietary components. Here we overview the
2 Phospholipase A2 29
Fig. 2.3 Several examples of sPLA2-driven lipid networks. (a) sPLA2-V in M2 macrophages
facilitates the Th2 response and that in airway epithelial cells degrades lung surfactant. sPLA2-X
from the airway epithelium acts on eosinophils to augment LTC4 generation. Accordingly, the two
sPLA2s independently promote asthma. (b) sPLA2-III released from immature MCs acts on bro-
blasts to promote L-PGDS-dependent generation of PGD2, which in turn acts on the PGD2 receptor
DP1 on MCs to promote MC maturation. Accordingly, PLA2G3 facilitates MC-dependent ana-
phylaxis. Activation of cPLA2 in mature MCs is highlighted in Fig. 2.1. (c) In lymph nodes,
sPLA2-IID in DCs hydrolyzes PE to release DHA, which is then converted to the pro-resolving
lipid mediator resolving D1 (RvD1) to sequester Th1 immunity. Accordingly, sPLA2-IID amelio-
rates Th1-dependent contact dermatitis
2.4.2 sPLA2-IB
2.4.3 sPLA2-IIA
sPLA2-IIA is the only isozyme detectable in the circulation, particularly under path-
ological conditions. It is often referred to as inammatory sPLA2 as its levels in
sera or inammatory exudates correlate with the severity of inammatory diseases,
and it is robustly induced by pro-inammatory stimuli in various cells [42].
However, the precise role of sPLA2-IIA in inammation remains debatable, as a
natural mutation in its gene (Pla2g2a) in C57BL/6 and 129Sv mice [43] prevents
adequate evaluation of its functions by gene targeting. So far, therefore, most of the
in vivo functions of sPLA2-IIA have currently been addressed mainly using Pla2g2a-
transgenic mice.
Pla2g2a-transgenic mice have skin abnormalities manifested by hair loss and
epidermal hyperplasia [44] and by increased carcinogen-induced skin cancer [45].
In line with clinical evidence that the serum level of sPLA2-IIA correlates with car-
diovascular diseases [46], Pla2g2a-transgenic mice develop advanced atheroscle-
rotic lesions [47]. Given that atherosclerosis represents chronic inammation in the
aorta, sPLA2-IIA can be regarded as a pro-inammatory enzyme in atherosclerosis.
The most probable physiological role of sPLA2-IIA is degradation of bacterial
membranes, thereby providing a rst line of antimicrobial defense [48]. sPLA2-IIA
is capable of hydrolyzing phosphatidylethanolamine (PE) and phosphatidylglycerol
in marked preference to PC, which can account for the preferential action of this
enzyme on bacteria rather than on mammalian cells. Accordingly, Pla2g2a-
transgenic mice or wild-type mice treated with sPLA2-IIA are resistant to pneumo-
nia and sepsis following bacterial infection [49]. For this reason, sPLA2-IIA is often
referred to as a bactericidal sPLA2.
Mouse strains with natural disruption of the Pla2g2a gene (see foregoing) are
more sensitive to intestinal tumorigenesis [50]. Transgenic transfer of the Pla2g2a
gene into these strains reduces the incidence of intestinal polyposis [51]. Thus,
sPLA2-IIA appears to have an antitumorigenic role in the gastrointestinal tract.
Presumably, bactericidal sPLA2-IIA may affect the gastrointestinal microora,
thereby inuencing tumor development. On the other hand, sPLA2-IIA expression
is positively correlated with the malignancy of prostate cancer [52], revealing dis-
tinct impacts of sPLA2-IIA on different types of cancer. Recently, the mutated
Pla2g2a allele in the C57BL/6 mouse strain was delivered into the BALB/c mouse
strain to produce Pla2g2a/ BALB/c mice. Autoantibody-induced arthritis is atten-
uated in these Pla2g2a/ mice relative to Pla2g2a-sufcent mice, whereas it is
conversely aggravated in Pla2g2a-transgenic mice [53]. This study has provided the
2 Phospholipase A2 31
2.4.4 sPLA2-IID
2.4.5 sPLA2-IIE
2.4.6 sPLA2-V
tipping the immune balance toward an Th2/M2 state that counteracts adipose tissue
inammation. Mechanistically, sPLA2-V-driven oleate and linoleate from PC in
LDL dampen M1 macrophage polarization by saturated fatty acids (e.g., palmitate),
probably through attenuation of endoplasmic reticulum stress. Clinically also,
sPLA2-V expression in human visceral adipose tissue inversely correlates with
plasma LDL levels. These studies underscore the physiological relevance of lipo-
protein hydrolysis by sPLA2s, highlight two adipocyte-driven metabolic sPLA2s
(sPLA2-IIE and sPLA2-V) as integrated regulators of immune and metabolic
responses, and bring about a paradigm shift toward a better understanding of the
roles of the sPLA2 family as a metabolic coordinator.
2.4.7 sPLA2-X
cells. Rather, the possibility that paracrine sPLA2-X may alter the properties of
inammatory cells should be taken into account. Because sPLA2-X is abundantly
expressed in the gut epithelium, it is likely that the decreased digestion and absorp-
tion of dietary and biliary phospholipids are eventually linked to reduced fat accu-
mulation in adipose tissue of Pla2g10/ mice [79], a situation similar to
Pla2g1b/ mice (see foregoing).
sPLA2-X is most abundantly expressed in the testis, where it is stored in acro-
somes (secretory granules) in the head of sperm cells [82]. Pla2g10/ spermatozoa
display an impaired acrosome reaction and low fertility despite showing a normal
number and motility [83, 82]. Thus, sPLA2-X plays a specic role in sperm activa-
tion, boosting the acrosome reaction by producing LPC from sperm membranes in
a paracrine or autocrine manner. Last, a striking skin phenotype characterized by
alopecia in Pla2g10-transgenic mice points to a unique role of sPLA2-X in hair
homeostasis [84]. Although grossly the coat hairs of Pla2g10/ mice appear nor-
mal, they have ultrastructural abnormalities including a hypoplasic outer root sheath
and reduced melanin granules in their hair follicles.
2.4.8 sPLA2-III
sPLA2-III, an atypical sPLA2, more closely resembles bee venom sPLA2 rather than
other mammalian sPLA2s [85]. Transgenic overexpression of sPLA2-III in mice
with an ApoE/ background results in increased atherosclerosis from accelerated
LDL hydrolysis and increased TXA2 synthesis [86]. These mice also develop sys-
temic inammation as they age because of elevated eicosanoid formation [87].
Thus, beyond the overexpression strategy, sPLA2-III has a pro-inammatory
potential.
sPLA2-III is highly expressed in the epididymal epithelium. Studies using mice
lacking sPLA2-III (Pla2g3/) have revealed that epididymal sPLA2-III acts on
immature sperm cells passing through the duct in a paracrine manner to regulate
phospholipid remodeling. During epididymal transit of spermatozoa, PC in the
sperm membrane undergoes a dramatic shift in its acyl groups from oleate, linole-
ate, and AA to docosapentaenoic acid (DPA) and DHA, and the increased propor-
tion of DPA/DHA consequently contributes to increased sperm membrane uidity
and thereby sperm motility. In Pla2g3/ mice, this sperm membrane remodeling is
severely compromised. Accordingly, spermatozoa from Pla2g3/ mice have a low
DPA/DHA content, aberrant acrosomes and agella with an abnormal axoneme
conguration, and display hypomotility and reduced fertility [88]. Thus, the two
reproductive sPLA2s (sPLA2-III and sPLA2-X), which are expressed in different
locations within the male genital organs, exert nonredundant but interrelated func-
tions in two major steps of male fertility, the former during sperm maturation in the
epididymis and the latter during capacitation and the acrosome reaction, likely after
ejaculation in the uterus and oviduct.
2 Phospholipase A2 35
With the growing list of knockout and transgenic mouse strains for PLA2s, much
progress has been made in delineating the physiological functions of each PLA2. It
is now becoming obvious that cPLA2 is a central regulator of AA metabolism, sup-
ported by the view that the molecular evolution of cPLA2 coincided with that of
eicosanoid receptors when vertebrates evolved, that the iPLA2 family is a funda-
mental regulator of membrane homeostasis and energy metabolism, and that indi-
vidual sPLA2s exert unique and tissue-specic biological functions by acting on
extracellular phospholipids, which include adjacent cell membranes, noncellular
lipid components, and foreign phospholipids such as those in microbes and the diet.
The diversity of target phospholipids and products may explain why each PLA2
family contains many isoforms. Further advances in this research eld and their
integration for therapeutic applications are likely to benet from improved, time-
and space-resolved lipidomics technology that will allow monitoring of individual
PLA2s and their associated forms of lipid metabolism within specic tissue niches.
Hopefully, the next decade will yield a comprehensive map of the PLA2-driven lipid
networks, which will allow the therapeutic application of inhibitors for some PLA2s
central to human diseases.
Acknowledgments This work was supported by grants-in aid for Scientic Research from the
Ministry of Education, Culture, Sports, Science and Technology of Japan and CREST from the
Japan Science and Technology Agency (JST).
36 M. Murakami and Y. Taketomi
References
30. Li H, Zhao Z, Wei G, Yan L, Wang D, Zhang H, Sandusky GE, Turk J, Xu Y (2010) Group VIA
phospholipase A2 in both host and tumor cells is involved in ovarian cancer development.
FASEB J 24(10):41034116. doi:10.1096/fj.10-161356
31. Yan W, Jenkins CM, Han X, Mancuso DJ, Sims HF, Yang K, Gross RW (2005) The highly
selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by puried calcium-
independent phospholipase A2: identication of a novel enzymatic mediator for the genera-
tion of a key branch point intermediate in eicosanoid signaling. J Biol Chem
280(29):2666926679. doi:10.1074/jbc.M502358200
32. Mancuso DJ, Sims HF, Han X, Jenkins CM, Guan SP, Yang K, Moon SH, Pietka T, Abumrad
NA, Schlesinger PH, Gross RW (2007) Genetic ablation of calcium-independent phospholi-
pase A2 leads to alterations in mitochondrial lipid metabolism and function resulting in a
decient mitochondrial bioenergetic phenotype. J Biol Chem 282(48):3461134622.
doi:10.1074/jbc.M707795200
33. Song H, Wohltmann M, Bao S, Ladenson JH, Semenkovich CF, Turk J (2010) Mice decient
in group VIB phospholipase A2 (iPLA2) exhibit relative resistance to obesity and metabolic
abnormalities induced by a Western diet. Am J Physiol Endocrinol Metab 298(6):E1097
E1114. doi:10.1152/ajpendo.00780.2009
34. Mancuso DJ, Sims HF, Yang K, Kiebish MA, Su X, Jenkins CM, Guan S, Moon SH, Pietka T,
Nassir F, Schappe T, Moore K, Han X, Abumrad NA, Gross RW (2010) Genetic ablation of
calcium-independent phospholipase A2 prevents obesity and insulin resistance during high fat
feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation. J Biol
Chem 285(47):3649536510. doi:10.1074/jbc.M110.115766
35. Mancuso DJ, Han X, Jenkins CM, Lehman JJ, Sambandam N, Sims HF, Yang J, Yan W, Yang
K, Green K, Abendschein DR, Saftz JE, Gross RW (2007) Dramatic accumulation of triglyc-
erides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in
transgenic myocardium expressing human calcium-independent phospholipase A2. J Biol
Chem 282(12):92169227. doi:10.1074/jbc.M607307200
36. Mancuso DJ, Kotzbauer P, Wozniak DF, Sims HF, Jenkins CM, Guan S, Han X, Yang K, Sun
G, Malik I, Conyers S, Green KG, Schmidt RE, Gross RW (2009) Genetic ablation of calcium-
independent phospholipase A2 leads to alterations in hippocampal cardiolipin content and
molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunc-
tion. J Biol Chem 284(51):3563235644. doi:10.1074/jbc.M109.055194
37. Bione S, DAdamo P, Maestrini E, Gedeon AK, Bolhuis PA, Toniolo D (1996) A novel
X-linked gene, G4.5. is responsible for Barth syndrome. Nat Genet 12(4):385389.
doi:10.1038/ng0496-385
38. Huggins KW, Boileau AC, Hui DY (2002) Protection against diet-induced obesity and obesity-
related insulin resistance in group 1B PLA2-decient mice. Am J Physiol Endocrinol Metab
283(5):E994E1001. doi:10.1152/ajpendo.00110.2002
39. Hollie NI, Konaniah ES, Goodin C, Hui DY (2014) Group 1B phospholipase A2 inactivation
suppresses atherosclerosis and metabolic diseases in LDL receptor-decient mice.
Atherosclerosis 234(2):377380. doi:10.1016/j.atherosclerosis.2014.03.027
40. Hollie NI, Hui DY (2011) Group 1B phospholipase A2 deciency protects against diet-induced
hyperlipidemia in mice. J Lipid Res 52(11):20052011. doi:10.1194/jlr.M019463
41. Labonte ED, Kirby RJ, Schildmeyer NM, Cannon AM, Huggins KW, Hui DY (2006) Group
1B phospholipase A2-mediated lysophospholipid absorption directly contributes to postpran-
dial hyperglycemia. Diabetes 55(4):935941. doi:10.2337/diabetes.55.04.06.db05-1286
42. Scott DL, White SP, Browning JL, Rosa JJ, Gelb MH, Sigler PB (1991) Structures of free and
inhibited human secretory phospholipase A2 from inammatory exudate. Science
254(5034):10071010
43. MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD, Buchberg AM (1995) The
secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modier of
ApcMin-induced intestinal neoplasia. Cell 81(6):957966. doi:10.1016/0092-8674(95)90015-2
2 Phospholipase A2 39
44. Grass DS, Felkner RH, Chiang MY, Wallace RE, Nevalainen TJ, Bennett CF, Swanson ME
(1996) Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia
in the absence of inammatory inltrate. J Clin Invest 97(10):22332241. doi:10.1172/
JCI118664
45. Mulherkar R, Kirtane BM, Ramchandani A, Mansukhani NP, Kannan S, Naresh KN (2003)
Expression of enhancing factor/phospholipase A2 in skin results in abnormal epidermis and
increased sensitivity to chemical carcinogenesis. Oncogene 22(13):19361944. doi:10.1038/
sj.onc.1206229
46. Kugiyama K, Ota Y, Takazoe K, Moriyama Y, Kawano H, Miyao Y, Sakamoto T, Soejima H,
Ogawa H, Doi H, Sugiyama S, Yasue H (1999) Circulating levels of secretory type II phospho-
lipase A2 predict coronary events in patients with coronary artery disease. Circulation
100(12):12801284. doi:10.1161/01.CIR.100.12.1280
47. Ivandic B, Castellani LW, Wang XP, Qiao JH, Mehrabian M, Navab M, Fogelman AM, Grass
DS, Swanson ME, de Beer MC, de Beer F, Lusis AJ (1999) Role of group II secretory phos-
pholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in trans-
genic mice expressing group IIa phospholipase A2. Arterioscler Thromb Vasc Biol
19(5):12841290. doi:10.1161/01.ATV.19.5.1284
48. Weinrauch Y, Elsbach P, Madsen LM, Foreman A, Weiss J (1996) The potent anti-
Staphylococcus aureus activity of a sterile rabbit inammatory uid is due to a 14-kD phos-
pholipase A2. J Clin Invest 97(1):250257. doi:10.1172/JCI118399
49. Laine VJ, Grass DS, Nevalainen TJ (1999) Protection by group II phospholipase A2 against
Staphylococcus aureus. J Immunol 162(12):74027408
50. Cormier RT, Hong KH, Halberg RB, Hawkins TL, Richardson P, Mulherkar R, Dove WF,
Lander ES (1997) Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigen-
esis. Nat Genet 17(1):8891. doi:10.1038/ng0997-88
51. Kennedy BP, Soravia C, Moffat J, Xia L, Hiruki T, Collins S, Gallinger S, Bapat B (1998)
Overexpression of the nonpancreatic secretory group II PLA2 messenger RNA and protein in
colorectal adenomas from familial adenomatous polyposis patients. Cancer Res
58(3):500503
52. Mirtti T, Laine VJ, Hiekkanen H, Hurme S, Rowe O, Nevalainen TJ, Kallajoki M, Alanen K
(2009) Group IIA phospholipase A as a prognostic marker in prostate cancer: relevance to
clinicopathological variables and disease-specic mortality. APMIS 117(3):151161.
doi:10.1111/j.1600-0463.2008.00002.x
53. Boilard E, Lai Y, Larabee K, Balestrieri B, Ghomashchi F, Fujioka D, Gobezie R, Coblyn JS,
Weinblatt ME, Massarotti EM, Thornhill TS, Divangahi M, Remold H, Lambeau G, Gelb MH,
Arm JP, Lee DM (2010) A novel anti-inammatory role for secretory phospholipase A2 in
immune complex-mediated arthritis. EMBO Mol Med 2(5):172187. doi:10.1002/
emmm.201000072
54. Boudreau LH, Duchez AC, Cloutier N, Soulet D, Martin N, Bollinger J, Pare A, Rousseau M,
Naika GS, Levesque T, Laamme C, Marcoux G, Lambeau G, Farndale RW, Pouliot M,
Hamzeh-Cognasse H, Cognasse F, Garraud O, Nigrovic PA, Guderley H, Lacroix S, Thibault
L, Semple JW, Gelb MH, Boilard E (2014) Platelets release mitochondria serving as substrate
for bactericidal group IIA-secreted phospholipase A2 to promote inammation. Blood
124(14):21732183. doi:10.1182/blood-2014-05-573543
55. Miki Y, Yamamoto K, Taketomi Y, Sato H, Shimo K, Kobayashi T, Ishikawa Y, Ishii T,
Nakanishi H, Ikeda K, Taguchi R, Kabashima K, Arita M, Arai H, Lambeau G, Bollinger JM,
Hara S, Gelb MH, Murakami M (2013) Lymphoid tissue phospholipase A2 group IID resolves
contact hypersensitivity by driving antiinammatory lipid mediators. J Exp Med 210(6):1217
1234. doi:10.1084/jem.20121887
56. von Allmen CE, Schmitz N, Bauer M, Hinton HJ, Kurrer MO, Buser RB, Gwerder M,
Muntwiler S, Sparwasser T, Beerli RR, Bachmann MF (2009) Secretory phospholipase A2-IID
is an effector molecule of CD4+CD25+ regulatory T cells. Proc Natl Acad Sci U S A
106(28):1167311678. doi:10.1073/pnas.0812569106
40 M. Murakami and Y. Taketomi
71. Henderson WR Jr, Oslund RC, Bollinger JG, Ye X, Tien YT, Xue J, Gelb MH (2011) Blockade
of human group X secreted phospholipase A2 (GX-sPLA2)-induced airway inammation and
hyperresponsiveness in a mouse asthma model by a selective GX-sPLA2 inhibitor. J Biol Chem
286(32):2804928055. doi:10.1074/jbc.M111.235812
72. Lai Y, Oslund RC, Bollinger JG, Henderson WR Jr, Santana LF, Altemeier WA, Gelb MH,
Hallstrand TS (2010) Eosinophil cysteinyl leukotriene synthesis mediated by exogenous
secreted phospholipase A2 group X. J Biol Chem 285(53):4149141500. doi:10.1074/jbc.
M110.153338
73. Kelvin AA, Degousee N, Banner D, Stefanski E, Leomicronn AJ, Angoulvant D, Paquette SG,
Huang SS, Danesh A, Robbins CS, Noyan H, Husain M, Lambeau G, Gelb M, Kelvin DJ,
Rubin BB (2014) Lack of group X secreted phospholipase A2 increases survival following
pandemic H1N1 inuenza infection. Virology 454-455:7892. doi:10.1016/j.virol.2014.01.030
74. Hallstrand TS, Lai Y, Ni Z, Oslund RC, Henderson WR Jr, Gelb MH, Wenzel SE (2011)
Relationship between levels of secreted phospholipase A2 groups IIA and X in the airways and
asthma severity. Clin Exp Allergy 41(6):801810. doi:10.1111/j.1365-2222.2010.03676.x
75. Watanabe K, Fujioka D, Saito Y, Nakamura T, Obata JE, Kawabata K, Watanabe Y, Mishina H,
Tamaru S, Hanasaki K, Kugiyama K (2012) Group X secretory PLA2 in neutrophils plays a
pathogenic role in abdominal aortic aneurysms in mice. Am J Physiol Heart Circ Physiol
302(1):H95H104. doi:10.1152/ajpheart.00695.2011
76. Fujioka D, Saito Y, Kobayashi T, Yano T, Tezuka H, Ishimoto Y, Suzuki N, Yokota Y, Nakamura
T, Obata JE, Kanazawa M, Kawabata K, Hanasaki K, Kugiyama K (2008) Reduction in myo-
cardial ischemia/reperfusion injury in group X secretory phospholipase A2-decient mice.
Circulation 117(23):29772985. doi:10.1161/CIRCULATIONAHA.107.743997
77. Ait-Oufella H, Herbin O, Lahoute C, Coatrieux C, Loyer X, Joffre J, Laurans L, Ramkhelawon
B, Blanc-Brude O, Karabina S, Girard CA, Payre C, Yamamoto K, Binder CJ, Murakami M,
Tedgui A, Lambeau G, Mallat Z (2013) Group X secreted phospholipase A2 limits the develop-
ment of atherosclerosis in LDL receptor-null mice. Arterioscler Thromb Vasc Biol 33(3):466
473. doi:10.1161/ATVBAHA.112.300309
78. Zack M, Boyanovsky BB, Shridas P, Bailey W, Forrest K, Howatt DA, Gelb MH, de Beer FC,
Daugherty A, Webb NR (2011) Group X secretory phospholipase A2 augments angiotensin
II-induced inammatory responses and abdominal aortic aneurysm formation in apoE-
deficient mice. Atherosclerosis 214(1):5864. doi:10.1016/j.atherosclerosis.2010.08.054
79. Sato H, Isogai Y, Masuda S, Taketomi Y, Miki Y, Kamei D, Hara S, Kobayashi T, Ishikawa Y,
Ishii T, Ikeda K, Taguchi R, Ishimoto Y, Suzuki N, Yokota Y, Hanasaki K, Suzuki-Yamamoto
T, Yamamoto K, Murakami M (2011) Physiological roles of group X-secreted phospholipase
A2 in reproduction, gastrointestinal phospholipid digestion, and neuronal function. J Biol
Chem 286(13):1163211648. doi:10.1074/jbc.M110.206755
80. Li X, Shridas P, Forrest K, Bailey W, Webb NR (2010) Group X secretory phospholipase A2
negatively regulates adipogenesis in murine models. FASEB J 24(11):43134324. doi:10.1096/
fj.10-154716
81. Shridas P, Bailey WM, Talbott KR, Oslund RC, Gelb MH, Webb NR (2011) Group X secretory
phospholipase A2 enhances TLR4 signaling in macrophages. J Immunol 187(1):482489.
doi:10.4049/jimmunol.1003552
82. Escofer J, Jemel I, Tanemoto A, Taketomi Y, Payre C, Coatrieux C, Sato H, Yamamoto K,
Masuda S, Pernet-Gallay K, Pierre V, Hara S, Murakami M, De Waard M, Lambeau G, Arnoult
C (2010) Group X phospholipase A2 is released during sperm acrosome reaction and controls
fertility outcome in mice. J Clin Invest 120(5):14151428. doi:10.1172/JCI40494
83. Escofer J, Pierre VJ, Jemel I, Munch L, Boudhraa Z, Ray PF, De Waard M, Lambeau G,
Arnoult C (2011) Group X secreted phospholipase A2 specically decreases sperm motility in
mice. J Cell Physiol 226(10):26012609. doi:10.1002/jcp.22606
84. Yamamoto K, Taketomi Y, Isogai Y, Miki Y, Sato H, Masuda S, Nishito Y, Morioka K, Ishimoto
Y, Suzuki N, Yokota Y, Hanasaki K, Ishikawa Y, Ishii T, Kobayashi T, Fukami K, Ikeda K,
Nakanishi H, Taguchi R, Murakami M (2011) Hair follicular expression and function of group
42 M. Murakami and Y. Taketomi
Shuntaro Hara
3.1 Introduction
Glycerophospholipids
PLA2
Arachidonic Acid
PGH2
PGES PGDS
mPGES-1
PGIS TXS
mPGES-2 H-PGDS PGFS
cPGES L-PGDS
Fig. 3.1 Prostanoid biosynthetic pathway. Arachidonic acid released from membrane glycero-
phospholipids by phospholipase A2 (PLA2) enzymes is then supplied to either of the two cyclooxy-
genase (COX) isozymes, COX-1 or COX-2. The COX metabolite prostaglandin (PG) H2 is then
converted to each prostanoid [PGE2, PGD2, PGF2, PGI2, and thromboxane (TX) A2] by specific
PG terminal synthases
3.2.1 mPGES-1
mPGES-1 was identified as the first PGES by Jakobsson et al. [6]. Murakami et al.
also cloned rat and mouse orthologues of this protein and showed that this enzyme
is identical to a membrane-associated PGES, which our group had originally
detected in lipopolysaccharide (LPS)-stimulated macrophages [7]. mPGES-1 con-
sists of 152 or 153 amino acids and belongs to the MAPEG (membrane-associated
proteins involved in eicosanoid and glutathione metabolism) family. Among PGES
enzymes, only mPGES-1 is markedly induced by pro-inflammatory stimuli, is
downregulated by antiinflammatory glucocorticoids, and is functionally coupled
with COX-2 in marked preference to COX-1. Because both COX-2 and mPGES-1
are present in the perinuclear membrane, colocalization of these two enzymes in the
same subcellular compartment may allow efficient transfer of the unstable substrate
PGH2 between them. Steady-state expression of mPGES-1 in normal tissues is very
low. Induction of mPGES-1 expression has been observed in various processes in
which COX-2-derived PGE2 has been shown to play a critical role, such as inflam-
mation, fever, pain, tissue repair, and cancer [5, 8].
46
mPGES-1 KO mice were established in 2002 and have been used to demonstrate
the involvement of mPGES-1 in various kinds of diseases [9, 10]. mPGES-1 KO
mice are generally protected against a variety of inflammatory disease phenotypes,
including collagen- or anti-collagen antibody-induced arthritis, LPS-induced bone
loss, and antigen-induced edema [1012]. In a collagen-induced arthritis model,
reduced inflammation in the mPGES-1 KO mice was associated with a failure to
produce antibody against type II collagen, suggesting that mPGES-1 has a role in
the development of a humoral immune response [13]. We further found that
mPGES-1 KO mice displayed significantly reduced accumulation of exudate and
impaired leukocyte migration into the pleural cavity during carrageenan-induced
paw edema formation [5]. The formation of inflammatory granulation tissue and
attendant angiogenesis in the dorsum induced by subcutaneous implantation of a
cotton thread were also significantly reduced in mPGES-1 KO mice [11].
Furthermore, mPGES-1 KO mice exhibited reductions in pain, fever, and other
symptoms associated with inflammatory diseases [14, 15]. It is noteworthy that
genetic deletion of mPGES-1 in mice does not adversely affect cardiovascular func-
tions. These studies suggested the possibility that pharmacological targeting of
mPGES-1 may ultimately prove to less toxic and perhaps more effective than the
traditional NSAIDs for controlling acute inflammatory diseases. New drug candi-
dates have recently been developed for targeting mPGES-1. Some of them have
been shown to suppress inflammatory reactions in animal models. Xu et al. reported
that when tested in the guinea pig and a knock-in mouse expressing human mPGES-1,
MF63 inhibited LPS-induced pyresis, hyperalgesia, and iodoacetate-induced osteo-
arthritic pain, although it did not cause gastrointestinal toxic effects [16]. Leclerc
et al. reported that their compound II attenuated both the acute and delayed inflam-
matory responses in rat adjuvant-induced arthritis [17].
The role of mPGES-1-derived PGE2 in brain diseases, including ischemic injury
and several neurodegenerative diseases, has also been established in models using
mPGES-1 KO mice. In mPGES-1 KO mice, infarction, edema, and apoptotic cell
death in the cortex after ischemia were all reduced compared with those in wild-
type (WT) mice [18]. The behavioral neurological dysfunctions observed after isch-
emia in WT mice were also significantly ameliorated in mPGES-1 KO mice.
Furthermore, mPGES-1 KO mice had less severe symptoms of experimental auto-
immune encephalomyelitis [19]. We further found that mPGES-1 deletion reduced
the accumulation of microglia around senile plaques and attenuated learning impair-
ments in Tg2576 mice, a transgenic Alzheimers disease mouse model [20].
It has also been shown that whereas the selective inhibition or KO of COX-2
accelerated thrombogenesis and elevated blood pressure in mice, the deletion of
mPGES-1 had no such effect and restrained atherogenesis, the proliferative response
to vascular injury, and angiotensin-induced aortic aneurysm formation in mice [21
23]. mPGES-1 inhibitors are thus expected to be applicable as therapeutic agents for
inflammatory neurological or cardiovascular diseases.
In addition to COX-2, mPGES-1 levels are increased within a number of human
cancers, and the tumorigenic potential of mPGES-1 has been suggested by several
48 S. Hara
3.2.2 mPGES-2
mPGES-2 was initially purified from a microsomal fraction of bovine heart, and
cDNAs encoding human and monkey homologues were subsequently identified
[31]. mPGES-2 is a 41-kDa protein consisting of 378 to 385 amino acids that is
structurally distinct from mPGES-1. mPGES-2 has an N-terminal hydrophobic
domain, followed by a glutaredoxin/thioredoxin homology region, in which the
consensus thioredoxin homology sequence of Cys110-X-X-Cys113 is present.
mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteo-
lytic removal of the N-terminal hydrophobic domain leads to the formation of a
mature cytosolic enzyme. When transfected in several cell lines, mPGES-2 is cou-
pled with both COX-1 and COX-2, leading to PGE2 production [32]. The transcript
for mPGES-2 is more abundantly distributed in the brain, heart, skeletal muscle,
kidney, and liver than in other tissues; this differs from the expression profile of
mPGES-1. mPGES-2 expression is rather constitutive in various cells and tissues
and is not elevated appreciably during inflammation or tissue damage. This finding
suggests that the operation of these two enzymes is not always redundant, but rather
that both enzymes exhibit tissue-specific functions.
mPGES-2 KO mice showed no specific phenotype and no alteration in PGE2
levels in several tissues (including liver, kidney, heart, and brain) or in LPS-
stimulated macrophages [33]. These results suggest that mPGES-2 is not involved
in PGE2 synthesis under the physiological and pathological conditions tested thus
far. However, the possibility of tissue-specific or particular pathological roles of
mPGES-2 has not yet been ruled out.
3.2.3 cPGES
Our group purified cPGES as a cytosolic form of PGES from LPS-treated rat brains,
and sequence analysis of the 23-kDa purified protein revealed that it is identical to
the heat shock protein 90 (Hsp90)-associated protein p23, which had been origi-
nally implicated as a cofactor for the molecular chaperone function of Hsp90 [34].
cPGES is directly associated with and phosphorylated by protein kinase CK2. In
activated cells, CK2-directed phosphorylation of cPGES occurs in parallel with
increased cPGES enzymatic activity and PGE2 production, and these processes are
facilitated by interaction with Hsp90 [35].
cPGES is expressed ubiquitously and in abundance in the cytosol of various tis-
sues and cells. Cotransfection and antisense experiments have indicated that cPGES
is capable of converting COX-1-, but not COX-2-, derived PGH2 to PGE2 in cells,
particularly during the immediate PGE2 biosynthetic response elicited by Ca2+-
evoked stimuli [34]. Localization of cPGES in the cytosol may allow coupling with
proximal COX-1 in the ER in preference to distal COX-2 in the perinuclear enve-
lope, although other regulatory mechanisms could also be involved.
50 S. Hara
PGD synthase (PGDS) catalyzes the isomerization of PGH2 to PGD2 and occurs in
two distinct types [3]. One is hematopoietic PGDS (H-PGDS), which is found in
mast cells, Th2 cells, and microglia, and the other is lipocalin-type PGDS (L-PGDS),
which is localized in the brain, male genital organs, and cardiovascular tissues,
including the human heart.
3.3.1 H-PGDS
H-PGDS was originally purified from rat spleen by Christ-Hazelhof and Nugteren
as a 26-kDa, cytosolic, monomeric glutathione-requiring enzyme [40]. Sequences
of full-length cDNAs for the human and mouse H-PGDS were obtained by Kanaoka
et al. [41]. The cDNA encodes a protein composed of 199 amino acid residues,
which is identified as a vertebrate homologue of class glutathione-S-transferase.
The N-terminal methionine is cleaved from the mature protein. The X-ray crystal
structure analysis of the human recombinant H-PGDS revealed that this enzyme is
a 45- to 49-kDa homodimeric protein that binds one molecule of reduced glutathi-
one per monomer and one Mg2+ ion per dimer [41].
H-PGDS-derived PGD2 is involved in a variety of allergic and nonallergic disor-
ders. H-PGDS is expressed in infiltrated leukocytes in the nasal mucosa of patients
with polyposis or allergic rhinitis, and in necrotic muscle fibers of patients with
Duchennes muscular dystrophy and polymyositis. In addition to gene deletion of
3 Prostaglandin Terminal Synthases as Novel Drug Targets 51
3.3.2 L-PGDS
L-PGDS was isolated from the rat brain as a 26-kDa glutathione-independent PGDS
[48]. The cDNA for L-PGDS was isolated from the rat brain by Urade et al. [49] and
subsequently from humans and many other mammalian species, as well as from
nonmammals such as chickens, frogs, and fish. The L-PGDS cDNA encodes a pro-
tein composed of 189 to 190 amino acid residues. L-PGDS is posttranslationally
52 S. Hara
PGE2 and PGF2 were the first prostanoids to be isolated from human semen.
However, despite the long history of research on the physiological and pathological
functions of PGF2, the identity of PGF synthase (PGFS), which catalyzes PGH2 to
PGF2in vivo, is unclear. Some enzymes belonging to the aldo-keto reductase (AKR)
superfamily have been shown to exhibit PGFS activity [3, 57].
3 Prostaglandin Terminal Synthases as Novel Drug Targets 53
PGI synthase (PGIS) catalyzes the isomerization of PGH2 to PGI2. Ullrich and
coworkers were the first to provide spectral evidence that PGIS is a cytochrome
P450, but unlike most P450s, PGIS does not require NADPH and O2 as cosubstrates
[58]. We collaborated with Ullrichs research group to purify and characterize PGIS
from bovine aorta, and then isolated cDNA for PGIS and designated this enzyme as
CYP8A1 in the P450 family [59]. The PGIS cDNA encodes a 56-kDa protein con-
sisting of 500 to 501 amino acid residues. In addition to mPGES-1, PGIS is func-
tionally coupled with COX-2 in marked preference to COX-1, although this enzyme
is constitutively expressed in vascular cells [60].
PGIS-derived PGI2 is a strong vasodilator that inhibits the growth of vascular
smooth muscle cells and is also the most potent endogenous inhibitor of platelet
aggregation. Therefore, it has been considered to play an important role in cardio-
vascular diseases. We showed that overexpression of PGIS prevents neointimal for-
mation after carotid balloon injury in rats [61]. Iwai et al. identified a repeat
polymorphism in the promoter region of the human PGIS gene that is associated
with promoter activity [62]. They further showed that this repeat polymorphism
might be a risk factor for higher pulse pressure and consequently a risk factor for
systolic hypertension in the Japanese population. The blood pressure of PGIS KO
mice was significantly higher than that of WT mice [63]. Furthermore, PGIS KO
mice developed ischemic renal disorders, including nephrosclerosis and renal
infarction.
A role of PGIS in inflammatory disease and carcinogenesis has also been sug-
gested. Pulmonary-specific PGIS-overexpressing mice were chemoprotected from
developing lung tumors in a smoke-exposure model [64].
3.6 TX Synthase
TX synthase (TXS) catalyzes the isomerization of PGH2 to TXA2 with parallel pro-
duction of malondialdehyde and 12-hydroxyheptadecatrienoic acid (12-HHT). In
addition to PGIS, Ullrich and coworkers purified and characterized TXS from
human platelets as a cytochrome P450 enzyme [65]. The cDNA for TXS was iso-
lated from human platelets [66]. The TXS cDNA encodes a 60-kDa protein com-
posed of 533 amino acid residues. Although both TXS and PGIS belong to the
cytochrome P450 family, they share only 15 % sequence identity. TXS was desig-
nated as CYP5 in the P450 family.
TXA2 is a potent stimulator of platelet activation and aggregation and vascular
constriction. Gene deletion of TXS has been shown to cause a mild hemostatic
defect and to protect mice against arachidonate-induced shock and death [67]. TXS
inhibitors were originally considered to be promising antiplatelet agents, but clini-
cal trials of various inhibitors yielded unsatisfactory results when compared with
54 S. Hara
low-dose aspirin. Nonetheless, TXS inhibitors have been evaluated for other dis-
eases involving TXA2, such as bronchial asthma and pulmonary hypertension [68].
Very recently, 12-HHT, the other TXS-derived metabolite, was found to act on
BLT2 receptors [69]. Specifically, Liu et al. reported that 12-HHT promoted
epidermal wound healing by accelerating keratinocyte migration via the BLT2
receptors [70].
3.7 Conclusion
It has become apparent there are multiple PG terminal enzymes in mammalian cells
and that distinct PG terminal enzymes may control the spatial and temporal produc-
tion of prostanoids in different pathophysiological events in particular tissues and
cells. Further investigation into the biochemical properties, transcriptional regula-
tion, and in vitro and in vivo functions of each PG terminal enzyme may illuminate
the potential utility of clinically targeting these enzymes.
References
1. Smith WL, DeWitt DL, Garavito RM (2000) Cyclooxygenases: structural, cellular, and
molecular biology. Annu Rev Biochem 69:145182
2. Kudo I, Murakami M (2002) Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat
69:358
3. Smith WL, Urade Y, Jakobsson PJ (2011) Enzymes of the cyclooxygenase pathways of
prostanoid biosynthesis. Chem Rev 111:58215865
4. Hara S, Kudo I (2006) COX-2 inhibitors and the risk of cardiovascular events. Jpn Med Assoc
J 49:276278
5. Hara S, Kamei D, Sasaki Y et al (2010) Prostaglandin E synthases: understanding their
pathophysiological roles through mouse genetic models. Biochimie 92:651659
6. Jakobsson PJ, Thorn S, Morgenstern R et al (1999) Identification of human prostaglandin E
synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential
novel drug target. Proc Natl Acad Sci U S A 96:72207225
7. Murakami M, Naraba H, Tanioka T et al (2000) Regulation of prostaglandin E2 biosynthesis
by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclo-
oxygenase-2. J Biol Chem 275:3278332792
8. Samuelsson B, Mogenstern R, Jakobsson PJ (2007) Membrane prostaglandin E synthase-1: a
novel therapeutic target. Pharmacol Rev 59:207224
9. Uematsu S, Matsumoto M, Takeda K et al (2002) Lipopolysaccharide-dependent prostaglan-
din E2 production is regulated by the glutathione-dependent prostaglandin E2 synthase gene
induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. J Immunol 168:58115816
10. Trebino CE, Stock JL, Gibbons CP et al (2003) Impaired inflammatory and pain responses in
mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci U S A
100:90449049
11. Kamei D, Yamakawa K, Takegoshi Y et al (2004) Reduced pain hypersensitivity and
inflammation in mice lacking microsomal prostaglandin E synthase-1. J Biol Chem
279:3368433695
3 Prostaglandin Terminal Synthases as Novel Drug Targets 55
4.1 Introduction
Fig. 4.1 Prostanoid synthesis and its receptors. PGH2 is produced from arachidonic acid by
COX-1/2, and is subsequently converted to different prostanoids by the action of their respective
synthases. Prostanoids act on specific receptors on the plasma membrane and elicit changes in the
levels of second messengers (cAMP or Ca2+)
roles and functional mechanisms of prostanoids at the molecular level [2]. It has
been known that prostanoids regulate fever responses at the time of organism infec-
tion. Recently, functional mechanisms of the fever response by prostanoids in the
CNS have been clarified, and it has also been shown that prostanoids are deeply
involved in the development of many neurodegenerative diseases including
Alzheimers disease. In this review, we summarize these new physiological and
pathophysiological roles and functional mechanisms of prostanoids in the CNS and
discuss their possibilities as therapeutic targets.
Then, how does the EP3 receptor in the POA induce a fever response? Nakamura
et al. showed that the EP3 receptor is expressed on the surface of neuronal cell bod-
ies in the POA [31]. Moreover, they revealed that most EP3 receptor-positive neu-
rons in the POA are also positive for glutamate decarboxylase 67, that is, they are
inhibitory GABAergic neurons. They also revealed that administration of the
GABAA agonist muscimol into the raphe nucleus abolishes the PGE2-induced fever
response [32]. Tsuchiya et al. isolated EP3-positive POA neurons from PGE2-
administered mice and examined PGE2-induced gene expression changes. They
revealed that PGE2 decreased the expression of the GABAA receptor in POA neu-
rons [33]. PGE2 is therefore thought to decrease GABAA receptor expression by
acting on EP3 receptors on POA neurons and to attenuate the inhibitory control of
GABAergic neurons to the raphe pallidus nucleus, leading to sympathetic nerve-
mediated thermogenesis (Fig. 4.2).
Interestingly, a recent report demonstrated that stress signals activate dorsome-
dial hypothalamic neurons, which provide direct glutamatergic input to sympathetic
premotor neurons in the medullary raphe region to drive thermogenesis [34]. Thus,
inflammation-induced and stress-induced thermogenesis share a common output
pathway but have very different input pathways (Fig. 4.2).
LPS activates innate immunity not only in peripheral tissues but also in the CNS
during bacterial infection [35]. Microglia are mainly responsible for the host defense
response in the CNS, but excessive activation of microglia induces a severe inflam-
matory state, leading to neurotoxicity [36]. Breyer and his colleagues revealed that
neuronal damage induced by intracerebral LPS injection is blocked by NSAIDs
treatment or EP2 receptor gene deficiency [37]. Moreover, they showed that EP2
receptor deficiency in microglia inhibits LPS-induced neuronal apoptosis in a co-
culture system of neurons and microglia [38]. Recently, Andreasson et al. revealed
that LPS induces the expression of the EP4 receptor in microglia. Moreover, they
showed that an EP4 selective agonist inhibits the LPS-induced inflammatory
response, and this inflammatory response is delayed in microglia-specific EP4-
deficient mice [39]. These results demonstrated that PGE2 produced by LPS stimuli
activates innate immunity via the EP2 receptor of microglia in the early phase, and
then inhibits the excessive inflammatory response by acting on the EP4 receptor in
the late phase (Fig. 4.3). Both EP2 and EP4 receptors couple to the Gs protein and
have similar signaling pathways: stimulation of adenylyl cyclase or the
Gs-independent -arrestin pathway [21, 40]. In this case, EP2 receptor signaling
induces neuroinflammation, whereas EP4 receptor signaling mediates antiinflam-
matory effects. It remains to be elucidated as to how such different outcomes are
elicited downstream of EP2 and EP4 receptors.
64 T. Inazumi and Y. Sugimoto
Fig. 4.3 Regulation of microglial activation by PGE2 in the inflammatory response. In the early
phase, LPS-induced PGE2 activates innate immunity via microglial EP2. In contrast, in the late
phase, EP4 expression is upregulated and EP4 inhibits inflammation
O OH
OH
2-Arachidonoylglycerol O
JZL184
MAGL (MAGL inhibitor)
COOH
Arachidonic acid
Cyclooxygenase NSAIDs
PGE synthase
O
COOH
PGE2 Neurodegenerative
diseases
OH OH
Fig. 4.4 Synthesis of arachidonic acid and PGE2 in the brain. In the brain, MAGL converts
2-arachidonoylgycerol to arachidonic acid, which is further oxidized into PGE2 by cyclooxygenase
and PGE synthase. The MAGL inhibitor JZL184 was found to decrease brain PGE2 levels and
ameliorate the neuropathological phenotypes of neurodegenerative diseases
4.6 Perspectives
References
1. Tai HH, Ensor CM, Tong M, Zhou H, Yan F (2002) Prostaglandin catabolizing enzymes.
Prostaglandins Other Lipid Mediat 68-69:483493
2. Sugimoto Y, Narumiya S (2007) Prostaglandin E receptors. J Biol Chem 282:1161311617
66 T. Inazumi and Y. Sugimoto
3. Andersen NH, Eggerman TL, Harker LA, Wilson CH, De B (1980) On the multiplicity of
platelet prostaglandin receptors. I. Evaluation of competitive antagonism by aggregometry.
Prostaglandins 19:711735
4. Gardiner PJ, Collier HO (1980) Specific receptors for prostaglandins in airways. Prostaglandins
19:819841
5. Hirata M, Hayashi Y, Ushikubi F, Yokota Y, Kageyama R, Nakanishi S, Narumiya S (1991)
Cloning and expression of cDNA for a human thromboxane A2 receptor. Nature
349:617620
6. Sugimoto Y, Namba T, Honda A, Hayashi Y, Negishi M, Ichikawa A, Narumiya S (1992)
Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol
Chem 267:64636466
7. Watabe A, Sugimoto Y, Honda A, Irie A, Namba T, Negishi M, Ito S, Narumiya S, Ichikawa
A (1993) Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E recep-
tor. J Biol Chem 268:2017520178
8. Honda A, Sugimoto Y, Namba T, Watabe A, Irie A, Negishi M, Narumiya S, Ichikawa A
(1993) Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J
Biol Chem 268:77597762
9. Katsuyama M, Nishigaki N, Sugimoto Y, Morimoto K, Negishi M, Narumiya S, Ichikawa A
(1995) The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern
blot analysis. FEBS Lett 372:151156
10. Hirata M, Kakizuka A, Aizawa M, Ushikubi F, Narumiya S (1994) Molecular characterization
of a mouse prostaglandin D receptor and functional expression of the cloned gene. Proc Natl
Acad Sci U S A 91:1119211196
11. Namba T, Oida H, Sugimoto Y, Kakizuka A, Negishi M, Ichikawa A, Narumiya S (1994)
cDNA cloning of a mouse prostacyclin receptor: multiple signaling pathways and expression
in thymic medulla. J Biol Chem 269:99869992
12. Sugimoto Y, Hasumoto K, Namba T, Irie A, Katsuyama M, Negishi M, Kakizuka A, Narumiya
S, Ichikawa A (1994) Cloning and expression of a cDNA for mouse prostaglandin F receptor.
J Biol Chem 269:13561360
13. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura K,
Nakamura M, Takano S, Nagata K (2001) Prostaglandin D2 selectively induces chemotaxis in
T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J
Exp Med 193:255261
14. Tang DG, Grossi IM, Tang KQ, Diglio CA, Honn KV (1995) Inhibition of TPA and
12(S)-HETE-stimulated tumor cell adhesion by prostacyclin and its stable analogs: rationale
for their antimetastatic effects. Int J Cancer 60:418425
15. Tabata H, Tanaka S, Sugimoto Y, Kanki H, Kaneko S, Ichikawa A (2002) Possible coupling of
prostaglandin E receptor EP1 to TRP5 expressed in Xenopus laevis oocytes. Biochem Biophys
Res Commun 298:398402
16. Ito S, Sakamoto K, Mochizuki-Oda N, Ezashi T, Miwa K, Okuda-Ashitaka E, Shevchenko VI,
Kiso Y, Hayaishi O (1993) Prostaglandin F2 receptor is coupled to Gq in cDNA-transfected
Chinese hamster ovary cells. Biochem Biophys Res Commun 200:756762
17. Dorn GW, Becker MW (1993) Thromboxane A2 stimulated signal transduction in vascular
smooth muscle. J Pharmacol Exp Ther 265:447456
18. Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A,
Narumiya S (1993) Alternative splicing of C-terminal tail of prostaglandin E receptor subtype
EP3 determines G-protein specificity. Nature 365:166170
19. Hatae N, Yamaoka K, Sugimoto Y, Negishi M, Ichikawa A (2002) Augmentation of receptor-
mediated adenylyl cyclase activity by Gi-coupled prostaglandin receptor subtype EP3 in a G
subunit-independent manner. Biochem Biophys Res Commun 290:162168
20. Yamaoka K, Yano A, Kuroiwa K, Morimoto K, Inazumi T, Hatae N, Tabata H, Segi-Nishida
E, Tanaka S, Ichikawa A, Sugimoto Y (2009) Prostaglandin EP3 receptor superactivates ade-
nylyl cyclase via the Gq/PLC/Ca2+ pathway in a lipid raft-dependent manner. Biochem Biophys
Res Commun 389:678682
4 Pathophysiological Roles of Prostanoid Receptors in the Central Nervous System 67
21. Buchanan FG, Gorden DL, Matta P, Shi Q, Matrisian LM, DuBois RN (2006) Role of -arrestin
1 in the metastatic progression of colorectal cancer. Proc Natl Acad Sci U S A
103:14921497
22. Kluger MJ (1991) Fever: role of pyrogens and cryogens. Physiol Rev 71:93127
23. Itami T, Ema M, Kanoh S (1986) Antipyretic mechanism of indomethacin in rabbits.
J Pharmacobiodyn 9:271275
24. Cao C, Matsumura K, Yamagata K, Watanabe Y (1997) Involvement of cyclooxygenase-2 in
LPS-induced fever and regulation of its mRNA by LPS in the rat brain. Am J Physiol
272:R1712R1725
25. Sehic E, Szkely M, Ungar AL, Oladehin A, Blatteis CM (1996) Hypothalamic prostaglandin
E2 during lipopolysaccharide-induced fever in guinea pigs. Brain Res Bull 39:391399
26. Saigusa T, Iriki M (1988) Regional differentiation of sympathetic nerve activity during fever
caused by intracerebroventricular injection of PGE2. Pflugers Arch 411:121125
27. Ushikubi F, Segi E, Sugimoto Y, Murata T, Matsuoka T, Kobayashi T, Hizaki H, Tuboi K,
Katsuyama M, Ichikawa A, Tanaka T, Yoshida N, Narumiya S (1998) Impaired febrile response
in mice lacking the prostaglandin E receptor subtype EP3. Nature 395:281284
28. Sugimoto Y, Shigemoto R, Namba T, Negishi M, Mizuno N, Narumiya S, Ichikawa A (1994)
Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse
nervous system. Neuroscience 62:919928
29. Leibbrandt A, Penninger JM (2008) RANK/RANKL: regulators of immune responses and
bone physiology. Ann N Y Acad Sci 1143:123150
30. Hanada R, Leibbrandt A, Hanada T, Kitaoka S, Furuyashiki T, Fujihara H, Trichereau J,
Paolino M, Qadri F, Plehm R, Klaere S, Komnenovic V, Mimata H, Yoshimatsu H, Takahashi
N, von Haeseler A, Bader M, Kilic SS, Ueta Y, Pifl C, Narumiya S, Penninger JM (2009)
Central control of fever and female body temperature by RANKL/RANK. Nature
462:505509
31. Nakamura K, Kaneko T, Yamashita Y, Hasegawa H, Katoh H, Ichikawa A, Negishi M (1999)
Immunocytochemical localization of prostaglandin EP3 receptor in the rat hypothalamus.
Neurosci Lett 260:117120
32. Nakamura K, Matsumura K, Kaneko T, Kobayashi S, Katoh H, Negishi M (2002) The rostral
raphe pallidus nucleus mediates pyrogenic transmission from the preoptic area. J Neurosci
22:46004610
33. Tsuchiya H, Oka T, Nakamura K, Ichikawa A, Saper CB, Sugimoto Y (2008) Prostaglandin E2
attenuates preoptic expression of GABAA receptors via EP3 receptors. J Biol Chem
283:1106411071
34. Kataoka N, Hioki H, Kaneko T, Nakamura K (2014) Psychological stress activates a dorsome-
dial hypothalamus-medullary raphe circuit driving brown adipose tissue thermogenesis and
hyperthermia. Cell Metab 20:346358
35. Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT (2007) Systemic
LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55:453462
36. Lehnardt S (2010) Innate immunity and neuroinflammation in the CNS: the role of microglia
in Toll-like receptor-mediated neuronal injury. Glia 58:253263
37. Montine TJ, Milatovic D, Gupta RC, Valyi-Nagy T, Morrow JD, Breyer RM (2002) Neuronal
oxidative damage from activated innate immunity is EP2 receptor-dependent. J Neurochem
83:463470
38. Shie FS, Montine KS, Breyer RM, Montine TJ (2005) Microglial EP2 is critical to neurotoxic-
ity from activated cerebral innate immunity. Glia 52:7077
39. Shi J, Johansson J, Woodling NS, Wang Q, Montine TJ, Andreasson K (2010) The prostaglan-
din E2 E-prostanoid 4 receptor exerts anti-inflammatory effects in brain innate immunity.
J Immunol 184:72077218
40. Chun KS, Lao HC, Trempus CS, Okada M, Langenbach R (2009) The prostaglandin receptor
EP2 activates multiple signaling pathways and -arrestin1 complex formation during mouse
skin papilloma development. Carcinogenesis 30:16201627
41. Murakami M, Kudo I (2002) Phospholipase A2. J Biochem 131:285292
68 T. Inazumi and Y. Sugimoto
42. Rosenberger TA, Villacreses NE, Contreras MA, Bonventre JV, Rapoport SI (2003) Brain lipid
metabolism in the cPLA2 knockout mouse. J Lipid Res 44:109117
43. Nomura DK, Morrison BE, Blankman JL, Long JZ, Kinsey SG, Marcondes MC, Ward AM,
Hahn YK, Lichtman AH, Conti B, Cravatt BF (2011) Endocannabinoid hydrolysis generates
brain prostaglandins that promote neuroinflammation. Science 334:809813
44. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010) Mechanisms underlying
inflammation in neurodegeneration. Cell 140:918934
45. Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot S, Vila M, Jackson-Lewis V,
Przedborski S (2003) Cyclooxygenase-2 is instrumental in Parkinsons disease neurodegen-
eration. Proc Natl Acad Sci U S A 100:54735478
46. McKee AC, Carreras I, Hossain L, Ryu H, Klein WL, Oddo S, LaFerla FM, Jenkins BG,
Kowall NW, Dedeoglu A (2008) Ibuprofen reduces A, hyperphosphorylated tau and memory
deficits in Alzheimer mice. Brain Res 1207:225236
47. Piro JR, Benjamin DI, Duerr JM, Pi Y, Gonzales C, Wood KM, Schwartz JW, Nomura DK,
Samad TA (2012) A dysregulated endocannabinoid-eicosanoid network supports pathogenesis
in a mouse model of Alzheimers disease. Cell Rep 1:617623
Chapter 5
Lipoxygenases: A Chronological Perspective
on the Synthesis of S and R Fatty Acid
Hydroperoxides
Alan R. Brash
the structurefunction of LOX enzymes [2]. The perspective here is on the LOX-
catalyzed synthesis of R and S conguration fatty acid hydroperoxides. In historical
context, I describe discovery of S- and R-LOX enzymes and nally explain how we
approached our current studies on the vital role of 12R-LOX in mammalian skin.
There are numerous and occasionally very abundant LOX enzymes in plants, and all
the early history of the LOX enzyme familyas well as many of the concepts of
their mechanism of actionstemmed from studies with the enzymes in soybeans.
Originally detected in the late 1920s and 1930s as an oxidizing activity in soybean
our [3], and named lipoxidase in 1932 [4], by 1940 it was recognized as an activ-
ity that reacted polyunsaturated fatty acids (PUFA) with molecular oxygen (O2) to
form fatty acid peroxides [5, 6]. By 1947 the work of Nobel laureate Hugo Theorell
with Ralph Holman and ke keson resulted in purication and crystallization of
the main enzyme in soybean our [7], now known as the L-1 isozyme or soybean
LOX-1. It is commercially available and is by far the most studied and most utilized
LOX enzyme. It happens to use arachidonic acid, EPA, and DHA as excellent sub-
strates, along with the fatty acids it would encounter in plants (linoleic, -linolenic,
and -linolenic acids), so this greatly enhances its utility as a model LOX enzyme.
The Theorell preparation of soybean LOX-1 reacted with linoleic acid at 330
turnovers/s [7], around the top attainable rate for this enzyme, which is one of the
most efcient known. In fact, for reasons that currently remain obscure, the best that
many other LOX enzymes can manage is 10100 times or even 1000 times slower
than this. All the mammalian enzymes fall into the relatively slow category.
The reaction of soybean LOX with linoleic acid was outlined by Bergstrm and
Holman in 1948 [8], and noting the conjugation of the double bonds in association
with the substrate oxygenation and the likeness to the reactions of lipid peroxida-
tion, which Bergstrm earlier had helped characterize [9]. This similarity to lipid
peroxidation for a time raised a somewhat misleading concept. It was recognized
that lipid peroxidation requires generation of a radical to start the process and then
it is self-generating [10]: the radical is not lost as more and more molecules are
peroxidized, hence the term autoxidation. Around the 1940s and early 1950s it
was considered possible that the soybean lipoxygenase generated a radical and then
the process continued out of enzymatic control. A study of the enzymology by Al
Tappel, Boyer, and Lundberg put paid to this misconception: they showed that
5 Lipoxygenases: A Chronological Perspective on the Synthesis of S and R Fatty 71
soybean LOX could follow MichaelisMenten kinetics and that the enzyme initiates
each turnover of substrate to product [11]. Still, in modern days there remains some-
thing partly akin to the old concept: LOX enzymes require activation (generally an
oxidation of the resting ferrous enzyme (Fe2+) to ferric (Fe3+)) and only then do
repetitive cycles of turnover occur (see [12, 13]). The difference in this modern
understanding is that once activated, the LOX enzyme remains in control of each
turnover of substrate to product.
The nding in 1971 that aspirin inhibits prostaglandin synthesis and the known
effects of aspirin on blood platelet function [22] directed attention to the metabo-
lism of arachidonic acid in platelets. A brilliant series of experiments by Mats
Hamberg, Svensson and Samuelsson at the Karolinska Institutet not only revealed
the existence of thromboxane A2 of the cyclooxygenase pathway and its powerful
pro-aggregatory effect on platelets [23], they also uncovered the rst LOX enzyme
in animal tissues, arachidonate 12S-lipoxygenase (product of the ALOX12 gene)
[24]; platelet 12-LOX was discovered independently by Diederik Nugteren of
Unilever [25]. We know from the work of Shozo Yamamotos group and others that
this platelet-type 12-LOX enzyme is widespread in many tissues [26, 27].
At much the same time Samuel Rapoports group in Berlin was investigating a
peroxidizing enzyme in peripheral blood reticulocytes induced by severe anemia, and
reporting that it could oxygenate PUFA esteried in phospholipids [28]; this enzyme
was identied as an arachidonate 15S-lipoxygenase [29, 30], and is now designated as
the ALOX15 gene product, 15-LOX-1. Most mammals express the ALOX15 gene
primarily as a 12-LOX, given the helpful designation leukocyte-type 12-LOX by
Shozo Yamamoto [26, 27], and the enzyme is often referred to as a 12/15-LOX.
By 1976 the search for lipid mediators in mammalian leukocytes led to the dis-
covery by Pierre Borgeat, Hamberg and Samuelsson at the Karolinska Institutet of
5S-lipoxygenase (from the ALOX5 gene) [31]. Soon the role of 5-LOX as the rst
committed step in leukotriene synthesis was revealed, along with the identity of the
peptide leukotrienes as the slow-reacting substance of anaphylaxis, a major lipid
mediator with bronchiolar contractile activity in asthma [3234]. The development
of leukotriene receptor antagonists and their clinical efcacy conrmed the impor-
tant role of 5-LOX and the leukotrienes in asthma and other inammatory diseases
[35]. In more recent times the antitheses of these actions have also been appreciated
with the recognition by Charles Serhan and colleagues of the involvement of vari-
ous LOX products (lipoxins, resolvins, protectins) in promoting the resolution
phase of inammation [36].
For 20 years after the discovery in the mid-1970s of 12-LOX, 15-LOX-1, and
5-LOX, these three enzymes stood alone at the forefront of lipoxygenase patho-
physiological studies in mammals [26, 37]. They each synthesize an S-conguration
fatty acid hydroperoxide, the same as all the other known LOX enzymes of plants
and animals at the time. It took until the late 1990s to recognize that three additional
LOX genes occur in humans and other mammals: 15-LOX-2, 12R-LOX, and
epidermal-LOX3 (eLOX3) (Fig. 5.1).
5 Lipoxygenases: A Chronological Perspective on the Synthesis of S and R Fatty 73
In a curious nding at the time, in 1979 van Os, Rijke-Schilder, and Vliegenthart
reported that the soybean LOX-2 enzyme formed not only 13S-HPODE from lin-
oleic acid (same as the well-known soybean LOX-1 enzyme), but also 9R-HPODE,
the rst description of a specic LOX-catalyzed R-hydroperoxide formation, and
later understood in terms of the fundamental basis of S and R stereocontrol in lipox-
ygenases (see following).
My interest in R-LOX biochemistry was sparked by a couple of unexpected
occurrences. The rst eye-opening event was a telephone conversation in 1985 with
Gordon Bundy, a chemist at the Upjohn Company in Kalamazoo, Michigan. As an
aside to the topic of our conversation on leukotrienes, Gordon told me he had uncov-
ered R-HPETE synthesis from an unusual source and he asked me if I had ever
heard of a lipoxygenase that synthesized a fatty acid R-hydroperoxide and did I
know of any reason this was not possible? I replied that it did seem possible, noting
that the cyclooxygenase enzyme initiates prostaglandin synthesis with an
11R-oxygenation of arachidonic acid [38]. Gordon Bundys discovery, reported in
the Journal of Biological Chemistry in 1986, described the synthesis of 8R-HPETE
from arachidonic acid in the Caribbean coral Pseudoplexaura porosa [39].
My second introduction to R-LOX activity stemmed from the persistent letter
writing of Laurent Meijer of the CNRS research laboratories in Roscoff, France,
asking me to prepare 8R-HETE and 8S-HETE so he could test their activities in
inducing the reinitiation of meiosis in starsh oocytes. Laurent had identied ara-
chidonic acid and particularly 8-HETE as potent inducers of oocyte maturation
(unpublished at the time, but related to the results of Meijer et al. [40]), and he had
noticed that we resolved the 8-HETE enantiomers in a study of the mechanisms of
autoxidation [41]. I prepared 8R- and 8S-HETE, and Laurent identied 8R-HETE as
the active principle in inducing starsh oocyte maturation; I characterized the
synthesis of specically 8R-HETE in starsh oocyte homogenates, published in the
Journal of Biological Chemistry in 1986 [42]. Later, Molly Hughes provided much
circumstantial evidence for the existence of an 8R-HETE-specic G protein-coupled
receptor (GPCR) in starsh oocytes [43].
Just before the Bundy and Meijer publications, E.J. Corey, Peter Lansbury, and
Yasuji Yamada described the synthesis of a prostaglandin-related cyclopentenone
fatty acid in the Japanese coral Clavularia viridis and postulated its synthesis via
8R-HPETE [44]. Bundys and Coreys work had a background in the earlier discov-
ery that the Caribbean coral Plexaura homomalla contained masses of prostaglandins
74 A.R. Brash
(~3 % of the dry weight) [45, 46]. During the 1970s this coral was used as a source
of prostaglandins (hence the Upjohn interest in coral), and for biochemical interest
the possibility had arisen that the coral prostaglandins were formed via a non-cyclo-
oxygenase route [47, 48].
I decided to investigate the metabolism of arachidonic acid in the prostaglandin-
containing coral Plexaura homomalla: this led to the discovery of 8R-LOX activity
in the coral as well as transformation of 8R-HPETE to an extremely unstable but
covalently complete epoxide known as an allene oxide [49]. Allene oxides can
cyclize to give a prostaglandin-like 5-membered carbon ring, and this is a key step
in the biosynthesis of fatty acid cyclopentenones in corals [50] and of jasmonic acid
biosynthesis in plants [51, 52]. Despite the apparent similarity of the allene oxide-
derived cyclopentenone to true prostaglandins, through many productive collabora-
tions with Nigulas Samel and colleagues of Tallinn University of Technology in
Estonia, the basis of coral prostaglandin synthesis was shown to be a cyclooxygenase-
catalyzed pathway. This work led to the cloning and characterization of the coral
cyclooxygenases, which are related quite closely (~50 % sequence identity) to
mammalian COX-1 and COX-2 [5355].
By the late 1980s the synthesis of R-HETEs was identied from many sources, mostly
invertebrate animals [49, 5658]. One human connection was known, the occurrence
of high concentrations of 12-HETE in the hyperproliferative and inammatory skin of
psoriasis, described in 1975 by Hammarstrm, Hamberg, and Samuelsson in collabo-
ration with the dermatology group of Voorhees in Michigan [59]. In 1986 Pat Woollard
presented the unexpected nding that the psoriatic 12-HETE is predominantly of the
12R conguration [60]. At the time, only cytochrome P450s were known to synthe-
size 12R-HETE, albeit as one of a complex mixture of epoxy and HETE products
[61], and the concept of P450 involvement in mammalian 12R-HETE synthesis (mis-
takenly) dominated the thinking for the next decade.
Fig. 5.2 Perspective view of the CH2 hydrogens and available positions for oxygenation in lin-
oleic and arachidonic acids. As shown on linoleate, the 9S and 13R positions are on the same face
of the substrate (front face in this view) at opposite ends of the pentadiene, with 9R/13S on the back
face. The same principles apply to arachidonate: for example, the green pentadiene with CH2
hydrogens at C7 has the 5S and 9R positions facing towards the viewer with 5R/9S at the back. For
clarity, the positions around the CH2 at C10 and C13 are not labeled
By the early 2000s there were many S-LOX sequences available, although only four
from R-LOX. Gianguido Coffa in my laboratory made extensive R/S LOX enzyme
chimeras and selected mutations in attempts to identify amino acid residue(s) that
76 A.R. Brash
12S
Fe Fe
C4H 9 C4H 9
HS HS
8R HR HR
8S
Fe Fe (CH 2)2CO2H
(CH 2)2CO2H
HR
HR HS
HS 12R
C4H 9
C4H 9
Fig. 5.3 The principle of R or S stereo control in lipoxygenases. In this example, formation of four
different products is represented in lipoxygenase active sites of related structure. Top panels: The
LOX non-heme iron removes the pro-S hydrogen from C10 (curved arrow), and O2 reacts on the
opposite face of the substrate (antarafacial to H-abstraction) to form either the 8R-hydroperoxide
(left side) or 12S-hydroperoxide (right side). Lower panels: The fatty acid binds in the reverse
head-to-tail orientation, allowing removal of the pro-R hydrogen from C10, followed by oxygen-
ation on the opposite face of the substrate to form the 8S-hydroperoxide (right) or 12R-hydroperoxide
(left) (Reproduced from Brash et al. [62] with permission)
in a LOX from Olea europaea (common olive; accession no. EU678670); the
expressed enzyme synthesizes 9S-HPODE along with 13R-HPODE [73], exactly as
predicted by the model. The recent report from the Newcomer laboratory of the rst
crystal structure of a lipoxygenasesubstrate complex [68] paves the way to a
detailed understanding of stereo control in the LOX enzyme superfamily [2].
The eicosanoid group in the German Cancer Centre in Heidelberg led by Gerhard
Frstenberger and Peter Krieg has a long-standing interest in skin LOX enzymes
and their role in carcinogenesis and epidermal proliferation. By the late 1990s they
had cloned three epidermal LOX genes distinct from the mammalian 15-LOX-1,
12S-LOX, and 5-LOX known at the time [7477]. The rst of these epidermal LOX
enzymes was identied in my laboratory by Mitsuo Jisaka and in Heidelberg as
8S-LOX in the mouse [74, 78] (its human homologue being 15-LOX-2, which we
had cloned a couple of years beforehand [79]). The second epidermal LOX was
identied by Bill Boeglin in my laboratory as (human) 12R-LOX [80]. Colin Funk
and colleagues later described both the human and murine enzymes [81] and Peter
Krieg and colleagues found that the murine 12R-LOX reacts only with fatty acid
esters [82], an intriguing and biologically signicant property in relation to its pro-
posed role in the epidermis (see following). The third epidermal enzyme described
by Frstenberger and Krieg was also an enigmaat the time no catalytic activity
could be ascribed [75]and its name has endured as epidermal lipoxygenase-3, or
eLOX3 (gene ALOXE3).
isomerase. Subsequently, LOX gene knockout studies in mice established the role
in barrier function for both 12R-LOX and eLOX3 [86, 87]; in each case the knock-
out is neonatal lethal because of severe water loss within 510 h of birth. Human
babies with inactivating mutations in 12R-LOX or eLOX3 survive and develop the
hyperproliferative scaly skin of ichthyosis, presumably in attempts to counteract the
defective barrier.
When animals [88] or humans [89] are nutritionally decient in essential fatty acids,
there is a permeability barrier defect, and they develop a scaly skin. Various topi-
cally applied fatty acids have the ability to reverse this skin defect, and the structural
requirements for active fatty acids have been extensively examined. By testing
twenty different fatty acids, Houtsmuller and van der Beek identied the minimal
structural requirement as including a pair of cis double bonds in the 6 and 9 posi-
tions, as in linoleic acid [90]. Changing the position of this pair of double bonds in
the fatty acid, removal of one of them or substitution with a trans double bond, or
adding a CH2 between the bonds eliminated activity [90]. The strong inference from
these studies is that having double bonds alone is insufcient to relieve the skin
symptoms of essential fatty acid deciency. By all appearances the key structural
features of an active fatty acid match the substrate specicity of a LOX enzyme that
utilizes the 96 pair of double bonds.
A conceptual model of how 12R-LOX, eLOX3, and essential fatty acids are involved
in sealing the epidermal barrier was developed by Yuxiang Zheng in my laboratory.
The only EFA in the mammalian outer epidermis where the barrier resides is lin-
oleic acid [91], and it is esteried to the omega-hydroxy group of a unique,
epidermal-specic ceramide, EOS (esteried omega-hydroxy sphingosine) [92].
Yuxiang showed that the linoleate moiety in EOS can be oxidized by 12R-LOX and
further transformed by eLOX3 to a specic epoxy-alcohol derivative [93]. She
detected traces of these oxidized ceramides in pig epidermis and in the epidermis of
neonatal mice, and the products were absent in the epidermis of 12R-LOX knockout
mice [93]. Most signicantly, the knockout mice also had an almost complete
absence of omega-hydroxy ceramide covalently bound to protein. It is known that a
fraction of EOS is de-esteried and the free omega-OH is coupled to a layer of
polymerized protein on the outer surface of the corneocytes, replacing the original
plasma membrane, and forming a covalently bound lipid coating named the corneo-
cyte lipid envelope or CLE [94]. The CLE lies between the polymerized protein and
5 Lipoxygenases: A Chronological Perspective on the Synthesis of S and R Fatty 79
extracellular lamellar lipids, and potentially functions as a scaffold that holds the
two other parts together as a complete and intact barrier. Taking all the facts together,
we propose that LOX-catalyzed oxidation of the linoleate ester in EOS is required
to facilitate enzymatic hydrolysis of the ester, thus freeing OS for coupling to pro-
tein (Fig. 5.4) [93, 95].
In EFA deciency, oleate (not a LOX substrate) replaces linoleate as the fatty
acid ester in EOS [91]. Because it is not a LOX substrate it cannot be oxidized and
therefore it cannot be hydrolyzed, resulting in a deciency in free OS; the predic-
tion, therefore, is that this should lead to decreased levels of covalently bound
ceramides in the barrier layer, which is exactly what is found [96]. On the other
hand, in LOX deciency, in either knockout mice or aficted human families, the
Glc
O Gln
Omega-hydroxyacyl-Sphingosine (OS) C
H2N
HO
O OH
HO CH2
N
H n
Transglutaminase-1
Covalently bound ceramide
O Glu
HO
O C
HO CH2 O
N
H n
Cornified
Corneocyte Lipid Envelope (CLE) Envelope (CE),
cross-linked protein
oxidizing enzymes are missing, with ultimately the same consequences: no oxida-
tion, therefore no hydrolysis of the linoleate ester, therefore a lack of covalently
bound ceramides, an epidermal barrier defect, and the resulting phenotype [93, 95].
There is a good chance that understanding the basis of the role of 12R-LOX and
eLOX in epidermal function will lead to improved treatments for patients aficted
with LOX-dependent ichthyoses. In these patients it is predicted that the fundamen-
tal problem with the skin barrier is a defect in the covalently bound lipid envelope
resulting from the lack of available free omega-hydroxy ceramide. As topical appli-
cations can be effective, the prediction is that topical OS should provide the missing
substrate and ameliorate the skin symptoms. In the near future, this may emerge as
another example of basic research resulting in a translational impact.
Acknowledgments This work is supported by grants from the NIH (GM-15431, GM-74888, and
AR-51968).
References
40. Meijer L, Guerrier P, Maclouf J (1984) Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic
acids, eicosapentaenoic acid, and phospholipase A2 induce starsh oocyte maturation. Dev
Biol 106:368378
41. Brash AR, Porter AT, Maas RL (1985) Investigation of the selectivity of hydrogen abstraction
in the non-enzymatic formation of hydroxyeicosatetraenoic acids and leukotrienes by autoxi-
dation. J Biol Chem 260:42104216
42. Meijer L, Brash AR, Bryant RW, Ng K, Maclouf J, Sprecher H (1986) Stereospecic induction
of starsh oocyte maturation by (8R)-hydroxyeicosatetraenoic acid. J Biol Chem
261:1704017047
43. Hughes MA (1993) Role of 8(R)-hydroxyeicosatetraenoic acid (8(R)-HETE) in induction of
starsh oocyte maturation. Ph.D. Vanderbilt University, Nashville
44. Corey EJ, Lansbury PT Jr, Yamada Y (1985) Identication of a new eicosanoid from in vitro
biosynthetic experiments with Clavularia viridis. Implications for the biosynthesis of clavulo-
nes. Tetrahedron Lett 26:41714174
45. Weinheimer AJ, Spraggins RL (1969) The occurrence of two new prostaglandin derivatives
(15-epi-PGA2 and its acetate, methyl ester) in the gorgonian Plexaura homomalla. Tetrahedron
Lett 59:51855188
46. Schneider WP, Hamilton RD, Rhuland LE (1972) Occurrence of esters of (15S)-prostaglandin
A2 and E2 in coral. J Am Chem Soc 94:21222123
47. Corey EJ, Washburn WN, Chen JC (1973) Studies on the prostaglandin A2 synthetase complex
from Plexaura homomalla. J Am Chem Soc 95:20542055
48. Corey EJ, Ensley, HE, Hamberg M, Samuelsson B (1975) Disparate pathways of prostaglan-
din biosynthesis in coral and mammalian systems. J Chem Soc Chem Commun 277278
49. Brash AR, Baertschi SW, Ingram CD, Harris TM (1987) On non-cyclooxygenase prostaglan-
din synthesis in the sea whip coral Plexaura homomalla: an 8(R)-lipoxygenase pathway leads
to formation of an -ketol and a racemic prostanoid. J Biol Chem 262:1582915839
50. Corey EJ, Matsuda SPT, Nagata R, Cleaver MB (1988) Biosynthesis of 8-R-HPETE and pre-
clavulone A from arachidonate in several species of Caribbean coral. A widespread route to
marine prostanoids. Tetrahedron Lett 29:25552558
51. Hamberg M (1988) Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: identication of an
allene oxide cyclase. Biochem Biophys Res Commun 156:543550
52. Feussner I, Wasternack C (2002) The lipoxygenase pathway. Annu Rev Plant Biol
53:275297
53. Koljak R, Jrving I, Kurg R, Boeglin WE, Varvas K, Valmsen K, Ustav M, Brash AR, Samel
N (2001) The basis of prostaglandin synthesis in coral. Molecular cloning and expression of a
cyclooxygenase from the Arctic soft coral Gersemia fruticosa. J Biol Chem 276:70337040
54. Valmsen K, Jrving I, Boeglin WE, Varvas K, Koljak R, Pehk T, Brash AR, Samel N (2001)
The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: molecular clon-
ing and expression of a novel cyclooxygenase. Proc Natl Acad Sci U S A 98:77007705
55. Valmsen K, Boeglin WE, Jrving J, Schneider C, Varvas K, Brash AR, Samel N (2004)
Structural and functional comparison of 15S- and 15R-specic cyclooxygenases from the coral
Plexaura homomalla. Eur J Biochem 271:35333538
56. Hawkins DJ, Brash AR (1987) Eggs of the sea urchin, Strongylocentrotus purpuratus, contain
a prominent (11R) and (12R) lipoxygenase activity. J Biol Chem 262:76297634
57. Hawkins DJ, Brash AR (1989) Mechanism of biosynthesis of 11R-and
12R-hydroxyeicosatetraenoic acids by eggs of the sea urchin Strongylocentrotus purpuratus.
FEBS Lett 247:912
58. Corey EJ, dAlarcao M, Matsuda SPT, Lansbury PT Jr (1987) Intermediacy of 8-(R)-HPETE
in the conversion of arachidonic acid to pre-clavulone A by Clavularia viridis. Implications for
the biosynthesis of marine prostanoids. J Am Chem Soc 109:289290
59. Hammarstrm S, Hamberg M, Samuelsson B, Duell EA, Stawiski M, Voorhees JJ (1975)
Increased concentrations of nonesteried arachidonic acid, 12 L-hydroxy-5,8,10,14-
eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 in epidermis of psoriasis. Proc
Natl Acad Sci U S A 72:51305134
5 Lipoxygenases: A Chronological Perspective on the Synthesis of S and R Fatty 83
79. Brash AR, Jisaka M, Boeglin WE, Chang MS (1997) Molecular cloning of a second human
15S-lipoxygenase and its murine homologue, an 8S-lipoxygenase: their relationship to other
mammalian lipoxygenases. In: Pace-Asciak CR, Nigam S (eds) Lipoxygenases and their prod-
ucts: biological functions. Plenum Press, New York, pp 2936
80. Boeglin WE, Kim RB, Brash AR (1998) A 12R-lipoxygenase in human skin: mechanistic
evidence, molecular cloning and expression. Proc Natl Acad Sci U S A 95:67446749
81. Sun D, McDonnell M, Chen X-S, Lakkis MM, Li H, Isaacs SN, Elsea SH, Patel PI, Funk CD
(1998) Human 12(R)-lipoxygenase and the mouse ortholog. Molecular cloning, expression,
and gene chromosomal assignment. J Biol Chem 273:3354033547
82. Krieg P, Siebert M, Kinzig A, Bettenhausen R, Marks F, Frstenberger G (1999) Murine
12(R)-lipoxygenase: functional expression, genomic structure and chromosomal localization.
FEBS Lett 446:142148
83. Jobard F, Lefvre C, Karaduman A, Blanchet-Bardon C, Emre S, Weissenbach J, zgc M,
Lathrop M, Prudhomme JF, Fischer J (2002) Lipoxygenase-3 (ALOXE3) and 12(R)-
lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma
(NCIE) linked to chromosome 17p13.1. Hum Mol Genet 11:107113
84. Yu Z, Schneider C, Boeglin WE, Marnett LJ, Brash AR (2003) The lipoxygenase gene
ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase. Proc Natl
Acad Sci U S A 100:91629167
85. Yu Z, Schneider C, Boeglin WE, Brash AR (2006) Human and mouse eLOX3 have distinct
substrate specicities: implications for their linkage with lipoxygenases in skin. Arch Biochem
Biophys 455:188196
86. Moran JL, Qiu H, Turbe-Doan A, Yun Y, Boeglin WE, Brash AR, Beier DR (2007) A mouse
mutation in the 12(R)-lipoxygenase, Alox12b, disrupts formation of the epidermal permeabil-
ity barrier. J Invest Dermatol 127:18931897
87. Epp N, Furstenberger G, Muller K, de Juanes S, Leitges M, Hausser I, Thieme F, Liebisch G,
Schmitz G, Krieg P (2007) 12R-lipoxygenase deciency disrupts epidermal barrier function. J
Cell Biol 177:173182
88. Burr GO, Burr MM (1929) A new deciency disease produced by the rigid exclusion of fat
from the diet. J Biol Chem 82:345367
89. Hansen AE, Haggard ME, Boelsche AN, Adam DJ, Wiese HF (1958) Essential fatty acids in
infant nutrition. III. Clinical manifestations of linoleic acid deciency. J Nutr 66:565576
90. Houtsmuller UMT, van der Beek A (1981) Effects of topical application of fatty acids. Prog
Lipid Res 20:219224
91. Hansen HS (1986) The essential nature of linoleic acid in mammals. Trends Biochem Sci
11:263265
92. Uchida Y, Holleran WM (2008) Omega-O-acylceramide, a lipid essential for mammalian sur-
vival. J Dermatol Sci 51:7787
93. Zheng Y, Yin H, Boeglin WE, Elias PM, Crumrine D, Beier DR, Brash AR (2011)
Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed
role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope. J
Biol Chem 286:2404624056
94. Elias PM, Gruber R, Crumrine D, Menon G, Williams ML, Wakeeld JS, Holleran WM,
Uchida Y (2014) Formation and functions of the corneocyte lipid envelope (CLE). Biochim
Biophys Acta 1841:314318
95. Muoz-Garcia A, Thomas CP, Keeney DS, Zheng Y, Brash AR (2014) The importance of the
lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier. Biochim Biophys Acta
1841:401408
96. Meguro S, Arai Y, Masukawa Y, Uie K, Tokimitsu I (2000) Relationship between covalently
bound ceramides and transepidermal water loss (TEWL). Arch Dermatol Res 292:463468
Chapter 6
Leukotriene B4 Receptors
Leukotriene B4 (LTB4) is a classical lipid mediator that activates and attracts neutro-
phils [1, 2]. LTB4 is produced mainly in leukocytes, including neutrophils and mac-
rophages, and it contains three conjugated double bonds (a triene), explaining the
origin of the name leukotriene. LTB4 is an arachidonic acid metabolite that is
produced by two enzymes, 5-lipoxygenase (5-LO) and leukotriene A4 hydrolase
(LTA4H) [3]. Arachidonic acid is converted into 5-hydroperoxyeicosatetraenoic
acids (5-HpETE) and subsequently into LTA4. 5-LO mediates these reactions in
functional cooperation with 5-lipoxygenase-activating protein (FLAP) [3]. When
cells are exposed to certain stimuli (e.g., Ca2+ inux), cytoplasmic 5-LO is activated
and translocates to the nuclear membrane. FLAP binds to 5-LO to form an active
complex on the nuclear membrane, and this complex catabolizes arachidonic acid
into 5-HpETE and subsequently to LTA4. LTA4 is either converted to LTB4 by
LTA4H or conjugated to glutathione to generate leukotriene C4 (LTC4) by LTC4 syn-
thase (LTC4S). LTB4 is exported from the cytoplasm to the extracellular space in an
energy-dependent manner. In humans, LTB4 export is mediated by the ATP-binding
cassette (ABC) family transporter ABCC4/MRP4 [4]. The biosynthesis and trans-
port pathway of LTB4 is summarized in Fig. 6.1.
Degradation of LTB4 is catalyzed by -oxidation and subsequent -oxidation, or
by the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase (12HDH/
15oPGR) pathway [3]. In the -oxidation pathway, LTB4 is metabolized into
20-hydroxy-LTB4 (20-OH-LTB4), and subsequently into 20-carboxy-LTB4
(20-COOH-LTB4) [5]. The cytochrome P450 family protein CYP4F3A (CYP4F18 in
mouse) is a human neutrophil LTB4--hydroxylase that is induced by retinoic acid
and phorbol ester stimulation. This enzyme efciently oxidizes the methyl moiety
of LTB4 at the -terminus. In neutrophils, 20-OH-LTB4 is further oxidized by
CYP4F3A into 20-COOH-LTB4 through the 20-oxo-LTB4 intermediate. In other
tissues (e.g., liver), an alternative pathway produces 20-COOH-LTB4 from
20-OH-LTB4; in the liver, the enzymes involved in LTB4 oxidation are alcohol dehy-
drogenase (ADH) and aldehyde dehydrogenase (ALDH). Although 20-OH-LTB4
binds fairly well to leukotriene B4 receptor 1 (BLT1), 20-COOH-LTB4 is not an
active ligand of BLT1. A recent report showed that patients with SjgrenLarsson
syndrome, who have mutations in the fatty aldehyde dehydrogenase (FALDH) gene,
exhibited high urinary levels of LTB4 and 20-OH-LTB4, indicating the importance
of the ADH/ALDH pathway in LTB4 metabolism. In some tissues, LTB4 can be
converted into 12-oxo-LTB4, a reaction catalyzed by the enzyme 12HDH/
15oPGR [6]. 12HDH/15oPGR recognizes the structural motif [R-CH(OH)-
(trans)-CH = CH-R] of LTB4, and oxidizes the 12(R) hydroxyl group into the
12-oxo moiety. Biosynthesized 12-oxo-LTB4 is unable to activate BLT1 signaling.
A recent report showed that LTB4 is extensively metabolized by the 12HDH/15oPGR
pathway in human keratinocytes. Because CYP4F activity is very low in the human
keratinocytes, -oxidized LTB4 metabolite is rarely observed in these cells. The
degradation pathway of LTB4 is summarized in Fig. 6.2.
Fig. 6.1 Biosynthesis and export pathway of LTB4 and 12-HHT. LTB4 is synthesized from arachi-
donic acid by 5-LO, FLAP, and LTA4H. LTB4 is exported by MRP4, at least in humans, and recog-
nized by high-afnity receptor BLT1. On the other hand, production of 12-HHT is catalyzed by
COX-1, COX-2, and TxA2S. 12-HHT is exported and recognized by BLT2. PM plasma
membrane
88 T. Koga and T. Yokomizo
Fig. 6.2 Degradation pathway of LTB4. Two pathways inactivate LTB4. The -oxidation pathway
is catalyzed by CYP4F3A. Its product, 20-hydroxy-LTB4, still has the ability to activate BLT1. To
inactivate 20-hydroxy-LTB4, 20-carboxylation by either CYP4F3A (in human neutrophils) or
ADH/ALDH (in hepatocytes) is required. Alternatively, the 12HDH/15oPGR pathway is also
important for inactivation of LTB4. Its metabolite, 12-oxo-LTB4, is inactive. PM plasma
membrane
Fig. 6.3 Phylogenic tree of G protein-coupled receptors (GPCRs) for lipid mediators. Among all
GPCRs, BLT1 and BLT2 receptors are the most closely related to each other
6 Leukotriene B4 Receptors 89
osteoclasts with LTB4 increases osteoclastic activity, and BLT1 deciency attenu-
ates osteoporosis after ovariectomy in mice [31]. In summary, although BLT1 was
initially identied as a LTB4 receptor expressed in neutrophils, recent reports reveal
that it is expressed in various cell types, including neutrophils, eosinophils, macro-
phages, dendritic cells, differentiated T cells, smooth muscle cells, osteoclasts, and
adipocytes, and that it plays important roles in acute and chronic inammation and
in immune diseases.
BLT2 was identied as a low-afnity receptor for LTB4; the gene that encodes this
protein is localized in the proximal promoter region of BLT1 on human chromo-
some 14 [15]. Similar to BLT1, BLT2 is also coupled to two classes of G proteins
(Gi and Gq), through which it activates the MAPK (ERK and p38), NADP oxidase
(NOX)-reactive oxygen species (ROS), and NF-B signaling pathways [32]. We
recently identied 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) as
an endogenous ligand for BLT2. 12-HHT is a fatty acid with 17 carbons, and its
physiological role has not been elucidated. 12-HHT is also a metabolite of arachi-
donic acid, which is biosynthesized during blood coagulation primarily by two
enzymes, cyclooxygenase (COX) and thromboxane A2 synthase (TxA2S). In addi-
tion to this pathway, we recently identied a nonenzymatic production of 12-HHT
from PGH2 by using pharmacological and genetic inhibition of TxA2S in vitro and
in vivo [33]. 12-HHT is present in intestine, and BLT2 is highly expressed in intes-
tinal epithelial cells [34, 35]. BLT2 plays protective roles against dextran sulfate-
induced colitis by enhancing barrier function in intestinal epithelial cells [36]. BLT2
is also expressed in human and mouse skin. BLT2 deciency delays epidermal
wound healing by slowing keratinocyte migration without affecting broblast dif-
ferentiation or keratinocyte proliferation [37]. 12-HHT is mainly produced by plate-
lets in wound areas, and it activates BLT2 expressed in epidermal keratinocytes.
Through the NF-B pathway, activated keratinocytes induce tumor necrosis factor-,
which upregulates matrix metalloproteinase (MMP) 9 in an autocrine or paracrine
manner. Although BLT2 is primarily expressed in epithelial cells, a recent report
demonstrated that BLT2 is also expressed in Th2 cells; furthermore, BLT2-decient
mice exhibit enhanced eosinophil accumulation and IL-13 and IL-4 production in a
Th2-dependent murine asthma model [38]. Furthermore, several studies showed
that BLT2 is involved in progression of various cancers, including bladder, ovarian,
prostate, and breast cancers [32]. BLT2 is upregulated in human bladder cancer,
ovarian carcinoma, breast cancer, renal cell carcinoma, lung carcinoma, and other
tumors [39]. In addition, cell transformation induced by constitutively active mutant
Ras might be BLT2 dependent [39]. BLT2-mediated cancer progression is associ-
ated with the NOX-ROS-NF-B signaling pathway, which promotes cancer cell
survival and invasion through induction of androgen receptor and MMP9,
respectively.
6 Leukotriene B4 Receptors 91
The biosynthesis and degradation pathways of LTB4, with the exception of the
export mechanism of LTB4 to the extracellular space, have been claried in detail.
Because transporters are promising therapeutic targets, detailed studies of MRP4 by
MRP4-decient mice are required. On the other hand, the roles of leukotriene B4
receptors have been extensively investigated in both BLT1 and BLT2 systemically
decient mice. However, because many studies found that BLT1 and BLT2 are
expressed in various tissues and cell types, the cell-specic role of those receptors
should be elucidated using conditional-knockout mice. Future studies should focus
on determining which cells produce LTB4, which cells express BLT1 in vivo, and
the clinical relevance of the LTB4BLT1 and 12-HHT-BLT2 pathways.
References
13. Goodarzi K, Goodarzi M, Tager AM, Luster AD, von Andrian UH (2003) Leukotriene B4 and
BLT1 control cytotoxic effector T cell recruitment to inamed tissues. Nat Immunol
4:965973
14. Tager AM, Luster AD (2003) BLT1 and BLT2: the leukotriene B(4) receptors. Prostaglandins
Leukot Essent Fatty Acids 69:123134
15. Back M, Dahlen S-E, Drazen JM, Evans JF, Serhan CN, Shimizu T, Yokomizo T, Rovati GE
(2011) International Union of Basic and Clinical Pharmacology. LXXXIV: leukotriene recep-
tor nomenclature, distribution, and pathophysiological functions. Pharmacol Rev 63:539584
16. Chou RC, Kim ND, Sadik CD, Seung E, Lan Y, Byrne MH, Haribabu B, Iwakura Y, Luster AD
(2010) Lipid-cytokine-chemokine cascade drives neutrophil recruitment in a murine model of
inammatory arthritis. Immunity 33:266278
17. Sadik CD, Kim ND, Iwakura Y, Luster AD (2012) Neutrophils orchestrate their own recruit-
ment in murine arthritis through C5aR and Fc-gamma-R signaling. Proc Natl Acad Sci U S A
109:E3177E3185
18. Sumida H, Yanagida K, Kita Y, Abe J, Matsushima K, Nakamura M, Ishii S, Sato S, Shimizu
T (2014) Interplay between CXCR2 and BLT1 facilitates neutrophil inltration and resultant
keratinocyte activation in a murine model of imiquimod-induced psoriasis. J Immunol
192:43614369
19. Patnode ML, Bando JK, Krummel MF, Locksley RM, Rosen SD (2014) Leukotriene B4
amplies eosinophil accumulation in response to nematodes. J Exp Med 211:12811288
20. Okamoto F, Saeki K, Sumimoto H, Yamasaki S, Yokomizo T (2010) Leukotriene B4 augments
and restores Fc gamma-Rs-dependent phagocytosis in macrophages. J Biol Chem
285:4111341121
21. Serezani CH, Lewis C, Jancar S, Peters-Golden M (2011) Leukotriene B4 amplies NF-kappaB
activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression. J Clin
Invest 121:671682
22. Wang Z, Filgueiras LR, Wang S, Serezani APM, Peters-Golden M, Jancar S, Serezani CH
(2014) Leukotriene B4 enhances the generation of proinammatory microRNAs to promote
MyD88-dependent macrophage activation. J Immunol 192:23492356
23. ONeill LAJ, Bowie AG (2007) The family of ve: TIR-domain-containing adaptors in Toll-
like receptor signalling. Nat Rev Immunol 7:353364
24. Del Prete A, Shao W-H, Mitola S, Santoro G, Sozzani S, Haribabu B (2007) Regulation of
dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for
LTB4 in up-regulation of CCR7 expression and function. Blood 109:626631
25. Terawaki K, Yokomizo T, Nagase T, Toda A, Taniguchi M, Hashizume K, Yagi T, Shimizu T
(2005) Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness
and Th2-type immune responses. J Immunol 175:42174225
26. Kihara Y, Yokomizo T, Kunita A, Morishita Y, Fukayama M, Ishii S, Shimizu T (2010) The
leukotriene B4 receptor, BLT1, is required for the induction of experimental autoimmune
encephalomyelitis. Biochem Biophys Res Commun 394:673678
27. Wang L, Zhao L, Lv J, Yin Q, Liang X, Chu Y, He R (2012) BLT1-dependent alveolar recruit-
ment of CD4(+)CD25(+) Foxp3(+) regulatory T cells is important for resolution of acute lung
injury. Am J Respir Crit Care Med 186:989998
28. Heller EA, Liu E, Tager AM, Sinha S, Roberts JD, Koehn SL, Libby P, Aikawa ER, Chen JQ,
Huang P, Freeman MW, Moore KJ, Luster AD, Gerszten RE (2005) Inhibition of atherogene-
sis in BLT1-decient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruit-
ment. Circulation 112:578586
29. Back M, Bu D-X, Branstrom R, Sheikine Y, Yan Z-Q, Hansson GK (2005) Leukotriene B4
signaling through NF-kappaB-dependent BLT1 receptors on vascular smooth muscle cells in
atherosclerosis and intimal hyperplasia. Proc Natl Acad Sci U S A 102:1750117506
30. Hirata K, Wada K, Murata Y, Nakajima A, Yamashiro T, Kamisaki Y (2013) Critical role of
leukotriene B4 receptor signaling in mouse 3 T3-L1 preadipocyte differentiation. Lipids
Health Dis 12:122
6 Leukotriene B4 Receptors 93
31. Hikiji H, Ishii S, Yokomizo T, Takato T, Shimizu T (2009) A distinctive role of the leukotriene
B4 receptor BLT1 in osteoclastic activity during bone loss. Proc Natl Acad Sci U S A
106:2129421299
32. Cho N-K, Joo Y-C, Wei JD, Park JI, Kim J-H (2013) BLT2 is a pro-tumorigenic mediator dur-
ing cancer progression and a therapeutic target for anti-cancer drug development. Am J Cancer
Res 3:347355
33. Matsunobu T, Okuno T, Yokoyama C, Yokomizo T (2013) Thromboxane A synthase-
independent production of 12-hydroxyheptadecatrienoic acid, a BLT2 ligand. J Lipid Res
54:29792987
34. Iizuka Y, Yokomizo T, Terawaki K, Komine M, Tamaki K, Shimizu T (2005) Characterization
of a mouse second leukotriene B4 receptor, mBLT2: BLT2-dependent ERK activation and cell
migration of primary mouse keratinocytes. J Biol Chem 280:2481624823
35. Okuno T, Iizuka Y, Okazaki H, Yokomizo T, Taguchi R, Shimizu T (2008) 12(S)-
Hydroxyheptadeca-5Z, 8E, 10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2.
J Exp Med 205:759766
36. Iizuka Y, Okuno T, Saeki K, Uozaki H, Okada S, Misaka T, Sato T, Toh H, Fukayama M,
Takeda N, Kita Y, Shimizu T, Nakamura M, Yokomizo T (2010) Protective role of the leukot-
riene B4 receptor BLT2 in murine inammatory colitis. FASEB J 24:46784690
37. Liu M, Saeki K, Matsunobu T, Okuno T, Koga T, Sugimoto Y, Yokoyama C, Nakamizo S,
Kabashima K, Narumiya S, Shimizu T, Yokomizo T (2014) 12-Hydroxyheptadecatrienoic acid
promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2
receptor. J Exp Med 211:10631078
38. Matsunaga Y, Fukuyama S, Okuno T, Sasaki F, Matsunobu T, Asai Y, Matsumoto K, Saeki K,
Oike M, Sadamura Y, Machida K, Nakanishi Y, Kubo M, Yokomizo T, Inoue H (2013)
Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia. FASEB J
27:33063314
39. Yoo M-H, Song H, Woo C-H, Kim H, Kim J-H (2004) Role of the BLT2, a leukotriene B4
receptor, in Ras transformation. Oncogene 23:92599268
Chapter 7
Platelet-Activating Factor (PAF) in Infectious
Diseases
Satoshi Ishii
S. Ishii (*)
Department of Immunology, Akita University Graduate School of Medicine,
Akita 010-8543, Japan
e-mail: [email protected]
cPLA2 and LPCAT2 act sequentially seems reasonable for the effective production
of PAF in response to extracellular stimuli. Following secretion into the extracellular
space, PAF exerts its bioactivity on a variety of cells through a specific G protein-
coupled receptor (see following).
Deacetylation of PAF is catalyzed by plasma PAF acetylhydrolase, which results in
its conversion into lyso-PAF [14]. This enzyme appears to be secreted mainly by mac-
rophages [15, 16] and circulates in association with LDL and HDL particles [17].
Thus, PAF released into the extracellular space can be rapidly inactivated to prevent
excessive and uncontrolled activation of the specific receptor for PAF (PAFR).
7.2 PAFR
Using an expression cloning method with Xenopus oocytes, Prof. Takao Shimizu
and his research team isolated a cDNA clone encoding PAFR from a guinea pig
lung cDNA library in 1991 [18]. Subsequent cross-hybridization screening studies
identified PAFR orthologues from humans [19], rats [20], and mice [21].
Consistently, these PAFRs were found to be single polypeptides composed of
approximately 340 amino acids. Similar to other G protein-coupled receptors, PAFR
possesses seven transmembrane domains. A single receptor subtype is currently
thought to mediate all the actions of PAF. Of note, PAFR can couple to both Gq/11
and Gi/o proteins, which initiate distinct signals (Fig. 7.2). This trait enables the
receptor to simultaneously activate various kinases and phospholipases. The former
involves MAPK, PKC, phosphatidylinositol 3-kinase (PI3K), and protein tyrosine
kinase (PTK), and the latter includes phospholipase C (PLC) and cPLA2.
Northern hybridization experiments have revealed the distribution of PAFR
mRNA expression in several species. In guinea pigs [18], PAFR mRNA is most
abundant in leukocytes. Furthermore, the spleen and lung contain detectable
amounts of PAFR mRNA. In humans, the PAFR message has been detected in neu-
trophils [19], monocytes [22], monocyte/CD34+ cell-derived dendritic cells [23],
and umbilical vein endothelial cells (HUVECs) [24]. As in guinea pigs, the human
lung has been shown to express high levels of PAFR mRNA [25, 26]. Rat PAFR
mRNA can be detected in the spleen, small intestine, and lung [20], as well as in
microglia [27] and mesangial cells [28]. Mouse peritoneal macrophages express a
significant amount of PAFR mRNA. In mouse tissues, abundant expression of PAFR
mRNA has been observed in the spleen, lung, and small intestine [21]. Altogether,
these observations indicate that tissues rich in myeloid cells commonly contain high
levels of PAFR mRNA.
Specific receptors for PAF have been identified in numerous tissues and cells
through the use of 3H-labeled PAFR agonists and antagonists. The first binding
experiment utilizing [3H]-PAF was conducted in washed human platelets [29].
Consistent with the data obtained by Northern hybridization, specific receptors for
PAF have been detected on neutrophils, macrophages, mononuclear leukocytes,
eosinophils, and Kupffer cells (see [30] for review). In addition to these myeloid
98 S. Ishii
Fig. 7.2 Schematic representation of intracellular signaling events coupled to PAF receptor. This
scheme represents an overlay of signaling pathways leading to intracellular Ca2+ increase and
mitogen-activated protein kinase (MAPK) activation. Some of the signaling events may be cell-
type specific. Dashed lines indicate the pathways that need to be further characterized. PKCs
include conventional and novel PKC isozymes, which are activated by Ca2+ and DAG. PTKs
include Src and Tec family tyrosine kinases as well as receptor tyrosine kinases. AA arachidonic
acid, AC adenylate cyclase, DAG diacylglycerol, IP3 inositol 1,4,5-trisphosphate, MEK MAP
kinase kinase, LT leukotriene, PG prostaglandin, TX thromboxane. See text for other
abbreviations
cells, tracheal epithelial cells [31] and HUVECs [32] also have shown specific
binding activity to PAF. Although these cultured cells were assayed intact, mem-
brane fractions were used to demonstrate specific PAFRs in tissues or to eliminate
the intracellular degradation of PAF. Such specimens were prepared from the lung
[33] and spleen [34].
published during the past dozen years or so will be reviewed that offer insight into
the protective or deleterious roles of the PAF/PAFR axis in infectious disease.
The characteristic features of each bacterial pathogen are diverse in terms of the
mode of infection transmission, colonization procedure, and mechanism of patho-
genesis. Phosphorylcholine is a critical moiety of PAF and is also a component of
the cell walls of Streptococcus pneumoniae [38] and nontypeable Haemophilus
influenzae [39].
Susceptibility to S. pneumoniae infection is reportedly observed after inflammatory
activation of A549 alveolar epithelial cells and HUVECs in vitro [40], likely caused by
upregulation of PAFR expression at the cell surface. The in vivo relevance of the inter-
action between pneumococcal phosphorylcholine and PAFR was evident in a model of
pneumococcal pneumonia showing that PAFR-KO mice had an attenuated inflamma-
tory response, reduced bacterial numbers, and an improved outcome [41].
It should be noted that the interaction between phosphorylcholine and PAFR
does not affect murine host defense during nontypeable H. influenzae pneumonia
[42]; PAFR-KO and WT mice display similar bacterial counts, myeloperoxidase
100 S. Ishii
activity, and histopathology in the lung. These results suggest that receptors other
than PAFR are responsible for the regulation of innate immune responses to non-
typeable H. influenzae infection despite in vitro data indicating a significant role of
the phosphorylcholinePAFR interaction in the invasion of pulmonary epithelial
cells [43].
7.3.4 Conclusions
In this review, the involvement of the PAF/PAFR axis in several models of infec-
tious diseases is described. These experimental infection studies revealed several
distinctive functions of PAFR in host defense, depending on the pathogenic mecha-
nisms of infection. Outcomes for each study are summarized in Table 7.1.
By facilitating phagocytosis and killing of bacteria, PAF appears to affect the
ability of phagocytes (likely mainly neutrophils) to address pulmonary infections
with K. pneumoniae [36] and P. aeruginosa [37]. Most likely because of different
characteristic features of pathogens, the other pulmonary infections with extracel-
lular bacteria, that is, S. pneumoniae [41] and H. influenzae [42], appear to develop
independently of PAFR-activated phagocytes. It is of interest that PAFR-mediated
activation of phagocytes (likely mainly macrophages) is also responsible for host
resistance to the intracellular protozoan parasites L. amazonensis [54] and T. cruzi
[55]. NO produced in PAFR-activated macrophages may be a key effector molecule
of the intracellular parasite killing. Alveolar macrophages serve as the major host
cell niche for the growth and survival of M. tuberculosis, and NO also has mycobac-
tericidal activity in mice [62]. However, PAFR-KO mice have normal protective
immune responses to M. tuberculosis [45], the reason for which remains unclear.
PAF exerts different functions during intracellular infection with dengue and
influenza A viruses [51, 52]. PAF-producing cells, such as phagocytes, in the
infected tissues appear to release a large amount of PAF, which aberrantly activates
PAFR in an autocrine/paracrine manner and causes tissue injury and, in some
instances, death. A similar mechanism may underlie the pathogenesis of cerebral
malaria [58], pneumococcal pneumonia [41], and periodontitis [46], although the
104 S. Ishii
Table 7.1 Characteristics of the experimental infection studies using platelet-activating receptor
(PAFR)-knockout (KO) mice
Effects of PAF/PAFR
on
Pathogen Disease Responsible immune
Pathogens burden symptoms mechanisms References
Klebsiella pneumoniae PAF activates neutrophil [36]
phagocytosis.
Pseudomonas aeruginosa PAF activates neutrophil [37]
phagocytosis.
Streptococcus pneumoniae PAFR anchors S. pneumoniae [41]
to epithelial and endothelial
cells.
Haemophilus influenzae N/Aa [42]
Mycobacterium tuberculosis N/A [45]
Aggregatibacter PAF enhances the [46]
actinomycetemcomitans differentiation and activity of
osteoclasts in inflamed
periodontal tissues.
Dengue virus PAF induces [51]
thrombocytopenia,
hyperpermeability,
hemoconcentration, and Th1
cytokine production.
Influenza virus PAF induces lung injury with [52]
increased neutrophil
recruitment and Th1 cytokine
production.
Leishmania amazonensis PAF stimulates macrophages [54]
and Th1 polarization.
Trypanosoma cruzi PAF stimulates macrophages. [55]
Plasmodium berghei PAF contributes to vascular [58]
permeability and CD8+ T cell
activation in the brain.
Strongyloides venezuelensis PAF stimulates Th2- [61]
predominant intestinal
inflammation.
a
N/A, not applicable
latter two are caused by infection with extracellular pathogens. Thus, PAFR could
be a useful therapeutic target to interfere with the aggravation of these diseases. It is
worth noting that treatment of mice with PAFR antagonists prevented, at least in
part, the progression of infection with these pathogens [51, 52, 58, 40, 63].
Equivalent studies in humans are required to understand the potential role of PAFR
blockade in the amelioration of disease symptoms.
7 Platelet-Activating Factor (PAF) in Infectious Diseases 105
References
1. Nakanaga K, Hama K, Aoki J (2010) Autotaxinan LPA producing enzyme with diverse func-
tions. J Biochem 148(1):1324
2. Maceyka M, Harikumar KB, Milstien S, Spiegel S (2012) Sphingosine-1-phosphate signaling
and its role in disease. Trends Cell Biol 22(1):5060
3. Ishii S, Shimizu T (2000) Platelet-activating factor (PAF) receptor and genetically engineered
PAF receptor mutant mice. Prog Lipid Res 39(1):4182
4. Shimizu T (2009) Lipid mediators in health and disease: enzymes and receptors as therapeutic
targets for the regulation of immunity and inflammation. Annu Rev Pharmacol Toxicol
49:123150
5. Snyder F (1995) Platelet-activating factor: the biosynthetic and catabolic enzymes. Biochem J
305:689705
6. Prescott SM, Zimmerman GA, Stafforini DM, McIntyre TM (2000) Platelet-activating factor
and related lipid mediators. Annu Rev Biochem 69:419445
7. Uozumi N, Kume K, Nagase T, Nakatani N, Ishii S, Tashiro F, Komagata Y, Maki K, Ikuta K,
Ouchi Y, Miyazaki J, Shimizu T (1997) Role of cytosolic phospholipase A2 in allergic response
and parturition. Nature 390(6660):618622
8. Shindou H, Ishii S, Uozumi N, Shimizu T (2000) Roles of cytosolic phospholipase A(2) and
platelet-activating factor receptor in the Ca-induced biosynthesis of PAF. Biochem Biophys
Res Commun 271(3):812817
9. Shindou H, Hishikawa D, Nakanishi H, Harayama T, Ishii S, Taguchi R, Shimizu T (2007) A
single enzyme catalyzes both platelet-activating factor production and membrane biogenesis
of inflammatory cells. Cloning and characterization of acetyl-CoA:LYSO-PAF acetyltransfer-
ase. J Biol Chem 282(9):65326539
10. Morimoto R, Shindou H, Oda Y, Shimizu T (2010) Phosphorylation of lysophosphatidylcho-
line acyltransferase 2 at Ser34 enhances platelet-activating factor production in endotoxin-
stimulated macrophages. J Biol Chem 285(39):2985729862
11. Morimoto R, Shindou H, Tarui M, Shimizu T (2014) Rapid production of platelet-activating
factor is induced by protein kinase c alpha-mediated phosphorylation of lysophosphatidylcho-
line acyltransferase 2 protein. J Biol Chem 289(22):1556615576
12. Nalefski EA, Sultzman LA, Martin DM, Kriz RW, Towler PS, Knopf JL, Clark JD (1994)
Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory
Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain. J Biol
Chem 269(27):1823918249
13. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ (1993) cPLA2 is phosphorylated
and activated by MAP kinase. Cell 72(2):269278
14. Tjoelker LW, Wilder C, Eberhardt C, Stafforini DM, Dietsch G, Schimpf B, Hooper S, Le
Trong H, Cousens LS, Zimmerman GA, Yamada Y, McIntyre TM, Prescott SM, Gray PW
106 S. Ishii
32. Korth RM, Hirafuji M, Benveniste J, Russo MF (1995) Human umbilical vein endothelial
cells: specific binding of platelet-activating factor and cytosolic calcium flux. Biochem
Pharmacol 49(12):17931799
33. Hwang SB, Lam MH, Shen TY (1985) Specific binding sites for platelet activating factor in
human lung tissues. Biochem Biophys Res Commun 128(2):972979
34. Bito H, Nakamura M, Honda Z-I, Izumi T, Iwatsubo T, Seyama Y, Ogura A, Kudo Y, Shimizu
T (1992) Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+
in hippocampal neurons. Neuron 9(2):285294
35. Ishii S, Kuwaki T, Nagase T, Maki K, Tashiro F, Sunaga S, Cao WH, Kume K, Fukuchi Y,
Ikuta K, Miyazaki J, Kumada M, Shimizu T (1998) Impaired anaphylactic responses with
intact sensitivity to endotoxin in mice lacking a platelet-activating factor receptor. J Exp Med
187(11):17791788
36. Soares AC, Pinho VS, Souza DG, Shimizu T, Ishii S, Nicoli JR, Teixeira MM (2002) Role of
the platelet-activating factor (PAF) receptor during pulmonary infection with gram negative
bacteria. Br J Pharmacol 137(5):621628
37. van Zoelen MAD, Florquin S, Meijers JCM, De Beer R, De Vos AF, De Boer OJ, Van Der Poll
T (2008) Platelet-activating factor receptor contributes to host defense against Pseudomonas
aeruginosa pneumonia but is not essential for the accompanying inflammatory and procoagu-
lant response. J Immunol 180(5):33573365
38. Fischer W, Behr T, Hartmann R, Peter-Katalinic J, Egge H (1993) Teichoic acid and lipotei-
choic acid of Streptococcus pneumoniae possess identical chain structures. A reinvestigation
of teichoid acid (C polysaccharide). Eur J Biochem 215(3):851857
39. Risberg A, Masoud H, Martin A, Richards JC, Moxon ER, Schweda EK (1999) Structural
analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient
strain of Haemophilus influenzae Rd. Eur J Biochem 261(1):171180
40. Cundell DR, Gerard NP, Gerard C, Idanpaan-Heikkila I, Tuomanen EI (1995) Streptococcus
pneumoniae anchor to activated human cells by the receptor for platelet-activating factor.
Nature 377(6548):435438
41. Rijneveld AW, Weijer S, Florquin S, Speelman P, Shimizu T, Ishii S, Van Der Poll T (2004)
Improved host defense against pneumococcal pneumonia in platelet-activating factor receptor-
deficient mice. J Infect Dis 189(4):711716
42. Branger J, Wieland CW, Florquin S, Maris NA, Pater JM, Speelman P, Shimizu T, Ishii S, Van
Der Poll T (2004) Platelet-activating factor receptor-deficient mice show an unaltered clear-
ance of nontypeable Haemophilus influenzae from their respiratory tract. Shock
22(6):543547
43. Swords WE, Buscher BA, Ver Steeg Ii K, Preston A, Nichols WA, Weiser JN, Gibson BW,
Apicella MA (2000) Non-typeable Haemophilus influenzae adhere to and invade human
bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor. Mol
Microbiol 37(1):1327
44. van Crevel R, Ottenhoff TH, van der Meer JW (2002) Innate immunity to Mycobacterium
tuberculosis. Clin Microbiol Rev 15(2):294309
45. Weijer S, Leemans JC, Florquin S, Shimizu T, Ishii S, Van Der Poll T (2003) Host response of
platelet-activating factor receptor-deficient mice during pulmonary tuberculosis. Immunology
109(4):552556
46. Madeira MF, Queiroz-Junior CM, Costa GM, Werneck SM, Cisalpino D, Garlet GP, Teixeira
MM, Silva TA, Souza DG (2013) Platelet-activating factor receptor blockade ameliorates
Aggregatibacter actinomycetemcomitans-induced periodontal disease in mice. Infect Immun
81(11):42444251
47. Emingil G, Cinarcik S, Baylas H, Huseyinov A (2001) Levels of platelet-activating factor in
gingival crevicular fluid and gingival tissue in specific periodontal diseases. J Periodontol
72(8):10321037
48. Hikiji H, Ishii S, Shindou H, Takato T, Shimizu T (2004) Absence of platelet-activating factor
receptor protects mice from osteoporosis following ovariectomy. J Clin Invest 114(1):8593
108 S. Ishii
49. Rothman AL (2004) Dengue: defining protective versus pathologic immunity. J Clin Invest
113(7):946951
50. Yang KD, Lee CS, Shaio MF (1995) A higher production of platelet activating factor in ex vivo
heterologously secondary dengue-2 virus infections. Acta Microbiol Immunol Hung
42(4):403407
51. Souza DG, Fagundes CT, Sousa LP, Amaral FA, Souza RS, Souza AL, Kroon EG, Sachs D,
Cunha FQ, Bukin E, Atrasheuskaya A, Ignatyev G, Teixeira MM (2009) Essential role of
platelet-activating factor receptor in the pathogenesis of dengue virus infection. Proc Natl
Acad Sci U S A 106(33):1413814143
52. Garcia CC, Russo RC, Guabiraba R, Fagundes CT, Polidoro RB, Tavares LP, Salgado APC,
Cassali GD, Sousa LP, Machado AV, Teixeira MM (2010) Platelet-activating factor receptor
plays a role in lung injury and death caused by influenza A in mice. PLoS Pathog 6(11):e1001171
53. Sibley LD (2011) Invasion and intracellular survival by protozoan parasites. Immunol Rev
240(1):7291
54. Santiago HC, Braga Pires MF, Souza DG, Roff E, Crtes DF, Tafuri WL, Teixeira MM, Vieira
LQ (2006) Platelet activating factor receptor-deficient mice present delayed interferon-
upregulation and high susceptibility to Leishmania amazonensis infection. Microbes Infect
8(11):25692577
55. Talvani A, Santana G, Barcelos LS, Ishii S, Shimizu T, Romanha J, Silva JS, Soares MBP,
Teixeira MM (2003) Experimental Trypanosoma cruzi infection in platelet-activating factor
receptor-deficient mice. Microbes Infect 5(9):789796
56. van der Heyde HC, Nolan J, Combes V, Gramaglia I, Grau GE (2006) A unified hypothesis for
the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to micro-
circulatory dysfunction. Trends Parasitol 22(11):503508
57. Lou J, Lucas R, Grau GE (2001) Pathogenesis of cerebral malaria: recent experimental data
and possible applications for humans. Clin Microbiol Rev 14(4):810820
58. Lacerda-Queiroz N, Rodrigues DH, Vilela MC, Rachid MA, Soriani FM, Sousa LP, Campos
RDL, Quesniaux VFJ, Teixeira MM, Teixeira AL (2012) Platelet-activating factor receptor is
essential for the development of experimental cerebral malaria. Am J Pathol 180(1):246255
59. Brooker S (2010) Estimating the global distribution and disease burden of intestinal nematode
infections: adding up the numbersa review. Int J Parasitol 40(10):11371144
60. Sato Y, Toma H (1990) Strongyloides venezuelensis infections in mice. Int J Parasitol
20(1):5762
61. Negro-Corra D, Souza DG, Pinho V, Barsante MM, Souza ALS, Teixeira MM (2004)
Platelet-activating factor receptor deficiency delays elimination of adult worms but reduces
fecundity in Strongyloides venezuelensis-infected mice. Infect Immun 72(2):11351142
62. MacMicking JD, North RJ, LaCourse R, Mudgett JS, Shah SK, Nathan CF (1997) Identification
of nitric oxide synthase as a protective locus against tuberculosis. Proc Natl Acad Sci U S A
94(10):52435248
63. Cabellos C, MacIntyre DE, Forrest M, Burroughs M, Prasad S, Tuomanen E (1992) Differing
roles for platelet-activating factor during inflammation of the lung and subarachnoid space.
The special case of Streptococcus pneumoniae. J Clin Invest 90(2):612618
64. Ichinose M, Hara N, Sawada M, Maeno T (1994) A flow cytometric assay reveals an enhance-
ment of phagocytosis by platelet activating factor in murine peritoneal macrophages. Cell
Immunol 156(2):508518
65. Au BT, Teixeira MM, Collins PD, Williams TJ (2001) Blockade of PAF receptors controls
interleukin-8 production by regulating the activation of neutrophil CD11/CD18. Eur J
Pharmacol 425(1):6571
Chapter 8
Lysophospholipid Mediators: Their Receptors
and Synthetic Pathways
8.1 Introduction
studies have identified multiple receptors and synthetic enzymes specific to each
LPL. From the studies on their receptors and producing enzymes together with stud-
ies on LPLs themselves, the pathophysiological roles have been identified espe-
cially for LPA, S1P, LysoPS, and LPI. Thus, these LPLs are now known as lipid
mediators. In this chapter, we review the receptors and synthetic enzymes for each
LPL and summarize their pathophysiological function.
potential V1 (TRPV1), but we shall not consider these in this chapter. After their
discovery, multiple nomenclatures have been proposed. In this chapter, we princi-
pally follow the nomenclature proposed recently by Kihara et al. [1], except that
LysoPS receptors are noted as LPSx. In all cases, receptor proteins are noted as
LP(X)(y), whereas receptor genes are LP(X)R(y), where X and X denote the head
group of LPLs and y and y denote the order of discovery.
In 1989, van Corven et al. reported that LPA-induced cell proliferation is G protein
(Gi) dependent and indicated that the LPA receptor was GPCR [2]. In 1996, Hecht
et al. first reported that EDG2 was a cellular receptor for LPA [3]. More recently,
Noguchi et al. reported that P2Y9 reacted specifically with LPA [4]. To date there
are six GPCRs for LPA belonging to either of two clusters: one is the EDG family
members consisting of LPA1/EDG2, LPA2/EDG4, and LPA3/EDG7, and the other is
the P2Y family members consisting of LPA4/P2Y9, LPA5/GPR92, and LPA6/P2Y5.
These LPA receptors are expressed by particular cells in different organs and acti-
vate multiple intracellular signaling pathways, which accounts for their many bio-
logical functions. Here, we summarize recent advances in the studies of LPA
receptors and their physiological function obtained mainly from analyses of knock-
out (KO) mice and human genetic diseases.
LPA1 is the first identified LPA receptor. It was identified by Chun and colleagues in
1996 [3]. LPA1 is expressed ubiquitously in various tissues. It couples to multiple G
proteins (Gi, Gq, G12/13) to activate various intracellular signaling pathways.
Several studies have shown the pathophysiological functions of LPA1. (1) LPA1
KO mice have defects in the development of parts of the central nervous system
such as the cerebral cortex and olfactory neurons. (2) LPA1 has a role in the adult
peripheral nervous system. LPA1 KO mice are resistant to injury-induced demyelin-
ation of the spinal cord and less sensitive to neuropathic pain [5, 6]. (3) LPA1 has
been linked to fibrotic disease in multiple organs. LPA1 promotes pulmonary fibro-
sis and renal fibrosis by mediating fibroblast migration and vascular permeability,
suggesting that LPA1 could be a promising new therapeutic target in fibrosis [7].
LPA1 KO mice were also reported to be resistant to bleomycin-induced dermal
fibrosis [8]. (4) LPA1 KO mice show cranial and rib cage deformities and low bone
mass [9]. We found that LPA1 KO mice exhibited abnormal chondrocyte arrange-
ment in cartilage tissue throughout the body, including the cranial bone
(unpublished).
112 K. Kano et al.
Two years after identification of LPA1, the second LPA receptor LPA2 was discov-
ered by virtue of its homology to LPA1 [10]. LPA2 is coupled with G proteins similar
to LPA1 (Gi, Gq, G12/13). LPA2 KO mice exhibit no obvious phenotypic abnor-
malities under normal conditions, and it is speculated that LPA2 acts redundantly
with LPA1 [11]. However, colon cancer induced by azoxymethane and dextran sul-
fate sodium was dramatically reduced in LPA2 KO mice [12]. In addition, dendritic
cells from LPA2 KO mice were found to induce T-cell proliferation and interleukin
(IL)-13 production more efficiently than their wild-type (WT) counterparts, and
LPA2 KO mice developed greater allergen-induced lung inflammation in ovalbumin-
challenged asthma [13], revealing the immunomodulatory roles of LPA2.
LPA3 is the third LPA receptor identified by our group in 1999 [14]. Interestingly,
LPA3 appears to discriminate among different LPA species (classes and positions of
fatty acid), and LPA3 is fully activated by 2-acyl-LPA with unsaturated fatty acids
such as 18:1, 18:2, and 20:4. LPA3 is known to couple to mainly Gq and Gi.
Female LPA3 KO mice exhibited implantation failure, as revealed by delayed
implantation and crowded implantation sites [15]. As a result, LPA3 KO mice
showed decreased litter size and shared placenta. In the mouse uterus, LPA3 is
expressed specifically in the endometrial epithelium in a progesterone-dependent
fashion during the peri-implantation period or menstrual cycle. This manner of
expression is also observed in humans, sheep, and pigs [16, 17]. On the other hand,
clinical observations revealed that LPA3 expression is low in the secretory endome-
trium of patients with recurrent implantation failure [18]. These findings suggest
that LPA3 is essential for normal pregnancy in a wide range of mammals.
LPA4 is a novel type of LPA receptor classified in the P2Y receptor family that is
very distant from Edg family LPA receptors. Noguchi et al. identified this receptor
by screening for orphan GPCRs using a large number of lipids in 2003 [4]. LPA4 is
coupled with various G proteins including Gs, Gq, Gi, and G12/13.
LPA4 deficiency in C57BL/6 mice was reported to be partially embryonically
lethal [19]. LPA4-deficient embryo displayed hemorrhage or edema in many organs
as a result of abnormal development of blood and lymphatic vessels. Mouse embryos
deficient in autotaxin (ATX), an LPA-producing enzyme, showed profound vascular
defects and died around embryonic day 9.510.5 [20]. Although the phenotype of
ATX-deficient mice was more severe than that of LPA4 KO mice, some of the phe-
notypes observed in ATX-deficient mice might be attributed to LPA4. Recently, we
found that zebrafish embryos showed aberrant segmental vascular development
8 Lysophospholipid Mediators: Their Receptors and Synthetic Pathways 113
upon knockdown of both LPA4 and LPA1 [21], again suggesting that LPA4 is one of
the candidates in vascular development by ATX-LPA signaling.
GPR92/LPA5 was shown to be an LPA receptor by two groups in 2006 [22, 23].
LPA5 was more strongly activated by alkyl-LPA than by acyl-LPA. Because alkyl-
LPA robustly aggregates human platelets, it has been speculated that LPA-induced
platelet activation and aggregation are mediated through LPA5 [24]. Although LPA5
did react with LPA, it also responds to a dietary protein hydrolysate (peptone) at
nanomolar (nM) concentrations [25] and to farnesyl pyrophosphate (FPP) [26]. So,
further studies are needed to reveal the real endogenous ligand for LPA5 and how
LPA5 distinguishes different ligands.
LPA5 is expressed in platelets, small intestine, and dorsal root ganglia. LPA5 KO
mice, which are used as a sciatic nerve ligation model, are apparently normal, but
not sensitive to injury-induced neuropathic pain [27]. As already described, LPA1
KO mice were also insensitive to neuropathic pain. However, LPA5 seems to have a
downstream signaling pathway that is mechanistically distinct from the LPA1 path-
way, because nerve injury induced demyelination in LPA5 KO mice but not in LPA1
KO mice.
LPA6 (P2Y5) is also a member of the P2Y family. It has high sequence homology to
LPA4. Patients who have mutations in LPAR6 or LIPH, a gene coding an LPA-
producing enzyme (PA-PLA1), show the same congenital hypotrichia phenotypes
[28, 29], which strongly suggests that P2Y5 is an LPA receptor (and PA-PLA1 is
an LPA-producing enzyme). However, conventional GPCR assays including Ca2+
and cAMP assays failed to detect LPA-induced activation of P2Y5. In 2009,
Yanagida et al. found that in B103 stably expressing P2Y5, LPA induced significant
neurite regression, and the LPA effect was blocked by ROCK inhibitor [30]. On the
other hand, we found that PA-PLA1 KO mice exhibited wavy hairs, which resem-
bled hairs in mutant mice defective in tumor necrosis factor--converting enzyme
(TACE), transforming growth factor- (TGF-), and epidermal growth factor recep-
tor (EGFR). In addition, we showed that LPA as well as recombinant PA-PLA1
itself induced P2Y5- and TACE-mediated ectodomain shedding of TGF- in
HEK293 cells [31]. These findings clearly showed that P2Y5 is a receptor for
LPA. Now, P2Y5 is recognized as the sixth LPA receptor, LPA6.
LPA6 showed a marked preference for 2-acyl LPA with unsaturated fatty acid
[30] and primarily coupled with G12/13. In addition to being expressed in hair fol-
licles, LPA6 is highly expressed in endothelial cells of blood vessels [32], which
suggests that LPA6 is involved in endothelial functions such as blood vessel
formation.
114 K. Kano et al.
LPA receptors other than those described here have been proposed: these include
GPR87 and P2Y10 because cells stably expressing these GPCRs responded to
LPA. However, further studies are needed to confirm these findings.
Several orphan GPCRs have been recently identified as LysoPS receptors, including
GPR34, P2Y10, A630033H20, and GPR174, all of which are P2Y family members.
According to the nomenclature of lysophospholipid receptors, we propose that
GPR34, P2Y10, A630033H20, and GPR174 be designated as LPS1, LPS2, LPS2-
like (LPS2L), and LPS3, respectively. These four LysoPS receptors are encoded by
the X chromosome and are highly expressed in immune cells, but their functions are
largely unknown.
In 2006, Sugo et al. identified GPR34 as the first receptor that is specific to LysoPS
[33]. They showed that LysoPS inhibited forskolin-stimulated cAMP accumulation,
promoted incorporation of [35S]-GTPS, and induced phosphorylation of ERK in
GPR34-expressing cells. The response was completely abolished by treatment of
pertussis toxin (PTX), indicating that GPR34 couples to Gi/Go-type G proteins.
Under normal breeding condition, GPR34 KO mice show no remarkable abnor-
malities in development, reproductive potential, size of organs, histology, and blood
parameters, although they were slightly less active in the open field test and more
active in the light arena in a lightdark test [34]. GPR34 is highly expressed in
mononuclear cells of the immune system, suggesting that it has a role in the immune
response. Accordingly, GPR34 KO mice (1) showed an increased delayed-type
hypersensitivity response, (2) were more susceptible toward a disseminating
Cryptococcus neoformans infection, and (3) accumulated significantly fewer granu-
locytes/macrophages in the spleen after methylated bovine serum albumin (BSA)
immunization. They produced more cytokines (TNF-, GM-CSF, interferon
(IFN)-). These results show that GPR34 is important in appropriate immune
response. Further studies are needed to elucidate what types of cells are involved.
but not with other G proteins. In 2008, it was reported that a fusion protein of
P2Y10 and G16 was activated by LPA and S1P [36]. However, our TGF- shedding
assay showed that P2Y10 is a LysoPS-specific receptor [35]. Neither LPA nor S1P
induced P2Y10-dependent response, even in the presence of G16. P2Y10 is highly
expressed in thymus and spleen, suggesting that it has a role in the immune system.
However, the in vivo role of P2Y10 is unknown.
The role of LPI was first demonstrated in 1986, when it was shown to stimulate the
release of insulin from pancreatic cells [43]. Subsequent studies found that LPI is
produced in various cell systems and that it induces a number of cellular events [44].
LPI is known as an agonist for two GPCRs (GPR55 and GPR119). However,
GPR119 is activated by not only LPI, but also by other lysophospholipids including
lysophosphatidylcholine [45, 46], so that in this section, we focus on GPR55 as an
LPI-specific receptor.
8.2.3.1 GPR55
Human GPR55 is 319 amino acids in length, and its gene maps to human chromo-
some 2q37. A database search for sequences similar to human GPR55 revealed that
GPR55 is conserved among vertebrates from fish to mammals. The closest homo-
logues to GPR55, as judged by amino acid homology, are LPA6/P2Y5 (29 %), LPA4/
GPR23 (30 %), GPR35 (27 %), and the chemokine receptor CCR4 (23 %). GPR55
was once proposed as the third receptor for cannabinoid. However, to date, the most
potent ligand identified for GPR55 is LPI [47]. Although GPR55 clearly interacts
with certain cannabinoid ligands that interact with cannabinoid receptors such as
CB1 and CB2, it is not clear whether CB1/CB2-independent cannabinoid actions
are mediated by GPR55.
GPR55 appears primarily to be coupled with G13. GPR55 is expressed in osteo-
clasts, and GPR55 activation in the cells results in osteoclastogenesis, cell polariza-
tion, and bone resorption [48]. Knockout of GPR55 in male mice significantly
increased the numbers of osteoclasts. These findings indicate that the LPIGPR55
axis affects differentiation and proliferation of osteoclasts and thus regulates bone
metabolism. GPR55 KO mice were also resistant to mechanical hyperalgesia
associated with Freunds complete adjuvant (FCA)-induced inflammation or partial
nerve ligation [49]. In GPR55 KO female mice, the onset of experimentally autoim-
mune encephalomyelitis was delayed, and the symptoms were less severe than
those in WT mice [50].
Phospholipid
PLA1 PLA2
2-acyl-lysophospholipid 1-acyl-lysophospholipid
lysoPLD (Autotaxin)
Phosphatidic acid
(PA)
Fig. 8.1 Lysophosphatidic acid (LPA)-producing pathway. There are two pathways of LPA pro-
duction. First, lysophospholipids generated by phospholipase A1 (PLA1) or PLA2 reaction are sub-
sequently converted to LPA by lysophospholipase D (lysoPLD) reaction. Second, phosphatidic
acid (PA) is converted to LPA by the PLA1 or PLA2 reaction. In extracellular LPA production,
mainly two phospholipases, autotaxin (ATX)/lysoPLD and phosphatidic acid-specific phospholi-
pase A1 (PA-PLA1), are crucial
At least two pathways have been postulated for bioactive lipid LPA production. In
the first pathway, lysophospholipids are produced by phospholipase A1 (PLA1) or
phospholipase A2 (PLA2) and then converted to LPA by lysophospholipase D (lyso-
PLD). In the other pathway, phosphatidic acid is hydrolyzed by PLA1 or PLA2,
generating LPA. Extracellular LPA production mainly involves two phospholipases,
autotaxin (ATX)/lysoPLD and phosphatidic acid-selective phospholipase A1
(PA-PLA1). Inside the cell, LPA is generated as intermediates by addition of fatty
acyl-CoA to glycerol-3-phosphate in the course of de novo phospholipid biosynthe-
sis. This LPA-producing pathway is conserved in lower organisms such as
Escherichia coli that do not have LPA receptors. Currently, there is no evidence that
this pathway contributes to the extracellular functions of LPA production. In this
section, we describe the latest findings in LPA-producing enzymes, especially ATX
and PA-PLA1.
118 K. Kano et al.
ATX, although the role of ATX in these tumors is unknown. Mice overexpressing
ATX exhibit a higher rate of the initiation and metastasis of breast cancer, which
suggests that ATX is related to the initiation and progression of cancer [68].
Although the synthetic pathway of LPA is well known, the synthetic pathways for
LysoPS and LPI remained to be solved. Analyses of these LPLs in tissues and cells
by a recently developed LC-MS/MS-based method revealed that they are mixtures
of 1-acyl-LPLs and 2-acyl-LPLs, indicating that both PLA1 and PLA2 are involved.
120 K. Kano et al.
Rat platelets express two extracellular PLA enzymes, secretory PLA2 group IIA
(sPLA2IIA) and PS-specific PLA1 (PS-PLA1), and secrete them upon activation
(Fig. 8.2). In the course of activation of rat platelets, sPLA2-IIA and PS-PLA1 are
thought to participate in production of 1-acyl LysoPS and 2-acyl LysoPS,
respectively.
PS-PLA1 is stored in granules in rat platelets and is secreted into the medium
when activated. Although PS-PLA1 is structurally homologous to triglyceride (TG)
lipase, it selectively hydrolyzes PS and does not have lipase activity for TG [74].
The amino acid sequence of PS-PLA1 showed about 30 % identity to an LPA-
producing enzyme, PA-PLA1, and PS-PLA1 expression is dramatically induced at
the mRNA and protein levels by various inflammatory stimuli. For these reasons,
PS-PLA1 is the most likely candidate for an in vivo LysoPS-producing enzyme.
Because secretory PLA2 and PS-PLA1 are secreted proteins, they should act on
PS extracellularly. However, PS exists in the inner leaflet of the plasma membrane.
Recently, TMEM16F was identified as a scramblase that triggers exposure of PS in
activated platelets [75]. Although LysoPS exists at only several nanomoles (nM) in
mouse plasma, it exists at about 100 nM in mouse serum, and especially LysoPS
species with unsaturated fatty acids are abundant. These observations suggest that
LysoPS is produced as follows: PS is exposed outside the activated platelets by
TMEM16F during serum preparation or in wound sites and then deacylated by a
PLA1 such as PS-PLA1.
O O
HOOC P
Phosphatidylserine O O O
OH O
PS NH2
O
O O O
HOOC P HOOC P
O O O O O OH
OH OH OH O
NH2 NH2
O
1-acyl-LysoPS 2-acyl-LysoPS
8.4 Conclusion
In contrast to LPA, which is relatively well characterized, LysoPS and LPI are just
beginning to be investigated. Receptors for LPI and LysoPS have only recently been
identified. In the future, physiological and pathophysiological roles of LPI and
LysoPS as novel lysophospholipid mediators will be elucidated through functional
analysis of these receptors.
References
3. Hecht JH, Weiner JA, Post SR, Chun J (1996) Ventricular zone gene-1 (vzg-1) encodes a lyso-
phosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex.
J Cell Biol 135:10711083
4. Noguchi K, Ishii S, Shimizu T (2003) Identification of p2y9/GPR23 as a novel G protein-
coupled receptor for lysophosphatidic acid, structurally distant from the Edg family. J Biol
Chem 278:2560025606
5. Estivill-Torrus G, Llebrez-Zayas P, Matas-Rico E, Santin L, Pedraza C, De Diego I, Del Arco
I, Fernandez-Llebrez P, Chun J, De Fonseca FR (2008) Absence of LPA1 signaling results in
defective cortical development. Cereb Cortex 18:938950
6. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H (2004) Initiation of neuropathic
pain requires lysophosphatidic acid receptor signaling. Nat Med 10:712718
7. Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, Polosukhin V, Wain J,
Karimi-Shah BA, Kim ND et al (2008) The lysophosphatidic acid receptor LPA1 links pulmo-
nary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med
14:4554
8. Castelino FV, Seiders J, Bain G, Brooks SF, King CD, Swaney JS, Lorrain DS, Chun J, Luster
AD, Tager AM (2011) Amelioration of dermal fibrosis by genetic deletion or pharmacologic
antagonism of lysophosphatidic acid receptor 1 in a mouse model of scleroderma. Arthritis
Rheum 63:14051415
9. Gennero I, Laurencin-Dalicieux S, Conte-Auriol F, Briand-Mesange F, Laurencin D, Rue J,
Beton N, Malet N, Mus M, Tokumura A et al (2011) Absence of the lysophosphatidic acid
receptor LPA1 results in abnormal bone development and decreased bone mass. Bone
49:395403
10. An S, Bleu T, Hallmark OG, Goetzl EJ (1998) Characterization of a novel subtype of human
G protein-coupled receptor for lysophosphatidic acid. J Biol Chem 273:79067910
11. Contos JJ, Ishii I, Fukushima N, Kingsbury MA, Ye X, Kawamura S, Brown JH, Chun J (2002)
Characterization of lpa(2) (Edg4) and lpa(1)/lpa(2) (Edg2/Edg4) lysophosphatidic acid recep-
tor knockout mice: signaling deficits without obvious phenotypic abnormality attributable to
lpa(2). Mol Cell Biol 22:69216929
12. Lin S, Wang D, Iyer S, Ghaleb AM, Shim H, Yang VW, Chun J, Yun CC (2009) The absence
of LPA2 attenuates tumor formation in an experimental model of colitis-associated cancer.
Gastroenterology 136:17111720
13. Emo J, Meednu N, Chapman TJ, Rezaee F, Balys M, Randall T, Rangasamy T, Georas SN
(2012) Lpa2 is a negative regulator of both dendritic cell activation and murine models of
allergic lung inflammation. J Immunol 188:37843790
14. Bandoh K, Aoki J, Hosono H, Kobayashi S, Kobayashi T, Murakami-Murofushi K, Tsujimoto
M, Arai H, Inoue K (1999) Molecular cloning and characterization of a novel human G-protein-
coupled receptor, EDG7, for lysophosphatidic acid. J Biol Chem 274:2777627785
15. Ye X, Hama K, Contos JJ, Anliker B, Inoue A, Skinner MK, Suzuki H, Amano T, Kennedy G,
Arai H et al (2005) LPA3-mediated lysophosphatidic acid signalling in embryo implantation
and spacing. Nature 435:104108
16. Ziecik AJ, Waclawik A, Bogacki M (2008) Conceptus signals for establishment and mainte-
nance of pregnancy in pigs: lipid signaling system. Exp Clin Endocrinol Diabetes
116:443449
17. Liszewska E, Reinaud P, Dubois O, Charpigny G (2012) Lysophosphatidic acid receptors in
ovine uterus during estrous cycle and early pregnancy and their regulation by progesterone.
Domest Anim Endocrinol 42:3142
18. Achache H, Tsafrir A, Prus D, Reich R, Revel A (2010) Defective endometrial prostaglandin
synthesis identified in patients with repeated implantation failure undergoing in vitro fertiliza-
tion. Fertil Steril 94:12711278
19. Sumida H, Noguchi K, Kihara Y, Abe M, Yanagida K, Hamano F, Sato S, Tamaki K, Morishita
Y, Kano MR et al (2010) LPA4 regulates blood and lymphatic vessel formation during mouse
embryogenesis. Blood 116:50605070
8 Lysophospholipid Mediators: Their Receptors and Synthetic Pathways 123
20. Tanaka M, Okudaira S, Kishi Y, Ohkawa R, Iseki S, Ota M, Noji S, Yatomi Y, Aoki J, Arai H
(2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by produc-
ing lysophosphatidic acid. J Biol Chem 281:2582225830
21. Yukiura H, Hama K, Nakanaga K, Tanaka M, Asaoka Y, Okudaira S, Arima N, Inoue A,
Hashimoto T, Arai H et al (2011) Autotaxin regulates vascular development via multiple lyso-
phosphatidic acid (LPA) receptors in zebrafish. J Biol Chem 286:4397243983
22. Lee CW, Rivera R, Gardell S, Dubin AE, Chun J (2006) GPR92 as a new G12/13- and
Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5. J Biol Chem
281:2358923597
23. Kotarsky K, Boketoft A, Bristulf J, Nilsson NE, Norberg A, Hansson S, Owman C, Sillard R,
Leeb-Lundberg LM, Olde B (2006) Lysophosphatidic acid binds to and activates GPR92, a G
protein-coupled receptor highly expressed in gastrointestinal lymphocytes. J Pharmacol Exp
Ther 318:619628
24. Williams JR, Khandoga AL, Goyal P, Fells JI, Perygin DH, Siess W, Parrill AL, Tigyi G,
Fujiwara Y (2009) Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor
indicates role in human platelet activation. J Biol Chem 284:1730417319
25. Choi S, Lee M, Shiu AL, Yo SJ, Aponte GW (2007) Identification of a protein hydrolysate
responsive G protein-coupled receptor in enterocytes. Am J Physiol Gastrointest Liver Physiol
292:G98G112
26. Oh DY, Yoon JM, Moon MJ, Hwang JI, Choe H, Lee JY, Kim JI, Kim S, Rhim H, ODell DK
et al (2008) Identification of farnesyl pyrophosphate and N-arachidonylglycine as endogenous
ligands for GPR92. J Biol Chem 283:2105421064
27. Lin ME, Rivera RR, Chun J (2012) Targeted deletion of LPA5 identifies novel roles for lyso-
phosphatidic acid signaling in development of neuropathic pain. J Biol Chem
287:1760817617
28. Kazantseva A, Goltsov A, Zinchenko R, Grigorenko AP, Abrukova AV, Moliaka YK, Kirillov
AG, Guo Z, Lyle S, Ginter EK et al (2006) Human hair growth deficiency is linked to a genetic
defect in the phospholipase gene LIPH. Science 314:982985
29. Shimomura Y, Wajid M, Ishii Y, Shapiro L, Petukhova L, Gordon D, Christiano AM (2008)
Disruption of P2RY5, an orphan G protein-coupled receptor, underlies autosomal recessive
woolly hair. Nat Genet 40:335339
30. Yanagida K, Masago K, Nakanishi H, Kihara Y, Hamano F, Tajima Y, Taguchi R, Shimizu T,
Ishii S (2009) Identification and characterization of a novel lysophosphatidic acid receptor,
p2y5/LPA(6). J Biol Chem 284:1773117741
31. Inoue A, Arima N, Ishiguro J, Prestwich GD, Arai H, Aoki J (2011) LPA-producing enzyme
PA-PLA(1)alpha regulates hair follicle development by modulating EGFR signalling. EMBO
J 30:42484260
32. Ren Y, Guo L, Tang X, Apparsundaram S, Kitson C, Deguzman J, Fuentes ME, Coyle L,
Majmudar R, Allard J et al (2013) Comparing the differential effects of LPA on the barrier
function of human pulmonary endothelial cells. Microvasc Res 85:5967
33. Sugo T, Tachimoto H, Chikatsu T, Murakami Y, Kikukawa Y, Sato S, Kikuchi K, Nagi T,
Harada M, Ogi K et al (2006) Identification of a lysophosphatidylserine receptor on mast cells.
Biochem Biophys Res Commun 341:10781087
34. Liebscher I, Muller U, Teupser D, Engemaier E, Engel KM, Ritscher L, Thor D, Sangkuhl K,
Ricken A, Wurm A et al (2011) Altered immune response in mice deficient for the G protein-
coupled receptor GPR34. J Biol Chem 286:21012110
35. Inoue A, Ishiguro J, Kitamura H, Arima N, Okutani M, Shuto A, Higashiyama S, Ohwada T,
Arai H, Makide K et al (2012) TGFalpha shedding assay: an accurate and versatile method for
detecting GPCR activation. Nat Methods 9:10211029
36. Murakami M, Shiraishi A, Tabata K, Fujita N (2008) Identification of the orphan GPCR,
P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor. Biochem
Biophys Res Commun 371:707712
124 K. Kano et al.
37. Sugita K, Yamamura C, Tabata K, Fujita N (2013) Expression of orphan G-protein coupled
receptor GPR174 in CHO cells induced morphological changes and proliferation delay via
increasing intracellular cAMP. Biochem Biophys Res Commun 430:190195
38. Frasch SC, Fernandez-Boyanapalli RF, Berry KZ, Leslie CC, Bonventre JV, Murphy RC,
Henson PM, Bratton DL (2011) Signaling via macrophage G2A enhances efferocytosis of
dying neutrophils by augmentation of Rac activity. J Biol Chem 286:1210812122
39. Murakami N, Yokomizo T, Okuno T, Shimizu T (2004) G2A is a proton-sensing G-protein-
coupled receptor antagonized by lysophosphatidylcholine. J Biol Chem 279:4248442491
40. Obinata H, Hattori T, Nakane S, Tatei K, Izumi T (2005) Identification of 9-hydroxyoc-
tadecadienoic acid and other oxidized free fatty acids as ligands of the G protein-coupled
receptor G2A. J Biol Chem 280:4067640683
41. Iwashita M, Makide K, Nonomura T, Misumi Y, Otani Y, Ishida M, Taguchi R, Tsujimoto M,
Aoki J, Arai H et al (2009) Synthesis and evaluation of lysophosphatidylserine analogues as
inducers of mast cell degranulation. Potent activities of lysophosphatidylthreonine and its
2-deoxy derivative. J Med Chem 52:58375863
42. Chang HW, Inoue K, Bruni A, Boarato E, Toffano G (1988) Stereoselective effects of lyso-
phosphatidylserine in rodents. Br J Pharmacol 93:647653
43. Metz SA (1986) Lysophosphatidylinositol, but not lysophosphatidic acid, stimulates insulin
release. A possible role for phospholipase A2 but not de novo synthesis of lysophospholipid in
pancreatic islet function. Biochem Biophys Res Commun 138:720727
44. Pineiro R, Falasca M (2012) Lysophosphatidylinositol signalling: new wine from an old bottle.
Biochim Biophys Acta 1821:694705
45. Andersson DA, Nash M, Bevan S (2007) Modulation of the cold-activated channel TRPM8 by
lysophospholipids and polyunsaturated fatty acids. J Neurosci 27:33473355
46. Soga T, Ohishi T, Matsui T, Saito T, Matsumoto M, Takasaki J, Matsumoto S, Kamohara M,
Hiyama H, Yoshida S et al (2005) Lysophosphatidylcholine enhances glucose-dependent insu-
lin secretion via an orphan G-protein-coupled receptor. Biochem Biophys Res Commun
326:744751
47. Yamashita A, Oka S, Tanikawa T, Hayashi Y, Nemoto-Sasaki Y, Sugiura T (2013) The actions
and metabolism of lysophosphatidylinositol, an endogenous agonist for GPR55. Prostaglandins
Other Lipid Mediat 107:103116
48. Whyte LS, Ryberg E, Sims NA, Ridge SA, Mackie K, Greasley PJ, Ross RA, Rogers MJ
(2009) The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone
mass in vivo. Proc Natl Acad Sci U S A 106:1651116516
49. Staton PC, Hatcher JP, Walker DJ, Morrison AD, Shapland EM, Hughes JP, Chong E, Mander
PK, Green PJ, Billinton A et al (2008) The putative cannabinoid receptor GPR55 plays a role
in mechanical hyperalgesia associated with inflammatory and neuropathic pain. Pain
139:225236
50. Sisay S, Pryce G, Jackson SJ, Tanner C, Ross RA, Michael GJ, Selwood DL, Giovannoni G,
Baker D (2013) Genetic background can result in a marked or minimal effect of gene knockout
(GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multi-
ple sclerosis. PLoS One 8, e76907
51. Stracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, Schiffmann E, Liotta LA (1992)
Identification, purification, and partial sequence analysis of autotaxin, a novel motility-
stimulating protein. J Biol Chem 267:25242529
52. Tokumura A, Majima E, Kariya Y, Tominaga K, Kogure K, Yasuda K, Fukuzawa K (2002)
Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing
enzyme, as autotaxin, a multifunctional phosphodiesterase. J Biol Chem 277:3943639442
53. Umezu-Goto M, Kishi Y, Taira A, Hama K, Dohmae N, Takio K, Yamori T, Mills GB, Inoue
K, Aoki J et al (2002) Autotaxin has lysophospholipase D activity leading to tumor cell growth
and motility by lysophosphatidic acid production. J Cell Biol 158:227233
8 Lysophospholipid Mediators: Their Receptors and Synthetic Pathways 125
54. Hausmann J, Kamtekar S, Christodoulou E, Day JE, Wu T, Fulkerson Z, Albers HM, van
Meeteren LA, Houben AJ, van Zeijl L et al (2011) Structural basis of substrate discrimination
and integrin binding by autotaxin. Nat Struct Mol Biol 18:198204
55. Nishimasu H, Okudaira S, Hama K, Mihara E, Dohmae N, Inoue A, Ishitani R, Takagi J, Aoki
J, Nureki O (2011) Crystal structure of autotaxin and insight into GPCR activation by lipid
mediators. Nat Struct Mol Biol 18:205212
56. van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, van Rooijen MA, Pradere JP, Pettit TR,
Wakelam MJ, Saulnier-Blache JS, Mummery CL et al (2006) Autotaxin, a secreted lysophos-
pholipase D, is essential for blood vessel formation during development. Mol Cell Biol
26:50155022
57. Tanaka M, Kishi Y, Takanezawa Y, Kakehi Y, Aoki J, Arai H (2004) Prostatic acid phosphatase
degrades lysophosphatidic acid in seminal plasma. FEBS Lett 571:197204
58. Masuda A, Nakamura K, Izutsu K, Igarashi K, Ohkawa R, Jona M, Higashi K, Yokota H,
Okudaira S, Kishimoto T et al (2008) Serum autotaxin measurement in haematological malig-
nancies: a promising marker for follicular lymphoma. Br J Haematol 143:6070
59. Nakamura K, Igarashi K, Ide K, Ohkawa R, Okubo S, Yokota H, Masuda A, Oshima N,
Takeuchi T, Nangaku M et al (2008) Validation of an autotaxin enzyme immunoassay in
human serum samples and its application to hypoalbuminemia differentiation. Clin Chim Acta
388:5158
60. Masuda A, Fujii T, Iwasawa Y, Nakamura K, Ohkawa R, Igarashi K, Okudaira S, Ikeda H,
Kozuma S, Aoki J et al (2011) Serum autotaxin measurements in pregnant women: application
for the differentiation of normal pregnancy and pregnancy-induced hypertension. Clin Chim
Acta 412:19441950
61. Kremer AE, Martens JJWW, Kulik W, Rueff F, Kuiper EMM, van Buuren HR, van Erpecum
KJ, Kondrackiene J, Prieto J, Rust C et al (2010) Lysophosphatidic acid is a potential mediator
of cholestatic pruritus. Gastroenterology 139:10081018
62. Kanda H, Newton R, Klein R, Morita Y, Gunn MD, Rosen SD (2008) Autotaxin, an ectoen-
zyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary
lymphoid organs. Nat Immunol 9:415423
63. Nakasaki T, Tanaka T, Okudaira S, Hirosawa M, Umemoto E, Otani K, Jin S, Bai Z, Hayasaka
H, Fukui Y et al (2008) Involvement of the lysophosphatidic acid-generating enzyme autotaxin
in lymphocyte-endothelial cell interactions. Am J Pathol 173:15661576
64. Zhang Y, Chen YC, Krummel MF, Rosen SD (2012) Autotaxin through lysophosphatidic acid
stimulates polarization, motility, and transendothelial migration of naive T cells. J Immunol
189:39143924
65. Inoue M, Ma L, Aoki J, Chun J, Ueda H (2008) Autotaxin, a synthetic enzyme of lysophospha-
tidic acid (LPA), mediates the induction of nerve-injured neuropathic pain. Mol Pain 4:6
66. Oikonomou N, Thanasopoulou A, Tzouvelekis A, Harokopos V, Paparountas T, Nikitopoulou
I, Witke W, Karameris A, Kotanidou A, Bouros D et al (2009) Gelsolin expression is necessary
for the development of modelled pulmonary inflammation and fibrosis. Thorax 64:467475
67. Hama K, Aoki J, Fukaya M, Kishi Y, Sakai T, Suzuki R (2004) Lysophosphatidic acid and
autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA(1). J Biol
Chem 279:1763417639
68. Liu SY, Umezu-Goto M, Murph M, Lu YL, Liu WB, Zhang F, Yu SX, Stephens LC, Cui XJ,
Murrow G et al (2009) Expression of autotaxin and lysophosphatidic acid receptors increases
mammary tumorigenesis, invasion, and metastases. Cancer Cell 15:539550
69. Sonoda H, Aoki J, Hiramatsu T, Ishida M, Bandoh K, Nagai Y, Taguchi R, Inoue K, Arai H
(2002) A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic
acid. J Biol Chem 277:3425434263
70. Shinkuma S, Akiyama M, Inoue A, Aoki J, Natsuga K, Nomura T, Arita K, Abe R, Ito K,
Nakamura H et al (2010) Prevalent LIPH founder mutations lead to loss of P2Y5 activation
ability of PA-PLA1alpha in autosomal recessive hypotrichosis. Hum Mutat 31:602610
126 K. Kano et al.
71. Ali G, Chishti MS, Raza SI, John P, Ahmad W (2007) A mutation in the lipase H (LIPH) gene
underlie autosomal recessive hypotrichosis. Hum Genet 121:319325
72. Tariq M, Azhar A, Baig SM, Dahl N, Klar J (2012) A novel mutation in the lipase H gene
underlies autosomal recessive hypotrichosis and woolly hair. Sci Rep 2:730
73. Pasternack SM, von Kugelgen I, Al Aboud K, Lee YA, Ruschendorf F, Voss K, Hillmer AM,
Molderings GJ, Franz T, Ramirez A et al (2008) G protein-coupled receptor P2Y5 and its
ligand LPA are involved in maintenance of human hair growth. Nat Genet 40:329334
74. Sato T, Aoki J, Nagai Y, Dohmae N, Takio K, Doi T, Arai H, Inoue K (1997) Serine
phospholipid-specific phospholipase A that is secreted from activated platelets. A new mem-
ber of the lipase family. J Biol Chem 272:21922198
75. Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling
by TMEM16F. Nature 468:834838
76. Imae R, Inoue T, Kimura M, Kanamori T, Tomioka NH, Kage-Nakadai E, Mitani S, Arai H
(2010) Intracellular phospholipase A1 and acyltransferase, which are involved in
Caenorhabditis elegans stem cell divisions, determine the sn-1 fatty acyl chain of phosphati-
dylinositol. Mol Biol Cell 21:31143124
77. Higgs HN, Han MH, Johnson GE, Glomset JA (1998) Cloning of a phosphatidic acid-
preferring phospholipase A1 from bovine testis. J Biol Chem 273:54685477
78. Mariggio S, Sebastia J, Filippi BM, Iurisci C, Volonte C, Amadio S, De Falco V, Santoro M,
Corda D (2006) A novel pathway of cell growth regulation mediated by a PLA2alpha-derived
phosphoinositide metabolite. FASEB J 20:25672569
Chapter 9
Sphingolipid Metabolism via Sphingosine
1-Phosphate and Its Role in Physiology,
Pathology, and Nutrition
Akio Kihara
Sphingolipids are one of the major eukaryotic lipid species and have a role in a
variety of physiological functions, including embryogenesis, organogenesis, skin
barrier formation, neural functions, cell adhesion, recognition of bacterial toxins
A. Kihara (*)
Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University,
Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan
e-mail: [email protected]
lacto-series, and neolacto-series (Fig. 9.1). SM3, which contains a sulfate group at
the 3-position of the galactose residue, is also produced from LacCer.
GalCer is produced by the Cer galactosyltransferase CGT/UGT8. Some GalCer
is further converted to sulfatide, GM4, or the gala-series GSLs (Fig. 9.1). GalCer
and sulfatide are enriched in myelin. Accordingly, Cgt-knockout mice, which pro-
duce neither GalCer nor sulfatide, exhibit neural symptoms such as severe general-
ized tremors and mild ataxia [15].
Sphingomyelin, the most abundant sphingolipid in mammals, can interact with
cholesterol to form lipid microdomains in the plasma membrane [16]. Sphingomyelin
synthases catalyze the formation of sphingomyelin by transferring the phosphocho-
line moiety of phosphatidylcholine to Cer. Two sphingomyelin synthases with dif-
fering intracellular localizations exist in mammals; SMS1 is localized to the
trans-Golgi network whereas SMS2 is localized to the plasma membrane [17].
Sms1-knockout mice exhibit impaired insulin secretion, immune function, and hear-
ing [1820], whereas Sms2-knockout mice are resistant to high-fat diet-induced
obesity and atherosclerosis [21, 22].
Cer can also be metabolized to Cer 1-phosphate (C1P) by the Cer kinase CerK
(Fig. 9.1). C1P is involved in the regulation of inflammation, stimulation of phago-
cytosis by neutrophils, degranulation of mast cells, and obesity [1, 23]. However,
significant amounts of C1P are still present in CerK-knockout mice, strongly sug-
gesting the existence of another C1P-generating pathway [24].
The polar head groups of sphingolipids are removed by a series of hydrolases, pro-
ducing Cer that is then degraded to sphingosine and a fatty acid by ceramidase.
Mutations in the genes encoding these hydrolases result in sphingolipid storage
diseases (SLSDs), also known as sphingolipidoses. Because the hydrolases respon-
sible for SLSs are localized to the lysosomes, SLSDs are classified as lysosomal
diseases. To date, about 10 SLSDs with about 40 genetically distinct forms have
been reported [25, 26]. For example, mutations in the lysosomal forms of cerami-
dase (acid ceramidase) and sphingomyelinase (acid sphingomyelinase) genes are
the underlying cause of Farber disease and NiemannPick disease (type A and B),
respectively [26, 27] (Fig. 9.2). Another form of NiemannPick disease (type C) is
caused by mutations in NPC-1 or NPC-2 [28]. NPC-1 and NPC-2 are thought to be
involved in the removal of cholesterol from endosomes, in their roles as a choles-
terol transporter and a cholesterol-binding protein, respectively [26, 28].
Furthermore, it has been suggested that the interaction of sphingomyelin with cho-
lesterol obstructs the removal of cholesterol from endosomes [26]. Accordingly,
patients with NiemannPick disease type A or B exhibit accumulation of choles-
terol as well as sphingomyelin [29].
Fig. 9.2 Sphingolipid degradation pathways and related disorders. In the sphingolipid degrada-
tion pathway, the polar head groups of complex sphingolipids are removed by the successive
actions of lysosomal hydrolases. The resulting Cer is then converted to sphingosine, which is either
recycled for sphingolipid synthesis or converted to S1P. When S1P is irreversibly degraded by S1P
lyase to trans-2-hexadecenal, it is metabolized to glycerophospholipids via trans-2-hexadecenoic
acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA
132 A. Kihara
After the degradation of Cer by ceramidase, the resultant fatty acids are converted
to acyl-CoAs and used in the synthesis of a variety of lipids such as glycerophos-
pholipids, triglycerides, and sphingolipids or in energy production via -oxidation.
On the other hand, sphingosine, another degradation product of Cer, is recycled
for sphingolipid synthesis or metabolized to glycerophospholipids via sphingo-
sine 1-phosphate (S1P). The latter pathway, referred to as the S1P-metabolic path-
way hereafter, is the sole route for the removal of the sphingosine (LCB)
component of sphingolipids. Therefore, the S1P-metabolic pathway is important
for the maintenance of sphingolipid homeostasis: indeed, this pathway is con-
served in eukaryotes.
Sphingosine/LCB is phosphorylated to S1P/LCB 1-phosphate (LCBP) by sphin-
gosine/LCB kinases. Mammals contain two sphingosine kinases: SPHK1 and
SPHK2 [31, 32]. Extracellular S1P acts as a lipid mediator by binding to one of the
five S1P receptors (S1P1S1P5). The functions of S1P as a lipid mediator are par-
ticularly important in the immune and vascular systems. Taking advantage of its
role in immune system, the sphingosine analogue fingolimod (FTY720) has been
developed to treat multiple sclerosis [33]. Fingolimod is a pro-drug: its functional
form is fingolimod phosphate, which binds to all S1P receptors with the exception
9 Sphingolipid Metabolism via Sphingosine 1-Phosphate 133
of S1P2 [34]. Only vertebrates and chordates possess S1P receptors, yet all
eukaryotes produce S1P/LCBP. Thus, in evolutionary terms, the role of S1P/LCBP
in sphingolipid metabolism greatly precedes its role as a lipid mediator [35].
The first irreversible (i.e., committed) step of the S1P-metabolic pathway is the
cleavage of S1P between the C2 and C3 positions by the S1P lyase SPL/SGPL1
[36]. This reaction produces the fatty aldehyde trans-2-hexadecenal and phospho-
ethanolamine. Spl-knockout mice die approximately 1 month after birth, and exhibit
abnormalities in the lung, heart, urinary tract, and bone, as well as displaying
enhanced pro-inflammatory responses, myeloid cell hyperplasia, and aberrant lipid
homeostasis in the liver and brain [3740], thus clearly indicating the importance of
the S1P-metabolic pathway. The S1P metabolite phosphoethanolamine is converted
to CDP-ethanolamine, which is then used for synthesis of the glycerophospholipid
phosphatidylethanolamine. On the other hand, trans-2-hexadecenal is converted to
palmitoyl-CoA via trans-2-hexadecenoic acid and trans-2-hexadecenoyl-CoA [41,
42]. The majority of palmitoyl-CoA is then incorporated into glycerophospholipids
[35, 4143]. All these reactions of the S1P-metabolic pathway occur in the ER [35].
The conversion of trans-2-hexadecenal to trans-2-haxadecenoic acid is cata-
lyzed by the fatty aldehyde dehydrogenase ALDH3A2 [41]. Mutations in the
ALDH3A2 gene cause the neurocutaneous disorder SjgrenLarsson syndrome
(SLS). ALDH3A2 exhibits activity toward a broad range of aliphatic aldehydes
with medium- and long-chain lengths in vitro [44]. A variety of ALDH3A2 sub-
strates are generated via the metabolism of lipids such as fatty alcohol, leukotriene
B4, and phytol (other than S1P) and through lipid peroxidation [45]. Aldehyde mol-
ecules are reactive and can form Schiff bases with the amino groups of biomole-
cules. Furthermore, ,-unsaturated aldehydes, to which trans-2-hexadecenal
belongs, can react with general nucleophiles (e.g., lysine, cysteine, and histidine
side chains) via a 1,4-Michael addition [46]. Therefore, accumulated aldehydes are
thought to cause SLS by reacting with important cellular biomolecules and impair-
ing their functions. However, the primary aldehyde responsible for the pathology of
SLS is still unknown. Both the activities of the S1P-producing enzyme (sphingosine
kinase) and the degradation enzyme (S1P lyase) are high in most tissues [47, 48],
suggesting that S1P metabolism is an active pathway that occurs ubiquitously and
continuously in cells. Therefore, the S1P metabolite trans-2-hexadecenal appears to
be produced constitutively at a relatively high level. In addition, both ALDH3A2
and trans-2-hexadecenal coexist in the ER [35], suggesting that trans-2-hexadecenal
may be a major ALDH3A2 substrate. Thus, it is possible that impairment of the
S1P-metabolic pathway is one of the major causes of SLS pathology.
Fatty acids must be converted to their active acyl-CoA form by acyl-CoA synthe-
tases (ACSs) for further metabolism. Humans possess 26 ACSs, which are classified
into six subfamiliesACSS, ACSM, ACSL, ACSVL, ACSF, and ACSBGbased
on their substrate specificity and sequence similarity [49]. Among them, the ACSL
subfamily members (ACSL1, -3, -4, -5, -6), which exhibit activity toward long-
chain fatty acids, have central roles in the conversion of trans-2-hexadecanoic acid
to trans-2-hexadecenoyl-CoA in the S1P-metabolic pathway [41, 43].
134 A. Kihara
The function of S1P as a lipid mediator has attracted significant attention and has
been analyzed extensively. Although research on the function of S1P as a metabolic
intermediate has slowed since the identification of S1P as an intermediate in the
sphingolipid degradation pathway in the late 1960s [60], the recent elucidation of
the entire S1P-metabolic pathway provides welcome information allowing further
analysis of the pathophysiological functions of this pathway. It is possible that
impairment of the S1P-metabolic pathway is responsible for the pathology of SLS
and other disorders. Recent analyses of Spl-knockout mice also emphasize the phys-
iological importance of the S1P-metabolic pathway. Future studies will doubtless
reveal additional physiological, pathological, and nutritional functions of the S1P-
metabolic pathway.
9 Sphingolipid Metabolism via Sphingosine 1-Phosphate 135
References
19. Dong L, Watanabe K, Itoh M, Huan CR, Tong XP, Nakamura T, Miki M, Iwao H, Nakajima A,
Sakai T, Kawanami T, Sawaki T, Masaki Y, Fukushima T, Fujita Y, Tanaka M, Yano M,
Okazaki T, Umehara H (2012) CD4+ T-cell dysfunctions through the impaired lipid rafts ame-
liorate concanavalin A-induced hepatitis in sphingomyelin synthase 1-knockout mice. Int
Immunol 24:327337
20. Lu MH, Takemoto M, Watanabe K, Luo H, Nishimura M, Yano M, Tomimoto H, Okazaki T,
Oike Y, Song WJ (2012) Deficiency of sphingomyelin synthase-1 but not sphingomyelin syn-
thase-2 causes hearing impairments in mice. J Physiol 590:40294044
21. Mitsutake S, Zama K, Yokota H, Yoshida T, Tanaka M, Mitsui M, Ikawa M, Okabe M, Tanaka
Y, Yamashita T, Takemoto H, Okazaki T, Watanabe K, Igarashi Y (2011) Dynamic modifica-
tion of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and
type 2 diabetes. J Biol Chem 286:2854428555
22. Liu J, Huan C, Chakraborty M, Zhang H, Lu D, Kuo MS, Cao G, Jiang XC (2009) Macrophage
sphingomyelin synthase 2 deficiency decreases atherosclerosis in mice. Circ Res
105:295303
23. Gomez-Muoz A, Gangoiti P, Arana L, Ouro A, Rivera IG, Ordoez M, Trueba M (2013) New
insights on the role of ceramide 1-phosphate in inflammation. Biochim Biophys Acta
1831:10601066
24. Boath A, Graf C, Lidome E, Ullrich T, Nussbaumer P, Bornancin F (2008) Regulation and
traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to gluco-
sylceramide and sphingomyelin. J Biol Chem 283:85178526
25. Sillence DJ, Platt FM (2003) Storage diseases: new insights into sphingolipid functions.
Trends Cell Biol 13:195203
26. Schulze H, Sandhoff K (2014) Sphingolipids and lysosomal pathologies. Biochim Biophys
Acta 1841:799810
27. Park JH, Schuchman EH (2006) Acid ceramidase and human disease. Biochim Biophys Acta
1758:21332138
28. Storch J, Xu Z (2009) Niemann-Pick C2 (NPC2) and intracellular cholesterol trafficking.
Biochim Biophys Acta 1791:671678
29. Vanier MT (1983) Biochemical studies in Niemann-Pick disease. I. Major sphingolipids of
liver and spleen. Biochim Biophys Acta 750:178184
30. Tamargo RJ, Velayati A, Goldin E, Sidransky E (2012) The role of saposin C in Gaucher dis-
ease. Mol Genet Metab 106:257263
31. Kohama T, Olivera A, Edsall L, Nagiec MM, Dickson R, Spiegel S (1998) Molecular cloning
and functional characterization of murine sphingosine kinase. J Biol Chem 273:2372223728
32. Liu H, Sugiura M, Nava VE, Edsall LC, Kono K, Poulton S, Milstien S, Kohama T, Spiegel S
(2000) Molecular cloning and functional characterization of a novel mammalian sphingosine
kinase type 2 isoform. J Biol Chem 275:1951319520
33. Bigaud M, Guerini D, Billich A, Bassilana F, Brinkmann V (2014) Second generation S1P
pathway modulators: research strategies and clinical developments. Biochim Biophys Acta
1841:745758
34. Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, Thornton R, Shei GJ,
Card D, Keohane C, Rosenbach M, Hale J, Lynch CL, Rupprecht K, Parsons W, Rosen H
(2002) Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.
Science 296:346349
35. Kihara A (2014) Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glyc-
erophospholipids. Biochim Biophys Acta 1841:766772
36. Zhou J, Saba JD (1998) Identification of the first mammalian sphingosine phosphate lyase
gene and its functional expression in yeast. Biochem Biophys Res Commun 242:502507
37. Vogel P, Donoviel MS, Read R, Hansen GM, Hazlewood J, Anderson SJ, Sun W, Swaffield J,
Oravecz T (2009) Incomplete inhibition of sphingosine 1-phosphate lyase modulates immune
system function yet prevents early lethality and non-lymphoid lesions. PLoS One 4:e4112
9 Sphingolipid Metabolism via Sphingosine 1-Phosphate 137
38. Bektas M, Allende ML, Lee BG, Chen W, Amar MJ, Remaley AT, Saba JD, Proia RL (2010)
Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis in liver. J Biol Chem
285:1088010889
39. Allende ML, Bektas M, Lee BG, Bonifacino E, Kang J, Tuymetova G, Chen W, Saba JD, Proia
RL (2011) Sphingosine-1-phosphate lyase deficiency produces a pro-inflammatory response
while impairing neutrophil trafficking. J Biol Chem 286:73487358
40. Hagen-Euteneuer N, Lutjohann D, Park H, Merrill AH Jr, van Echten-Deckert G (2012)
Sphingosine 1-phosphate (S1P) lyase deficiency increases sphingolipid formation via recy-
cling at the expense of de novo biosynthesis in neurons. J Biol Chem 287:91289136
41. Nakahara K, Ohkuni A, Kitamura T, Abe K, Naganuma T, Ohno Y, Zoeller RA, Kihara A
(2012) The Sjgren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the
sphingosine 1-phosphate degradation pathway. Mol Cell 46:461471
42. Wakashima T, Abe K, Kihara A (2014) Dual functions of the trans-2-enoyl-CoA reductase
TER in the sphingosine 1-phosphate metabolic pathway and in fatty acid elongation. J Biol
Chem 289:2473624748
43. Ohkuni A, Ohno Y, Kihara A (2013) Identification of acyl-CoA synthetases involved in the
mammalian sphingosine 1-phosphate metabolic pathway. Biochem Biophys Res Commun
442:195201
44. Kelson TL, Secor McVoy JR, Rizzo WB (1997) Human liver fatty aldehyde dehydrogenase:
microsomal localization, purification, and biochemical characterization. Biochim Biophys
Acta 1335:99110
45. Rizzo WB (2011) The role of fatty aldehyde dehydrogenase in epidermal structure and func-
tion. Dermatoendocrinology 3:9199
46. Catal A (2009) Lipid peroxidation of membrane phospholipids generates hydroxy-alkenals
and oxidized phospholipids active in physiological and/or pathological conditions. Chem Phys
Lipids 157:111
47. Fukuda Y, Kihara A, Igarashi Y (2003) Distribution of sphingosine kinase activity in mouse
tissues: contribution of SPHK1. Biochem Biophys Res Commun 309:155160
48. Ikeda M, Kihara A, Igarashi Y (2004) Sphingosine-1-phosphate lyase SPL is an endoplasmic
reticulum-resident, integral membrane protein with the pyridoxal 5-phosphate binding domain
exposed to the cytosol. Biochem Biophys Res Commun 325:338343
49. Watkins PA, Maiguel D, Jia Z, Pevsner J (2007) Evidence for 26 distinct acyl-coenzyme A
synthetase genes in the human genome. J Lipid Res 48:27362750
50. Vesper H, Schmelz EM, Nikolova-Karakashian MN, Dillehay DL, Lynch DV, Merrill AH Jr
(1999) Sphingolipids in food and the emerging importance of sphingolipids to nutrition. J Nutr
129:12391250
51. Imaizumi K, Tominaga A, Sato M, Sugano M (1992) Effects of dietary sphingolipids on levels
of serum and liver lipids in rats. Nutr Res 12:543548
52. Dillehay DL, Webb SK, Schmelz EM, Merrill AH Jr (1994) Dietary sphingomyelin inhibits
1,2-dimethylhydrazine-induced colon cancer in CF1 mice. J Nutr 124:615620
53. Schmelz EM, Dillehay DL, Webb SK, Reiter A, Adams J, Merrill AH Jr (1996) Sphingomyelin
consumption suppresses aberrant colonic crypt foci and increases the proportion of adenomas
versus adenocarcinomas in CF1 mice treated with 1,2-dimethylhydrazine: implications for
dietary sphingolipids and colon carcinogenesis. Cancer Res 56:49364941
54. Kobayashi T, Shimizugawa T, Osakabe T, Watanabe S, Okuyama H (1997) A long-term feed-
ing of sphingolipids affected the levels of plasma cholesterol and hepatic triacylglycerol but
tissue phospholipids and sphingolipids. Nutr Res 17:111114
55. Duan J, Sugawara T, Sakai S, Aida K, Hirata T (2011) Oral glucosylceramide reduces
2,4-dinitrofluorobenzene induced inflammatory response in mice by reducing TNF- levels
and leukocyte infiltration. Lipids 46:505512
56. Duan J, Sugawara T, Hirose M, Aida K, Sakai S, Fujii A, Hirata T (2012) Dietary sphingolip-
ids improve skin barrier functions via the upregulation of ceramide synthases in the epidermis.
Exp Dermatol 21:448452
138 A. Kihara
57. Zhang Y, Cheng Y, Hansen GH, Niels-Christiansen LL, Koentgen F, Ohlsson L, Nilsson A,
Duan RD (2011) Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a
study on enzyme knockout mice. J Lipid Res 52:771781
58. Kono M, Dreier JL, Ellis JM, Allende ML, Kalkofen DN, Sanders KM, Bielawski J, Bielawska
A, Hannun YA, Proia RL (2006) Neutral ceramidase encoded by the Asah2 gene is essential
for the intestinal degradation of sphingolipids. J Biol Chem 281:73247331
59. Zhao Y, Kalari SK, Usatyuk PV, Gorshkova I, He D, Watkins T, Brindley DN, Sun C, Bittman
R, Garcia JG, Berdyshev EV, Natarajan V (2007) Intracellular generation of sphingosine
1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphin-
gosine kinase 1. J Biol Chem 282:1416514177
60. Stoffel W, Sticht G (1967) Metabolism of sphingosine bases. I. Degradation and incorporation
of [3-14C]erythro-DL-dihydrosphingosine and [7-3H2]erythro-DL-sphingosine into sphingolip-
ids of rat liver. Hoppe Seylers Z Physiol Chem 348:941943
Chapter 10
Fatty Acids Receptors
10.1 Introduction
Free fatty acids (FFAs) are not only essential dietary nutrients but they also act as
signaling molecules in various physiological functions. The nuclear receptors per-
oxisome proliferator-activated receptors (PPARs) and fatty acid-binding proteins
(FABPs) are known to act as sensors of FFAs. They maintain homeostasis under
A. Hirasawa (*)
Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical
Sciences, Kyoto University, Kyoto 606-8501, Japan
Institute for Integrated Medical Sciences, Tokyo Womens Medical University,
Tokyo 162-8666, Japan
e-mail: [email protected]
M. Takeuchi T. Hara A. Hirata S. Tanabe N. Umeda
Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical
Sciences, Kyoto University, Kyoto 606-8501, Japan
10.2 FFAR1
FFAR1, FFAR2, FFAR3, and GPR42, which is thought to be a pseudogene, are all
GPCRs of the rhodopsin family located within a gene cluster on the human 19q13 chro-
mosome. GPR42 only exists in the family Hominidae and cannot be detected in species
below gibbons. The members of this subfamily share approximately 3040 % identity,
with the exception that human GPR42 (hGPR42) differs from human FFAR3 (hFFAR3)
at only six amino acid positions [3]. These ndings suggest that hGPR42 arose as the
result of a gene duplication of hFFAR3 that occurred after the gibbons branched off from
the superfamily Hominoidea [3]. Recent advances in genomic analysis of various spe-
cies have revealed that FFAR1, FFAR2, and FFAR3 are widely conserved throughout
vertebrates from shes to mammals (with the exception of birds). In amphibians, rep-
tiles, and mammals, FFAR1, FFAR2, and FFAR3 create a family of genes in tandem
sequence with shared synteny (Fig. 10.1). However, similar genomic structures cannot
be found in birds, at least among genomically analyzed species such as pigeons and
chickens. On the other hand, we have found multiple clusters of these genes existing in
Teleostei, which suggests that multiplication of these genes occurred after the divergence
of amphibians. We have also found that only one homologous gene exists in cartilagi-
nous shes. To date, the function of FFARs have only been investigated in mammals,
and we lack information about their expression and physiological function in other spe-
cies. Thus, the connection between these receptors and physiological functions are not
fully understood and should become a matter of great interest in the upcoming years.
Fig. 10.1 Schematic representation of genomic structures around free fatty acids receptors (FFAR)14 of multiple animal species, and synteny analysis of the
FFAR4 locus between Homo sapiens and Xenopus tropicalis genomes. The homology genes of FFAR1 and FFAR4 are compared and aligned in each species.
Black or white boxes indicate genes that have been studied or not, respectively. Synteny analysis using Synteny Database [4] reveals syntenic conservation of
many neighbor genes around FFAR4 between human and Xenopus (cluster ID#1424337 according to the Synteny Database)
143
144 A. Hirasawa et al.
a synthetic FFAR1 antagonist, GW1100, has been identied and its antagonistic
activities were examined via in both in vitro and in vivo studies [1, 19, 22, 53]. The
antidiabetic thiazolidinediones troglitazone and rosiglitazone, and the experimental
anti-obesity compound MEDICA16, also activate FFAR1 [2, 16]. Very recently, the
X-ray crystallography of co-crystals of a novel compound, TAK-875 (fasiifam),
and FFAR1 was performed, and the state of the ligand binding to the receptor was
revealed [42]. It was reported that binding sites other than those predicted in recent
models exist and that TAK-875 could act as a partial agonist or an allosteric ligand.
In the same paper, a mass spectrometry-based ligand-binding assay system for
FFAR1 was also established, and the FFARligand interaction has been studied in
great detail.
Free fatty acids have so far been known to exert versatile effects on pancreatic
-cells. Chronic exposure to high levels of FFAs results in the impairment of -cell
function and secretory capacity, whereas acute administration of FFAs stimulates
insulin release. FFAs are considered to be important for maintaining basal insulin
secretion as well as increasing glucose-stimulated insulin secretion when fasting [1,
7, 8, 13, 43, 44], but the mechanisms of these phenomena have not been explained.
Itoh et al. showed that long-chain free fatty acids amplied glucose-stimulated insu-
lin secretion from pancreatic -cells via activation of FFAR1 [24]. Because inhibi-
tion of FFAR1 expression with small interfering RNA (siRNA) resulted in quenching
of FFA-stimulated insulin secretion, FFAR was presumed to be involved in this
pathway. In contrast to the decrease in FFA-stimulated insulin secretion observed in
FFAR1-decient -cells, FFAR1-decient mice actually exhibited resistance to
obesity-induced hyperinsulinemia, hepatic steatosis, hypertriglyceridemia,
increased hepatic glucose output, hyperglycemia, and glucose intolerance. Steneberg
et al. have indicated that both acute and chronic effects of FFAs were mediated by
FFAR1 [44]. In contrast, overexpression of FFAR1 in -cells by methods via the
mouse Ipf1/Pdx1 promoter impaired -cell function and resulted in hyperinsu-
linemia and diabetes [44]. Moreover, FFAR1 was found to regulate glucose-
stimulated insulin secretion by overexpressing FFAR1 under the control of mouse
insulin II promoter [33]. On the basis of these studies, we speculate that FFAR1 is
involved in an essential pathway that connects obesity and type 2 diabetes.
Furthermore, there have been several studies reporting genomic polymorphisms
in human FFAR1. One type of polymorphisms, D175N, has the same EC50 but lower
maximum response compared to the wild-type receptor. This polymorphism, how-
ever, has no relevance to changes in insulin secretion [15]. Another polymorphism,
R211H, shows no difference in primary response, but results from laboratory data
comparison suggests its involvement in insulin secretion [34]. Moreover, -cell
response to FFAs is quenched in the polymorphism G180S because of impaired
mechanisms in increasing intracellular Ca2+ concentration [52].
146 A. Hirasawa et al.
These results have raised a great deal of interest in FFAR1 as a potential target
for novel drugs in metabolic diseases such as type 2 diabetes. Various experimental
models have identied chemical compounds that display agonistic or antagonistic
activity, and their physiological and pharmacological functions are being examined.
One such compound was TAK-875 (fasiglifam, mentioned earlier), an orally avail-
able, potent, and selective agonist of FFAR1 [39]. This agent was tested in a phase
III clinical trial for the potential treatment of type 2 diabetes mellitus, but the trial
was cancelled because of an undesired side effect. Another FFAR1 agonist,
JTT-851, has completed phase II clinical trials and is anticipated to become the rst
therapeutic drug to target FFAR1.
10.3 FFAR4
Using a receptor internalization assay [11], medium- to long-chain FFAs were iden-
tied as endogenous ligands of FFAR4. Saturated FFAs (C14-18) and unsaturated
FFAs (C16-22) activate FFAR4. Although some have claimed that FFAR4 was a
selective receptor for -3 polyunsaturated fatty acids (PUFAs), it is now widely
accepted that both -3 and -6 PUFAs act as agonists. A variety of PUFAs, regard-
less of -3 or -6 species, can activate FFAR4 in the micromolar concentration
range [18]. The ligand proles for FFAR4 are similar to those for FFAR1; however,
the amino acid homology between FFAR4 and FFAR1 is only 10 %.
FFAR1 and FFAR4 have no homology in structure, although some ligands activate
both receptors. FFAR4 has been experimentally proved to function as a receptor in
only mammals. Nonetheless, the FFAR4 gene is conserved in vertebrates from
Coelacanthiformes to mammals, and the neighboring genomic structures are also
quite similar, as shown in the comparison of the human and Silurana tropicalis
genomes (Fig. 10.1). However, differing from FFAR1, FFAR2, and FFAR3, which
prevail throughout vertebrates, the FFAR4 gene cannot be seen in teleost sh, with
an exception of the family Cichlidae. Sequences similar to FFAR4 are detected in
Cichlidae genomes, but the reason for this exception is unknown. Multiplication of
genes such as those in FFAR1, FFAR2, and FFAR3 are also not observed in FFAR4.
Comparing the FFAR4 orthologues including Cichlidae, we found that the chief
amino acid sequences are well conserved among species. The amino acid residue
equivalent to human R99 (described later in this chapter), which is important for the
interaction of FFAR4 and FFAs, is conserved in all species, strongly suggesting that
the function is conserved as well (Fig. 10.3).
10 Fatty Acids Receptors 147
I
Homo sapiens
Mus musculus
Gallus gallus
Python bivittatus
Xenopus tropicalis
Oreochromis niloticus
II III
Homo sapiens
Mus musculus
Gallus gallus
Python bivittatus
Xenopus tropicalis
Oreochromis niloticus
IV V
Homo sapiens
Mus musculus
Gallus gallus
Python bivittatus
Xenopus tropicalis
Oreochromis niloticus
VI VII
Homo sapiens
Mus musculus
Gallus gallus
Python bivittatus
Xenopus tropicalis
Oreochromis niloticus
VII
Homo sapiens
Mus musculus
Gallus gallus
Python bivittatus
Xenopus tropicalis
Oreochromis niloticus
Fig. 10.3 Sequence alignment of FFAR4 in multiple animal species. The alignment is obtained by
multiple sequence alignment for six homology protein sequences. Amino acid sequences corre-
sponding to FFAR4 from different animal species were aligned using the NCBI COBALT algo-
rithm [37]. Transmembrane helix regions are shown between black bars. Highly conserved
residues are highlighted by yellow
Both PUFAs and synthetic ligands induced a rise in cytosolic free Ca2+ in FFAR4-
overexpressing HEK293 cells, suggesting that FFAR4 is coupled with the Gq pro-
tein family. Recently, Shah et al. showed that PUFA-induced depolarization induced
by the monovalent cation-specic transient receptor potential channel type M5
(TRPM5) is related to intracellular Ca2+ rise as well as CCK secretion from STC-1
cells, suggesting that TRPM5 plays a crucial role in FFAR4 signaling in STC-1 cells
[41]. Oh et al. showed that a FFAR4 agonist exerts anti-inammatory effects through
-arrestin 2 signaling in monocytic RAW 264.7 cells and primary macrophages
[36]. FFAR4 can also induce the activation of ERK1/2 under certain conditions and
activation of PI3-kinase and the serine/threonine protein kinase Akt in FFAR4-
expressing cells [17, 25].
148 A. Hirasawa et al.
Fig. 10.4 Single-nucleotide polymorphisms (SNPs) in human FFAR4 and homology model of
FFAR4. (a) Representation of secondary structure of FFAR4. Locus of its SNPs (R67C, R270H)
and an interaction site (R99) are marked. (b) FFAR4 homology model docked with NCG21
Because the three-dimensional structure of FFAR4 has not yet been elucidated by
X-ray crystallography, structureactivity relationship studies are being conducted
by combining site-directed mutagenesis and homology modeling. The results of the
site-directed mutagenesis showed that the R99 residue is signicant for the ligand
binding for FFAR4, and the amino acid sequences around R99 are well conserved
in aforementioned orthologues [47] (Figs. 10.3 and 10.4a). The calculation of the
docking simulation and homology model of FFAR4 revealed signicant correlation
between the calculated value of the hydrogen bond energy and ligand-induced
activity in many compounds, which led us to predict the activity of novel com-
pounds [47, 49]. To identify other natural ligands of FFAR4, we screened and iden-
tied a selective partial agonist among a series of natural compounds derived from
fruiting bodies of Albatrellus ovinus [17]. Depending on the experimental condi-
tions, this compound is also useful as an antagonist selective for FFAR4. In addi-
tion, based on the structure of the PPAR agonist thiazolidinediones, we synthesized
a series of compounds containing carboxylic acids and developed a selective ago-
nist using a homology model of FFAR4 [48] (Fig. 10.2). Hudson et al. have also
reported the synthesis of compounds selective for FFAR4 [20], and many patents of
compounds have been claimed [14]. The structureactivity relationship studies
combining site-directed mutagenesis and homology modeling of FFAR4 showed
that hydrophobic amino acid residues facing the ligand-binding pocket play an
important role in the binding of FFAR4 ligand [21]. These compounds might be
useful tools to monitor the physiological effects of FFAR4, and they might be poten-
tially useful in the development of novel drug candidates for the treatment of type 2
diabetes, obesity, and metabolic diseases.
10 Fatty Acids Receptors 149
Fig. 10.5 Obesity in FFAR4-decient mice fed a high-fat diet. (a) Body weight changes of wild-
type and FFAR4-decient mice fed a normal diet (ND) or a high-fat diet (HFD). All data represent
mean + SEM. **P < 0.01 versus the corresponding wild-type value. (b, c) Computed tomography
images of fat accumulation in wild-type (b) and FFAR4-decient (c) male mice fed a high-fat diet.
Fat depots are demarcated for illustration
We recently reported that dysfunctional FFAR4 led to obesity in both mice and
humans [23]. We found that FFAR4-decient mice fed a high-fat diet developed
obesity, glucose intolerance, and fatty liver along with decreased adipocyte differ-
entiation and lipogenesis and enhanced hepatic lipogenesis (Fig. 10.5). Insulin
resistance in these mice was associated with reduced insulin signaling and enhanced
inammation in adipose tissue. FFAR4 exon sequencing in obese subjects revealed
a deleterious nonsynonymous mutation (R270H) that inhibited FFAR4 signaling
activity (Fig. 10.4b). Furthermore, the R270H variant increases the risk of obesity
in European populations. Overall, this study demonstrates that the lipid sensor
FFAR4 has a key role in sensing dietary fat and, therefore, in the control of energy
balance in both humans and rodents.
Endogenous expression of FFAR4 has been identied in the intestine of humans
and mice. Our previous study showed that FFAR4-expressing cells were located in
the GLP-1-expressing enteroendocrine cells in the large intestine [18, 31].
Furthermore, the enteroendocrine cell line STC-1 also expressed FFAR4 endoge-
nously, and PUFA or synthetic ligand stimulation induced the secretion of GLP-1
and cholecystokinin (CCK) as well as the [Ca2+]i response [50]. These studies led us
to speculate the physiological function of FFAR4 in incretin secretion in vivo.
150 A. Hirasawa et al.
FFAR4 is also expressed in other cells and tissues. Oh et al. found endogenous
expression of FFAR4 in monocytic RAW 264.7 cells and in primary pro-
inammatory, M1-like macrophages. Matsumura et al. found the expression of
FFAR4 in taste buds [30]. In the lung, we found that FFAR4 protein was colocalized
with the Clara cell 10-kDa protein, a marker of Clara cells [31]. Further studies are
needed to reveal the physiological function of FFAR4 in the lung.
Taneera et al. performed a systems genomics approach to identify genes for type
2 diabetes, and FFAR4 was ranked among the top 20 possibly associated genes [51].
In this report, FFAR4 expression in human islets was positively correlated with
insulin secretion and insulin content and with lower HbA1c. Although inconsistent
with previous reports that FFAR1 is dominantly detected in mouse pancreatic
islets [33], these data suggest that FFAR4 can protect pancreatic islets from lipotox-
icity in humans.
Recently, it has become clear that FFAR4 is expressed in the pancreas and con-
tributes to glucagon secretion [46]. According to a report by Oh et al., continued
administration of an agonist selective for FFAR4 improved glucose tolerance and
insulin sensitivity in mice fed a high-fat diet [35]. However, they attributed their
results to a macrophage-mediated pathway and did not mention its relationship to
obesity; this is in disagreement with our results.
From the analyses of human and mice described here, there is no doubt that
FFAR4 is strongly involved in diet-induced obesity and acts as a lipid sensor that
maintains the balance of energy metabolism by controlling lipid biosynthesis [23].
Further investigation is anticipated to develop therapeutic drugs targeting FFAR4
for the treatment of obesity-related metabolic disorders.
10.4 Conclusion
Because of the low binding afnity of FFAs to FFARs, there was skepticism toward
FFARs when originally discovered. However, the experimental facts described here
conrmed that FFAs were indeed ligands for FFARs; thus, the names of the fatty acid
receptor family, which were originally the numbers of the orphan receptors (GPR40,
GPR120, etc.), are now ofcially changed to FFAR1, FFAR4, etc, respectively. [6,
45]. The ligands for some nutrient-sensing GPCRs bind with lower afnity (in the
micromolar or millimolar range) than that of classic high-afnity ligands, such as
hormones or growth factors, for their receptors. The fatty acid receptor family is con-
sidered to be a group of sensor molecules that detect FFAs of various lengths and
structures as natural ligands with binding constants comparable with their vivo con-
centrations. Among the FFARs, FFAR1 and FFAR4 are considered to be potential
drug targets for the treatment of metabolic diseases such as type 2 diabetes, because
their physiological functions are related to energy homeostasis. Further analysis of
FFARs may also be important to better understand the nutrient-sensing process and to
develop novel therapeutic compounds to treat metabolic diseases.
10 Fatty Acids Receptors 151
References
1. Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, Ellis C,
Elshourbagy NA, Goetz AS, Minnick DT, Murdock PR, Sauls HR, Shabon U, Spinage LD,
Strum JC, Szekeres PG, Tan KB, Way JM, Ignar DM, Wilson S, Muir AI (2003) The orphan
G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol
Chem 278(13):1130311311
2. Briscoe CP, Peat AJ, McKeown SC, Corbett DF, Goetz AS, Littleton TR, McCoy DC, Kenakin
TP, Andrews JL, Ammala C, Fornwald JA, Ignar DM, Jenkinson S (2006) Pharmacological
regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identica-
tion of agonist and antagonist small molecules. Br J Pharmacol 148(5):619628
3. Brown AJ, Goldsworthy SM, Barnes AA, Eilert MM, Tcheang L, Daniels D, Muir AI,
Wigglesworth MJ, Kinghorn I, Fraser NJ, Pike NB, Sturm JC, Steplewski KM, Murdock PR,
Holder JC, Marshall FH, Szekeres PG, Wilson S, Ignar DM, Foord SM, Wise A, Dowell SJ
(2003) The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propio-
nate and other short chain carboxylic acids. J Biol Chem 278:1131211319
4. Catchen JM, Conery JS, Postlethwait JH (2009) Automated identication of conserved syn-
teny after whole-genome duplication. Genome Res 19(8):14971505
5. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ (2001) Nuclear receptors and lipid physiol-
ogy: opening the X-les. Science 294(5548):18661870
6. Davenport AP, Harmar AJ (2013) Evolving pharmacology of orphan GPCRs: IUPHAR com-
mentary. Br J Pharmacol 170(4):693695
7. Dobbins RL, Chester MW, Daniels MB, McGarry JD, Stein DT (1998) Circulating fatty acids
are essential for efcient glucose-stimulated insulin secretion after prolonged fasting in
humans. Diabetes 47(10):16131618
8. Dobbins RL, Chester MW, Stevenson BE, Daniels MB, Stein DT, McGarry JD (1998) A fatty
acid-dependent step is critically important for both glucose- and non-glucose-stimulated insu-
lin secretion. J Clin Invest 101(11):23702376
9. Feng DD, Luo Z, Roh SG, Hernandez M, Tawadros N, Keating DJ, Chen C (2006) Reduction
in voltage-gated K+ currents in primary cultured rat pancreatic beta-cells by linoleic acids.
Endocrinology 147(2):674682
10. Fujiwara K, Maekawa F, Yada T (2005) Oleic acid interacts with GPR40 to induce Ca2+ signal-
ing in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release.
Am J Physiol Endocrinol Metab 289(4):E670E677
11. Fukunaga S, Setoguchi S, Hirasawa A, Tsujimoto G (2006) Monitoring ligand-mediated inter-
nalization of G protein-coupled receptor as a novel pharmacological approach. Life Sci
80(1):1723
12. Garrido DM, Corbett DF, Dwornik KA, Goetz AS, Littleton TR, McKeown SC, Mills WY,
Smalley TL, Briscoe CP, Peat AJ (2006) Synthesis and activity of small molecule GPR40
agonists. Bioorg Med Chem Lett 16(7):18401845
13. Gravena C, Mathias PC, Ashcroft SJ (2002) Acute effects of fatty acids on insulin secretion
from rat and human islets of Langerhans. J Endocrinol 173(1):7380
14. Halder S, Kumar S, Sharma R (2013) The therapeutic potential of GPR120: a patent review.
Expert Opin Ther Pat 23(12):15811590
15. Hamid YH, Vissing H, Holst B, Urhammer SA, Pyke C, Hansen SK, Glumer C, Borch-Johnsen
K, Jrgensen T, Schwartz TW, Pedersen O, Hansen T (2005) Studies of relationships between
variation of the human G protein-coupled receptor 40 gene and type 2 diabetes and insulin
release. Diabet Med 22(1):7480
16. Hara T, Hirasawa A, Sun Q, Koshimizu TA, Itsubo C, Sadakane K, Awaji T, Tsujimoto G
(2009) Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1).
Mol Pharmacol 75(1):8591
152 A. Hirasawa et al.
17. Hara T, Hirasawa A, Sun Q, Sadakane K, Itsubo C, Iga T, Adachi T, Koshimizu TA, Hashimoto
T, Asakawa Y, Tsujimoto G (2009) Novel selective ligands for free fatty acid receptors
GPR120 and GPR40. Naunyn Schmiedebergs Arch Pharmacol 380(3):247255
18. Hirasawa A, Tsumaya K, Awaji T, Katsuma S, Adachi T, Yamada M, Sugimoto Y, Miyazaki S,
Tsujimoto G (2005) Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion
through GPR120. Nat Med 11(1):9094
19. Hu H, He LY, Gong Z, Li N, Lu YN, Zhai QW, Liu H, Jiang HL, Zhu WL, Wang HY (2009) A
novel class of antagonists for the FFAs receptor GPR40. Biochem Biophys Res Commun
390(3):557563
20. Hudson BD, Shimpukade B, Mackenzie AE, Butcher AJ, Pediani JD, Christiansen E, Heathcote
H, Tobin AB, Ulven T, Milligan G (2013) The pharmacology of TUG-891, a potent and selec-
tive agonist of the free fatty acid receptor 4 (FFA4/GPR120), demonstrates both potential
opportunity and possible challenges to therapeutic agonism. Mol Pharmacol 84(5):710725
21. Hudson BD, Shimpukade B, Milligan G, Ulven T (2014) The molecular basis of ligand inter-
action at free fatty acid receptor 4 (FFA4/GPR120). J Biol Chem 289(29):2034520358
22. Humphries PS, Benbow JW, Bonin PD, Boyer D, Doran SD, Frisbie RK, Piotrowski DW,
Balan G, Bechle BM, Conn EL, Dirico KJ, Oliver RM, Soeller WC, Southers JA, Yang X
(2009) Synthesis and SAR of 1,2,3,4-tetrahydroisoquinolin-1-ones as novel G-protein-coupled
receptor 40 (GPR40) antagonists. Bioorg Med Chem Lett 19(9):24002403
23. Ichimura A, Hirasawa A, Poulain-Godefroy O, Bonnefond A, Hara T, Yengo L, Kimura I,
Leloire A, Liu N, Iida K, Choquet H, Besnard P, Lecoeur C, Vivequin S, Ayukawa K, Takeuchi
M, Ozawa K, Tauber M, Maffeis C, Morandi A, Buzzetti R, Elliott P, Pouta A, Jarvelin MR,
Korner A, Kiess W, Pigeyre M, Caiazzo R, Van Hul W, Van Gaal L, Horber F, Balkau B, Levy-
Marchal C, Rouskas K, Kouvatsi A, Hebebrand J, Hinney A, Scherag A, Pattou F, Meyre D,
Koshimizu TA, Wolowczuk I, Tsujimoto G, Froguel P (2012) Dysfunction of lipid sensor
GPR120 leads to obesity in both mouse and human. Nature 483(7389):350354
24. Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka
Y, Uejima H, Tanaka H, Maruyama M, Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura
F, Noguchi Y, Shinohara T, Hinuma S, Fujisawa Y, Fujino M (2003) Free fatty acids regulate
insulin secretion from pancreatic beta cells through GPR40. Nature 422(6928):173176
25. Katsuma S, Hatae N, Yano T, Ruike Y, Kimura M, Hirasawa A, Tsujimoto G (2005) Free fatty
acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendo-
crine cell line STC-1. J Biol Chem 280(20):1950719515
26. Kimura I, Inoue D, Maeda T, Hara T, Ichimura A, Miyauchi S, Kobayashi M, Hirasawa A,
Tsujimoto G (2011) Short-chain fatty acids and ketones directly regulate sympathetic nervous
system via G protein-coupled receptor 41 (GPR41). Proc Natl Acad Sci U S A
108(19):80308035
27. Kotarsky K, Nilsson NE, Flodgren E, Owman C, Olde B (2003) A human cell surface receptor
activated by free fatty acids and thiazolidinedione drugs. Biochem Biophys Res Commun
301(2):406410
28. Louet JF, Chatelain F, Decaux JF, Park EA, Kohl C, Pineau T, Girard J, Pegorier JP (2001)
Long-chain fatty acids regulate liver carnitine palmitoyl-transferase I gene (L-CPT I) expres-
sion through a peroxisome-proliferator-activated receptor alpha (PPARalpha)-independent
pathway. Biochem J 354(pt 1):189197
29. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS, Mackay
F, Artis D, Xavier RJ, Teixeira MM, Mackay CR (2009) Regulation of inammatory responses
by gut microbiota and chemoattractant receptor GPR43. Nature 461(7268):12821286
30. Matsumura S, Mizushige T, Yoneda T, Iwanaga T, Inoue K, Tsuzuki S, Fushiki T (2007) GPR
expression in the rat taste bud relating to fatty acid sensing. Biomed Res 28(1):4955
31. Miyauchi S, Hirasawa A, Iga T, Liu N, Itsubo C, Sadakane K, Hara T, Tsujimoto G (2009)
Distribution and regulation of protein expression of the free fatty acid receptor GPR120.
Naunyn Schmiedebergs Arch Pharmacol 379(4):427434
10 Fatty Acids Receptors 153
32. Miyauchi S, Hirasawa A, Ichimura A, Hara T, Tsujimoto G (2010) New frontiers in gut nutri-
ent sensor research: free fatty acid sensing in the gastrointestinal tract. J Pharmacol Sci
112(1):1924
33. Nagasumi K, Esaki R, Iwachidow K, Yasuhara Y, Ogi K, Tanaka H, Nakata M, Yano T,
Shimakawa K, Taketomi S, Takeuchi K, Odaka H, Kaisho Y (2009) Overexpression of
GPR40 in pancreatic beta-cells augments glucose-stimulated insulin secretion and improves
glucose tolerance in normal and diabetic mice. Diabetes 58(5):10671076
34. Ogawa T, Hirose H, Miyashita K, Saito I, Saruta T (2005) GPR40 gene Arg211His polymor-
phism may contribute to the variation of insulin secretory capacity in Japanese men. Metab
Clin Exp 54(3):296299
35. Ohda Y, Walenta E, Akiyama TE, Lagakos WS, Lackey D, Pessentheiner AR, Sasik R, Hah N,
Chi TJ, Cox JM, Powels MA, Di Salvo J, Sinz C, Watkins SM, Armando AM, Chung H, Evans
RM, Quehenberger O, McNelis J, Bogner-Strauss JG, Olefsky JM (2014) A Gpr120-selective
agonist improves insulin resistance and chronic inammation in obese mice. Nat Med
20(8):942947
36. Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W, Li P, Lu WJ, Watkins SM,
Olefsky JM (2010) GPR120 is an omega-3 fatty acid receptor mediating potent anti-
inammatory and insulin-sensitizing effects. Cell 142(5):687698
37. Papadopoulos JS, Agarwala R (2007) COBALT: constraint-based alignment tool for multiple
protein sequences. Bioinformatics 23(9):10731079
38. Remely M, Aumueller E, Merold C, Dworzak S, Hippe B, Zanner J, Pointner A, Brath H,
Haslberger AG (2014) Effects of short chain fatty acid producing bacteria on epigenetic regu-
lation of FFAR3 in type 2 diabetes and obesity. Gene 537(1):8592
39. Sasaki S, Kitamura S, Negoro N, Suzuki M, Tsujihata Y, Suzuki N, Santou T, Kanzaki N,
Harada M, Tanaka Y, Kobayashi M, Tada N, Funami M, Tanaka T, Yamamoto Y, Fukatsu K,
Yasuma T, Momose Y (2011) Design, synthesis, and biological activity of potent and orally
available G protein-coupled receptor 40 agonists. J Med Chem 54(5):13651378
40. Sauer LA, Dauchy RT, Blask DE (2000) Mechanism for the antitumor and anticachectic
effects of n-3 fatty acids. Cancer Res 60(18):52895295
41. Shah BP, Liu P, Yu T, Hansen DR, Gilbertson TA (2012) TRPM5 is critical for linoleic acid-
induced CCK secretion from the enteroendocrine cell line, STC-1. Am J Physiol Cell Physiol
302(1):C210C219
42. Srivastava A, Yano J, Hirozane Y, Kefala G, Gruswitz F, Snell G, Lane W, Ivetac A, Aertgeerts
K, Nguyen J, Jennings A, Okada K (2014) High-resolution structure of the human GPR40
receptor bound to allosteric agonist TAK-875. Nature 513(7516):124127
43. Stein DT, Esser V, Stevenson BE, Lane KE, Whiteside JH, Daniels MB, Chen S, McGarry JD
(1996) Essentiality of circulating fatty acids for glucose- stimulated insulin secretion in the
fasted rat. J Clin Invest 97(12):27282735
44. Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD, Edlund H (2005) The FFA receptor
GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse.
Cell Metab 1(4):245258
45. Stoddart LA, Smith NJ, Milligan G (2008) International Union of Pharmacology. LXXI. Free
fatty acid receptors FFA1, -2, and -3: pharmacology and pathophysiological functions.
Pharmacol Rev 60(4):405417
46. Suckow AT, Polidori D, Yan W, Chon S, Ma JY, Leonard J, Briscoe CP (2014) Alteration of the
glucagon axis in GPR120 (FFAR4) knockout mice: a role for GPR120 in glucagon secretion.
J Biol Chem 289(22):1575115763
47. Sun Q, Hirasawa A, Hara T, Kimura I, Adachi T, Awaji T, Ishiguro M, Suzuki T, Miyata N,
Tsujimoto G (2010) Structureactivity relationships of GPR120 agonists based on a docking
simulation. Mol Pharmacol 78(5):804810
48. Suzuki T, Igari S, Hirasawa A, Hata M, Ishiguro M, Fujieda H, Itoh Y, Hirano T, Nakagawa H,
Ogura M, Makishima M, Tsujimoto G, Miyata N (2008) Identication of G protein-coupled
receptor 120-selective agonists derived from PPARgamma agonists. J Med Chem
51(23):76407644
154 A. Hirasawa et al.
49. Takeuchi M, Hirasawa A, Hara T, Kimura I, Hirano T, Suzuki T, Miyata N, Awaji T, Ishiguro
M, Tsujimoto G (2013) FFA1-selective agonistic activity based on docking simulation using
FFA1 and GPR120 homology models. Br J Pharmacol 168(7):15701583
50. Tanaka T, Katsuma S, Adachi T, Koshimizu TA, Hirasawa A, Tsujimoto G (2008) Free fatty
acids induce cholecystokinin secretion through GPR120. Naunyn Schmiedebergs Arch
Pharmacol 377(4-6):523527
51. Taneera J, Lang S, Sharma A, Fadista J, Zhou Y, Ahlqvist E, Jonsson A, Lyssenko V, Vikman
P, Hansson O, Parikh H, Korsgren O, Soni A, Krus U, Zhang E, Jing XJ, Esguerra JL, Wollheim
CB, Salehi A, Rosengren A, Renstrom E, Groop L (2012) A systems genetics approach identi-
es genes and pathways for type 2 diabetes in human islets. Cell Metab 16(1):122134
52. Vettor R, Granzotto M, De Stefani D, Trevellin E, Rossato M, Farina MG, Milan G, Pilon C,
Nigro A, Federspil G, Vigneri R, Vitiello L, Rizzuto R, Baratta R, Frittitta L (2008) Loss-of-
function mutation of the GPR40 gene associates with abnormal stimulated insulin secretion by
acting on intracellular calcium mobilization. J Clin Endocrinol Metab 93(9):35413550
53. Zhang X, Yan G, Li Y, Zhu W, Wang H (2010) DC2601 in obese Zucker rats. Biomed
Pharmacother 64(9):647651
Chapter 11
Omega-3 Fatty Acid Metabolism
and Regulation of Inflammation
Abstract Increasing evidence from both human and animal studies has demon-
strated that omega-3 polyunsaturated fatty acids (PUFAs), primarily eicosapentae-
noic acid (EPA) and docosahexaenoic acid (DHA), can suppress inflammation and
play a beneficial role in a variety of inflammation-related human diseases, such as
inflammatory bowel disease, rheumatoid arthritis, asthma, cancer, and cardiovascu-
lar diseases. Omega-3 PUFAs serve as substrates for the production of potent bioac-
tive anti-inflammatory lipid mediators such as resolvins. Herein we review recent
advances in understanding the formation and action of these mediators, especially
focusing on the LC-ESI-MS/MS-based lipidomics approach and on recently identi-
fied bioactive compounds with potent anti-inflammatory properties.
11.1 Introduction
Polyunsaturated fatty acids (PUFAs) are essential in human nutrition and can be
divided into two subcategories, termed omega-3 and omega-6, based on the location
of their first double bond relative to the tail (omega) of the carbon chain. Omega-6
arachidonic acid is a common precursor of many eicosanoids, a family of bioactive
lipid mediators important in controlling inflammatory responses (Fig. 11.1).
Omega-3 PUFAs such as eicosapentaenoic acid (EPA) and docosahexaenoic acid
Y. Isobe
Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences,
Yokohama, Japan
M. Arita (*)
Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences,
Yokohama, Japan
Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
e-mail: [email protected]
Omega-6 Omega-3
COOH COOH
COOH
Fig. 11.1 Polyunsaturated fatty acid (PUFA)-derived mediators. Arachidonic acid is a metabolic
precursor of eicosanoids [e.g., prostaglandins (PGs) and leukotrienes (LTs)], which have distinct
roles as pro-inflammatory mediators. Omega-3 PUFAs prevent conversion of arachidonic acid into
pro-inflammatory eicosanoids via substrate competition. In addition, omega-3 PUFAs are con-
verted to bioactive metabolites such as resolvins and protectins with anti-inflammatory and pro-
resolving properties
(DHA), which are enriched in some fish oils, are believed to exert beneficial effects
on a wide range of human inflammatory disorders, including inflammatory bowel
disease, rheumatoid arthritis, and cardiovascular diseases [13]. In addition, studies
using omega-3 desaturase (fat-1) transgenic mice, which have abundant endoge-
nous omega-3 PUFAs, strongly support the idea that omega-3 PUFAs are protective
in inflammatory pathology [4, 5]. Omega-3 PUFAs prevent conversion of arachi-
donic acid (AA) to the pro-inflammatory eicosanoids by serving as an alternative
substrate for cyclooxygenase (COX) or lipoxygenase (LOX), resulting in the pro-
duction of less potent products. In addition, a number of enzymatically oxygenated
metabolites derived from omega-3 PUFAs were recently identified as anti-
inflammatory mediators. These compounds may contribute to the beneficial actions
that have been attributed to omega-3 PUFAs in human diseases.
17,18-EpETE
PGE2 PGD2
m/z 351 > 271
PD1
m/z 359 > 153
LTB4
m/z 335 > 195
18-HEPE
m/z 317 > 259
12-HEPE
m/z 317 > 179
8-HEPE
m/z 317 > 155
5-HEPE
m/z 317 > 115
11.5 Perspectives
The formation of endogenous autacoids derived from omega-3 PUFA may explain
in part the well-known, essential roles of the omega-3 PUFA in health and disease.
E-series reolvins are biosynthesized from a common precursor, 18-HEPE, and
12-OH-17,18-EpETE is formed from 17,18-EpETE. These oxidation reactions tar-
get the omega-3 double bond of EPA, which distinguishes it from omega-6 arachi-
donic acid. Also, as in EPA, our recent study has identified novel anti-inflammatory
metabolites generated via omega-3 oxidation of DHA [32]. Thus, we propose that
omega-3 oxidation is an important structural and metabolic feature for the anti-
inflammatory actions of omega-3 PUFA (Fig. 11.3). EPA is converted to 18-HEPE
by aspirin-acetylated COX-2 [7] or cytochrome P450 monooxygenase (CYP) [33].
Several CYPs, including the CYP1A, CYP2C, and CYP2J subfamily members, can
preferentially introduce a cis-epoxide at the omega-3 double bond of EPA to form
17,18-EpETE [3436]. Recent studies have demonstrated contributions of CYP1
enzymes to the resolvin-biosynthetic pathway, and there was increased neutrophil
recruitment in zymosan-induced peritoneal exudates of Cyp1a1/1a2/1b1 triple-
knockout mice [37]. Thus, cells expressing these enzymes are likely to be involved
in regulating inflammatory responses via local production of anti-inflammatory
metabolites. Further studies to understand lipid mediator biosynthesis and the
160 Y. Isobe and M. Arita
COX COOH
3-series PGs
5-series LTs
LOX
EPA
COOH COOH
OH O
PMNs
18-HEPE 17,18-EpETE
5-LOX
eosinophils
macrophages
12/15-LOX
etc.
OH OH
COOH
RvE1
COOH COOH
OH
OH O
HO OH OH
COOH
RvE2
RvE3 12-OH-17,18-EpETE
OH
Fig. 11.3 Novel anti-inflammatory metabolic pathways of eicosapentaenoic acid (EPA). EPA is
converted by COX or LOX to form 3-series PGs or 5-series LTs. In addition, recent studies uncov-
ered a novel EPA metabolic pathway via omega-3 hydroxylation or epoxygenation in vivo and
identified potent anti-inflammatory metabolites such as E-series resolvins and 12-OH-17,18-
EpETE. These metabolic pathways may contribute to the anti-inflammatory actions of omega-3
PUFAs in vivo
References
7. Serhan CN, Clish CB, Brannon J et al (2000) Novel functional sets of lipid-derived mediators
with anti-inflammatory actions generated from omega-3 fatty acids via cyclooxygenase
2-nonsteroidal anti-inflammatory drugs and transcellular processing. J Exp Med
192:11971204
8. Arita M, Bianchini F, Aliberti J et al (2005) Stereochemical assignment, anti-inflammatory
properties, and receptor for the omega-3 lipid mediator resolvin E1. J Exp Med 201:713722
9. Arita M, Yoshida M, Hong S et al (2005) Resolvin E1, an endogenous lipid mediator derived
from omega-3 eicosapentaenoic acid, protects against 2,4,6-trinitrobenzene sulfonic acid-
induced colitis. Proc Natl Acad Sci U S A 102:76717676
10. Schwab JM, Chiang N, Arita M et al (2007) Resolvin E1 and protectin D1 activate
inflammation-resolution programmes. Nature 447:869874
11. Ohira T, Arita M, Omori K et al (2010) Resolvin E1 receptor activation signals phosphoryla-
tion and phagocytosis. J Biol Chem 285:34513461
12. Connor KM, SanGiovanni JP, Lofqvist C et al (2007) Increased dietary intake of omega-3
polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med 13:868873
13. Campbell EL, MacManus CF, Kominsky DJ et al (2010) Resolvin E1-induced intestinal alka-
line phosphatase promotes resolution of inflammation through LPS detoxification. Proc Natl
Acad Sci U S A 107:1429814303
14. Xu ZZ, Zhang L, Liu T et al (2010) Resolvins RvE1 and RvD1 attenuate inflammatory pain
via central and peripheral actions. Nat Med 16:592597
15. Tjonahen E, Oh SF, Siegelman J et al (2006) Resolvin E2: identification and anti-inflammatory
actions. Pivotal role of human 5-lipoxygenase in resolvin E series biosynthesis. Chem Biol
13:11931202
16. Oh SF, Dona M, Fredman G et al (2012) Resolvin E2 formation and impact in inflammation
resolution. J Immunol 188:45274534
17. Isobe Y, Arita M, Matsueda S et al (2012) Identification and structure determination of novel
anti-inflammatory mediator resolvin E3, 17,18-dihydroxyeicosapentaenoic acid. J Biol Chem
287:1052510534
18. Urabe D, Todoroki H, Masuda K, Inoue M (2012) Total syntheses of four possible stereoiso-
mers of resolvin E3. Tetrahedron 68:32103219
19. Isobe Y, Arita M, Iwamoto R et al (2013) Stereochemical assignment and anti-inflammatory
properties of the omega-3 lipid mediator resolvin E3. J Biochem 153:355360
20. Yamashita A, Kawana K, Tomio K et al (2013) Increased tissue levels of omega-3 polyunsatu-
rated fatty acids prevents pathological preterm birth. Sci Rep 3:3113
21. Wu D, Molofsky AB, Liang HE et al (2011) Eosinophils sustain adipose alternatively activated
macrophages associated with glucose homeostasis. Science 332:243247
22. Molofsky AB, Nussbaum JC, Liang HE et al (2013) Innate lymphoid type 2 cells sustain vis-
ceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med
210:535549
23. Heredia JE, Mukundan L, Chen FM et al (2013) Type 2 innate signals stimulate fibro/adipo-
genic progenitors to facilitate muscle regeneration. Cell 153:376388
24. Yamada T, Tani Y, Nakanishi H et al (2011) Eosinophils promote resolution of acute peritonitis
by producing proresolving mediators in mice. FASEB J 25:561568
25. Tani Y, Isobe Y, Imoto Y et al (2014) Eosinophils control the resolution of inflammation and
draining lymph node hypertrophy through the proresolving mediators and CXCL13 pathway
in mice. FASEB J 28(9):40364043
26. Khn H, ODonnell VB (2006) Inflammation and immune regulation by 12/15-lipoxygenases.
Prog Lipid Res 45:334356
27. Merched AJ, Ko K, Gotlinger KH et al (2008) Atherosclerosis. Evidence for impairment of
resolution of vascular inflammation governed by specific lipid mediators. FASEB J
22:35953606
28. Krnke G, Katzenbeisser J, Uderhardt S et al (2009) 12/15-Lipoxygenase counteracts inflam-
mation and tissue damage in arthritis. J Immunol 183:33833389
162 Y. Isobe and M. Arita
29. Gronert K, Maheshwari N, Khan N et al (2005) A role for the mouse 12/15-lipoxygenase
pathway in promoting epithelial wound healing and host defense. J Biol Chem
280:1526715278
30. Uderhardt S, Herrmann M, Oskolkova OV et al (2012) 12/15-Lipoxygenase orchestrates the
clearance of apoptotic cells and maintains immunologic tolerance. Immunity 36:113
31. Kubota T, Arita M, Isobe Y et al (2014) Eicosapentaenoic acid is converted via -3 epoxygen-
ation to the anti-inflammatory metabolite 12-hydroxy-17,18-epoxyeicosatetraenoic acid.
FASEB J 28:586593
32. Yokokura Y, Isobe Y, Matsueda S et al (2014) Identification of 14,20-dihydroxy-
docosahexaenoic acid as a novel anti-inflammatory metabolite. J Biochem 156(6):315321
33. Arita M, Clish CB, Serhan CN (2005) The contributions of aspirin and microbial oxygenase to
the biosynthesis of anti-inflammatory resolvins. Novel oxygenase products from omega-3
polyunsaturated fatty acids. Biochem Biophys Res Commun 338:149157
34. Schwarz D (2004) Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1:
highly stereoselective formation of 17(R),18(S)-epoxyeicosatetraenoic acid. Biochem
Pharmacol 67:14451457
35. Lucas D, Goulitquer S, Marienhagen J et al (2010) Stereoselective epoxidation of the last
double bond of polyunsaturated fatty acids by human cytochromes P450. J Lipid Res
51:11251133
36. Arnold C, Markovic M, Blossey K et al (2010) Arachidonic acid-metabolizing cytochrome
p450 enzymes are targets of n-3 fatty acids. J Biol Chem 285:3272032733
37. Divanovic S, Dalli J, Jorge-Nebert LF et al (2013) Contributions of the three CYP1 monooxy-
genases to pro-inflammatory and inflammation-resolution lipid mediator pathways. J Immunol
191:33473357
Part II
Lipid Mediators in Drosophila
and Zebrafish
Chapter 12
Membrane Lipid Transporters in Drosophila
melanogaster
Abstract Membrane lipid transport within and across the membrane is mediated
by lipid transport machineries known as flippase, floppase, and scramblase. Flippase
translocates lipids from the exocytoplasmic to the cytoplasmic leaflet of cellular
membranes, floppase mediates the translocation of lipids in the opposite direction,
and scramblase facilitates bidirectional translocation of lipids. These specialized
lipid transport machineries are now demonstrated to have crucial roles in a variety
of biological processes, including lipid metabolism, immune response, apoptosis,
and neural function, in many mammalian species. The Drosophila melanogaster
genome contains orthologues to about 70 % of all human disease-associated genes,
and thus both traditional genetic approaches and more recent genome-wide screen-
ing techniques in Drosophila have been powerful tools for the study of lipid-related
processes. There are, however, many open questions about the structure and func-
tion of lipids and their transport machineries in Drosophila. In this review, we sum-
marize the functions of flippase, floppase, and scramblase from several species, and
discuss the roles of these lipid transporters in D. melanogaster.
12.1 Introduction
The fruit fly Drosophila melanogaster has played a central role in establishing the
link between genetics and embryology and has provided a useful model system for
cell biology, physiology, immunology, social biology, and population genetics.
Although evolutionarily distant from humans, the D. melanogaster genome con-
tains orthologues to around 70 % of all human disease-associated genes [1],
including genes whose disruption are responsible for a variety of human diseases
such as developmental disorders, cancer, neurological diseases, and metabolic dis-
orders [13]. Recent advances in Drosophila genetics have provided a powerful tool
for genome-wide screening of disease-associated genes and a pharmacological
approach for the development of anticancer drugs [4, 5]. Despite the continued
accumulation of knowledge about gene and protein function in Drosophila, how-
ever, there are many open questions about the structure and function of lipids in this
fly species. Drosophila has been shown to have a unique phospholipid composition
in which phosphatidylethanolamine (PE) is the dominant component, in contrast to
other animals and plants in which phosphatidylcholine (PC) is the major phospho-
lipid component [6]. Because of its sterol auxotrophy and facile genetic manipula-
tion, Drosophila also provides a unique system for the study of dietary sterol uptake
and the genetic basis of diet-induced metabolic disorders [7, 8]. A recent lipidomics
study identified more than 500 molecular species of lipids during Drosophila devel-
opment, and showed the dynamic remodeling of lipid species at specific stages dur-
ing development [9].
It is now known that lipid molecules are not tethered to the site of their synthesis,
but rather are actively transported and assembled into specific sites of cellular mem-
branes [10]. Specialized lipid transport machineries are now demonstrated to have
crucial roles in the formation of distinct membrane domains, which are involved in
highly localized remodeling of membrane structures as well as recruitment and acti-
vation of membrane proteins [11]. It is well established that phospholipids in bio-
logical membranes are distributed asymmetrically between the inner and outer
leaflets of the lipid bilayer. In many eukaryotic plasma membranes, aminophospho-
lipids such as PE and phosphatidylserine (PS) reside in the inner leaflet, whereas
choline-containing phospholipids, such as PC and sphingomyelin (SM), are localized
mainly in the outer leaflet [12]. This phospholipid asymmetry is generated and main-
tained in part by the three putative phospholipid transport machineries known as
phospholipid flippase, floppase, and scramblase [13, 14] (Fig. 12.1). Phospholipid
flippase translocates phospholipids from the exocytoplasmic (extracellular/luminal)
to the cytoplasmic leaflet of cellular membranes and is now known to belong to a
subfamily of P-type ATPases known as type IV P-type ATPases (P4-ATPases). Some
members of the ATP-binding cassette (ABC) transporters function as phospholipid
floppases, which translocate phospholipids from the cytoplasmic to the
A B C
Outer leaflet
Inner leaflet
Fig. 12.1 Lipid-transport machineries: flippase (a), floppase (b), and scramblase (c)
12 Membrane Lipid Transporters in Drosophila melanogaster 167
12.2 Flippase
Bovine ATPase II (currently designated as ATP8A1), which mediates the net trans-
fer of PS and PE from the outer to inner leaflets of the plasma membrane bilayer,
was identified as the first candidate for a phospholipid flippase [18]. A subsequent
genome search revealed that ATP8A1 and its closest yeast homologue, Drs2p,
were founding members of a novel subfamily of P-type ATPases known as
P4-ATPases [19]. P4-ATPases share structural similarity with other P-type
ATPases, including ten transmembrane-spanning segments and a P-type ATPase-
specific sequence motif, most notably the DKTGTLT sequence motif in which the
canonical aspartic acid is phosphorylated during the reaction cycle, and a nucleo-
tide (ATP)-binding site [20]. The unique structural feature of P4-ATPases is that
the conserved charged and polar amino acids in the transmembrane domains 4 and
6 of cation-transport ATPases are replaced with hydrophobic and aromatic amino
acid residues. Although it is not known how the P4-ATPases couple ATP hydroly-
sis and phospholipid translocation, recent studies by Grahams group proposed a
structural model for their phospholipid specificity and transport mechanism [21,
22]. It is also shown that the association of P4-ATPases with CDC50 family pro-
teins is required for their exit from the endoplasmic reticulum (ER) and for their
proper cellular localization [23, 24].
P4-ATPases represent the largest subfamily of P-type ATPases and are present
only in eukaryotic cells. In mammals, at least 14 members of P4-ATPases, desig-
nated ATP8A1 through ATP11C, and three CDC50 proteins (CDC50A, CDC50B,
and CDC50C) have been identified. Among the P4-ATPases expressed in
mammalian cells, ATP8A1, ATP8A2, ATP8B1, ATP8B3, ATP8B5, and ATP11C
have been implicated in the translocation of phospholipids. With regard to the bio-
logical functions of phospholipid flippases, studies using the budding yeast
Saccharomyces cerevisiae have demonstrated that P4-ATPases interact genetically
and directly with the components involved in clathrin-dependent vesicular transport
[25]. The translocation of aminophospholipids such as PS and PE creates an imbal-
ance in the numbers of phospholipids between the two leaflets, which increases the
membrane curvature and anionic phospholipid content of the cytosolic leaflet,
168 K. Nagao et al.
thereby driving the recruitment of functional molecules and the resulting vesicular
formation [26, 27]. Recent studies of mammalian P4-ATPases have also shown
their involvement in various cellular and pathophysiological events such as neural
function, bile salt secretion, acrosome reaction, B-cell differentiation, cell migra-
tion, and apoptosis [24, 28].
A CG4301 D CG31121
CG9981 E23
ATP11B ABCG2
ATP11A CG9663
ATP11C CG4822
ATP8B3 CG9664
ATP8B1 CG31689
CG14741 CG5853
ATP8B2 CG3164
ATP8B4 ATET
CG42321 ABCG1
ATP8A1 ABCG4
ATP8A2 CG17646
CG33298
CG32091
ATP10A
BR
ATP10B
ST
ATP10D
W
CG31729
CG11069
ATP9A
ABCG5
ATP9B
ABCG8
B CDC50C
dCDC50
CDC50A E TMEM16G
CDC50B TMEM16J
CG6938
C MDR50
TMEM16C
ABCB11 TMEM16D
ABCB5 TMEM16E
ABCB1 TMEM16F
ABCB4 TMEM16A
MDR49 TMEM16B
CG10226 SUBDUED
MDR65 CG10353
ABCB10
AXS
CG3156
TMEM16H
ABCB8
TMEM16K
CG1824
ABCB2
ABCB3 F PLSCR4
ABCB9 PLSCR3
ABCB6 PLSCR1
HMT1 PLSCR2
ABCB7 SCRAMB1
CG7955 SCRAMB2
Fig. 12.2 Phylogenic trees of lipid transporters in Homo sapiens and Drosophila melanogaster:
P4-ATPase (a), CDC50 (b), ABCB subfamily (c), ABCG subfamily (d), TMEM16 (e), PLSCR (f).
H. sapiens proteins are underlined
In contrast to the variation among the P4-ATPases, only a single CDC50 family
protein designated as dCDC50 (CG9947) has been identified in D. melanogaster.
The expression profile of dCDC50 was analyzed by using an antibody that we raised
against a synthetic peptide corresponding to the carboxyl terminus of the protein.
The expression of dCDC50 was detected throughout the developmental stages with
a prominent peak at the first-instar stage. In adult flies, dCDC50 was abundantly
expressed in gut and brain tissues, with ubiquitous expression detected in all organs
or body parts examined.
Ubiquitous gene silencing of the dCdc50 gene using the GAL4/UAS system
[38], in which a ubiquitous promoter from a Drosophila Tubulin84B gene and
12 Membrane Lipid Transporters in Drosophila melanogaster 171
12.3 Floppase
Fig. 12.3 Whole-mount preparation of a fat body from a last-instar larva with a dCdc50-silenced
somatic mosaic. dCdc50-silenced cells were randomly generated at the first-instar stage. Fat bod-
ies were dissected at the last-instar stage and observed with a Zeiss Axiovert 200 M fluorescent
microscope. Nuclei are visualized with DAPI (magenta). Note that dCdc50-silenced cells [marked
by green fluorescent protein (GFP), green] were indistinguishable from the neighboring wild-type
cells in both size and morphology. Bar 100 m
2. Secretion from the inner leaflet to extracellular environment. The substrate enters
the transporter from the inner leaflet as in model 1, but then exits the transporter
directly to an exogenous environment. Because the hydrophobic substrate read-
ily returns to the outer leaflet of the lipid bilayer, an acceptor is required for the
hydrophobic substrate to be solubilized in an exogenous environment.
3. Secretion from the outer leaflet to extracellular environment. In this model, the
substrate enters the transporter from the outer leaflet and exits the transporter
directly to an exogenous environment. An acceptor would also be required for
the substrate to be solubilized in an exogenous environment in this case. The
movement of substrates in this model can be called projection to the exoge-
nous environment.
Based on the sequence similarity of NBDs, ABC transporters could be divided into
eight subfamilies (ABCA to ABCH) [46, 47]. Although the D. melanogaster
genome encodes 56 ABC transporter genes (10 for ABCA, 8 for ABCB, 14 for
ABCC, 2 for ABCD, 1 for ABCE, 3 for ABCF, 15 for ABCG, 3 for ABCH), infor-
mation about the function and substrate of Drosophila ABC transporters is limited.
In this section, we summarize the functions of ABCG and ABCB subfamily mem-
bers that have been reported to be involved in the transport of hydrophobic
substrate.
Among the 15 ABCG subfamily members of D. melanogaster (Fig. 12.2d), the best
characterized members are W, BW, and ST (the gene products of white, brown, and
scarlet, respectively), which are involved in the determination of eye color [48].
Normally, the eye color of D. melanogaster is red-brown, because two color light-
screening pigments, xanthommatin (brown pigment) and a class of drosopterins
(red pigment), are deposited in membrane-bound granules in specialized pigment
cells in each ommatidium of the compound eye. Xanthommatin and drosopterins
are synthesized from tryptophan and guanosine triphosphate (GTP), respectively.
Two Drosophila ABC transporters, W and ST, form a heterodimer and mediate the
uptake of tryptophan and two intermediates of the xanthommatin pathway. W also
forms a heterodimer with BW to mediate the uptake of the drosopterin precursor
guanine. Therefore, W is required for the synthesis of both pigments, whereas ST
and BW are involved in the synthesis of xanthommatin and drosopterins, respec-
tively. Reflecting the role of each ABC transporter in pigment production, null
mutation of the white gene causes white eye color because of the lack of deposition
of either xanthommatin or drosopterins in the pigment cells, whereas flies with
174 K. Nagao et al.
deletion of the scarlet gene have red eyes and the null mutation of the brown gene
causes brown eyes.
Although three Drosophila ABCG subfamily proteins (W, BW, and ST) trans-
port hydrophilic compounds in eye pigment synthesis, mammalian ABCG subfam-
ily members mediate the secretion of hydrophobic molecules (e.g., ABCG1 and
ABCG4 mediate cholesterol efflux into HDL particles; the ABCG5-ABCG8 het-
erodimer transports cholesterol and sitosterol). There are 12 Drosophila ABCG
subfamily proteins in addition to the three transports for eye pigment synthesis.
Some of these proteins show high homology with mammalian lipid transporters and
are predicted to be lipid transporters. For example, ATET has high homology with
human ABCG1 (44 % identical) and human ABCG4 (41 % identical), but the trans-
port substrate of ATET is not clear [49]. E23 (CG3327) is involved in ecdyson-
mediated gene activation [50]. Ecdyson is a steroid hormone that regulates
developmental processes, and the concentration of ecdyson is drastically changed
during the developmental course. The temporal pattern of E23 transcript accumula-
tion is consistent with that of an ecdyson pulse during development, and the expres-
sion of E23 is robustly induced by ecdyson in cultured larval tissues, demonstrating
that E23 is an ecdyson-inducible ABC transporter. Ectopic expression of E23 sup-
presses the ecdyson-mediated transcriptional activation of the Eip74EF and Eip75B
genes, indicating that E23 may modulate the intracellular ecdyson concentration by
secreting ecdyson into the extracellular environment. Interestingly, the system for
induction of the putative ecdyson transporter E23 by ecdyson is similar to the induc-
tion mechanism of mammalian cholesterol transporters. For example, expression of
the cholesterol transporters ABCG1 and ABCG4 is induced by oxidized cholesterol
via activation of a nuclear receptor, liver X receptor (LXR) [51, 52]. Because ecdy-
son induces the transcription of several genes via binding to the nuclear receptor
ecdyson receptor (EcR) [53], mammals and Drosophila utilize similar mechanisms
for the modulation of cellular sterol levels. As E23 is a half-type transporter similar
to Drosophila W and mammalian ABCG1, E23 requires the formation of a dimer to
function as an active transporter. However, it is not clear whether E23 forms a
homodimer or a heterodimer with the other half-type transporter. Identification of a
dimer partner of E23 is important for understanding both the mechanism and
regulation of ecdyson transport during developmental processes. Thus, the
Drosophila ABCG subfamily transporter is also involved in the transport of hydro-
phobic compounds, and further research is needed to reveal the role of other ABCG
subfamily members.
that form dimers to serve as active transporters. Among them, two transporters have
been shown to mediate the transport of hydrophobic substrate. Because human
ABCB1 and ABCB4 secrete lipophilic xenobiotics and PC, respectively [54, 55], it
is plausible that Drosophila ABCB transporters mediate the transport of hydropho-
bic substrates.
The full-type transporter MDR65 is specifically localized at the humoral barrier
of the Drosophila central nervous system (CNS) of subperineural glia and is required
for protection against chemical attack [56]. Another full-type transporter, MDR49,
has been shown to be involved in the secretion of chemoattractant [57]. The migra-
tion of primordial germ cells from the site of origin to the somatic part of the gonad
is crucial for embryonic development. Chemoattractants are commonly secreted
through a classic, signal peptide-dependent pathway, but the secretion of a geranyl-
modified attractant requires MDR49. Among ABCB subfamily transporters of
Drosophila, the expression pattern and phenotype of the Mdr49 mutant are consis-
tent with a role in germ cell migration. Further, the overexpression of MDR49 and
HMGCR, an enzyme required for production of the geranylgeranyl moiety in insect
cell lines, enhances the migration of germ cells, demonstrating the role of the
MDR49 transporter in the secretion of the geranyl-modified attractant. Although the
transport mechanisms of MDR49 and MDR65 are not clear, these functions strongly
suggest the importance of Drosophila ABCB subfamily transporters in lipid trans-
port, as seen in mammalian cells.
12.4 Scramblase
12.5 Perspectives
References
1. Bier E (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev
Genet 6:923
2. Xu Y, Condell M, Plesken H, Edelman-Novemsky I, Ma J, Ren M, Schlame M (2006) A
Drosophila model of Barth syndrome. Proc Natl Acad Sci U S A 103:1158411588
3. Takeuchi K, Nakano Y, Kato U, Kaneda M, Aizu M, Awano W, Yonemura S, Kiyonaka S, Mori
Y, Yamamoto D, Umeda M (2009) Changes in temperature preferences and energy homeosta-
sis in dystroglycan mutants. Science 323:17401743
4. Dar AC, Das TK, Shokat KM, Cagan RL (2012) Chemical genetic discovery of targets and
anti-targets for cancer polypharmacology. Nature 486:8084
5. Lenz S, Karsten P, Schulz JB, Voigt A (2013) Drosophila as a screening tool to study human
neurodegenerative diseases. J Neurochem 127:453460
6. Pavlidis P, Ramaswami M, Tanouye MA (1994) The Drosophila easily shocked gene: a muta-
tion in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis. Cell
79:2333
7. Pospisilik JA, Schramek D, Schnidar H, Cronin SJ, Nehme NT, Zhang X, Knauf C, Cani PD,
Aumayr K, Todoric J, Bayer M, Haschemi A, Puviindran V, Tar K, Orthofer M, Neely GG,
Dietzl G, Manoukian A, Funovics M, Prager G, Wagner O, Ferrandon D, Aberger F, Hui CC,
Esterbauer H, Penninger JM (2010) Drosophila genome-wide obesity screen reveals hedgehog
as a determinant of brown versus white adipose cell fate. Cell 140:148160
8. Carvalho M, Sampaio JL, Palm W, Brankatschk M, Eaton S, Shevchenko A (2012) Effects of
diet and development on the Drosophila lipidome. Mol Syst Biol 8:600
9. Guan XL, Cestra G, Shui G, Kuhrs A, Schittenhelm RB, Hafen E, van der Goot FG, Robinett
CC, Gatti M, Gonzalez-Gaitan M, Wenk MR (2013) Biochemical membrane lipidomics dur-
ing Drosophila development. Dev Cell 24:98111
10. van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how
they behave. Nat Rev Mol Cell Biol 9:112124
11. Holthuis JC, Menon AK (2014) Lipid landscapes and pipelines in membrane homeostasis.
Nature 510:4857
178 K. Nagao et al.
34. Ha TS, Xia R, Zhang H, Jin X, Smith DP (2014) Lipid flippase modulates olfactory receptor
expression and odorant sensitivity in Drosophila. Proc Natl Acad Sci U S A 111:78317836
35. Liu YC, Pearce MW, Honda T, Johnson TK, Charlu S, Sharma KR, Imad M, Burke RE,
Zinsmaier KE, Ray A, Dahanukar A, de Bruyne M, Warr CG (2014) The Drosophila melano-
gaster phospholipid flippase dATP8B is required for odorant receptor function. PLoS Genet
10, e1004209
36. Ma Z, Liu Z, Huang X (2010) OSBP- and FAN-mediated sterol requirement for spermatogen-
esis in Drosophila. Development 137:37753784
37. Ma Z, Liu Z, Huang X (2012) Membrane phospholipid asymmetry counters the adverse effects
of sterol overloading in the Golgi membrane of Drosophila. Genetics 190:12991308
38. Elliott DA, Brand AH (2008) The GAL4 system: a versatile system for the expression of
genes. Methods Mol Biol 420:7995
39. Bischof J, Basler K (2008) Recombinases and their use in gene activation, gene inactivation,
and transgenesis. Methods Mol Biol 420:175195
40. Nagao K, Kimura Y, Mastuo M, Ueda K (2010) Lipid outward translocation by ABC proteins.
FEBS Lett 584:27172723
41. Hyde SC, Emsley P, Hartshorn MJ, Mimmack MM, Gileadi U, Pearce SR, Gallagher MP, Gill
DR, Hubbard RE, Higgins CF (1990) Structural model of ATP-binding proteins associated
with cystic fibrosis, multidrug resistance and bacterial transport. Nature 346:362365
42. Zhang DW, Graf GA, Gerard RD, Cohen JC, Hobbs HH (2006) Functional asymmetry of
nucleotide-binding domains in ABCG5 and ABCG8. J Biol Chem 281:45074516
43. Urbatsch IL, Beaudet L, Carrier I, Gros P (1998) Mutations in either nucleotide-binding site of
P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites. Biochemistry
37:45924602
44. Tombline G, Bartholomew LA, Urbatsch IL, Senior AE (2004) Combined mutation of cata-
lytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a
conformation that binds ATP and ADP tightly. J Biol Chem 279:3121231220
45. Hrycyna CA, Ramachandra M, Germann UA, Cheng PW, Pastan I, Gottesman MM (1999)
Both ATP sites of human P-glycoprotein are essential but not symmetric. Biochemistry
38:1388713899
46. Dean M, Rzhetsky A, Allikmets R (2001) The human ATP-binding cassette (ABC) transporter
superfamily. Genome Res 11:11561166
47. Dermauw W, Van Leeuwen T (2014) The ABC gene family in arthropods: comparative genom-
ics and role in insecticide transport and resistance. Insect Biochem Mol Biol 45:89110
48. Ewart GD, Howells AJ (1998) ABC transporters involved in transport of eye pigment precur-
sors in Drosophila melanogaster. Methods Enzymol 292:213224
49. Kuwana H, Shimizu-Nishikawa K, Iwahana H, Yamamoto D (1996) Molecular cloning and
characterization of the ABC transporter expressed in Trachea (ATET) gene from Drosophila
melanogaster. Biochim Biophys Acta 1309:4752
50. Hock T, Cottrill T, Keegan J, Garza D (2000) The E23 early gene of Drosophila encodes an
ecdysone-inducible ATP-binding cassette transporter capable of repressing ecdysone-mediated
gene activation. Proc Natl Acad Sci U S A 97:95199524
51. Kennedy MA, Venkateswaran A, Tarr PT, Xenarios I, Kudoh J, Shimizu N, Edwards PA (2001)
Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter
that produces a novel transcript encoding an alternative form of the protein. J Biol Chem
276:3943839447
52. Engel T, Lorkowski S, Lueken A, Rust S, Schluter B, Berger G, Cullen P, Assmann G (2001)
The human ABCG4 gene is regulated by oxysterols and retinoids in monocyte-derived macro-
phages. Biochem Biophys Res Commun 288:483488
53. Koelle MR, Talbot WS, Segraves WA, Bender MT, Cherbas P, Hogness DS (1991) The
Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor
superfamily. Cell 67:5977
180 K. Nagao et al.
54. Ueda K, Cardarelli C, Gottesman MM, Pastan I (1987) Expression of a full-length cDNA for
the human MDR1 gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc
Natl Acad Sci U S A 84:30043008
55. Morita SY, Kobayashi A, Takanezawa Y, Kioka N, Handa T, Arai H, Matsuo M, Ueda K (2007)
Bile salt-dependent efflux of cellular phospholipids mediated by ATP binding cassette protein
B4. Hepatology 46:188199
56. Mayer F, Mayer N, Chinn L, Pinsonneault RL, Kroetz D, Bainton RJ (2009) Evolutionary
conservation of vertebrate blood-brain barrier chemoprotective mechanisms in Drosophila. J
Neurosci 29:35383550
57. Ricardo S, Lehmann R (2009) An ABC transporter controls export of a Drosophila germ cell
attractant. Science 323:943946
58. Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ (1997) Molecular cloning of human
plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of
plasma membrane phospholipids. J Biol Chem 272:1824018244
59. Zhou Q, Zhao J, Wiedmer T, Sims PJ (2002) Normal hemostasis but defective hematopoietic
response to growth factors in mice deficient in phospholipid scramblase 1. Blood
99:40304038
60. Suzuki J, Fujii T, Imao T, Ishihara K, Kuba H, Nagata S (2013) Calcium-dependent phospho-
lipid scramblase activity of TMEM16 protein family members. J Biol Chem
288:1330513316
61. Acharya U, Edwards MB, Jorquera RA, Silva H, Nagashima K, Labarca P, Acharya JK (2006)
Drosophila melanogaster scramblases modulate synaptic transmission. J Cell Biol
173:6982
62. Wong XM, Younger S, Peters CJ, Jan YN, Jan LY (2013) Subdued, a TMEM16 family Ca2+-
activated Cl channel in Drosophila melanogaster with an unexpected role in host defense.
eLife 2, e00862
63. Kramer J, Hawley RS (2003) The spindle-associated transmembrane protein Axs identifies a
membranous structure ensheathing the meiotic spindle. Nat Cell Biol 5:261263
64. Mizuta K, Tsutsumi S, Inoue H, Sakamoto Y, Miyatake K, Miyawaki K, Noji S, Kamata N,
Itakura M (2007) Molecular characterization of GDD1/TMEM16E, the gene product respon-
sible for autosomal dominant gnathodiaphyseal dysplasia. Biochem Biophys Res Commun
357:126132
Chapter 13
Drosophila: A Model for Studying
Prostaglandin Signaling
Prostaglandins (PGs) are lipid signaling molecules that mediate a wide range of
physiological processes including reproduction, cardiovascular function and dis-
ease, pain and inflammation, and cancer development and progression (reviewed in
[1, 2]). Although PGs were identified more than 60 years ago, the study of PGs in
the genetic model system of Drosophila melanogaster (hereafter referred to as
Drosophila) has largely been restricted to the past decade. Here we review (1) the
A.J. Spracklen
Anatomy and Cell Biology Department, University of Iowa Carver College of Medicine,
Iowa City, IA 52242, USA
Current Address: Lineberger Comprehensive Cancer Center, Department of Biology,
University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
T.L. Tootle (*)
Anatomy and Cell Biology Department, University of Iowa Carver College of Medicine,
Iowa City, IA 52242, USA
e-mail: [email protected]
Prostaglandin (PG) synthesis is a multistep processes that begins with the release of
arachidonic acid (AA) from the glycerol backbone of membrane phospholipids
through the enzymatic activity of phospholipase A2 (PLA2). This free AA is then
converted into the PG precursor, PGH2, through the enzymatic activity of cyclooxy-
genase enzymes (in mammals, COX-1 and COX-2), which are the pharmacological
targets of nonsteroidal anti-inflammatory drugs (reviewed in [36]). Downstream of
COX enzymes, PGH2 is processed into the biologically active PGs (PGD2, PGE2,
PGF2, PGI2) and thromboxane (TXA2) through the activity of specific synthases
(PGD2: H-PGDS, L-PGDS; PGE2: mPGES-1, mPGES-2, cPGES; PGF2: AKR1B1;
PGI2: PGIS; TXA2: TXAS) [7]. These bioactive species then go on to serve as auto-
crine/paracrine signaling molecules.
Although PGs may induce MAPK signaling pathways [812], or serve as peroxi-
some proliferator-activated receptor-gamma (PPAR) nuclear hormone receptor
ligands [1316] independently of G protein-coupled receptors (GPCRs) (reviewed
in [17]), their most widely accepted and best understood mechanism of action is to
serve as ligands for specific GPCRs [18]. Each bioactive species of PG can bind to
and activate from one to four cognate GPCRs (PGD2: DP, CRTH2; PGE2: EP1, EP2,
EP3, EP4; PGF2: FP; PGI2: IP), which elicit their downstream effects through acti-
vation of G [17, 19] and, in some cases, G [20].
PGs are derived from the COX-dependent oxygenation of three long-chain polyun-
saturated fatty acids (PUFAs): arachidonic acid (AA, 20:4n-6), eicosapentaenoic
acid (EPA, 20:5n-3), and dihomo-gamma-linolenic acid (DGLA, 20:3n-6). In many
organisms, these long-chain PUFAs are acquired through the diet or through the
elongation/desaturation of the essential fatty acid, linoleic acid (LA, 18:2n-6).
Although some insects are capable of de novo synthesis of LA [21], there is little
evidence that this occurs in Drosophila [22, 23].
The presence of long-chain PUFAs and their biological significance in Drosophila
remains unclear. Early studies indicated that AA is not present in the fly, but its
13 Drosophila: A Model for Studying Prostaglandin Signaling 183
precursor, LA, is [2325]. Interestingly, Shen et al. found that when 22-carbon
PUFAs are supplied in the diet these lipids are readily converted to 20-carbon
PUFAs [25]. Thus, flies possess the machinery to utilize very long chain PUFAs.
Other more recent studies, conducted using liquid chromatography coupled to tan-
dem mass spectrometry (LC-MS/MS), suggest that 20-carbon PUFAs are present in
Drosophila. Using LC-MS/MS to analyze the fatty acid content of membrane phos-
pholipids in both whole adults and isolated adult testes, Steinhauer et al. reported
the presence of numerous phospholipid species containing AA in a wild-type fly
strain, ranging from 0.2 to 1.5 % of the total phospholipid class [26]. Other groups
also suggest 20-carbon PUFAs may be present in Drosophila [2729]. Together
these studies support the idea that the lipid precursors for PG synthesis are present
at low levels in Drosophila.
In 1986, Pages et al. found that Drosophila extracts incubated with AA can gen-
erate PGE2, PGF1, and PGF2, as detected by gas chromatographymass spectrom-
etry. Furthermore, endogenous PGE2 was detected in untreated extracts using high
performance liquid chromatographyradioimmunoassay [24]. These data were the
first to suggest that a COX-like activity may be conserved in Drosophila.
Given the finding by Pages et al. [24] and the highly conserved roles of PGs in
female reproduction [30, 31], we hypothesized that if PG synthesis and signaling
were conserved in Drosophila it would regulate oogenesis or follicle development.
Initially, we took advantage of the ability of mid-oogenesis stage 10B (S10B) fol-
licles or eggs to mature in in vitro culture to ask whether COX enzyme activity was
required to facilitate late-stage oogenesis [32]. These studies demonstrated that
COX-1-like activity is required for follicle maturation as COX-1 inhibitors, but not
COX-2 selective inhibitors, block follicle maturation in a dose-dependent manner
[32]. Importantly, this COX inhibitor-dependent block in development is rescued by
concomitant treatment with exogenous PGH2, PGF2, or fluprostenol, a stabilized
PGF2 analogue. These studies revealed that both COX-like activity and PGs are
required for late-stage follicle development in Drosophila [32].
The results of our pharmacological experiments [32], in combination with the previ-
ous findings of Pages et al. [24], strongly suggested the presence of a COX-like
enzyme in Drosophila. BLAST analysis [33] revealed Drosophila Pxt as a candi-
date COX-like enzyme [32]. Sequence alignment using MUltiple Sequence
Comparison by Log-Expectation (MUSCLE) [34, 35] reveals that Pxt is 26.76 %
identical to ovine COX-1 and that a number of key residues [36] are conserved
between COX-1 and Pxt. Most notably, the three critical residues for heme coordi-
nation in the peroxidase active site of COX-1 (Gln203, His207, and His388) are
conserved in Pxt (Gln396, His402, and His590). Interestingly, Pxt possesses a can-
didate COX catalytic residue (Pxt Tyr564 vs. COX-1 Tyr385), although there is no
184 A.J. Spracklen and T.L. Tootle
Table 13.1 Putative Drosophila homologues of prostaglandin (PG) synthesis and signaling
proteins defined by BLASTp
PG pathway Putative Drosophila Expression level during
component Function homologue mid-to-late oogenesisb
COX COX1-like Pxt High
COX-like? CG4009 Low, except high during S12
CG10211 Below detection
PGD2 synthases H-PGDS Gsts1 Low
L-PGDS n/a n/a
PGE2 synthases mPGES1 Mgst1 Medium
CG33178 Below detection
mPGES2 Su(P) (CG4086) Low
cPGES CG16817 High
CG9267 Medium
PGF2 synthases AKR1C3 CG6084a High
AKR1B1
PGI2 synthase PGIS n/a n/a
TXA2 synthase TBXAS n/a n/a
PG-like receptor GPCR CG7497 Below detection
15d-PGDH Degrades PGs Pdh Below detection
CG4086 Medium
a
Indicates existence of numerous other similar proteins in Drosophila; however, CG6084 exhibits
the highest homology
b
Expression during oogenesis determined by microarray analysis of staged wild-type follicles [37]
clear conservation of the residues that have been shown to be critical for substrate
binding through mutagenesis studies performed on mammalian COX enzymes.
Additionally, the residue that is the target of aspirin-mediated acetylation (Ser530)
is not clearly conserved.
Although the sequence homology between Pxt and COX-1 enzymes is not par-
ticularly striking, genetic loss of Pxt phenocopies the effects of COX inhibition.
Specifically, similar to wild-type follicles treated with COX inhibitors, pxt mutant
S10B follicles fail to complete maturation in vitro and exogenous PGs can restore
development [32]. Additionally, pxt mutants are female sterile, and this sterility can
be rescued by germline expression of mouse COX-1 [32]. Together, these data sug-
gest that Pxt is the Drosophila COX-like enzyme and that both COX-like activity
and PG signaling are required for Drosophila follicle maturation.
In addition to Pxt, Drosophila possesses putative homologues of other PG syn-
thesis and signaling components (identified by BLAST [33]). Table 13.1 summa-
rizes these candidates and their level of expression during mid-to-late oogenesis, as
revealed by our microarray analysis [37].
13 Drosophila: A Model for Studying Prostaglandin Signaling 185
Given the widely conserved roles of PGs in reproduction [30, 31] and our finding
that both COX activity and Pxt, the COX-like enzyme, mediate Drosophila follicle
development [32], we have continued to exploit the system of Drosophila oogenesis
to discover the specific activities of PGs. Thus, here we primarily discuss the
oogenic roles of PGs. Additionally, we briefly discuss functions of PGs in Drosophila
other than female reproduction.
Pxt is required for the coordination of eggshell gene expression throughout the end
of Drosophila oogenesis [37]. The somatic follicle cells secrete the eggshell. The
Drosophila eggshell consists of five structural layers, which are sequentially syn-
thesized in the following order: the vitelline membrane, the wax layer, and the cho-
rion, which consists of the inner chorion layer, the endochorion, and the exochorion
(Fig. 13.1a, d). Proper eggshell assembly requires tight temporal regulation of gene
expression for the eggshell structural components. As such, the expression of vitel-
line membrane components begins during Stage 8 (S8), peaks during S10, and
ceases by the end of S11, whereas the expression of chorion components occurs in
three distinct phases, spanning from S10 to S14: early, middle, and late [41].
In pxt mutants, the expression of many vitelline membrane genes is prolonged
while chorion gene expression is severely disrupted. The onset of expression of
some chorion genes is early, although the expression of other chorion genes is
186 A.J. Spracklen and T.L. Tootle
A B wt pxt-/- OE pxt
F G H
pxt-/-
C
S10B
DAPI EdU
F G H
wt pxt-/-
D E
vm
Follicle cells
Follicle
c
EdU
oocyte c
I J K
cells
S12
I J K
vm
oocy
te
wt pxt-/-
Fig. 13.1 Loss of Pxt in the soma results in defects in eggshell gene expression and eggshell for-
mation. (a, b) Images of laid eggs of the indicated genotypes. (d, e) Transmission electron micro-
graphs (Michael Sepanski) of the eggshell of S14 follicles (c chorion, vm vitelline membrane).
Bars 5 m. (fk) Confocal images of follicle cell nuclei from specified stages and genotypes
labeled with DAPI (blue in fk) to mark the nucleus and EdU (green in fk; white in fk) to label
the number and size of gene amplification sites. Loss of Pxt results in short eggs with defective
eggshells (b, c compared to a). In pxt mutants, the vitelline membrane is produced and fuses pre-
maturely (not shown), and the chorion of S14 follicles exhibits structural defects (e compared to
d). These eggshell defects are likely caused by the altered temporal regulation of eggshell gene
expression during mid-to-late oogenesis (not shown), which may be caused by the altered gene
amplification observed. When Pxt is lost, S10B follicle cells exhibit an increase in the number of
sites of gene amplification observed by EdU labeling (gg compared to ff), and during S12,
when most of the amplification has ceased in wild-type follicles (ii), multiple and larger sites of
amplification are observed in pxt mutants (jj). Conversely, overexpression of Pxt results in
decreased sites of amplification and an increase in the rate of elongation as separated replication
forks, indicated by double-bar EdU structures, are often observed (hh and kk, yellow arrows)
delayed in pxt mutants [37]. Additionally, the expression of some chorion genes
persists longer in pxt mutants than in wild-type [37]. These defects in temporal
regulation of gene expression ultimately lead to numerous eggshell abnormalities,
including a loss of vitelline membrane integrity and altered chorion production,
resulting in short, uneven dorsal appendages and chorion patterning defects [37]
(Fig. 13.1b, c compared to Fig. 13.1a, e compared to Fig. 13.1d). Although Pxt is
required in the germline for nurse cell actin remodeling and nurse cell dumping (see
following) [32], it is required in the soma (follicle cells) for temporal coordination
of eggshell gene expression [37].
One means by which PG signaling could regulate the timing of eggshell gene
expression is by affecting gene amplification of eggshell gene clusters. To promote
13 Drosophila: A Model for Studying Prostaglandin Signaling 187
the proper formation of the eggshell, the appropriate eggshell genes must be rapidly
transcribed at high rates during a strict temporal window. The eggshell-encoding
genes are organized into a few clusters throughout the Drosophila genome. These
gene clusters undergo gene amplification [42]. During gene amplification, particular
regions of the genome undergo multiple rounds of replication to increase the DNA
copy number of those regions. In nondividing cells, such as the Drosophila follicle
cells after S6, gene amplification can be visualized as spots of nucleotide analogue,
such as EdU incorporation. The size of the EdU spot generally corresponds to the
amount gene amplification. There are six characterized sites of gene amplification
in the Drosophila follicle cells [43]. During S10BS11, gene amplification is initiat-
ing and all the sites of amplification are visible (Fig. 13.1ff); by S12 it has shifted
to elongation and only a subset of the sites are visible (Fig. 13.1ii) [44].
Both loss of and overexpression of Pxt affect gene amplification during follicle
development. Follicles from pxt mutants exhibit an increase in both the visible num-
ber and size of amplification sites during both S10B and S12 (Fig. 13.1gg, j-j'
compared to Fig. 13.1ff, ii, respectively). Conversely, ubiquitous expression of
either Pxt or mouse COX-1 results in a reduction in the level of amplification as
decreased EdU incorporation is observed during both S10B and S12 (Fig. 13.1hh,
kk compared to Fig. 13.1ff, ii, respectively). Additionally, double-bar EdU
spots, indicative of replication forks [44], are easily observed in follicles overex-
pressing Pxt or mouse COX-1, suggesting increased elongation. These data indicate
the Pxt and PG signaling regulate the sites, level, and extent of elongation of gene
amplification (Tootle, Williams, and Spradling, unpublished observations).
Stage 9
GV
wt S9 pxtEY/f S9
Oocyte
B Phalloidin C Phalloidin
D Stage 10B
Fig. 13.2 Actin remodeling during Drosophila oogenesis requires the Drosophila COX-like
enzyme, Pxt. (a) Schematic detailing the cellular composition of a S9 follicle (GV germinal vesi-
cle). (bb, df) Maximum projections of three confocal slices of follicles, staged as indicated,
taken at 20. (cc) Single confocal slice of S9 follicle, taken at 20. Anterior is to the left. F-actin
(phalloidin) white, DNA (DAPI) cyan. (bb, ee) Wild type wt (yw). (cc, ff) pxtEY03052 mutant,
pxtEY. (d) Schematic detailing the cellular composition of a S10B follicle (GV germinal vesicle). S9
and S10B follicles consist of 16 germline-derived cells [1 oocyte (white) and 15 nurse cells (gray)]
that are surrounded by a somatic epithelium (a, c). During S9, the nurse cell cytoplasm is largely
devoid of actin filament structures aside from the cortical actin meshwork underlying the mem-
branes (bb). pxt mutants exhibit a range of actin remodeling defects during S9, including cortical
actin breakdown (not shown) and early actin remodeling, resulting in extensive early actin fila-
ments and actin aggregate structures (yellow arrows). (cc) During S10B, wild-type follicles rap-
idly undergo actin remodeling to generate a network of parallel actin filament bundles extending
from the nurse cell membranes toward the nuclei (ee). pxt mutants exhibit a range of actin
remodeling defects at S10B, ranging from mild defects in the number and distribution of actin fila-
ment bundles to a near complete loss of actin filament bundles (ff). Images are representative,
taken from multiple experiments. Scale Bars 50 m
13 Drosophila: A Model for Studying Prostaglandin Signaling 189
The studies of the roles of PGs in Drosophila have largely been limited to oogene-
sis, but PGs are likely to have additional functions in this organism. Indeed, pxt
mutant flies are sickly; they exhibit reduced viability, developmental delays, reduced
lifespan, motility defects, and abnormal fluid retention. Furthermore, COX inhibitor
studies have implicated PGs in Fascin-dependent neural morphogenesis and branch-
ing [64]. Additionally, it has recently been observed that loss of Pxt results in sperm
individualization defects (Josefa Steinhauer, personal communication); notably,
these defects may be caused by altered actin dynamics [65]. Thus, Drosophila pro-
vides a rich system to elucidate both functions and molecular mechanisms of PG
action in a variety of contexts.
The female reproductive functions of PG signaling are highly conserved. PGs regu-
late egg development and ovulation from insects [31] to mammals [30]. Indeed, PG
synthesis inhibitors cause fertility defects in women, likely the result of altered fol-
licle maturation and ovulation [6668]. Interestingly, COX2-dependent production
of PGE2 and PGF2 are implicated in mediating follicle development in mammals
[6971], whereas COX-1-dependent PGF2 mediates it in zebrafish [72], silkmoths
[73], and Drosophila [32]. It is important to determine if the molecular targets of
these PG signaling pathways are conserved across organisms.
13 Drosophila: A Model for Studying Prostaglandin Signaling 191
Both PGs [7481] and gene amplification [82, 83] are implicated in driving cancer
development and progression and are independently associated with poor patient
prognosis. We speculate that one means by which PGs may contribute to cancer is
by modulating gene amplification. In breast cancer, when the oncogene HER-2/neu
is highly expressed it is often caused by gene amplification [84]. Subbaramaiah
et al. found that the majority of HER-2/neu-positive tumor samples tested exhibit
high COX-2 levels [33]. It will be interesting to determine if there is mechanistic
association between PG signaling and gene amplification in cancer.
Numerous in vitro studies have implicated PG signaling in regulating the actin cyto-
skeleton. However, such studies have provided limited insight into the underlying
mechanisms of PG action. Multiple studies indicate that PGs induce changes in
cytoplasmic actin bundles by cAMP-dependent mechanisms. Indeed, PGE2- and
PGI2-induced actin stress fiber disassembly in human pulmonary artery endothelial
cells occurs via cAMP-dependent kinase (PKA) and nucleotide exchange proteins
directly activated by cAMP (Epac1)/Ras-related protein 1 (Rap1)-dependent activa-
tion of Rac [57]. In human umbilical vein endothelial cells, TXA2 inhibits, while
PGE2 promotes, v3-dependent cell adhesion and cell spreading by both PKA-
dependent Rac activation and Rac-independent activities [59]. PGE2 mediates actin
stress fiber disassembly in human aortic smooth muscle cells by PKA-dependent
decreases in focal adhesion kinase (FAK) phosphorylation [58]. PGs can also mod-
ulate the actin cytoskeleton via Rho GTPases. Specifically, PGE2 promotes actin
stress fiber assembly in rat IMCD cells [85], and PGF2 mediates filopodia retrac-
tion and actin stress fiber assembly in 293-EBNA cells via Rho activation [85, 86].
PGs also regulate actin cytoskeletal dynamics in vivo to control platelet activa-
tion and aggregation [87]. The major prostanoid produced in platelets is TXA2,
which serves as a potent activator of platelet aggregation [88]. Conversely, PGI2
[89], PGE1 [90], and PGD2 [91] inhibit platelet aggregation, and PGE2 may both
potentiate [90, 92] and inhibit platelet aggregation [93, 94]. The main activity of
these prostanoids is to regulate vasodilator-stimulated phosphoprotein (VASP), a
member of the Ena/VASP family of actin elongation factors. VASP is activated by
TXA2 and is inhibited through cAMP/cGMP-dependent phosphorylation down-
stream of PGI2 and PGE1 [95, 96]. The opposing actions of distinct prostanoids in
regulating VASP activity is strikingly similar to our findings on PG regulation of
Ena, the sole Drosophila Ena/VASP family member. Indeed, we find that PG signal-
ing inhibits Ena during S9, but likely promotes Ena activity during S10B [40].
192 A.J. Spracklen and T.L. Tootle
The study of PG signaling in Drosophila is in its infancy, yet it has already provided
key insights into how PGs regulate actin cytoskeletal remodeling. By taking advan-
tage of the genetic tools in Drosophila, further insights into the developmental and
homeostatic roles of PGs and their downstream signaling cascades can be uncov-
ered. Thus, this system is poised to truly advance our understanding of PG signal-
ing. In the future, it will be important to determine the extent to which the means of
PG signaling in Drosophila are conserved in higher organisms, both during normal
physiology, such as reproduction and tissue homeostasis, and disease, such as car-
diovascular diseases and cancer.
13 Drosophila: A Model for Studying Prostaglandin Signaling 193
References
22. Keith AD (1967) Fatty acid metabolism in Drosophila melanogaster: interaction between
dietary fatty acids and de novo synthesis. Comp Biochem Physiol 21(3):587600
23. Rapport EW, Stanley-Samuelson D, Dadd RH (1983) Ten generations of Drosophila melano-
gaster reared axenically on a fatty acid-free holidic diet. Arch Insect Biochem Physiol
1(3):243250
24. Pages M et al (1986) Cyclooxygenase and lipoxygenase-like activity in Drosophila melano-
gaster. Prostaglandins 32(5):729740
25. Shen LR et al (2010) Drosophila lacks C20 and C22 PUFAs. J Lipid Res 51(10):29852992
26. Steinhauer J et al (2009) Drosophila lysophospholipid acyltransferases are specifically
required for germ cell development. Mol Biol Cell 20(24):52245235
27. Carvalho M et al (2012) Effects of diet and development on the Drosophila lipidome. Mol
Syst Biol 8:600
28. Parisi M, Li R, Oliver B (2011) Lipid profiles of female and male Drosophila. BMC Res
Notes 4:198
29. Scheitz CJ et al (2013) Heritability and inter-population differences in lipid profiles of
Drosophila melanogaster. PLoS One 8(8):e72726
30. Kobayashi T, Narumiya S (2002) Function of prostanoid receptors: studies on knockout mice.
Prostaglandins Other Lipid Mediat 68-69:557573
31. Stanley D, Kim Y (2011) Prostaglandins and their receptors in insect biology. Front
Endocrinol (Lausanne) 2:105
32. Tootle TL, Spradling AC (2008) Drosophila Pxt: a cyclooxygenase-like facilitator of follicle
maturation. Development 135(5):839847
33. Subbaramaiah K et al (2002) Cyclooxygenase-2 is overexpressed in HER-2/neu-positive
breast cancer: evidence for involvement of AP-1 and PEA3. J Biol Chem
277(21):1864918657
34. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32(5):17921797
35. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and
space complexity. BMC Bioinformatics 5:113
36. Garavito RM, Malkowski MG, DeWitt DL (2002) The structures of prostaglandin endoper-
oxide H synthases-1 and -2. Prostaglandins Other Lipid Mediat 68-69:129152
37. Tootle TL et al (2011) Drosophila eggshell production: identification of new genes and coor-
dination by Pxt. PLoS One 6(5):e19943
38. Spradling AC (1993) Developmental genetics of oogenesis. In: Martinez-Arias B (ed) The
development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview,
pp 170
39. Groen CM et al (2012) Drosophila Fascin is a novel downstream target of prostaglandin
signaling during actin remodeling. Mol Biol Cell 23(23):45674578
40. Spracklen AJ et al (2014) Prostaglandins temporally regulate cytoplasmic actin bundle for-
mation during Drosophila oogenesis. Mol Biol Cell 25(3):397411
41. Cavaliere V et al (2008) Building up the Drosophila eggshell: first of all the eggshell genes
must be transcribed. Dev Dyn 237(8):20612072
42. Claycomb JM, Orr-Weaver TL (2005) Developmental gene amplification: insights into DNA
replication and gene expression. Trends Genet 21(3):149162
43. Kim JC et al (2011) Integrative analysis of gene amplification in Drosophila follicle cells:
parameters of origin activation and repression. Genes Dev 25(13):13841398
44. Claycomb JM et al (2004) Gene amplification as a developmental strategy: isolation of two
developmental amplicons in Drosophila. Dev Cell 6(1):145155
45. Hudson AM, Cooley L (2002) Understanding the function of actin-binding proteins through
genetic analysis of Drosophila oogenesis. Annu Rev Genet 36:455488
46. Bownes M, Hames BD (1978) Genetic analysis of vitellogenesis in Drosophila melanogas-
ter: the identification of a temperature-sensitive mutation affecting one of the yolk proteins. J
Embryol Exp Morphol 47:111120
13 Drosophila: A Model for Studying Prostaglandin Signaling 195
47. Bownes M, Hodson BA (1980) Mutant Fs(1) 1163 of Drosophila melanogaster alters yolk
protein secretion from the fat-body. Mol Gen Genet 180(2):411418
48. Gutzeit HO (1986) The role of microfilaments in cytoplasmic streaming in Drosophila folli-
cles. J Cell Sci 80:159169
49. Theurkauf WE et al (1992) Reorganization of the cytoskeleton during Drosophila oogenesis:
implications for axis specification and intercellular transport. Development 115(4):923936
50. Montell DJ, Yoon WH, Starz-Gaiano M (2012) Group choreography: mechanisms orches-
trating the collective movement of border cells. Nat Rev Mol Cell Biol 13(10):631645
51. Guild GM et al (1997) Actin filament cables in Drosophila nurse cells are composed of mod-
ules that slide passively past one another during dumping. J Cell Biol 138(4):783797
52. Huelsmann S, Ylanne J, Brown NH (2013) Filopodia-like actin cables position nuclei in
association with perinuclear actin in Drosophila nurse cells. Dev Cell 26(6):604615
53. Wheatley S, Kulkarni S, Karess R (1995) Drosophila nonmuscle myosin II is required for
rapid cytoplasmic transport during oogenesis and for axial nuclear migration in early
embryos. Development 121(6):19371946
54. Mahajan-Miklos S, Cooley L (1994) Intercellular cytoplasm transport during Drosophila
oogenesis. Dev Biol 165(2):336351
55. Cooley L, Verheyen E, Ayers K (1992) chickadee encodes a profilin required for intercellular
cytoplasm transport during Drosophila oogenesis. Cell 69(1):173184
56. Banan A et al (2000) Role of actin cytoskeleton in prostaglandin-induced protection against
ethanol in an intestinal epithelial cell line. J Surg Res 88(2):104113
57. Birukova AA et al (2007) Prostaglandins PGE2 and PGI2 promote endothelial barrier enhance-
ment via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res
313(11):25042520
58. Bulin C et al (2005) Differential effects of vasodilatory prostaglandins on focal adhesions,
cytoskeletal architecture, and migration in human aortic smooth muscle cells. Arterioscler
Thromb Vasc Biol 25(1):8489
59. Dormond O et al (2002) Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endo-
thelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.
J Biol Chem 277(48):4583845846
60. Kawada N, Klein H, Decker K (1992) Eicosanoid-mediated contractility of hepatic stellate
cells. Biochem J 285(pt 2):367371
61. Peppelenbosch MP et al (1993) Epidermal growth factor-induced actin remodeling is regu-
lated by 5-lipoxygenase and cyclooxygenase products. Cell 74(3):565575
62. Martineau LC et al (2004) p38 MAP kinase mediates mechanically induced COX-2 and PG
EP4 receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol
Renal Physiol 286(4):F693F701
63. Spracklen AJ, Tootle TL (2013) The utility of stage-specific mid-to-late Drosophila follicle
isolation. J Vis Exp 82:50493
64. Kraft R et al (2013) A cell-based fascin bioassay identifies compounds with potential anti-
metastasis or cognition-enhancing functions. Dis Model Mech 6(1):217235
65. Fabian L, Brill JA (2012) Drosophila spermiogenesis: big things come from little packages.
Spermatogenesis 2(3):197212
66. Akil M, Amos RS, Stewart P (1996) Infertility may sometimes be associated with NSAID
consumption. Br J Rheumatol 35(1):7678
67. Pall M, Friden BE, Brannstrom M (2001) Induction of delayed follicular rupture in the
human by the selective COX-2 inhibitor rofecoxib: a randomized double-blind study. Hum
Reprod 16(7):13231328
68. Smith G et al (1996) Reversible ovulatory failure associated with the development of lutein-
ized unruptured follicles in women with inflammatory arthritis taking non-steroidal anti-
inflammatory drugs. Br J Rheumatol 35(5):458462
69. Lim H et al (1997) Multiple female reproductive failures in cyclooxygenase 2-deficient mice.
Cell 91(2):197208
196 A.J. Spracklen and T.L. Tootle
94. Smith JP et al (2010) PGE2 decreases reactivity of human platelets by activating EP2 and
EP4. Thromb Res 126(1):e23e29
95. Aszodi A et al (1999) The vasodilator-stimulated phosphoprotein (VASP) is involved in
cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dis-
pensable for smooth muscle function. EMBO J 18(1):3748
96. Bearer EL et al (2000) VASP protects actin filaments from gelsolin: an in vitro study with
implications for platelet actin reorganizations. Cell Motil Cytoskeleton 47(4):351364
97. Troys M, Vandekerckhove J, Ampe C (2008) Actin and actin-binding proteins in cancer pro-
gression and metastasis. In: Remedios C, Chhabra D (eds) Actin-binding proteins and dis-
ease. Springer, New York, pp 229277
98. Li A et al (2010) The actin-bundling protein fascin stabilizes actin in invadopodia and poten-
tiates protrusive invasion. Curr Biol 20(4):339345
99. Qualtrough D et al (2009) The actin-bundling protein fascin is overexpressed in colorectal
adenomas and promotes motility in adenoma cells in vitro. Br J Cancer 101(7):11241129
100. Chen L et al (2010) Migrastatin analogues target fascin to block tumour metastasis. Nature
464(7291):10621066
101. Hall A (2009) The cytoskeleton and cancer. Cancer Metastasis Rev 28(1-2):514
102. Bae YH et al (2009) Loss of profilin-1 expression enhances breast cancer cell motility by
Ena/VASP proteins. J Cell Physiol 219(2):354364
103. Hu LD et al (2008) EVL (Ena/VASP-like) expression is up-regulated in human breast cancer
and its relative expression level is correlated with clinical stages. Oncol Rep
19(4):10151020
104. Bear JE, Gertler FB (2009) Ena/VASP: towards resolving a pointed controversy at the barbed
end. J Cell Sci 122(pt 12):19471953
105. Hashimoto Y, Parsons M, Adams JC (2007) Dual actin-bundling and protein kinase C-binding
activities of fascin regulate carcinoma cell migration downstream of Rac and contribute to
metastasis. Mol Biol Cell 18(11):45914602
106. Vignjevic D et al (2006) Role of fascin in filopodial protrusion. J Cell Biol 174(6):863875
107. Schoumacher M et al (2010) Actin, microtubules, and vimentin intermediate filaments coop-
erate for elongation of invadopodia. J Cell Biol 189(3):541556
108. Yoder BJ et al (2005) The expression of fascin, an actin-bundling motility protein, correlates
with hormone receptor-negative breast cancer and a more aggressive clinical course. Clin
Cancer Res 11(1):186192
109. Hashimoto Y, Skacel M, Adams JC (2005) Roles of fascin in human carcinoma motility and
signaling: prospects for a novel biomarker? Int J Biochem Cell Biol 37(9):17871804
110. Chan C et al (2010) Fascin expression predicts survival after potentially curative resection of
node-positive colon cancer. Am J Surg Pathol 34(5):656666
111. Hashimoto Y et al (2004) The prognostic relevance of fascin expression in human gastric
carcinoma. Oncology 67(3-4):262270
112. Lee TK et al (2007) Fascin over-expression is associated with aggressiveness of oral squa-
mous cell carcinoma. Cancer Lett 254(2):308315
113. Li X et al (2008) Aberrant expression of cortactin and fascin are effective markers for patho-
genesis, invasion, metastasis and prognosis of gastric carcinomas. Int J Oncol 33(1):6979
114. Okada K et al (2007) Fascin expression is correlated with tumor progression of extrahepatic
bile duct cancer. Hepatogastroenterology 54(73):1721
115. Gertler FB et al (1996) Mena, a relative of VASP and Drosophila enabled, is implicated in the
control of microfilament dynamics. Cell 87(2):227239
116. Gertler F, Condeelis J (2011) Metastasis: tumor cells becoming MENAcing. Trends Cell Biol
21(2):8190
117. Gurzu S et al (2013) The possible role of Mena protein and its splicing-derived variants in
embryogenesis, carcinogenesis, and tumor invasion: a systematic review of the literature.
Biomed Res Int 2013:365192
Chapter 14
Zebrafish as a Model Animal for Studying
Lysophosphatidic Acid Signaling
Abbreviations
ATX Autotaxin
Edg Endothelial differentiation gene
hpf Hours post fertilization
LPA Lysophosphatidic acid
LPC Lysophosphatidylcholine
MO Morpholino antisense oligonucleotide
S1P Sphingosine-1-phosphate
14.1 Introduction
Zebrafish are widely used for studies of vertebrate gene function. Approximately
70 % of human genes have at least one obvious zebrafish orthologue [15]. The virtu-
ally transparent embryos of this species, and the ability to accelerate genetic studies
by gene knockdown or overexpression, have led to the widespread use of zebrafish
14 Zebrafish as a Model Animal for Studying Lysophosphatidic Acid Signaling 201
in the detailed investigation of vertebrate gene function and, increasingly, the study
of human genetic disease. Fluorescent markers can be used in vivo to tag specific
cell types and visualize their location and migration during embryogenesis.
Moreover, well-developed videomicroscopic techniques have been available for
detailed analyses of developmental stages. Analyses of vascular formation using
mutants and antisense morpholino oligos (MOs), for example, have identified a
number of molecules involved in vasculature development, including growth fac-
tors, cell adhesion molecules, and transcription factors [16]. These analyses have
shown that the basic mechanisms of embryonic blood vessel formation are con-
served in vertebrates. In addition to the traditional forward genetics, injection of
morpholino oligonucleotides allows us to target gene knockdown more rapidly [17].
Moreover, new genome-editing tools such as TALENs (transcription activator-like
effector nucleases) [18] and CRISPR (clustered regularly interspaced short palin-
dromic repeats)/Cas [19] have also been applied to the zebrafish model, providing
exciting new opportunities for high-efficiency mutagenesis.
Table 14.1 Amino acid sequence homology between zebrafish and mammalian (human and
mouse) lysophosphatidic acid (LPA)-related genes
Homo sapiens (human) Mus musculus (mouse)
Amino acid sequence Amino acid sequence
Zebrafish Genes homology (%) Genes homology (%)
atxa ATX 66.7 Atx 66.1
atxb 66.6 66.1
papla1aa PAPLA1a 49.3 Papla1a 47.3
papla1ab 48.7 47.9
lpa1 LPAR1 89.3 Lpar1 90.1
lpa2a LPAR2 49.5 Lpar2 50.8
lpa2b 55.3 55.6
lpa3 LPAR3 62.0 Lpar3 61.1
lpa4 LPAR4 63.7 Lpar4 64.2
lpa5a LPAR5 32.0 Lpar5 33.5
lpa5b 28.5 29.2
lpa6a LPAR6 63.0 Lpar6 63.6
lpa6b 55.5 56.4
lpp1a LPP1 69.2 Lpp1 65.5
lpp1b 62.5 60.2
lpp2a LPP2 63.0 Lpp2 67.0
lpp2b 69.0 68.2
lpp3a LPP3 73.2 Lpp3 71.3
lpp3b 47.8 47.3
Autotaxin (ATX)-null mice die around embryonic day 9.510.5 with profound vas-
cular defects in both the yolk sac and embryo, and with aberrant neural tube forma-
tion [10, 11, 21]. A number of mutants and knockout mice have shown phenotypes
similar to those in ATX-knockout mice [22, 23]. However, the precise phenotypes
of these mice have not been determined because real-time observation of blood ves-
sel formation is impossible for mice. Introduction of mutant ATX, in which Thr210,
an amino acid responsible for the catalytic activity of ATX, was replaced with ala-
nine, could not rescue the phenotype, indicating that the product of ATX, that is,
LPA, is involved in embryonic vascular formation [24]. In addition, none of the LPA
receptor knockout mice has shown a similar phenotype [13, 25, 26], and thus, it
remains to be solved which LPA receptors are involved and how ATX regulates
embryonic vasculature in the early developmental stages.
As stated earlier, zebrafish has two ATX orthologues, both of which have been
shown to have lysophospholipase D activity to produce LPA. To examine the devel-
opment of embryonic blood vessels called intersegmental vessels (ISVs) in zebrafish
14 Zebrafish as a Model Animal for Studying Lysophosphatidic Acid Signaling 203
A B
C D
Fig. 14.1 Phylogenetic relationships of lysophosphatidic acid (LPA)-related genes [ATX (a), LPP
(b), LPAR (c), and PAPLA1A (d)] based on analyses of individual genes from zebrafish, mouse,
and human. Trees were all inferred using GENETYC-MAC Ver. 13.1.1
embryos, a transgenic line was used in which endothelial cells were labeled with
EGFP [27]. Injection of embryos with ATX MO caused ISVs to stall in mid-course
and to aberrantly connect to neighboring ISVs. The aberrant vascular network in
ATX-downregulated embryos is not caused by abnormal proliferation of endothelial
cells, because endothelial cells are differentiated and the number of the cells was
normal. It should be stressed here that the zebrafish system makes it possible to
204 J. Aoki and H. Yukiura
precisely analyze blood vessel formation, which is very difficult in mice. Another
important point was that the ISV phenotype has not been reported so far, indicating
that the ATXLPA system was a novel axis that regulates the embryonic blood ves-
sel formation.
Knocking out LPA receptors in mice revealed the cellular processes specific to
each of six LPA receptors, from brain development (LPA1) to hair follicle formation
(LPA6). None of the individual knockouts was lethal. As already stated, ATX down-
regulation resulted in embryonic lethality and impaired blood vessel formation in
both mice and zebrafish. Thus, it is possible that multiple LPA receptors redun-
dantly regulate the embryonic blood vessel formation, or that novel LPA receptor(s)
are involved. To suppress multiple LPA genes at a time in mice by crossing mice in
which different LPA receptors are knocked out would require much time and labor.
However, injecting zebrafish with MOs makes it possible to suppress multiple genes
at a time. Simultaneous downregulation of multiple LPA receptors in zebrafish
embryos revealed that LPA receptors have a redundant function in embryonic blood
vessel formation. Downregulation of lpa1 and lpa4 caused abnormalities of blood
vessel formation similar to those caused by atx downregulation. The phenotypic
similarity strongly suggests that the LPA receptors and ATX act in the same axis
governing embryonic blood vessel formation.
Because zebrafish embryos with a partially established vascular system can develop
for 7 days, other roles of ATX were uncovered by gene knockdown experiments
using MOs. ATX is secreted by cells from the floor plate of the hindbrain and stimu-
lates olig2-positive progenitor cells to differentiate into oligodendrocyte progeni-
tors [28]. Dorsal forerunner cells (DFCs) regulate the formation of the central organ
for establishing L-R asymmetry in zebrafish, called Kupffers vesicle (KV). ATX
LPA3 receptor signaling was found to induce calcium fluxes in DFCs, indicating that
LPA is a regulator of L-R asymmetry in zebrafish embryos [29]. Our preliminary
data suggest that ATX is also involved in the development of cartilage as ATX
knockdown results in malformation of cartilage in zebrafish embryos.
Nakanaga et al. accidentally found that when ATX was overexpressed in zebrafish
embryos by injecting atx mRNA, the embryos showed cardia bifida, a phenotype
induced by downregulation of S1P signaling [30]. A similar cardiac phenotype was
not induced when catalytically inactive ATX was introduced. The cardiac pheno-
type was synergistically enhanced when MOs against S1P receptor (s1pr2/mil) or
S1P transporter (spns2) were introduced together with atx mRNA. The Atx-induced
cardia bifida was prominently suppressed when embryos were treated with an MO
14 Zebrafish as a Model Animal for Studying Lysophosphatidic Acid Signaling 205
against LPA1. Thus, the study provided the first in vivo evidence of crosstalk
between LPA and S1P signaling.
We have also tried to use the zebrafish system to evaluate small compounds for drug
development. When zebrafish embryos injected with ATX mRNA were treated with
an LPA receptor antagonist (Ki16425) (by just adding the compound to water in
96-well plates in which the embryos develop), it dramatically suppressed the cardia
bifida phenotype [30]. The LPA antagonist was found to be active against zebrafish
LPA receptors. However, our compounds that had ATX-inhibitory activity did not
inhibit zebrafish ATX. Interestingly, overexpression of mammalian ATX instead of
zebrafish ATX in zebrafish embryos induced a similar cardia bifida phenotype, and
the phenotypes were efficiently suppressed by some of our ATX inhibitors specific
for mammalian ATX. It should be noted that only a small fraction of such com-
pounds suppressed the phenotype, even though all the compounds efficiently sup-
pressed the ATX activity in a test tube. Such compounds were also found to be
effective in vivo in mice. Thus our preliminary trial indicated that the zebrafish
system is a powerful tool for in vivo evaluation of small compounds. Because the
evaluation can be performed in 96-well plates, only small amounts of compounds
are required, which makes it possible to evaluate the compounds in a chemical
library in a first or second screening.
References
1. Contos JJ, Fukushima N, Weiner JA, Kaushal D, Chun J (2000) Requirement for the lpA1
lysophosphatidic acid receptor gene in normal suckling behavior. Proc Natl Acad Sci U S A
97:1338413389
2. Ye X et al (2005) LPA3-mediated lysophosphatidic acid signalling in embryo implantation and
spacing. Nature 435:104108
3. Sumida H et al (2010) LPA4 regulates blood and lymphatic vessel formation during mouse
embryogenesis. Blood 116:50605070
4. Pasternack SM et al (2008) G protein-coupled receptor P2Y5 and its ligand LPA are involved
in maintenance of human hair growth. Nat Genet 40:329334
5. Tager AM et al (2008) The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to
lung injury by mediating fibroblast recruitment and vascular leak. Nat Med 14:4554
6. Lin S et al (2009) The absence of LPA2 attenuates tumor formation in an experimental model
of colitis-associated cancer. Gastroenterology 136:17111720
7. Lin S, Lee SJ, Shim H, Chun J, Yun CC (2010) The absence of LPA receptor 2 reduces the
tumorigenesis by ApcMin mutation in the intestine. Am J Physiol Gastrointest Liver Physiol
299:G1128G1138
8. Deng W et al (2002) Lysophosphatidic acid protects and rescues intestinal epithelial cells from
radiation- and chemotherapy-induced apoptosis. Gastroenterology 123:206216
9. Aoki J, Inoue A, Okudaira S (2008) Two pathways for lysophosphatidic acid production.
Biochim Biophys Acta 1781:513518
206 J. Aoki and H. Yukiura
10. Tanaka M et al (2006) Autotaxin stabilizes blood vessels and is required for embryonic vascu-
lature by producing lysophosphatidic acid. J Biol Chem 281:2582225830
11. van Meeteren LA et al (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood
vessel formation during development. Mol Cell Biol 26:50155022
12. Kazantseva A et al (2006) Human hair growth deficiency is linked to a genetic defect in the
phospholipase gene LIPH. Science 314:982985
13. Inoue A et al (2011) LPA-producing enzyme PA-PLA(1)alpha regulates hair follicle develop-
ment by modulating EGFR signalling. EMBO J 30:42484260
14. Brindley DN, Pilquil C (2009) Lipid phosphate phosphatases and signaling. J Lipid Res
50(suppl):S225S230
15. Howe K et al (2013) The zebrafish reference genome sequence and its relationship to the
human genome. Nature 496:498503
16. Ellertsdottir E et al (2010) Vascular morphogenesis in the zebrafish embryo. Dev Biol
341:5665
17. Corey DR, Abrams JM (2001) Morpholino antisense oligonucleotides: tools for investigating
vertebrate development. Genome Biol 2: REVIEWS1015
18. Bedell VM et al (2012) In vivo genome editing using a high-efficiency TALEN system. Nature
491:114118
19. Hwang WY et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system.
Nat Biotechnol 31:227229
20. Yukiura H et al (2011) Autotaxin regulates vascular development via multiple lysophospha-
tidic acid (LPA) receptors in zebrafish. J Biol Chem 286:4397243983
21. Fotopoulou S et al (2010) ATX expression and LPA signalling are vital for the development of
the nervous system. Dev Biol 339:451464
22. Ruppel KM et al (2005) Essential role for Galpha13 in endothelial cells during embryonic
development. Proc Natl Acad Sci U S A 102:82818286
23. Kamijo H et al (2011) Impaired vascular remodeling in the yolk sac of embryos deficient in
ROCK-I and ROCK-II. Genes Cells 16:10121021
24. Ferry G et al (2007) Functional invalidation of the autotaxin gene by a single amino acid muta-
tion in mouse is lethal. FEBS Lett 581:35723578
25. Yang AH, Ishii I, Chun J (2002) In vivo roles of lysophospholipid receptors revealed by gene
targeting studies in mice. Biochim Biophys Acta 1582:197203
26. Lin ME, Rivera RR, Chun J (2012) Targeted deletion of LPA5 identifies novel roles for lyso-
phosphatidic acid signaling in development of neuropathic pain. J Biol Chem
287:1760817617
27. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using
transgenic zebrafish. Dev Biol 248:307318
28. Yuelling LW, Waggener CT, Afshari FS, Lister JA, Fuss B (2012) Autotaxin/ENPP2 regulates
oligodendrocyte differentiation in vivo in the developing zebrafish hindbrain. Glia
60:16051618
29. Lai SL et al (2012) Autotaxin/Lpar3 signaling regulates Kupffers vesicle formation and left-
right asymmetry in zebrafish. Development 139:44394448
30. Nakanaga K et al (2014) Overexpression of autotaxin, a lysophosphatidic acid-producing
enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebraf-
ish embryo. J Biochem 155:235241
Chapter 15
Sphingosine 1-Phosphate Signaling via
Transporters in Zebrafish and Mice
Abstract The bioactive lipid mediator sphingosine 1-phosphate (S1P) plays a piv-
otal role in various cellular functions, such as proliferation, migration, and differen-
tiation. S1P is intracellularly produced by sphingosine kinases and is released from
the cells. Subsequently, the secreted S1P associates with S1P receptors (S1PRs) on
a target cell surface, causing activation of downstream signaling pathways. The
zebrash (Danio rerio) is widely used as a vertebrate model organism to study the
processes of organogenesis and morphogenesis. Spns2 was originally identied as
an S1P transporter in zebrash; Spns2 regulates the migration of cardiac progeni-
tors via the S1PR2 receptor. Murine and human SPNS2 can also transport S1P from
the cells. In mice, SPNS2 enables transport of S1P from vascular endothelial cells
into the plasma and regulates lymphocyte egress from lymphoid organs. Recent
remarkable developments in genome-editing technologies, such as transcription
activator-like effector nucleases (TALENs) and the clustered regularly interspaced
short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 system, allow
researchers to introduce genomic modications in various model animals. In this
chapter, we review not only the physiological roles of S1P transporters in mammals
and zebrash but also the strategy for generating S1PR-knockout zebrash using
genome-editing technologies.
Y. Hisano (*)
Laboratory for Developmental Gene Regulation, Brain Science Institute,
Riken, Wako, Saitama 351-0198, Japan
e-mail: [email protected]
T. Nishi
Department of Cell Membrane Biology, Institute of Scientic and Industrial Research, Osaka
University, Ibaraki, Osaka 567-0047, Japan
Faculty of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871, Japan
A. Kawahara (*)
Laboratory for Developmental Biology, Center for Medical Education and Sciences,
Graduate School of Medical Science, University of Yamanashi,
Chimogatou 1110, Chuo, Yamanashi 409-3898, Japan
e-mail: [email protected]
15.1 Introduction
C17-S1P
S1P
0 4 8 12
Time [hr]
c d e
90
from erythrocytes [%]
S1P release
45
1 5
WT
KO
0
0 6 12 N.D.
0 0
Time [min] WT KO WT KO
Fig. 15.1 (a) Chinese hamster ovary (CHO) cells expressing mouse SphK1 and human SPNS2
were incubated for 2, 6, or 12 h. The amounts of released S1P were measured using high-
performance liquid chromatography (HPLC). (b) CHO/SphK1 cells (mock) or CHO/SphK1 cells
expressing mouse ABCA1, human ABCB1, mouse ABCC1, human ABCG2, or human SPNS2
were incubated for 2 h after sphingosine stimulation (5 M). Amounts of released S1P were mea-
sured using HPLC. C17-S1P served as an internal standard [27]. (c) Erythrocytes from wild-type
(WT) (squares) or Spns2-KO mice (circles) were incubated with [3H]sphingosine for 0, 4, or
10 min and the [3H]S1P released into the medium was quantied. (d) Platelets from WT or
Spns2-KO mice were incubated for 10 min in the presence of thrombin, and the S1P released into
the medium was measured using ultraperformance liquid chromatography with tandem mass spec-
trometry (UPLCMS/MS). (e) Vascular endothelial cells from the aorta of WT or Spns2-KO mice
were incubated for 4 h and the S1P released into the medium was measured using UPLCMS/
MS. In Spns2-KO endothelial cells, S1P in the medium was not detected (N.D.) [20]
several types of ABC transporters that utilize ATP hydrolysis as an energy source.
ABCA1, ABCB1, ABCC1, and ABCG2 were independently reported to be involved
in S1P release from several cell lines [2225]. Furthermore, S1P release from plate-
lets and erythrocytes is ATP dependent and is inhibited by glyburide (an ABCA1
transporter inhibitor) [11, 26]. As already described, SPNS2 functions physiologi-
cally as an S1P transporter [21] and belongs to the major facilitator superfamily
(MFS) but not the ABC transporter superfamily. In Chinese hamster ovary (CHO)
cells expressing SphK1, S1P is effectively synthesized and accumulated because of
the lack of an S1P export system. The introduction of SPNS2 into these cells induces
S1P release (Fig. 15.1). In contrast, introduction of ABCA1, ABCB1, ABCC1, or
ABCG2 does not stimulate the release of S1P, indicating that SPNS2 is an exclusive
S1P transporter capable of releasing S1P from the cells by itself (Fig. 15.1) [27]. If
210 Y. Hisano et al.
these ABC transporters are indeed involved in S1P release, they may require some
type of modication or cofactor(s) for successful transport of S1P.
Consistent with the results from the cell culture system, Spns2-knockout (KO)
mice exhibit a decreased plasma S1P level, at approximately 60 % of the level in
wild-type (WT) mice, whereas the plasma S1P level of Abca1-KO or Abcc1-KO
mice is not altered [14, 20, 28]. These data indicate that SPNS2 enables transport of
S1P from S1P-producing cells into the plasma. One important question is which cell
types carry SPNS2 that contributes to plasma S1P. We isolated platelets, erythro-
cytes, and endothelial cells from Spns2-KO mice and separately measured the
release of S1P by these cells. S1P release from Spns2-KO endothelial cells was
completely abolished, whereas this activity was not altered in Spns2-KO platelets
and erythrocytes (Fig. 15.1) [20]. In line with this observation, a knockdown of
SPNS2 by siRNA suppressed the S1P secretion in human umbilical vein endothelial
cells (HUVECs) and human pulmonary artery endothelial cells (HPAECs) [20]. In
fact, SPNS2 expression is detectable in the heart and lung endothelial cells by
means of an antibody [29].
The most remarkable feature of Spns2-KO mice is the depletion of peripheral
blood of circulating lymphocytes [20, 2831]. S1P signaling via S1PR1 regulates
the lymphocyte egress from lymphoid organs, such as the thymus, bone marrow,
and lymph nodes, into peripheral blood and lymph. Precursors from the bone mar-
row migrate to the thymus and mature into T cells in the medullary portion. S1PR1
expression is upregulated in the mature T cells, enabling them to exit from the thy-
mus [32]. S1P is believed to be a key regulator of the egress of mature T cells via
the activation of S1PR1. In the thymus of Spns2-KO mice, the population of mature
T cells is increased and that of immature T cells is decreased [20]. Mature T cells
isolated from Spns2-KO mice exhibit higher S1pr1 mRNA expression and stronger
migration mediated by S1P compared with WT cells [20]. These results suggest that
thymocytes of Spns2-KO mice can normally mature and migrate toward S1P, but
they cannot move from the thymus into the blood because of the lack of an SPNS2-
mediated S1P transport system (Fig. 15.2). Consequently, the number of circulating
T cells in the peripheral blood of Spns2-KO mice is signicantly reduced. Thus,
SPNS2 could be a novel target of immunosuppressive agents because SPNS2 par-
ticipates in the regulation of the number of circulating T cells.
Zebrash are known as small tropical sh (45 cm long) native to India. A zebrash
embryo is transparent and its development progresses rapidly. Most of the organs
start to function within several days after fertilization. One important point is that
organogenesis and morphogenesis are well conserved between zebrash and mam-
mals; therefore, zebrash is a convenient vertebrate model organism. A pair of
zebrash produces approximately 100200 eggs, allowing researchers to perform
genetic analysis such as screening of zebrash mutants produced by random muta-
genesis. In the forward genetic analysis, s1pr2 was identied as a gene responsible
15 Sphingosine 1-Phosphate Signaling via Transporters in Zebrash and Mice 211
Mice Zebrafish
Thymus
Spns2
S1PR1 S1PR2
myocardial
precursors
SPNS2
S1P
Fig. 15.2 Physiological roles of SPNS2. In mice, SPNS2 supplies S1P from vascular endothelial
cells to plasma, and regulates lymphocyte egress from lymphoid organs, such as the thymus and
bone marrow. S1P released by SPNS2 is believed to be recognized by S1PR1 on the surface of
lymphocytes. In zebrash, S1P released by Spns2 regulates cardiac progenitor migration by
S1PR2-mediated signaling. spns2 is strongly expressed in the yolk syncytial layer (YSL) [21]
for the miles apart mutation resulting in two hearts (cardia bifida) and tail blisters
[33]. In vertebrates, cardiac progenitors are bilaterally located in the lateral plate
mesoderm and migrate toward the midline where they merge to form a single primi-
tive heart tube. Although s1pr2 is expressed in the endoderm and cardiomyocytes,
s1pr2 mutants exhibit morphological defects in the anterior endoderm, which is
necessary for cardiac progenitor migration [33]. S1PR2 is believed to be a partner
of G12/13, Gi, and Gq heterotrimeric G protein that activates their downstream signal-
ing. In zebrash, G13 was shown to be downstream of S1PR2 and to regulate migra-
tion of cardiac progenitors via a RhoGEF-dependent pathway [34]. Transplantation
of endoderm decient in either S1PR2 or G13 into WT embryos results in the devel-
opment of two hearts; conversely, WT donor cells reverse the defects of a G13
knockdown [34]. These results suggest that activation of S1PR2 signaling in the
endoderm is indispensable for the migration of cardiac progenitors.
Recently, Stainiers group and ours independently identied other cardia bifida
mutants (two of hearts and ko157), which have two hearts and tail blisters identical
to those of the s1pr2 mutant (Fig. 15.3) [21, 35]. Both groups reported that the gene
responsible for the mutations encodes a novel membrane protein Spns2. We demon-
strated that Spns2 functions as an S1P transporter, as already mentioned. spns2
transcripts are easily detectable in an extraembryonic tissue called the yolk syncy-
tial layer (YSL), which is located immediately underneath the migrating cardiac
progenitors [21]. A knockdown of spns2 by antisense morpholino oligonucleotides
(MOs) in the YSL at the shield stage induced cardia bifida; the defects of the spns2
mutant were reversed by injection of spns2 mRNA into the YSL at the shield stage
[21]. These data suggest that Spns2 in the YSL is essential for the migration of car-
diac progenitors (Fig. 15.2). Nevertheless, it is still unclear how S1P supplied from
YSL is detected by S1PR2 in the endoderm or mesoderm.
212 Y. Hisano et al.
Fig. 15.3 Each spns2-mutant or s1pr2-KO embryo had two hearts (cardia bifida) and tail blisters.
The positions of the hearts in wild-type, spns2-mutant, and s1pr2-KO embryo at 1 day post fertil-
ization (1 dpf) are indicated by white arrowheads. Cardiac morphology (ventral view) was visual-
ized using monomeric red uorescent protein (mRFP) expression driven by the cardiac-specic
promoter of cmlc2 (gene of cardiac myosin light chain 2). Each spns2-mutant or s1pr2-KO embryo
exhibited tail blisters at 2 dpf (lateral view)
S1P signaling via Spns2 and S1PR2 was reported to be involved in lower-jaw
development [36]. S1PR2 in the endoderm is essential for lower-jaw development
just as is S1PR2 for the migration of cardiac progenitors; however, the origin of S1P
as supplied by Spns2 is unclear. We found that the Spns2S1PR2 signaling pathway
interacts with the cell adhesion molecule bronectin during both cardiac and lower-
jaw development [37]. Furthermore, expression of endothelin receptor A is down-
regulated in the spns2 mutant [37], suggesting that Spns2S1PR2 signaling affects
endothelin signaling involved in lower-jaw development. In situ hybridization anal-
ysis revealed that spns2 mRNA is expressed in somites, myocardial precursors, and
at the tip of the tail in addition to the YSL; these results indicate that Spns2 may
participate in regulation of other types of organogenesis [21].
During the past few years, tremendous innovations took place in the eld of genome-
editing technologies, such as zinc-nger nucleases (ZFNs), transcription activator-
like effector nucleases (TALENs), and the clustered regularly interspaced short
15 Sphingosine 1-Phosphate Signaling via Transporters in Zebrash and Mice 213
15.5 Conclusion
15.6 Methods
Platelets and erythrocytes were isolated from whole blood, which was collected
from mouse hearts using an acid citratedextrose solution as an anticoagulant.
Platelet-rich plasma (PRP) was separated by centrifugation at 100 g for 15 min at
room temperature. Platelets were prepared by centrifugation of PRP at 1000 g for
15 min at room temperature and were washed with buffer A (20 mM HEPES-NaOH
pH 7.4, 3.3 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 138 mM NaCl, and 1 mg/ml
glucose) containing 1 % bovine serum albumin (BSA). In the case of erythrocytes,
whole blood was centrifuged at 100 g for 15 min at room temperature and the pel-
leted erythrocytes were washed twice with buffer A containing 1 % BSA.
Endothelial cells were isolated from the mouse aorta, according to the previously
reported method with some modications [46]. The mouse aorta was cut at the
lower level of the abdominal aorta to release the blood and was perfused with
phosphate-buffered saline (PBS) containing heparin (1000 units/ml). Subsequently,
the aorta was excised between the aortic arch and the abdominal aorta and immersed
in 20 % fetal bovine serum (FBS)Dulbeccos modied Eagles medium (DMEM)
containing 100 units/ml heparin, 100 units/ml penicillin G, and 100 g/ml strepto-
mycin. A 24-gauge cannula was inserted into the proximal portion and the distal end
was closed with a silk thread. Subsequently, the aorta was briey washed with
serum-free DMEM and lled with a solution of type II collagenase (2 mg/ml). After
incubation for 45 min at 37 C, endothelial cells were removed from the aorta by
ushing it with 2 ml 20 % FBSDMEM. The collected endothelial cells were incu-
bated with 2 ml 20 % FBSDMEM and cultured in a plate coated with type I col-
lagen. After incubation for 2 h at 37 C, the cells were washed with warmed 20 %
FBSDMEM to remove smooth muscle cells, and cultured in the G medium [20 %
FBS, 100 units/ml penicillin G, 100 g/ml streptomycin, 2 mM l-glutamine, 1
nonessential amino acids, 1 sodium pyruvate, 25 mM HEPES pH 7.07.6, 100 g/ml
heparin, 100 g/l endothelial cell growth supplement (ECGS), and DMEM] until
conuent state.
15 Sphingosine 1-Phosphate Signaling via Transporters in Zebrash and Mice 215
The strategy for generating KO zebrash is shown in Fig. 15.4. First, genomic DNA
was isolated from 1 dpf (day post fertilization) embryos injected with TALENs or
gRNA plus Cas9. The genome-editing activity was evaluated via generation of het-
eroduplex bands in addition to homoduplex bands during polyacrylamide gel elec-
trophoresis (heteroduplex mobility assay, HMA). Next, potential F0 founders were
grown to adulthood and mated with WT zebrash. The germline transmission of
indels was conrmed by the formation of heteroduplex bands. F1 zebrash contain-
ing a mutant allele were identied by genotyping of genomic DNA prepared from
a X b target site
genome
WT WT
TALEN mRNA
or
gRNA + Cas9 mRNA PCR amplification
X
Heteroduplex
F0 founder WT
(2) Identification of
potential F0 founders Homoduplex
Fig. 15.4 (a) Strategy for preparation of KO zebrash using genome-editing technologies. (1)
Zebrash genomic DNA samples were prepared from 1 day post fertilization (dpf) embryos
injected with TALEN mRNA or gRNA plus Cas9 mRNA. Genome-editing activity was evaluated
as generation of insertion and/or deletion (indel) mutations in a heteroduplex mobility assay
(HMA). (2) Potential F0 founders that were capable of producing indel mutations were grown to
adulthood. We conrmed the germline transmission of indel mutations in F1 embryos by mating
the F0 founders with wild-type (WT) sh. (3) F1 zebrash containing mutant alleles were identied
by genotyping of genomic DNA samples prepared from n clips. At these three steps, we used
HMA to detect indel mutations induced by TALENs or gRNA plus Cas9. (b) The principle of
HMA. A genomic target region was amplied by means of PCR with locus-specic primers.
During polyacrylamide gel electrophoresis, homoduplexes are separated by molecular weight,
whereas heteroduplexes containing a mismatched region migrate more slowly than the homodu-
plexes because of the opened single-strand conguration. (1) PCR products from a WT genome
contain one homoduplex band of the expected size. (2) Among the PCR products from F0 embryos
injected with TALENs or gRNA plus Cas9, ladder bands (heteroduplex bands) appeared above the
homoduplex band because of various types of indel mutations in somatic cells. (3) When F0
founder containing a deletion allele is mated with WT zebrash, two homoduplex bands and two
heteroduplex bands among PCR products should appear in F1 embryos
15 Sphingosine 1-Phosphate Signaling via Transporters in Zebrash and Mice 217
Acknowledgements We thank Drs. S. Ota, N. Kobayashi, and A. Yamaguchi for valuable discus-
sion. This work was supported by the Funding Program for Next Generation World-Leading
Researchers (NEXT Program) and by the Japan Society for the Promotion of Science (JSPS).
References
1. Wymann MP, Schneiter R (2008) Lipid signalling in disease. Nat Rev Mol Cell Biol 9(2):162
176. doi:10.1038/nrm2335
2. Brinkmann V (2007) Sphingosine 1-phosphate receptors in health and disease: mechanistic
insights from gene deletion studies and reverse pharmacology. Pharmacol Ther 115(1):84
105. doi:10.1016/j.pharmthera.2007.04.006
3. Hisano Y, Nishi T, Kawahara A (2012) The functional roles of S1P in immunity. J Biochem
152(4):305311. doi:10.1093/jb/mvs090
4. Strub GM, Maceyka M, Hait NC, Milstien S, Spiegel S (2010) Extracellular and intracellular
actions of sphingosine-1-phosphate. Adv Exp Med Biol 688:141155.
doi:10.1007/978-1-4419-6741-1_10
5. Olivera A, Spiegel S (1993) Sphingosine-1-phosphate as second messenger in cell prolifera-
tion induced by PDGF and FCS mitogens. Nature 365(6446):557560. doi:10.1038/365557a0
6. Kihara A, Mitsutake S, Mizutani Y, Igarashi Y (2007) Metabolism and biological functions of
two phosphorylated sphingolipids, sphingosine 1-phosphate and ceramide 1-phosphate. Prog
Lipid Res 46(2):126144. doi:10.1016/j.plipres.2007.03.001
7. Chae SS, Proia RL, Hla T (2004) Constitutive expression of the S1P1 receptor in adult tissues.
Prostaglandins Other Lipid Mediat 73(1-2):141150. doi:10.1016/j.
prostaglandins.2004.01.006
8. Ishii I, Friedman B, Ye X, Kawamura S, McGiffert C, Contos JJ, Kingsbury MA, Zhang G,
Brown JH, Chun J (2001) Selective loss of sphingosine 1-phosphate signaling with no obvious
phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3. J Biol
Chem 276(36):3369733704. doi:10.1074/jbc.M10441200
9. Yatomi Y, Ruan F, Hakomori S, Igarashi Y (1995) Sphingosine-1-phosphate: a platelet-
activating sphingolipid released from agonist-stimulated human platelets. Blood
86(1):193202
218 Y. Hisano et al.
10. Herzog BH, Fu J, Wilson SJ, Hess PR, Sen A, McDaniel JM, Pan Y, Sheng M, Yago T, Silasi-
Mansat R, McGee S, May F, Nieswandt B, Morris AJ, Lupu F, Coughlin SR, McEver RP, Chen
H, Kahn ML, Xia L (2013) Podoplanin maintains high endothelial venule integrity by interact-
ing with platelet CLEC-2. Nature 502(7469):105109. doi:10.1038/nature12501
11. Kobayashi N, Nishi T, Hirata T, Kihara A, Sano T, Igarashi Y, Yamaguchi A (2006) Sphingosine
1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner. J Lipid
Res 47(3):614621. doi:10.1194/jlr.M500468-JLR200
12. Hanel P, Andreani P, Graler MH (2007) Erythrocytes store and release sphingosine 1-phosphate
in blood. FASEB J 21(4):12021209. doi:10.1096/fj.06-7433com
13. Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T (1999) The balance between
sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon
receptor I triggering. J Exp Med 190(1):18. doi:10.1084/jem.190.1.1
14. Lee Y-M, Venkataraman K, Hwang S-I, Han DK, Hla T (2007) A novel method to quantify
sphingosine 1-phosphate by immobilized metal afnity chromatography (IMAC).
Prostaglandins Other Lipid Mediat 84(3-4):154162. doi:10.1016/j.
prostaglandins.2007.08.001
15. Bassi R, Anelli V, Giussani P, Tettamanti G, Viani P, Riboni L (2006) Sphingosine-1-phosphate
is released by cerebellar astrocytes in response to bFGF and induces astrocyte proliferation
through Gi-protein-coupled receptors. Glia 53(6):621630. doi:10.1002/glia.20324
16. Anelli V, Bassi R, Tettamanti G, Viani P, Riboni L (2005) Extracellular release of newly syn-
thesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes. J Neurochem
92(5):12041215. doi:10.1111/j.1471-4159.2004.02955.x
17. Nieuwenhuis B, Luth A, Chun J, Huwiler A, Pfeilschifter J, Schafer-Korting M, Kleuser B
(2009) Involvement of the ABC-transporter ABCC1 and the sphingosine 1-phosphate receptor
subtype S1P(3) in the cytoprotection of human broblasts by the glucocorticoid dexametha-
sone. J Mol Med 87(6):645657. doi:10.1007/s00109-009-0468-x
18. Tann Z, Serrano-Sanchez M, Leiber D (2011) ATP-binding cassette ABCC1 is involved in
the release of sphingosine 1-phosphate from rat uterine leiomyoma ELT3 cells and late preg-
nant rat myometrium. Cell Signal 23(12):19972004. doi:10.1016/j.cellsig.2011.07.010
19. Pappu R, Schwab SR, Cornelissen I, Pereira JP, Regard JB, Xu Y, Camerer E, Zheng Y-W,
Huang Y, Cyster JG, Coughlin SR (2007) Promotion of lymphocyte egress into blood and
lymph by distinct sources of sphingosine-1-phosphate. Science 316(5822):295298.
doi:10.1126/science.1139221
20. Hisano Y, Kobayashi N, Yamaguchi A, Nishi T (2012) Mouse SPNS2 functions as a
sphingosine-1-phosphate transporter in vascular endothelial cells. PLoS One 7(6), e38941.
doi:10.1371/journal.pone.0038941
21. Kawahara A, Nishi T, Hisano Y, Fukui H, Yamaguchi A, Mochizuki N (2009) The sphingolipid
transporter spns2 functions in migration of zebrash myocardial precursors. Science
323(5913):524527. doi:10.1126/science.1167449
22. Sato K, Malchinkhuu E, Horiuchi Y, Mogi C, Tomura H, Tosaka M, Yoshimoto Y, Kuwabara
A, Okajima F (2007) Critical role of ABCA1 transporter in sphingosine 1-phosphate release
from astrocytes. J Neurochem 103(6):26102619. doi:10.1111/j.1471-4159.2007.04958.x
23. Honig SM, Fu S, Mao X, Yopp A, Gunn MD, Randolph GJ, Bromberg JS (2003) FTY720
stimulates multidrug transporter- and cysteinyl leukotriene-dependent T cell chemotaxis to
lymph nodes. J Clin Invest 111(5):627637. doi:10.1172/JCI16200
24. Mitra P, Oskeritzian CA, Payne SG, Beaven MA, Milstien S, Spiegel S (2006) Role of
ABCC1 in export of sphingosine-1-phosphate from mast cells. Proc Natl Acad Sci U S A
103(44):1639416399. doi:10.1073/pnas.0603734103
25. Takabe K, Kim RH, Allegood JC, Mitra P, Ramachandran S, Nagahashi M, Harikumar KB,
Hait NC, Milstien S, Spiegel S (2010) Estradiol induces export of sphingosine-1-phosphate
from breast cancer cells via ABCC1 and ABCG2. J Biol Chem 285(14):1047710486.
doi:10.1074/jbc.M109.064162
15 Sphingosine 1-Phosphate Signaling via Transporters in Zebrash and Mice 219
42. Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC (2007) p53
activation by knockdown technologies. PLoS Genet 3(5), e78. doi:10.1371/journal.
pgen.0030078
43. Ben Shoham A, Malkinson G, Krief S, Shwartz Y, Ely Y, Ferrara N, Yaniv K, Zelzer E (2012)
S1P1 inhibits sprouting angiogenesis during vascular development. Development. doi:10.1242/
dev.078550
44. Gaengel K, Niaudet C, Hagikura K, Lavina B, Muhl L, Hofmann JJ, Ebarasi L, Nystrom S,
Rymo S, Chen LL, Pang MF, Jin Y, Raschperger E, Roswall P, Schulte D, Benedito R, Larsson
J, Hellstrom M, Fuxe J, Uhlen P, Adams R, Jakobsson L, Majumdar A, Vestweber D, Uv A,
Betsholtz C (2012) The sphingosine-1-phosphate receptor S1PR1 restricts sprouting angio-
genesis by regulating the interplay between VE-cadherin and VEGFR2. Dev Cell 23(3):587
599. doi:10.1016/j.devcel.2012.08.005
45. Mendelson K, Zygmunt T, Torres-Vazquez J, Evans T, Hla T (2013) Sphingosine 1-phosphate
receptor signaling regulates proper embryonic vascular patterning. J Biol Chem 288(4):2143
2156. doi:10.1074/jbc.M112.427344
46. Kobayashi M, Inoue K, Warabi E, Minami T, Kodama T (2005) A simple method of isolating
mouse aortic endothelial cells. J Atheroscler Thromb 12(3):138142. doi:10.5551/jat.12.138
47. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purication. Can J
Physiol Pharmacol 37(8):911917. doi:10.1139/o59-099
Part III
Lipid Mediators and Diseases
Chapter 16
Lipid Mediator LPA-Induced Demyelination
and Self-Amplification of LPA Biosynthesis
in Chronic Pain Memory Mechanisms
Abstract Chronic pain is considered to have a memory process because of its long-
lasting nature even after the original cause such as nerve injury is resolved. This type
contrasts to the cases with acute pain, nociceptive or inflammatory pain, which van-
ishes without delay after the cessation of stimulation or inhibition of the original
inflammation. Lysophosphatidic acid (LPA) was identified to be a key initiator of neu-
ropathic pain, one of the representative types of chronic pain, via activation of multiple
machineries. Recent studies revealed that LPA induces LPA biosynthesis through
actions of microglia and interleukin-1. LPA1 and LPA3 receptor-mediated mecha-
nisms are involved in this self-amplification of LPA production. Neuropathic pain is
characterized as unique abnormal pain allodynia, in which gentle touch causes intense
pain. The functional switch in allodynia is reasonably explained by demyelination,
whose underlying mechanisms are also explained as downstream machineries of LPA
and its LPA1 receptor signaling. The conversion of tactile to intense pain caused by
demyelination may be involved in the long-lasting feed-forward machineries in neuro-
pathic pain. Recent reports describe the importance of endocannabinoids and new ara-
chidonic acid metabolites in the regulation of chronic pain. This chapter also describes
the possible relationships of LPA to these additional regulatory mechanisms.
Abbreviations
ATX autotaxin
BDNF brain-derived neurotrophic factor
CFA complete Freunds adjuvant
16.1 Introduction
Pain is classified into two groups, acute pain and chronic pain. Acute pain is further
classified into nociceptive pain and inflammatory pain. Nociceptive pain occurs
through an activation of unmyelinated C-fibers or myelinated A-fibers upon ther-
mal, mechanical, or chemical stimuli, whereas inflammatory pain occurs mainly
through C-fibers, which are activated by inflammatory mediators in response to
local inflammation. Both types of pain are caused by generation of action potentials
initiated through an activation of specific ion channels or receptors on nociceptive
endings [1]. The action potential later elicits a release of pain transmitters, such as
substance P (SP) or glutamate, and then causes excitatory postsynaptic potentials in
the superficial (lamina I and II) dorsal horn neurons of the spinal cord, whose fibers
ultimately cross the spinal cord and relay the signal to the contralateral side of thala-
mus and cerebral cortex through secondary synapse. Usually, this type of pain sen-
sation is transient and is abolished by the removal of the noxious signal with
nonsteroidal anti-inflammatory drugs (NSAIDs) or the opioid-induced suppression
of primary pain signals by driving the descending pain-inhibitory system through
noradrenergic or serotonergic neurons [2]. Innocuous or tactile perception occurs
through an activation of sensory organs (Merkel cells, Pacinian corpuscles, and hair
follicles) and associated myelinated A-fibers, which mostly innervate to the ipsi-
lateral side of medulla oblongata neurons, although some A-fibers innervate to the
deeper (lamina IV) dorsal horn cells, as do noxious fibers.
In chronic pain such as the neuropathic pain paradigm, on the other hand, the
damage to peripheral or central neurons in the pain pathway from the nociceptor
endings in the skin or internal organs to somatosensory cortex produces a contingent
16 Lipid Mediator LPA-Induced Demyelination and Self-Amplification of LPA 225
One of the anatomical and histochemical events in mice subjected to different types
of neuropathic pain model is demyelination [9], which occurs specifically in dorsal
root fibers following sciatic nerve injury [11]. Pharmacological and biochemical
analyses suggested that most positive actions, such as calcium channel Cav21
upregulation and sensory fiber-specific hyperalgesia, are closely related to myelin-
ated A-fibers [5]. Based on these findings, we successfully revealed that LPA is the
chemical signature in initiation of neuropathic pain including demyelination [46,
10, 12, 13]. Further confirmation has been obtained through neuropathic-like behav-
ior and focal demyelination in animals after intrathecal and intratrigeminal injec-
tions of LPA [1315].
Significant rapid demyelination of dorsal root fibers by partial sciatic nerve ligation
or intrathecal injection of LPA was abolished in Lpar1/ mice [11, 13]. LPA induced
downregulation of myelin-associated glycoprotein (MAG), and it was selectively
inhibited by calpain inhibitors [14]. MAG is restricted to the innermost membrane
facing the neuronal axon, and one of the MAG receptors is Nogo-66 receptor-1,
which is mostly found in a tripartite complex composed of transmembrane co-
receptors LINGO-1 and either p75NTR or TROY [20, 21]. As this postreceptor signal
uses RhoA-Rho-kinase (ROCK) signaling that inhibits tubulin and actin assembly
[22], the loss of MAG is supposed to lead to a sprouting of sensory A-fibers, thereby
possibly resulting in a formation of abnormal pain synapses in the spinal cord.
Interestingly, nerve injury-induced calpain activation in the dorsal root was abol-
ished in Lpar1/ mice, although inflammatogenic complete Freunds adjuvant
(CFA) did not induce calpain activation [14]. These findings were supported by
pharmacological studies in which pretreatment with the calpain inhibitor E-64d
[(2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester] or calpain
inhibitor X (Z-Leu-Abu-CONH-ethyl) abolished nerve injury-induced neuropathic
pain, but not CFA-induced pain [14]. Thus, it is suggested that the rapid downregu-
lation of myelin protein is caused by activated calpain following an elevation of
[Ca2+]i, possibly through an activation of LPA1 receptor-Gq/11-phospholipase C and
its subsequent production of inositol 1,4,5-trisphosphate.
LPA
LPA1-R Schwann cell
G12/13 Gq/11
PLC
Rho/ROCK
p IP3
IKK p MKK4
p
IB NF-B p300 JNK Ca2+
p300 Proteolysis
H3K9MT ac-NF-B Sox10 Egr2 Myelin proteins
Demyelination
Although LPA is able to cause global demyelination in the dorsal root, spinal nerve,
and sciatic nerve in ex vivo experiments, nerve injury-induced and LPA1 receptor-
dependent demyelination occurs specifically in the dorsal root remote from the injured
sciatic nerve [11]. This fact suggests that the amount of LPA required for causing
dorsal root demyelination after nerve injury may come from the spinal cord [11, 37].
NMDA alone did not [38]. This fact suggests that an intense pain signal is required
for LPA production. Recent studies have revealed that phosphatidylcholine is con-
verted to LPC by cytosolic phospholipase A2 (cPLA2) or calcium-independent PLA2
(iPLA2), both of which are regulated by Ca2+-related mechanisms. cPLA2 is acti-
vated by Ca2+ or phosphorylation by MAPK or protein kinase Cs [3941], whereas
iPLA2 is activated by the calcium influx factor, which is produced following Ca2+
depletion in the endoplasmic reticulum [42, 43]. Therefore, the activation of both
cPLA2 and iPLA2, which are predominantly expressed in neurons [4446], may be
induced by intense pain signals (SP and glutamate) after nerve injury. Using phar-
macological studies with cPLA2 or iPLA2 inhibitor, this view was confirmed by
recent studies that abolished neuropathic pain and LPA production [44, 47].
Cav2-1
DRG
EphrinB1 Primary afferent
Brain
(thalamus)
Demyelination SP/NK1
Glu/NMDA-R
GABAergic
Secondary spinal neuron
neuron
c/i PLA2
KCC2 ATX
LPA3
LPA
LPA1/3
Cytokines
Astrocyte
Our next question is what is the major machinery to cause exogenous LPA- or
endogenously amplified LPA-induced hyperalgesia. Regarding this issue, we are
also speculating on the involvement of LPA-stimulated microglia. In our previous
study, LPA caused an increase in the expression of brain-derived neurotrophic factor
(BDNF) in primary cultures of rat microglia, which express LPA3, but not LPA1 or
LPA2, receptors [51]. A previous study demonstrated that BDNF downregulates the
neuron-specific K+-Cl co-transporter KCC2, resulting in lower [Cl]i levels in
16 Lipid Mediator LPA-Induced Demyelination and Self-Amplification of LPA 231
neurons [52, 53]. Under this condition, GABA (or Glycine) receptor activation
causes depolarization from the efflux of Cl [52], and the phenomenon is called the
GABA (or Glycine) switch (Fig. 16.2). The current study using the microglia inhibi-
tor minocycline revealed that LPA-induced microglia activation is involved in early-
stage development, but not in the late-stage maintenance, of neuropathic pain [54].
In this study, the early treatment with minocycline abolished LPA-induced and
nerve injury-induced neuropathic pain, LPA synthesis and its underlying activation
of synthetic enzymes, and cPLA2 and iPLA2. As later treatment with minocycline
failed to attenuate established neuropathic pain, as previously reported [55], microg-
lia activation following LPA receptor signaling appears to be important for the ini-
tiation of neuropathic pain.
COOH
pro-inflammatory
HO OH
14,15-DHET
16.5 Conclusion
biosynthesis through activation of LPA3 receptor and microglia. Thus, LPA acts as
a reverse signal, and LPA-induced amplification of LPA biosynthesis is a key mech-
anism for the feed-forward system underlying sustained neuropathic pain. However,
LPA1 receptor-mediated signaling does not function in inflammatory pain. It is plau-
sible that endocannabinoids, which are rapidly and abundantly produced upon
inflammatory stimulation, may inhibit the presynaptic pain transmitter release and
repress the LPA production. As LPA not only stimulates the transcription of the
endocannabinoid-degrading enzyme [63] but also inhibits ATX enzyme activity
[64], the amplification of LPA production will be reactivated, although it will not
proceed further without limitation. Recent studies reveal that the LPA-LPA1 recep-
tor signaling is also observed in mechanisms underlying various chronic pain mod-
els. Discovery of the best inhibitors for LPA receptors or biosynthetic enzymes is
now needed.
References
13. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H (2004) Initiation of neuropathic
pain requires lysophosphatidic acid receptor signaling. Nat Med 10(7):712718. doi:10.1038/
nm1060 nm1060
14. Xie W, Uchida H, Nagai J, Ueda M, Chun J, Ueda H (2010) Calpain-mediated down-regulation
of myelin-associated glycoprotein in lysophosphatidic acid-induced neuropathic pain. J
Neurochem 113(4):10021011. doi:JNC6664
15. Ahn DK, Lee SY, Han SR, Ju JS, Yang GY, Lee MK, Youn DH, Bae YC (2009) Intratrigeminal
ganglionic injection of LPA causes neuropathic pain-like behavior and demyelination in rats.
Pain 146(12):114120. doi:S0304-3959(09)00395-9
16. OConnor AB, Schwid SR, Herrmann DN, Markman JD, Dworkin RH (2008) Pain associated
with multiple sclerosis: systematic review and proposed classification. Pain 137(1):96111.
doi:S0304-3959(07)00460-5
17. Scrivani SJ, Mathews ES, Maciewicz RJ (2005) Trigeminal neuralgia. Oral Surg Oral Med
Oral Pathol Oral Radiol Endod 100(5):527538. doi:S1079-2104(05)00487-7
18. Said G (2007) Diabetic neuropathy: a review. Nat Clin Pract Neurol 3(6):331340.
doi:ncpneuro0504
19. Pentland B, Donald SM (1994) Pain in the Guillain-Barre syndrome: a clinical review. Pain
59(2):159164. doi:0304-3959(94)90068-X
20. McDonald CL, Bandtlow C, Reindl M (2011) Targeting the Nogo receptor complex in diseases
of the central nervous system. Curr Med Chem 18(2):234244. doi:BSP/CMC/E-Pub/2011/
016
21. Llorens F, Gil V, del Rio JA (2011) Emerging functions of myelin-associated proteins during
development, neuronal plasticity, and neurodegeneration. FASEB J 25(2):463475.
doi:fj.10-162792
22. Ueda H (2011) Lysophosphatidic acid as the initiator of neuropathic pain. Biol Pharm Bull
34(8):11541158. doi:JST.JSTAGE/bpb/34.1154
23. Parkinson DB, Bhaskaran A, Arthur-Farraj P, Noon LA, Woodhoo A, Lloyd AC, Feltri ML,
Wrabetz L, Behrens A, Mirsky R, Jessen KR (2008) c-Jun is a negative regulator of myelina-
tion. J Cell Biol 181(4): 625637. doi:jcb.200803013
24. Aguilera C, Nakagawa K, Sancho R, Chakraborty A, Hendrich B, Behrens A (2011) c-Jun
N-terminal phosphorylation antagonises recruitment of the Mbd3/NuRD repressor complex.
Nature (Lond) 469(7329):231235. doi:nature09607
25. Marinissen MJ, Chiariello M, Tanos T, Bernard O, Narumiya S, Gutkind JS (2004) The small
GTP-binding protein RhoA regulates c-jun by a ROCK-JNK signaling axis. Mol Cell
14(1):2941. doi:S1097276504001534
26. Das KC, Muniyappa H (2010) c-Jun-NH2 terminal kinase (JNK)-mediates AP-1 activation by
thioredoxin: phosphorylation of cJun, JunB, and Fra-1. Mol Cell Biochem 337(1-2):5363.
doi:10.1007/s11010-009-0285-0
27. Muniyappa H, Das KC (2008) Activation of c-Jun N-terminal kinase (JNK) by widely used
specific p38 MAPK inhibitors SB202190 and SB203580: a MLK-3-MKK7-dependent mecha-
nism. Cell Signal 20(4):675683. doi:S0898-6568(07)00374-9
28. Tanaka T, Nishimura D, Wu RC, Amano M, Iso T, Kedes L, Nishida H, Kaibuchi K, Hamamori
Y (2006) Nuclear Rho kinase, ROCK2, targets p300 acetyltransferase. J Biol Chem
281(22):1532015329. doi:M510954200
29. Chen LF, Greene WC (2004) Shaping the nuclear action of NF-kappaB. Nat Rev Mol Cell Biol
5(5):392401 doi:10.1038/nrm1368 nrm1368
30. Chen Y, Wang H, Yoon SO, Xu X, Hottiger MO, Svaren J, Nave KA, Kim HA, Olson EN, Lu
QR (2011) HDAC-mediated deacetylation of NF-kappaB is critical for Schwann cell myelina-
tion. Nat Neurosci 14(4):437441. doi:nn.2780
31. Svaren J, Meijer D (2008) The molecular machinery of myelin gene transcription in Schwann
cells. Glia 56(14):15411551. doi:10.1002/glia.20767
16 Lipid Mediator LPA-Induced Demyelination and Self-Amplification of LPA 235
32. Kim HJ, Kim JG, Moon MY, Park SH, Park JB (2014) IkappaB kinase gamma/nuclear factor-
kappaB-essential modulator (IKKgamma/NEMO) facilitates RhoA GTPase activation, which,
in turn, activates Rho-associated KINASE (ROCK) to phosphorylate IKKbeta in response to
transforming growth factor (TGF)-beta 1. J Biol Chem 289(3):14291440. doi:M113.520130
33. Misu K, Yoshihara T, Shikama Y, Awaki E, Yamamoto M, Hattori N, Hirayama M, Takegami
T, Nakashima K, Sobue G (2000) An axonal form of Charcot-Marie-Tooth disease showing
distinctive features in association with mutations in the peripheral myelin protein zero gene
(Thr124Met or Asp75Val). J Neurol Neurosurg Psychiatry 69(6):806811
34. Ji RR, Baba H, Brenner GJ, Woolf CJ (1999) Nociceptive-specific activation of ERK in spinal
neurons contributes to pain hypersensitivity. Nat Neurosci 2(12):11141119. doi:10.1038/16040
35. Rosen LB, Ginty DD, Weber MJ, Greenberg ME (1994) Membrane depolarization and cal-
cium influx stimulate MEK and MAP kinase via activation of Ras. Neuron 12(6):12071221.
doi:0896-6273(94)90438-3
36. Matsumoto M, Xie W, Ma L, Ueda H (2008) Pharmacological switch in A beta-fiber
stimulation-induced spinal transmission in mice with partial sciatic nerve injury. Mol Pain
4:25. doi:1744-8069-4-25
37. Fujita R, Kiguchi N, Ueda H (2007) LPA-mediated demyelination in ex vivo culture of dorsal
root. Neurochem Int 50(2):351355. doi:S0197-0186(06)00283-X
38. Inoue M, Ma L, Aoki J, Ueda H (2008) Simultaneous stimulation of spinal NK1 and NMDA
receptors produces LPC which undergoes ATX-mediated conversion to LPA, an initiator of
neuropathic pain. J Neurochem 107(6):15561565. doi:JNC5725
39. Durstin M, Durstin S, Molski TF, Becker EL, Shaafi RI (1994) Cytoplasmic phospholipase A2
translocates to membrane fraction in human neutrophils activated by stimuli that phosphory-
late mitogen-activated protein kinase. Proc Natl Acad Sci U S A 91(8):31423146
40. Hirabayashi T, Murayama T, Shimizu T (2004) Regulatory mechanism and physiological role
of cytosolic phospholipase A2. Biol Pharm Bull 27(8):11681173. doi:JST.JSTAGE/
bpb/27.1168
41. Xing M, Tao L, Insel PA (1997) Role of extracellular signal-regulated kinase and PKC alpha
in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells. Am J Physiol 272(4 pt
1):C1380C1387
42. Csutora P, Zarayskiy V, Peter K, Monje F, Smani T, Zakharov SI, Litvinov D, Bolotina VM
(2006) Activation mechanism for CRAC current and store-operated Ca2+ entry: calcium influx
factor and Ca2+-independent phospholipase A2beta-mediated pathway. J Biol Chem
281(46):3492634935. doi:M606504200
43. Smani T, Zakharov SI, Csutora P, Leno E, Trepakova ES, Bolotina VM (2004) A novel mecha-
nism for the store-operated calcium influx pathway. Nat Cell Biol 6(2):113120. doi:10.1038/
ncb1089 ncb1089
44. Ma L, Nagai J, Chun J, Ueda H (2013) An LPA species (18:1 LPA) plays key roles in the self-
amplification of spinal LPA production in the peripheral neuropathic pain model. Mol Pain
9(1):29. doi:1744-8069-9-29
45. Kurusu S, Matsui K, Watanabe T, Tsunou T, Kawaminami M (2008) The cytotoxic effect of
bromoenol lactone, a calcium-independent phospholipase A2 inhibitor, on rat cortical neurons
in culture. Cell Mol Neurobiol 28(8):11091118. doi:10.1007/s10571-008-9287-9
46. Kishimoto K, Matsumura K, Kataoka Y, Morii H, Watanabe Y (1999) Localization of cyto-
solic phospholipase A2 messenger RNA mainly in neurons in the rat brain. Neuroscience
92(3):10611077. doi:S0306-4522(99)00051-2
47. Ma L, Uchida H, Nagai J, Inoue M, Aoki J, Ueda H (2010) Evidence for de novo synthesis of
lysophosphatidic acid in the spinal cord through phospholipase A2 and autotaxin in nerve
injury-induced neuropathic pain. J Pharmacol Exp Ther 333(2):540546. doi:jpet.109.164830
236 H. Ueda and H. Uchida
48. Milligan ED, Watkins LR (2009) Pathological and protective roles of glia in chronic pain. Nat
Rev Neurosci 10(1):2336. doi:nrn2533
49. Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat
Neurosci 10(11):13611368. doi:nn1992
50. Yano R, Ma L, Nagai J, Ueda H (2013) Interleukin-1beta plays key roles in LPA-induced
amplification of LPA production in neuropathic pain model. Cell Mol Neurobiol 33(8):1033
1041. doi:10.1007/s10571-013-9970-3
51. Fujita R, Ma Y, Ueda H (2008) Lysophosphatidic acid-induced membrane ruffling and brain-
derived neurotrophic factor gene expression are mediated by ATP release in primary microglia.
J Neurochem 107(1):152160. doi:JNC5599
52. Rivera C, Voipio J, Thomas-Crusells J, Li H, Emri Z, Sipila S, Payne JA, Minichiello L,
Saarma M, Kaila K (2004) Mechanism of activity-dependent downregulation of the neuron-
specific K-Cl cotransporter KCC2. J Neurosci 24(19):46834691. doi:10.1523/
JNEUROSCI.5265-03.2004 24/19/4683
53. Rivera C, Li H, Thomas-Crusells J, Lahtinen H, Viitanen T, Nanobashvili A, Kokaia Z,
Airaksinen MS, Voipio J, Kaila K, Saarma M (2002) BDNF-induced TrkB activation down-
regulates the K+Cl cotransporter KCC2 and impairs neuronal Cl extrusion. J Cell Biol
159(5):747752. doi:10.1083/jcb.200209011 jcb.200209011
54. Ma L, Nagai J, Ueda H (2010) Microglial activation mediates de novo lysophosphatidic acid
production in a model of neuropathic pain. J Neurochem 115(3):643653. doi:JNC6955
55. Raghavendra V, Tanga F, DeLeo JA (2003) Inhibition of microglial activation attenuates the
development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp
Ther 306(2):624630. doi:10.1124/jpet.103.052407 jpet.103.052407
56. Piomelli D, Sasso O (2014) Peripheral gating of pain signals by endogenous lipid mediators.
Nat Neurosci 17(2):164174. doi:nn.3612
57. Yao B, Mackie K (2009) Endocannabinoid receptor pharmacology. Curr Top Behav Neurosci
1:3763. doi:10.1007/978-3-540-88955-7_2
58. Agarwal N, Pacher P, Tegeder I, Amaya F, Constantin CE, Brenner GJ, Rubino T, Michalski
CW, Marsicano G, Monory K, Mackie K, Marian C, Batkai S, Parolaro D, Fischer MJ, Reeh
P, Kunos G, Kress M, Lutz B, Woolf CJ, Kuner R (2007) Cannabinoids mediate analgesia
largely via peripheral type 1 cannabinoid receptors in nociceptors. Nat Neurosci 10(7):870
879. doi:nn1916
59. Nagai J, Ueda H (2011) Pre-emptive morphine treatment abolishes nerve injury-induced lyso-
phospholipid synthesis in mass spectrometrical analysis. J Neurochem 118(2):256265.
doi:10.1111/j.1471-4159.2011.07297.x
60. Morisseau C, Hammock BD (2013) Impact of soluble epoxide hydrolase and epoxyeico-
sanoids on human health. Annu Rev Pharmacol Toxicol 53:3758. doi:10.1146/
annurev-pharmtox-011112-140244
61. Wagner K, Inceoglu B, Dong H, Yang J, Hwang SH, Jones P, Morisseau C, Hammock BD
(2013) Comparative efficacy of 3 soluble epoxide hydrolase inhibitors in rat neuropathic and
inflammatory pain models. Eur J Pharmacol 700(1-3):93101. doi:S0014-2999(12)01032-1
62. Inceoglu B, Wagner KM, Yang J, Bettaieb A, Schebb NH, Hwang SH, Morisseau C, Haj FG,
Hammock BD (2012) Acute augmentation of epoxygenated fatty acid levels rapidly reduces
pain-related behavior in a rat model of type I diabetes. Proc Natl Acad Sci U S A 109(28):11390
11395. doi:1208708109
63. Sordelli MS, Beltrame JS, Cella M, Gervasi MG, Perez Martinez S, Burdet J, Zotta E, Franchi
AM, Ribeiro ML (2012) Interaction between lysophosphatidic acid, prostaglandins and the
endocannabinoid system during the window of implantation in the rat uterus. PLoS One
7(9):e46059. doi:10.1371/journal.pone.0046059 PONE-D-12-19475
64. van Meeteren LA, Ruurs P, Christodoulou E, Goding JW, Takakusa H, Kikuchi K, Perrakis A,
Nagano T, Moolenaar WH (2005) Inhibition of autotaxin by lysophosphatidic acid and sphin-
gosine 1-phosphate. J Biol Chem 280(22):2115521161. doi:M413183200
Chapter 17
Vascular Endothelial S1P2 Receptor Limits
Tumor Angiogenesis and Hyperpermeability
Abbreviations
S1P Sphingosine-1-phosphate
SphK Sphingosine kinase
GPCR G protein-coupled receptor
PLC Phospholipase C
PI3K Phosphatidylinositol 3-kinase
PI3K-C2 Class II isoform of phosphatidylinositol 3-kinase
PAF Platelet-activating factor
eNOS Endothelial NO synthase
ERK Extracellular signal-regulated kinase
JNK Jun N-terminal kinase
MAPK Mitogen-activated protein kinase
PTEN Phosphatase and Tensin Homolog Deleted from Chromosome 10
HUVECs Human umbilical vein endothelial cells
VEGF Vascular endothelial growth factor
S1P1, S1P2, and S1P3 are widely expressed in most organs, mediating diverse actions
of S1P [13, 510, 21]. Differing from S1P1S1P3, the expression of S1P4 is
restricted to lymphoid tissues and the lung, and that of S1P5 to the brain (especially
oligodendrocytes), leukocytes, and spleen [1, 21].
The signaling mechanisms of S1P1S1P3 are better characterized compared with
S1P4 and S1P5. S1P1, S1P2, and S1P3 activate overlapping yet distinctive intracel-
lular signaling pathways (Fig. 17.1) [13, 7, 9, 1621, 3944]. S1P1 couples exclu-
sively to Gi to induce activation of Ras-ERK, class I PI3K-Akt, and small GTPase
Rac signaling pathways. S1P1 also moderately activates phospholipase C (PLC) and
mobilizes Ca2+ (Fig. 17.1) [16, 17, 3942]. In contrast to S1P1, S1P2 and S1P3 cou-
ple to multiple G proteins, such as Gq, Gi, and G12/13 [1820]. S1P2 stimulates Rho
via G12/13, PLC mainly via Gq, extracellular signal-regulated kinase (ERK) via Gi,
and Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)
via pertussis toxin (PTX)-insensitive G protein (Fig. 17.1) [18]. Similar to S1P2,
S1P3 also couples to Gq-mediated PLC stimulation, G12/13-mediated Rho stimula-
tion, and Gi-mediated ERK and Rac stimulation (Fig. 17.1) [19, 20]. Although S1P2
and S1P3 can similarly couple to Gq, Gi, and G12/13 when overexpressed, an obvious
difference in the two receptor subtypes exists in primary cells including mouse
embryonic broblasts (MEFs): S1P2 preferentially couples to the G12/13-Rho path-
way whereas S1P3 preferentially couples to the Gq-PLC pathway [43, 44].
Gi G12/13 Gq
Gi G12/13
Proliferation
Survival
eNOS eNOS
Fig. 17.1 Basic signaling mechanisms of S1P1, S1P2, and S1P3. S1P1 couples exclusively to Gi to
activate Ras-ERK and PI3K(class I p110)-Akt and -Rac, and also PLC-Ca2+ mobilization. S1P2
and S1P3 couple to multiple heterotrimeric G proteins. S1P2 couples most prominently to G12/13 to
stimulate the Rho-ROCK(Rho kinase)-PTEN pathway. S1P3 couples to Gq, stimulating PI3K(class I
p110)-Akt and PLC-Ca2+ mobilization
17 Vascular Endothelial S1P2 Receptor Limits Tumor 241
Because S1P1, S1P2, and S1P3 are widely expressed, an integrated outcome of
S1P signaling in a given cell type largely depends upon the relative expression lev-
els of the S1P receptor subtypes. In addition, crosstalk between S1P receptor signal-
ing and growth factor or cytokine receptor signaling has been reported.
These observations indicate that S1P1 mediates the barrier-protective effect of S1P
(Fig. 17.2). Unexpectedly, however, inducible deletion of S1P1 alleles in adult mice
(because of embryonic lethality in S1P1KO mice) did not compromise recovery
from histamine injection [46]. One possibility to explain the discrepancy between
the results obtained with different experimental models would be that relatively
gradual deletion of S1P1 function in inducible system allowed a compensatory res-
cue system to develop, whereas acute antagonism or downregulation of S1P1, which
rapidly became maximal, did not.
The maintenance of barrier integrity requires Gi protein [45]. S1P1 is exclusively
coupled to Gi to mediate multiple signaling pathways including Ras-ERK, PI3K-
Akt, and Rac (Fig. 17.1). Previous in vitro studies showed that Rac and another Rho
family GTPase Rho are both required for enhancing effect of S1P on VEcadherin
assembly at the adherens junction in HUVECs [50]. Because S1P1 does not activate
Rho, S1P receptor(s) that are capable of Rho activation, which include S1P2 and
S1P3, also seem to be involved in the maintenance of barrier integrity. However, it is
reported that genetic deletion of S1P3 does not compromise vascular barrier integ-
rity [46].
PI3KC2a
Gi G12/13 Gq
EC NO Hypotension
Shock
VSMC
cGMP VSM relaxation
Fig. 17.2 S1P1 and S1P2 protect vascular barrier function through distinctive mechanisms. S1P1
has a housekeeping function in endothelial barrier maintenance through mechanisms involving
Rac activation, and in a manner dependent upon the class II isoform of PI3K (PI3K-C2), which
is required for internalization of activated S1P1 receptor, endosomal Rac activation, and VEcad-
herin assembly at the adherens junction. Endothelial barrier disruptors such as PAF and histamine
act on Gq-coupled receptors to induce Ca2+ mobilization and Akt activation, which together lead to
excess activation of eNOS, resulting in S-nitrosylation of -catenin and barrier disruption. S1P2
suppresses Akt and eNOS through the G12/13-Rho-ROCK-PTEN pathway to prevent barrier disrup-
tion, acting as a guardian of endothelial barrier integrity. EC, endothelial cell; VSMC, vascular
smooth muscle cell
17 Vascular Endothelial S1P2 Receptor Limits Tumor 243
S1P1 mediates stimulatory effects of S1P on cell migration toward S1P, morphogen-
esis to form tube-like structures in vitro, and assembly of VEcadherin at adherens
junctions and consequent maintenance of endothelial barrier integrity. Global
S1P1KO mice and endothelial cell-specic conditional S1P1KO mice are embryonic
lethal because of defective vascular formation [58, 59]. Recent studies have dis-
closed that S1P1 in endothelial cells inhibits, rather than stimulates, sprouting angio-
genesis, which is otherwise excessive by the action of the angiogenic factor
VEGF-A. Endothelial cell S1P1 mediates promotion of VEcadherin localization
and assembly at adherens junctions, which suppresses tip cell formation and leads
to the formation of a stabilized vascular network [60, 61].
S1P2 is the rst chemorepellant GPCR to be identied that mediates inhibition of
cell migration through Rho and Rho-dependent inhibition of Rac [39]. In tumor
cells such as B16 melanoma cells, which express S1P2, S1P inhibits their migration,
invasion of extracellular matrix, and hematogenous metastasis in a tail vein injec-
tion model [12, 62, 63]. The expression level of endogenous S1P2 in cultured endo-
thelial cells is substantially low or barely detectable [64]. In S1P2LacZ/+ mice in which
LacZ gene was knocked in to S1P2 locus, the expression of S1P2 was identied in
both of vascular endothelial cells and smooth muscle cells in the vasculature of vari-
ous organs [57]. When overexpressed in endothelial cells, S1P2 mediated S1P-
induced inhibition of cell migration and tube formation in vitro and inhibition of
angiogenesis in Matrigel plug assay in vivo [65]. Overexpression of S1P2 in endo-
thelial cells resulted in decreased barrier function when challenged with thrombin
in vitro, for which Rho-ROCK-PTEN-dependent inhibition of Rac led to disassem-
bly of VEcadherin [66].
In the in vivo model of subcutaneous tumor inoculation, host S1P2 that is
expressed in both endothelial cells and angiogenic myeloid cells, the latter types of
cells being recruited from bone marrow to tumor stroma, inhibited tumor angiogen-
esis and tumor progression [57]. Lung microvascular endothelial cells derived from
S1P2KO mice showed enhanced activities of Rac and Akt but not ERK, and stimu-
lated cell proliferation, cell migration, and tube formation compared with those
derived from wild-type mice. S1P2-mediated inhibition of Akt is likely involved in
inhibition of endothelial cell proliferation. S1P2-mediated inhibition of Akt is the
consequence of Rho kinase-dependent activation of PTEN (3-specic phos-
phoinositide phosphatase). Rho kinase is dispensable, however, for S1P2-mediated
inhibition of Rac and cell migration, which are still observed in PTEN-decient
tumor cells [67]. S1P2KO mice develop deafness and ataxia from aberrant vascular
17 Vascular Endothelial S1P2 Receptor Limits Tumor 245
formation in the inner ear and loss of hair cells, and epileptic seizures and sudden
death in the weaning period in C57BL/6 background [6871].
S1P1/S1P2-double null and S1P1/S1P2/S1P3-triple null embryos show increas-
ingly more severe defects in vascular formation compared with single S1P1-null
embryos [72]. S1P3KO mice show the normal phenotype whereas S1P2/S1P3-double
null mice show partial embryonic and perinatal lethality [43, 44, 72]. Therefore, it
is possible that S1P2 and S1P3 have some functional redundancy in the regulation of
vascular formation.
S1P2 and S1P3 are involved also in the regulation of vascular tone [7375]. In vas-
cular smooth muscle cells, excitatory agonists induce Rho activation and potentiate
contraction through Rho kinase-dependent phosphorylation of the myosin-targeting
subunit MYPT1 of myosin phosphatase and consequent inhibition of the myosin
phosphatase activity [7681]. The Rho-Rho kinase-dependent mechanism also
involves phosphorylation of the myosin phosphatase inhibitor protein, CPI-17 [82].
Myosin phosphatase inhibition mediated by G12/13-dependent activation of Rho-Rho
kinase, together with activation of myosin light chain kinase by Gq/11-PLC-dependent
Ca2+ mobilization and Ca2+ inux, efciently increases sustained phosphorylation of
myosin light chain and consequent vascular contraction [80]. In addition, S1P2-
mediated contraction mechanism involves inhibition of eNOS as already described.
S1P3 couples moderately to Gi besides Gq/11 and G12/13 (Fig. 17.1) [19, 39, 43].
Differing from S1P2, S1P3 in endothelial cells rather stimulates eNOS via the Gi
pathway as does S1P1, leading to endothelial cell-dependent vasorelaxation.
Acknowledgments This work was supported by grants from the Ministry of Education, Science,
Sports and Culture of Japan, the Japan Society for the Promotion of Science, and grants for Core
Research for Evolutional Science and Technology from JST, and IPNU Research Promotion Fund.
17 Vascular Endothelial S1P2 Receptor Limits Tumor 247
References
1. Blaho VA, Hla T (2014) An update on the biology of sphingosine 1-phosphate receptors. J
Lipid Res 55:15961608
2. Takuwa Y, Ikeda H, Okamoto Y, Takuwa N, Yoshioka K (2013) Sphingosine-1-phosphate as a
mediator involved in development of brotic diseases. Biochim Biophys Acta 1831:185192.
doi:10.1016/j.bbalip.2012.06.008
3. Takuwa Y, Okamoto Y, Yoshioka K, Takuwa N (2012) Sphingosine-1-phosphate signaling in
physiology and diseases. Biofactors 38:329337. doi:10.1002/biof.1030
4. Cyster JG, Schwab SR (2012) Sphingosine-1-phosphate and lymphocyte egress from lym-
phoid organs. Annu Rev Immunol 30:6994. doi:10.1146/annurev-immunol-020711-075011
5. Skoura A, Hla T (2009) Lysophospholipid receptors in vertebrate development, physiology,
and pathology. J Lipid Res 50(suppl):S293S298. doi:10.1194/jlr.R800047-JLR200
6. Kono M, Allende ML, Proia RL (2008) Sphingosine-1-phosphate regulation of mammalian
development. Biochim Biophys Acta 1781:435441. doi:10.1016/j.bbalip.2008.07.001
7. Takuwa Y, Okamoto Y, Yoshioka K, Takuwa N (2008) Sphingosine-1-phosphate signaling and
biological activities in the cardiovascular system. Biochim Biophys Acta 1781:483488
8. Obinata H, Hla T (2012) Sphingosine 1-phosphate in coagulation and inammation. Semin
Immunopathol 34:7391. doi:10.1007/s00281-011-0287-3
9. Takuwa N, Du W, Kaneko E, Okamoto Y, Yoshioka K, Takuwa Y (2011) Tumor-suppressive
sphingosine-1-phosphate receptor-2 counteracting tumor-promoting sphingosine-1-phosphate
receptor-1 and sphingosine kinase 1: Jekyll Hidden behind Hyde. Am J Cancer Res
1:460481
10. Takuwa Y, Du W, Qi X, Okamoto Y, Takuwa N, Yoshioka K (2010) Roles of sphingosine-1-
phosphate signaling in angiogenesis. World J Biol Chem 1:298306. doi:10.4331/wjbc.v1.
i10.298
11. Zhang H, Desai NN, Olivera A, Seki T, Brooker G, Spiegel S (1991) Sphingosine-1-phosphate,
a novel lipid, involved in cellular proliferation. J Cell Biol 114:155167
12. Sadahira Y, Ruan F, Hakomori S, Igarashi Y (1992) Sphingosine 1-phosphate, a specic
endogenous signaling molecule controlling cell motility and tumor cell invasiveness. Proc Natl
Acad Sci U S A 89:96869690
13. Hla T, Maciag T (1990) An abundant transcript induced in differentiating human endothelial
cells encodes a polypeptide with structural similarities to G-protein-coupled receptors. J Biol
Chem 265:93089313
14. Okazaki H, Ishizaka N, Sakurai T, Kurokawa K, Goto K, Kumada M, Takuwa Y (1993)
Molecular cloning of a novel putative G protein-coupled receptor expressed in the cardiovas-
cular system. Biochem Biophys Res Commun 190:11041109
15. MacLennan AJ, Browe CS, Gaskin AA, Lado DC, Shaw G (1994) Cloning and characteriza-
tion of a putative G-protein coupled receptor potentially involved in development. Mol Cell
Neurosci 5:201209
16. Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T
(1998) Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.
Science 279:15521555
17. Okamoto H, Takuwa N, Gonda K, Okazaki H, Chang K, Yatomi Y, Shigematsu H, Takuwa Y
(1998) EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to mul-
tiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-
mitogen-activated protein kinase activation, and adenylate cyclase inhibition. J Biol Chem
273:2710427110
18. Gonda K, Okamoto H, Takuwa N, Yatomi Y, Okazaki H, Sakurai T, Kimura S, Sillard R, Harii
K, Takuwa Y (1999) The novel sphingosine 1-phosphate receptor AGR16 is coupled via per-
tussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways. Biochem J
337:6775
248 N. Takuwa et al.
37. Kharel Y, Mathews TP, Gellett AM, Tomsig JL, Kennedy PC, Moyer ML, Macdonald TL,
Lynch KR (2011) Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating
sphingosine 1-phosphate. Biochem J 440:345353. doi:10.1042/BJ20110817
38. Pham TH, Baluk P, Xu Y, Grigorova I, Bankovich AJ, Pappu R, Coughlin SR, McDonald DM,
Schwab SR, Cyster JG (2010) Lymphatic endothelial cell sphingosine kinase activity is
required for lymphocyte egress and lymphatic patterning. J Exp Med 207:1727. doi: 10.1084/
jem.20091619
39. Okamoto H, Takuwa N, Yokomizo T, Sugimoto N, Sakurada S, Shigematsu H, Takuwa Y
(2000) Inhibitory regulation of Rac activation, membrane rufing, and cell migration by the G
protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3. Mol Cell
Biol 20:92479261
40. Takuwa Y, Okamoto H, Takuwa N, Gonda K, Sugimoto N, Sakurada S (2001) Subtype-
specic, differential activities of the EDG family receptors for sphingosine-1-phosphate, a
novel lysophospholipid mediator. Mol Cell Endocrinol 177:311
41. Takuwa Y, Takuwa N, Sugimoto N (2002) The Edg family G protein-coupled receptors for
lysophospholipids: their signaling properties and biological activities. J Biochem
131:767771
42. Takuwa Y (2002) Subtype-specic differential regulation of Rho family G proteins and cell
migration by the Edg family sphingosine-1-phosphate receptors. Biochim Biophys Acta
1582:112120
43. Ishii I, Friedman B, Ye X, Kawamura S, McGiffert C, Contos JJ, Kingsbury MA, Zhang G,
Brown JH, Chun J (2001) Selective loss of sphingosine 1-phosphate signaling with no obvious
phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3. J Biol
Chem 276:3369733704
44. Ishii I, Ye X, Friedman B, Kawamura S, Contos JJ, Kingsbury MA, Yang AH, Zhang G, Brown
JH, Chun J (2002) Marked perinatal lethality and cellular signaling decits in mice null for the
two sphingosine 1-phosphate (S1P) receptors, S1P(2)/LP(B2)/EDG-5 and S1P(3)/LP(B3)/
EDG-3. J Biol Chem 277:2515225159
45. Camerer E, Regard JB, Cornelissen I, Srinivasan Y, Duong DN, Palmer D, Pham TH, Wong
JS, Pappu R, Coughlin SR (2009) Sphingosine-1-phosphate in the plasma compartment regu-
lates basal and inammation-induced vascular leak in mice. J Clin Invest 119:18711879
46. Olivera A, Eisner C, Kitamura Y, Dillahunt S, Allende L, Tuymetova G, Watford W, Meylan F,
Diesner SC, Li L, Schnermann J, Proia RL, Rivera J (2010) Sphingosine kinase 1 and sphin-
gosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock in mice. J Clin
Invest 120:14291440. doi:10.1172/JCI40659
47. Sanna MG, Wang SK, Gonzalez-Cabrera PJ, Don A, Marsolais D, Matheu MP, Wei SH, Parker
I, Jo E, Cheng WC, Cahalan MD, Wong CH, Rosen H (2006) Enhancement of capillary leak-
age and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo. Nat Chem Biol
2:434441
48. Marsolais D, Rosen H (2009) Chemical modulators of sphingosine-1-phosphate receptors as
barrier-oriented therapeutic molecules. Nat Rev Drug Discov 8:297307. doi:10.1038/nrd2356
49. Brinkmann V, Cyster JG, Hla T (2004) FTY720: sphingosine 1-phosphate receptor-1 in the
control of lymphocyte egress and endothelial barrier function. Am J Transplant 4:10191025
50. Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk M, Volpi M, Shaa RI, Hla T
(1999) Vascular endothelial cell adherens junction assembly and morphogenesis induced by
sphingosine-1-phosphate. Cell 99:301312
51. Cui H, Okamoto Y, Yoshioka K, Du W, Takuwa N, Zhang W, Asano M, Shibamoto T, Takuwa
Y (2013) Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through
suppression of endothelial nitric oxide synthase in mice. J Allergy Clin Immunol 132:1205
1214.e9. doi:10.1016/j.jaci.2013.07.026
52. Oskeritzian CA, Price MM, Hait NC, Kapitonov D, Falanga YT, Morales JK, Ryan JJ, Milstien
S, Spiegel S (2010) Essential roles of sphingosine-1-phosphate receptor 2 in human mast cell
250 N. Takuwa et al.
68. MacLennan AJ, Benner SJ, Andringa A, Chaves AH, Rosing JL, Vesey R, Karpman AM,
Cronier SA, Lee N, Erway LC, Miller ML (2006) The S1P2 sphingosine 1-phosphate receptor
is essential for auditory and vestibular function. Hear Res 220:3848
69. Kono M, Belyantseva IA, Skoura A, Frolenkov GI, Starost MF, Dreier JL, Lidington D, Bolz
SS, Friedman TB, Hla T, Proia RL (2007) Deafness and stria vascularis defects in S1P2
receptor-null mice. J Biol Chem 282:1069010696
70. Herr DR, Grillet N, Schwander M, Rivera R, Mller U, Chun J (2007) Sphingosine 1-phosphate
(S1P) signaling is required for maintenance of hair cells mainly via activation of S1P2. J
Neurosci 27:14741478
71. MacLennan AJ, Carney PR, Zhu WJ, Chaves AH, Garcia J, Grimes JR, Anderson KJ, Roper
SN, Lee N (2001) An essential role for the H218/AGR16/Edg-5/LP(B2) sphingosine
1-phosphate receptor in neuronal excitability. Eur J Neurosci 14:203209
72. Kono M, Mi Y, Liu Y, Sasaki T, Allende ML, Wu YP, Yamashita T, Proia RL (2004) The
sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during
embryonic angiogenesis. J Biol Chem 279:2936729373
73. Ohmori T, Yatomi Y, Osada M, Kazama F, Takafuta T, Ikeda H, Ozaki Y (2003) Sphingosine
1-phosphate induces contraction of coronary artery smooth muscle cells via S1P2. Cardiovasc
Res 58:170177
74. Lorenz JN, Arend LJ, Robitz R, Paul RJ, MacLennan AJ (2007) Vascular dysfunction in S1P2
sphingosine 1-phosphate receptor knockout mice. Am J Physiol Regul Integr Comp Physiol
292:R440R446
75. Murakami A, Takasugi H, Ohnuma S, Koide Y, Sakurai A, Takeda S, Hasegawa T, Sasamori J,
Konno T, Hayashi K, Watanabe Y, Mori K, Sato Y, Takahashi A, Mochizuki N, Takakura N
(2010) Sphingosine 1-phosphate (S1P) regulates vascular contraction via S1P3 receptor:
investigation based on a new S1P3 receptor antagonist. Mol Pharmacol 77:704713.
doi:10.1124/mol.109.061481
76. Noda M, Yasuda-Fukazawa C, Moriishi K, Kato T, Okuda T, Kurokawa K, Takuwa Y (1995)
Involvement of rho in GTP gamma S-induced enhancement of phosphorylation of 20 kDa
myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS
Lett 367:246250
77. Takuwa Y (1996) Regulation of vascular smooth muscle contraction. The roles of Ca2+, protein
kinase C and myosin light chain phosphatase. Jpn Heart J 37:793813
78. Nagumo H, Sasaki Y, Ono Y, Okamoto H, Seto M, Takuwa Y (2000) Rho kinase inhibitor
HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells. Am
J Physiol Cell Physiol 278:C57C65
79. Sakurada S, Okamoto H, Takuwa N, Sugimoto N, Takuwa Y (2001) Rho activation in excit-
atory agonist-stimulated vascular smooth muscle. Am J Physiol Cell Physiol 281:C571C578
80. Takuwa Y (2003) Regulation of the Rho signaling pathway by excitatory agonists in vascular
smooth muscle. Adv Exp Med Biol 538:6775
81. Takagi T, Okamoto Y, Tomita S, Sato A, Yamaguchi S, Takuwa Y, Watanabe G (2011)
Intraradial administration of fasudil inhibits augmented Rho kinase activity to effectively
dilate the spastic radial artery during coronary artery bypass grafting surgery. J Thorac
Cardiovasc Surg 142:e59e65. doi:10.1016/j.jtcvs.2011.01.055
82. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, Nakano T (2000)
Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phospha-
tase, by Rho-kinase. FEBS Lett 475:197200
83. Posor Y, Eichhorn-Gruenig M, Puchkov D, Schneberg J, Ullrich A, Lampe A, Mller R,
Zarbakhsh S, Gulluni F, Hirsch E, Krauss M, Schultz C, Schmoranzer J, No F, Haucke V
(2013) Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.
Nature 499:233237. doi:10.1038/nature12360
252 N. Takuwa et al.
Masataka Majima
18.1 Angiogenesis
We are complex multicellular organisms, and all cells in the body require a nely
controlled supply of oxygen and nutrients [13]. The diffusion of oxygen in the tis-
sues is limited to 100200 m, and a highly developed vascular system has evolved
to ensure that all cells are within this distance of a supply of oxygen. Angiogenesis,
the process of new blood vessel development from preexisting vasculature, is indis-
pensable to maintain the integrity of the body. This function involves endothelial
cell division, selective degradation of the basement membrane and the surrounding
extracellular matrix, endothelial cell migration, and the formation of a tubular struc-
ture. Once blood vessels have been established, the endothelial cells undergo tissue-
specic changes to generate functionally active vessels. During embryogenesis,
blood vessels are formed by the differentiation of endothelial cell precursors (angio-
blasts), which associate to form primitive vessels. This process is called vasculo-
genesis [3, 4].
Angiogenesis is subject to a complex control system with pro-angiogenic and
antiangiogenic factors [3, 4]. In adults, angiogenesis is tightly controlled by this
angiogenic balance, that is, a physiological balance between the stimulatory and
inhibitory signals for blood vessel formation. In normal status, the formation of new
blood vessels occurs during wound healing, organ regeneration, and in the female
reproductive system during ovulation, menstruation, and the formation of the pla-
centa. It is also an important factor in several pathological processes such as tumor
growth, rheumatoid arthritis, diabetic retinopathy, and psoriasis.
The arachidonic acid (AA) cascade is the biosynthetic pathway that involves the
release of the n-6 polyunsaturated fatty acid AA from the sn-2 position of mem-
brane phospholipids by a phospholipase A2 (PLA2) enzyme, and its subsequent
metabolism to bioactive prostaglandins (PGs), thromboxanes, leukotrienes, and
epoxy fatty acids, by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome
P450 epoxygenase enzymes, coupled to specic terminal synthases. AA and its end
products are involved in both physiological and pathological processes. Although
pharmacological inhibitors can be used to investigate the role of key enzymes
involved in AA release and metabolism of AA in physiological and pathological
models, the lack, in some cases, of specic inhibitors or of a complete pharmaco-
logical inhibition, and standardization of dosing paradigms complicate the studies.
Two COX isoforms have been identied: COX-1 is constitutively expressed in vari-
ous tissues, whereas COX-2 is induced by mitogens, cytokines, and tumor promot-
ers [6]. COX regulates the formation of an unstable endoperoxide intermediate,
PGH2, which, in turn, is metabolized to PGD2, PGE2, PGF2, PGI2, and thrombox-
ane (TX)A2 by cell-specic isomerases and synthases. Prostanoids formed are
immediately released outside the cell; they are either chemically or metabolically
unstable, and thus prostanoids are believed to work only locally, near their site of
production. PGI2 and TXA2 spontaneously degrade into inactive compounds under
physiological conditions, and other PGs are enzymatically inactivated during a sin-
gle passage through the lung. PGD2 and PGE2 are slowly dehydrated in biological
uids containing serum albumin to be the cyclopentenone PGs.
Prostanoids exert their actions via membrane receptors on the surface of target cells.
Genes for each of these receptors have been disrupted and the corresponding knock-
out mice have been produced [7]. Furthermore, with the use of cloned receptors,
agonists and antagonists highly selective for each of the four EP subtypes have been
developed. Eight types and subtypes of membrane prostanoid receptors are con-
served in mammals, from mouse to human [7]: the PGD receptor (DP), four sub-
types of the PGE receptor (EP1, EP2, EP3, EP4), the PGF receptor (FP), the PGI
receptor (IP), and the TXA receptor (TP). All receptors are G protein-coupled
rhodopsin-type receptors with seven transmembrane domains, and each is encoded
by different genes. There are several splice variants in the EP3, FP, and TP recep-
tors, which differ only in their C-terminal tails. Among the eight types and subtypes,
the IP, DP, EP2, and EP4 receptors mediate a cAMP rise, whereas the TP, FP, and
256 M. Majima
Table 18.1 Roles of prostaglandins (PGs) revealed by studies using prostaglandin receptor
knockout mice
PGs Receptors Pathophysiological roles
PGD2 DP A mediator of allergic asthma
PGE2 EP1 Inhibition of gastric motor activity
EP2 Ovulation and fertilization, salt-sensitive hypertension
EP3 A mediator of febrile response, acid-induced bicarbonate secretion in
duodenum, urinary concentration, mast cell activation, angiogenesis,
lymphangiogenesis
EP4 Closure of ductus arteriosus, bone resorption, lymphangiogenesis
PGI2 IP Antithrombotic function, adaptive cytoprotection of stomach, acetic
acid-induced writhing reaction
PGF2 FP An essential inducer of labor
TXA2 TP Hemostasis, endotoxicin-induced hepatic microcirculatory dysfunction,
lymphangiogenesis
EP1 receptors induce calcium mobilization. EP3 has several splice variants, which
increase or decrease cAMP levels and induce calcium mobilization. The effects of
prostanoids on these G protein-coupled signaling pathways are reported to be
changed in a ligand concentration-dependent manner. PGI2 analogues bind to IP and
activate adenylate cyclase via Gs in a dose-dependent manner, but higher concentra-
tions of the ligand couple to IP and activate phospholipase C to mobilize calcium
ions via Gq. There are four subtypes of the receptor for PGE2, although the other
prostanoids each have only a single receptor. The homology of amino acid sequences
between different types of the receptors within each functional group is much higher
than that found among the four PGE receptor subtypes. The phylogenetic tree
derived from receptor homologies indicates that prostanoid receptors originated
from the primitive PGE receptor. Other PGs and TX receptors subsequently evolved
from functionally related PGE receptor subtypes by gene duplication. The roles of
PGs in various physiological and pathophysiological processes have been claried
with mice decient in each prostanoid receptor. The ndings, including ours, with
use of knockout mice are summarized in Table 18.1. In this review article, we dis-
cuss the signicance of the ndings related to angiogenesis together with lymphan-
giogenesis in vivo.
Fig. 18.1 Delay in wound healing in EP3 knockout mice. Typical appearance of wounds 7 days
after wounding. Surgical wounds were made on the backs of EP3 receptor knockout mice (EP3/)
and of their wild-type counterparts (WT). Original diameter of the wounds was 8 mm. One divi-
sion on the scale below the wound represents 1 mm (Cited from Kamoshita et al. [13] with
permission)
of stimulators and inhibitors that favor either vascular growth or regression [9]. It
has been reported that E-type PGs have a pro-angiogenic activity in corneal tests
[10] and in the chorioallantoic membrane (CAM) technique [11]. Further, Form and
Auerbach reported that PGE2 strongly induced angiogenesis on the CAM of 8-day-
old chicken embryos, but PGA2, PGF2, and a derivative of TXA2 did not. A report
[12] described that the endothelial migration was mediated by COX-2. This experi-
ment was performed in vitro using conuent monolayer endothelial cells stimulated
with PMA, and the authors also reported that corneal angiogenesis was suppressed
by a COX-2 inhibitor, suggesting the involvement of COX-2 products in vivo. These
results suggest that endogenous PGs regulate angiogenesis not only in physiological
conditions but also in pathological states. The roles of COX-2-derived PGs in wound
healing and the PG receptor signaling relevant to wound-induced angiogenesis were
reported recently [13]. When full-thickness skin wounds were created in mice, and
angiogenesis in wound granulation tissues was estimated, wound closure and reepi-
thelization were delayed in mice treated with COX-2 inhibitors. The wound closure
and reepithelization in EP3 receptor knockout mice (EP3/) were signicantly
delayed compared with wild-type mice (WT) (Fig. 18.1), whereas those in EP1/,
EP2/, and EP4/ were not delayed. Wound-induced angiogenesis in EP3/ was
signicantly inhibited compared with that in WT (Fig. 18.2). Reduced wound-
induced angiogenesis in EP3/ was accompanied by poor development of granula-
tion tissues under the wound. VEGF expression in wound granulation tissues in
EP3/ was markedly less than that in WT. Wound closure in WT was delayed sig-
nicantly by a VEGF-neutralizing antibody compared with control IgG. Wound-
induced angiogenesis and wound closure were signicantly suppressed in
EP3/ bone marrow transplantation mice, compared with those in WT bone marrow
258 M. Majima
Fig. 18.2 Reduced angiogenesis in wound granulation tissues in EP3 knockout mice. (a)
Immunohistochemical localization of CD31 in wound granulation tissues isolated from mice 7
days after wounding. (b) Vascular density in wound granulation tissues isolated from mice 3 and 7
days after wounding (Cited from Kamoshita et al. [13] with permission)
emphasize here that the models must be carefully selected according to the interests
of researchers. Analysis of a tumor implantation model in some knockout mice is
suitable for observing the host stromal responses that facilitate tumorigenesis,
because the lack of a given receptor is observable in the host stroma, although tumor
cells may, or may not, express the receptors in question, depending on the cell lines
implanted. In tumor implantation experiments, differences in phenotype such as
tumor growth and tumor-associated angiogenesis in mice are highly dependent on
receptor signaling in the host. The models of other categories are developed to see
the effect of PG receptor signaling on tumor cells themselves (tumor cell-autonomous
effect), as induced by the mutation in tumor epithelial cells in addition to the host
stromal effect. The most successful example of the latter is the marked reduction of
polyp number in Apc716 mice (a model of FAP) against a COX-2/ background, in
comparison with control animals [15]. In COX-2/ mice, COX-2 was decient both
in polyp epithelial cells and in stromal cells.
It was previously reported that PGE2 can promote colorectal cancer growth, in
part through the activation of the PGE2 receptor subtypes EP1 and EP4. Experiments
using knockout mice with a colon carcinogen, azoxymethane, revealed the signi-
cant suppression of aberrant crypt foci in EP1-decient mice together with EP4-
decient mice. The suppression was limited in both cases, suggesting the possible
involvement of other receptors or mechanisms. Moreover, aberrant crypt formation
represents an initial step in carcinogenesis, and many events precede the develop-
ment of colon cancer. It is likely that PGs are also involved in other steps and mech-
anisms. The expression of COX-2, but not COX-1, is elevated in many colorectal
cancers, and the protein has been localized to both stromal and epithelial compart-
ments. One mechanism by which elevated COX-2 promotes carcinogenesis is
through stimulation of tumor-associated angiogenesis.
In tumor implantation models, the involvement of PGE2-EP receptor signaling in
tumor-induced angiogenesis was tested [28]. It was reported that among the four
subtypes of EP receptor knockout mice, in IP/, and in their WT counterparts,
tumor-associated angiogenesis in EP3/ mice was suppressed by nearly 80 %,
although partial reduction of angiogenesis was observed in EP2/ mice [28].
Histological examination of tumors formed in EP3/ mice revealed a low level of
vascularization and distinct boundaries with the surrounding normal tissue. In spite
of the implantation of the same number of tumor cells, differences in tumor growth
and tumor-associated angiogenesis were observed in these mice, strongly suggest-
ing that the EP3 receptor in the host, not on the tumor cells, has a major, indeed, the
critical role in tumor-induced angiogenesis and tumor growth. Staining for COX-2
was apparent in tumors together with the stromal cells and endothelial cells [28]. In
WT mice, VEGF was abundant in the surrounding stromal cells, whose major com-
ponents were Mac-1 and CD3-negative broblast-like cells. Expression of VEGF
was markedly reduced in EP3/ mice. Gel shift assays revealed that AP-1 may be
important in VEGF expression and angiogenesis [28], although other factors, such
as HIF-1, whose activation was related to EP1, EP2, and EP4 signaling [29, 30],
were not ruled out.
It was reported that angiogenesis and growth of polyps were EP2-dependent
when the APC gene was mutated [22, 23]. The reports of those authors stated that
18 Roles of Prostaglandins in Regulation of Pathological Angiogenesis 261
the major elements that express COX-2 are stromal cells around the intestinal pol-
yps. The tumor stromal cells that produced VEGF so as to facilitate angiogenesis
and tumor growth were CD3 and Mac-1 double-negative broblasts [28]. Fibroblasts
exhibit heterogeneity in various biological parameters including PG-generating and
receptor systems [3133]. The authors of the foregoing report [23] did not show the
microvessel density in EP3/ with APC mutation, and the reduction percentage of
angiogenesis in APC-mutated EP2/ mice was approximately 30 % at best. The
major EP receptor expressed in the subcutaneous tissues of WT mice was EP3 [28],
which was not expressed in the intestine [22]. These ndings suggest that tumor-
associated angiogenesis may be regulated in a site-specic fashion, and that it may
be related to the heterogeneity of the stromal broblasts. It was reported that EP2-
null mice bearing subcutaneous tumor cells exhibited cancer-associated immunode-
ciency and dendritic cell abnormalities, but surprisingly had no effects on
tumor-associated angiogenesis and VEGF induction, in spite of a partial reduction
in tumor growth. This nding indicates that intracellular signaling linked to EP2
receptor activation also may be heterogeneous.
The host microenvironment is believed to inuence tumor progression [34, 35].
As already mentioned, PGs may be one of the important determinants of tumor
host communications. Examination of human colorectal cancer has revealed marked
COX-2 expression not only in cancer cells but also in the stroma that surrounds
them [36]. COX-2-decient mice also exemplify the signicance of stromal
COX-2 in tumor-induced angiogenesis [37]. The study using EP3/ mice revealed
that COX-2-expressing stromal cells around the tumors, or the tumor cells them-
selves, or both, synthesize and release PGE2 into the tumor microenvironment; and
that PGE2 then acts on the stromal cells that express EP3 receptor to induce pro-
angiogenic factor production and consequent angiogenesis (Fig. 18.3). EP3 receptor
signaling is important in angiogenesis promotion, but it cannot be ruled out that EP2
receptor signaling may facilitate angiogenesis synergistically.
The lymphatic vasculature forms a network of vessels that drain interstitial uid
from tissues and return this uid to the blood. Lymphangiogenesis, the formation of
lymphatic vessels from preexisting lymphatic vessels, is important in homeostasis
of interstitial uids, metabolism, and immunity. Recent evidence, however, indi-
cates that lymphangiogenesis, similar to angiogenesis, also occurs during certain
inammatory and autoimmune conditions. Psoriasis, a chronic inammatory skin
disease, is characterized by extensive lymphangiogenesis [38], and lymphatic
hyperplasia is frequently observed in rejected renal transplants from patients with
this condition [25]. Furthermore, bacterial infection of the airway epithelia of mice
induced intense lymphangiogenesis and upregulated VEGF-C and VEGF-D [39].
After inammation has been resolved, new lymphatic networks form a drainage
system for uid and immune cells.
262 M. Majima
Fig. 18.3 Targets for controlling tumor-associated angiogenesis regulated by endogenous PGs.
Stromal EP3 signaling is a key regulator of tumor angiogenesis and tumor growth. COX-2 inhibi-
tion and EP3 blockade are effective in preventing tumor growth and angiogenesis. Controlling host
stromal function by modication of inammatory signaling relevant to tumor angiogenesis may
also become a useful strategy to treat solid tumors such as cancers
As discussed, highly selective EP antagonists such as EP3 and EP2 receptor antago-
nists therefore exhibit benecial actions on the stromal cells and may be a good
choice as novel therapeutic tools against cancer. Administration of an EP3 antago-
nist to the tumor-bearing mice signicantly suppressed tumor-associated angiogen-
esis and tumor growth in WT mice [28]. By contrast, administration of neither an
EP1 nor an EP4 antagonist, both previously developed [21, 42], did so. Furthermore,
such a preventive effect of an EP3 antagonist was not seen in EP3/ mice [28], sug-
gesting that EP3 receptors expressed on the tumor cells themselves did not exhibit
any signicant function in tumor-associated angiogenesis or tumor growth, because
the EP3 antagonist administered may have effectively blocked the EP3 receptors on
the tumor cells (Fig. 18.3). These facts conrmed the result obtained in EP3/ mice,
namely, that EP3 receptor signaling acts predominantly on the host stroma. This
signaling on the stromal cells was relevant to the induction of the potent pro-
angiogenic growth factor VEGF, and upregulated VEGF certainly has a pro-
angiogenic action and facilitates tumor growth (Fig. 18.3) [28]. A highly selective
EP3 antagonist therefore exhibits preventive action on the stromal cells and is
expected to become a novel therapeutic tool against cancer.
Inammation is a local protective response to harmful stimuli. Recent results have
expanded the concept that inammation is an important factor in facilitating tumor
growth [43]. In fact, many cancers arise from the sites of infection, chronic irritation,
and inammation. PGs are the major pro-inammatory mediators and increase
inammation induced by various chemical mediators. The tumor stromal reaction can
be characterized by the proliferation of tissues including broblasts, which can facili-
tate angiogenesis and probably lymphangiogenesis. The results obtained from the
sponge implantation model can support the signicance of the proliferation or inl-
tration, or both, of inammatory cells in the site where angiogenesis occurred
[4449]. Stromal broblasts may be derived from the bone marrow [43], and from
tumor-associated macrophages as a paradigm for polarized M2 mononuclear phago-
cytes [50]. Targeting tumor angiogenesis with exogenous genes to tumor angiogene-
sis was performed by transplantation of genetically modied hematopoietic stem
cells [51]. Thus, transplantation of EP receptor-null bone marrow cells may provide
the means for targeted inhibition of tumor-associated angiogenesis. Control of the
inammatory responses in the tumor microenvironment where EP receptor-express-
ing cells are accumulating is also likely to be a novel therapeutic approach against
cancer, which now annually causes some 550,000 deaths in the United States. [52].
264 M. Majima
References
1. Folkman J (2007) Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug
Discov 6(4):273286
2. Carmeliet P (2005) Angiogenesis in life, disease and medicine. Nature 438(7070):932936
3. Ferrara N, Kerbel RS (2005) Angiogenesis as a therapeutic target. Nature
438(7070):967974
4. Jain RK (2003) Molecular regulation of vessel maturation. Nat Med 9(6):685693
5. Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of angiogenic switch during
tumorigenesis. Cell 86:353364
6. Katori M, Majima M (2000) Cyclooxygenase-2: its rich diversity of roles and possible applica-
tion of its selective inhibitors. Inamm Res 49:367392
7. Narumiya S et al (1999) Prostanoid receptors: structures, properties, and function. Physiol Rev
79:11931226
8. Sivakumar B, Harry LE, Paleolog EM (2004) Modulating angiogenesis: more vs. less. JAMA
292:972977
9. Neal MS (2001) Angiogenesis: is it the key to controlling the healing process? J Wound Care
10:281287
10. Ziche M, Jones J, Gullino PM (1982) Role of prostaglandin E1 and copper in angiogenesis. J
Natl Cancer Inst 69:475482
11. Spisni E, Manica F, Tomasi Y (1992) Involvement of prostanoids in the regulation of angio-
genesis by polypeptide growth factors. Prostaglandins Leukot Essent Fatty Acids 47:111115
12. Daniel TO et al (1999) Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endo-
thelial migration and angiogenesis. Cancer Res 59:45744577
13. Kamoshita E et al (2007) Recruitment of a prostaglandin E receptor subtype, EP3-expressing
bone marrow cells is crucial in wound-induced angiogenesis. Am J Pathol 169(4):14581472
14. Smalley W, Dubois RN (1997) Colorectal cancer and non steroidal anti-inammatory drugs.
Adv Pharmacol 39:120
15. Ohshima M et al (1996) Suppression of intestinal polyposis in Apc716 knockout mice by inhi-
bition of cyclooxygenase 2 (COX-2). Cell 87:803809
16. Norwood VF et al (2000) Postnatal development and progression of renal dysplasia in cyclo-
oxygenase-2 null mice. Kidney Int 58:22912300
17. Marx J (2001) Anti-inammatories inhibit cancer growthbut how? Science 291:581582
18. Prescott SM, White RL (1996) Self-promotion? Intimate connections between APC and pros-
taglandin H synthase-2. Cell 87:783786
19. Shiff SJ, Rigas B (1997) Nonsteroidal anti-inammatory drugs and colorectal cancer: evolving
concepts of their chemopreventive actions. Gastroenterology 113:19921998
20. Watanabe K et al (2000) Inhibitory effect of a prostaglandin E receptor subtype EP1 selective
antagonist, ONO-8713, on development of azoxymethane-induced aberrant crypt foci in mice.
Cancer Lett 156:5761
21. Watanabe K (1999) Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis.
Cancer Res 59:50935096
22. Sonoshita M et al (2001) Acceleration of intestinal polyposism through prostaglandin receptor
EP2 in Apc(Delta 716) knockout mice. Nat Med 7:10481051
23. Seno H et al (2002) Cyclooxygenase 2- and prostaglandin E2 receptor EP2-dependent angio-
genesis in Apc(Delta716) mouse intestinal polyps. Cancer Res 62:506511
24. Yang L et al (2003) Cancer-associated immunodeciency and dendritic cell abnormalities
mediated by the prostaglandin EP2 receptor. J Clin Invest 111:727735
18 Roles of Prostaglandins in Regulation of Pathological Angiogenesis 265
19.1 Introduction
Aortic aneurysm is the 13th leading cause of death in the United States, with
approximately 15,000 deaths per year [10]. Abdominal aortic aneurysm (AAA) is
the most common type, comprising more than 80 % of all aortic aneurysms [10].
19 Eicosanoids and Aortic Aneurysm 269
Among the prostanoids, PGE2 is one of the major products generated by the actions
of COX on AA and is well known to be an important mediator of inflammation,
fever, and pain [28, 29]. In human AAA tissues, COX-2-dependent PGE2 synthesis
is induced during the development of the disease [22, 30]. In aneurysm walls, COX-2
is widely expressed in macrophages and SMCs along with locally synthesized PGE2
[22]. PGE2 synthesized by macrophages and SMCs increases the production of
MMPs [31, 32] and stimulates the production of cytokines [22]. In addition to the
presence of PGE2 in AAA, it has been demonstrated that selective COX-2 inhibition,
as induced by celecoxib or genetic disruption of COX-2, decreased angiotensin
II-induced AAA formation in ApoE/ mice [33, 34]. Microsomal PGE2 synthase-1
(mPGES-1) is an inducible enzyme that catalyzes the isomerization of the COX
product PGH2 into PGE2 and is expressed in response to inflammatory cytokines
[35]. A study using deletion of mPGES-1 has also demonstrated that the inhibition
of COX-2-PGE2 decreased the rate of angiotensin II-induced AAA in ApoE/ mice
[36]. Progressed AAA is frequently associated with atherosclerosis. The biosynthe-
sis of PGE2 is increased in human atherosclerotic plaques [37] and has been impli-
cated in atherosclerotic plaque rupture as well [38]. This accumulating evidence
from both human and murine models of AAA suggests that COX-2-PGE2 signaling
is greatly involved in the pathogenesis of AAA.
19 Eicosanoids and Aortic Aneurysm 271
The biological effects of PGE2 depend on the prostanoid EP receptor subtypes EP1
through EP4 [28]. Although it has been well recognized that EP2, EP3, and EP4 are
present in vascular SMCs, EP4 is thought to play a pivotal role in AAA progression
[39]. Bayston et al. examined the expression of EPs in human AAA tissues and
found that EP2, EP3, and EP4 were expressed in AAA explants and that positive
immunoreaction against EP4 was detected primarily in macrophages. Because EP4
is a predominant PGE2 receptor expressed in macrophages [40], EP4 expression
appears to be increased during the development of AAA, contemporaneous with the
infiltration of macrophages.
In addition to macrophages, we have demonstrated through immunohistochemi-
cal examination that EP4 is abundantly expressed in the SMCs of human AAA
explants [2]. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)
has revealed that the expression levels of EP4 mRNA are greater in primary cultures
of SMCs from human AAA tissues than in those from normal aortae [2]. Interestingly,
EP4 expression and elastic fiber degradation were both enhanced in aneurysmal
areas compared to normal areas. Statistical analysis revealed that a significant cor-
relation existed between EP4 expression level and the degree of elastic fiber degra-
dation in eight human AAA tissue samples [2]. These studies of expression of PGE2
receptors suggest that EP4 is highly expressed in both macrophages and SMCs in
AAA.
Based on the expression profiles of prostanoid receptors in AAA, two groups inde-
pendently examined the role of EP4 signaling in AAA in murine models. They
obtained similar results, indicating that pharmacological inhibition of the EP4
receptor with ONO-AE3-208 significantly inhibited progression and rupture rate of
angiotensin II-induced AAA [2, 3]. Cao et al. demonstrated that the expression
levels of macrophage inflammatory protein (MIP)-1, interleukin-17 (IL-17), and
the pan-T-cell marker CD90.2 were lower in mouse aortae treated with
ONO-AE3-208 than in untreated aortae. Furthermore, in ONO-AE3-208-treated
aortae, activation of MMP2 and MMP9, both of which degrade elastic fibers, was
attenuated [3]. Not only pharmacological inhibition of EP4 but also global gene
deletion of the EP4 receptor significantly inhibited the progression of angiotensin
II- or calcium chloride-induced mouse AAA in which MMP2 and MMP9 activation
was attenuated [2]. These data showing EP4-mediated proteolytic activation sug-
gest that EP4 signaling promotes elastic fiber degradation.
We further examined the contribution of EP4 signaling to elastogenesis using
fetal arteries and found that activation of the EP4 receptor in SMCs inhibited elastic
fiber formation by inducing the degradation of lysyl oxidase, which crosslinks
272 U. Yokoyama et al.
elastin [41]. Previous reports have demonstrated that aortic aneurysms and coronary
dissections were related to a disturbance in lysyl oxidase expression in animal mod-
els and humans [42, 43]. Therefore, EP4 signaling may inhibit newly synthesized
elastic fiber formation in AAAs as well.
Expression of EP4 is not restricted to macrophages and SMCs, but is also found
in vascular endothelial cells [44]. In vascular endothelial cells, PGE2 may have a
role in angiogenesis. Activation of the EP4 receptor promoted in vitro tube forma-
tion of microvascular endothelial cells through protein kinase A (PKA) catalytic
subunit--mediated upregulation of endothelial nitric oxide synthase (eNOS) or
VEGF production [45, 46]. In vivo experiments have indicated EP4-mediated
angiogenesis as well [44, 45, 47]. Although the role of EP4 in the endothelial cells
of AAA remains unclear, available evidence suggests that EP4 signaling may con-
tribute to angiogenesis in atherosclerotic intima or adventitia.
The foregoing data, in particular those obtained in experiments using global
deletion or pharmacological inhibition of EP4, suggest that PGE2-EP4 signaling
promotes AAA progression. However, EP4 seems to have a contrasting function in
macrophages. To understand the cell type-specific effects of EP4, Tang et al. used
hypercholesterolemic low density lipoprotein receptor knockout mice transplanted
with either wild-type or EP4-deficient bone marrow and treated them with angioten-
sin II. When EP4 signaling was inhibited only in bone marrow-derived cells, inflam-
mation and angiotensin II-induced AAA formation were enhanced [1]. This change
most likely occurred because PGE2 in blood cells had an anti-inflammatory effect,
especially through reducing MCP-1 production [1].
Despite these conflicting results obtained with murine AAA models, some studies
using human AAA explants have suggested that PGE2-EP4 contributes to AAA
progression through pro-inflammatory and proteolytic actions. Bayston et al. dem-
onstrated that human AAA explants and macrophages isolated from aneurysm
biopsy specimens secreted large amounts of IL-6 [48]. This IL-6 secretion was
reduced in the presence of indomethacin, but the simultaneous addition of exoge-
nous PGE2 or 11-deoxy PGE1 partially reversed the indomethacin inhibition,
whereas the EP2 agonist butaprost or the EP1/3 agonist sulprostone had no effect
[48]. We have reported on EP4-mediated IL-6 production in human AAA explants
and SMCs isolated from human aneurysmal specimens [2] as well. Several reports
have demonstrated that IL-6 is produced by EP4 stimulation in various cell types
including macrophages, neutrophils, and fibroblasts [39]. Although downstream
signaling of EP4 has not been reported to contribute to IL-6 production in AAA,
Chen et al. have suggested that intracellular signaling events involving protein
kinase A (PKA), protein kinase C (PKC), p38 mitogen-activated protein kinase
(MAPK), and nuclear factor-kappa B (NF-B) contribute to IL-6 induction by EP4
stimulation [49].
19 Eicosanoids and Aortic Aneurysm 273
To examine the role of EP4 on proteolytic activation, we used human AAA explants
and SMCs isolated from human aneurysmal specimens, and found that EP4 stimula-
tion by ONO-AE1-329 increased MMP2 activation in both SMCs and vascular walls
of human AAA [2]. Furthermore, inhibition of EP4 signaling with the EP4 antagonist
ONO-AE3-208 reduced MMP2 activation and IL-6 production in cultured human
AAA explants [2]. Based on previous reports, the inhibition of EP4 is predicted to
rather enhance MCP-1 production in human AAA. In practice, however, inhibition of
EP4 did not enhance MCP-1 production in cultured human AAA explants treated with
EP4 antagonist ONO-AE3-208 at doses ranging from 1 to 100 nM [2].
Some clinical trials related to AAA have been conducted during the past decade.
Because early reports strongly suggested that the COX-2-PGE2 pathway was
responsible for AAA progression, COX-2 inhibitors were expected to inhibit
AAA. Contrary to these expectations, the administration of selective COX-2 inhibi-
tors actually increased the frequency of adverse cardiovascular events [50, 51].
These results may indicate the non-selective inhibition of prostanoid production.
Nonetheless, the inhibition of pathophysiological COX-2-dependent PGE2 signal-
ing would still remain an attractive therapeutic strategy. Selective inhibition of EP4
signaling may be a means of preventing aortic aneurysm formation. Further studies
will be required to clarify the possibility of systemic administration of an EP4
antagonist as a pharmacological therapeutic strategy in AAA.
LTs are pro-inflammatory lipid mediators derived from the 5-LO pathway of AA
metabolism. They are known to recruit and activate leukocytes at sites of inflamma-
tion [52, 53]. 5-LO, which acts in concert with 5-LO-activating protein (FLAP), is
a rate-limiting enzyme in the production of LTs. During the past decade, the roles of
the 5-LO-LT pathway in AAA have been extensively examined.
It is well recognized that 5-LO are abundantly expressed and downstream LTs
are robustly produced in human atherosclerotic plaques [54] and associated with
plaque instability [55]. In addition, Zhao et al. have demonstrated that 5-LO is
highly expressed in the macrophages of the adventitia in a cholate-containing ath-
erogenic diet-induced AAA model in ApoE/ mice [4]. Similar findings have been
reported using human AAA tissues. Biochemical analysis revealed an overexpres-
sion of 5-LO, FLAP, and LTC4 synthase, but not of LTA4 hydrolase, in the human
AAA wall [6]. These three proteins, required for CysLTs biosynthesis, are expressed
in the medial and adventitial layers of the AAA wall and are co-localized with
immune cells including macrophages and neutrophils [6]. The specific receptor for
LTB4 is BLT1, a high-affinity receptor expressed in leukocytes, vascular SMCs, and
endothelial cells that also mediates chemotaxis [56]. It has been demonstrated that
production of LTB4 and mRNA expression of BLT1 were increased in angiotensin
II-induced AAA in ApoE/ mice [7].
274 U. Yokoyama et al.
In accordance with these data on expression, other evidence has also demonstrated
that the 5-LO pathways including LTB4 and CysLTs are involved in the progression
of AAA. Funk and coworkers found a protective effect of 5-LO gene deletion
against cholate-containing atherogenic diet-induced AAA in ApoE/ mice [4].
Using a zymogram assay, the authors demonstrated that MMP2 activity in aneurys-
mal aortae and plasma levels of MIP-1 were attenuated by 5-LO gene deletion. An
in vitro experiment suggested the involvement of LTD4 in macrophage-derived
MIP-1 and endothelial cell-derived MIP-2 [4]. Another line of study also empha-
sized the role of CysLTs. Di Gennaro et al. demonstrated using HPLC that human
AAA explants converted AA into significant quantities of CysLTs and, to a lesser
extent, LTB4 [6]. In organ culture of human AAA explants, administration of LTD4
increased MMP2 activation, which was inhibited by the selective CysLT1 antago-
nist montelukast [6]. These data from mice and humans suggest that CysLTs such
as LTD4 play an important role in AAA. In keeping with the data on expression in
AAA, these data indicate that the 5-LO pathway contribute to adventitial inflamma-
tion in the vascular wall.
Although some papers have emphasized the role of CysLTs rather than LTB4, it
has been demonstrated that genetic deletion or pharmacological inhibition of BLT1
receptor has a protective effect on angiotensin II-induced AAA [7, 9]. In these stud-
ies, diminished AAA formation in BLT1-deficient mice was associated with signifi-
cant reductions in MMP2 and MMP9 and with the infiltration of macrophages and
CD4+ T cells [7]. Stimulation of LTB4, but not LTD4, increased MCP-1, MIP-2, and
IL-8 proteins in freshly isolated human monocytes [7]. Pharmacological inhibition
of BLT1, which can be achieved by CP-105696, decreased macrophage infiltration
and MMP2 activation [9]. Using human AAA explants, Houard et al. recently pro-
posed a role for LTB4, derived from neutrophils within the intraluminal thrombus,
as a chemotactic factor in AAA [8].
As recent research has identified the importance of inflammation via the 5-LO path-
way as a critical step in the initiation and perpetuation of atherosclerosis [57], drugs
that inhibit the 5-LO pathway are the subject of current vascular research. In a phase
II trial of the 5-LO inhibitor atreleuton, new coronary plaques were observed in
27.8 % of the placebo group but only 4.8 % of the treatment group [58]. Together
with two other phase II trials, these phase II findings on atreleuton demonstrated
inhibition of LTs, lowering of hsCRP, and potential for plaque stabilization.
Investigation of the FLAP inhibitor celiflapon was examined in a phase III trial in
2006 [58]. Despite the relatively positive results that have emerged from these
19 Eicosanoids and Aortic Aneurysm 275
clinical trials on atherosclerosis, no clinical trial using the 5-LO pathway to inhibit
AAA has been reported.
The effects of a compound that inhibits both the PGE2 and 5-LO pathways in
murine models of AAA have been recently reported [59]. A pirinixic acid derivative
called LP105 potently inhibited 5-LO [60], COX-1, and mPGES-1 [61]. The
researchers further demonstrated that LP105 interfered with the development of
AAA in an angiotensin II-induced model in ApoE/ mice [59]. In this model,
MMP9 and the expression levels of several inflammatory cytokines including IL-6,
IL-1, and TNF- were decreased by the administration of LP105.
19.5 Conclusions
Tunica
MMP-2 media
IL-6 SMC
PGE2
EP4
LTD4
Vasa
Vasorum LTB4 MIP-1
EP4
VEGF MIP-2 BLT1 CysLT1
MCP-1 Adventitia
Angiogenesis IL-8 Macrophage
Fig. 19.1 Schematic diagram of the roles of eicosanoids in the progression of abdominal aortic
aneurysm (AAA). Inflammatory infiltrates such as macrophages are enriched in the tunica adven-
titia of human AAA tissue. The expression of the PGE2 receptor EP4 is upregulated in smooth
muscle cells (SMCs) in AAA. EP4 signaling upregulates secretion of IL-6 in SMCs and induces
activation of the elastic fiber-degrading enzyme MMP2. Stimulation of microvascular endothelial
cells with EP4 induces VEGF expression and may contribute to the increased rate of neovascular-
ization into the tunica media. Adventitial macrophages express the LT receptors CysLT1 and BLT1.
LTD4 via CysLT1 upregulates MIP-1 expression, which may promote T-cell recruitment. LTB4 via
BLT1 induces secretion of several cytokines by macrophages, e.g., IL-8, MCP-1, and MIP2
276 U. Yokoyama et al.
activation and to promote degradation of elastic fibers in the medial layer. CysLTs
such as LTD4 and LTB4 are suggested to contribute to inflammatory response and
proteolytic activation in the adventitia. Chronic inflammation of both medial and
adventitial layers might synergistically exacerbate AAA.
Based on these findings, the regulation of PGE2 and 5-LO-LT signaling may be
useful for exploring pharmacological therapeutic strategies to treat or prevent AAA,
a condition for which no pharmacological treatment is currently available. Further
experimental and clinical studies are needed to determine the potential therapeutic
strategies targeting these drugs in AAA.
Acknowledgments This work was supported by grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (U.Y. and Y.I.), a Grant-in-Aid for Scientific Research on
Innovative Areas (U.Y.: 1123116514; YI, 22136009), the fund for Creation of Innovation Centers
for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation
Systems from the Ministry of Education, Culture, Sports, Science and Technology (U.Y.), the
Yokohama Foundation for Advanced Medical Science (U.Y.), the Vehicle Racing Commemorative
Foundation (U.Y.), and the Takeda Science Foundation (U.Y. and Y.I.).
References
13. Ruddy JM, Jones JA, Ikonomidis JS (2013) Pathophysiology of thoracic aortic aneurysm
(TAA): is it not one uniform aorta? Role of embryologic origin. Prog Cardiovasc Dis
56(1):6873
14. MacSweeney ST, Powell JT, Greenhalgh RM (1994) Pathogenesis of abdominal aortic aneu-
rysm. Br J Surg 81(7):935941
15. Guo DC et al (2006) Pathogenesis of thoracic and abdominal aortic aneurysms. Ann N Y Acad
Sci 1085:339352
16. MacSweeney ST et al (1993) High prevalence of unsuspected abdominal aortic aneurysm in
patients with confirmed symptomatic peripheral or cerebral arterial disease. Br J Surg
80(5):582584
17. Freestone T et al (1995) Inflammation and matrix metalloproteinases in the enlarging abdomi-
nal aortic aneurysm. Arterioscler Thromb Vasc Biol 15(8):11451151
18. Campa JS, Greenhalgh RM, Powell JT (1987) Elastin degradation in abdominal aortic aneu-
rysms. Atherosclerosis 65(1-2):1321
19. Longo GM et al (2002) Matrix metalloproteinases 2 and 9 work in concert to produce aortic
aneurysms. J Clin Invest 110(5):625632
20. Thompson RW, Parks WC (1996) Role of matrix metalloproteinases in abdominal aortic aneu-
rysms. Ann N Y Acad Sci 800:157174
21. Bobryshev YV, Lord RS (2001) Vascular-associated lymphoid tissue (VALT) involvement in
aortic aneurysm. Atherosclerosis 154(1):1521
22. Walton LJ et al (1999) Inhibition of prostaglandin E2 synthesis in abdominal aortic aneurysms:
implications for smooth muscle cell viability, inflammatory processes, and the expansion of
abdominal aortic aneurysms. Circulation 100(1):4854
23. Treska V et al (2002) Inflammation in the wall of abdominal aortic aneurysm and its role in the
symptomatology of aneurysm. Cytokines Cell Mol Ther 7(3):9197
24. Tieu BC et al (2009) An adventitial IL-6/MCP1 amplification loop accelerates macrophage-
mediated vascular inflammation leading to aortic dissection in mice. J Clin Invest
119(12):36373651
25. Holmes DR et al (1995) Medial neovascularization in abdominal aortic aneurysms: a histo-
pathologic marker of aneurysmal degeneration with pathophysiologic implications. J Vasc
Surg 21(5):761771; discussion 771772
26. Choke E et al (2006) Abdominal aortic aneurysm rupture is associated with increased medial
neovascularization and overexpression of proangiogenic cytokines. Arterioscler Thromb Vasc
Biol 26(9):20772082
27. Kaneko H et al (2011) Role of vascular endothelial growth factor-A in development of abdom-
inal aortic aneurysm. Cardiovasc Res 91(2):358367
28. Woodward DF, Jones RL, Narumiya S (2011) International Union of Basic and Clinical
Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress.
Pharmacol Rev 63(3):471538
29. Sugimoto Y, Narumiya S (2007) Prostaglandin E receptors. J Biol Chem 282(16):1161311617
30. Holmes DR et al (1997) Prostaglandin E2 synthesis and cyclooxygenase expression in abdomi-
nal aortic aneurysms. J Vasc Surg 25(5):810815
31. Khan KM, Howe LR, Falcone DJ (2004) Extracellular matrix-induced cyclooxygenase-2 reg-
ulates macrophage proteinase expression. J Biol Chem 279(21):2203922046
32. Corcoran ML et al (1992) Interleukin 4 inhibition of prostaglandin E2 synthesis blocks inter-
stitial collagenase and 92-kDa type IV collagenase/gelatinase production by human mono-
cytes. J Biol Chem 267(1):515519
33. King VL et al (2006) Selective cyclooxygenase-2 inhibition with celecoxib decreases angio-
tensin II-induced abdominal aortic aneurysm formation in mice. Arterioscler Thromb Vasc
Biol 26(5):11371143
34. Gitlin JM et al (2007) Genetic deficiency of cyclooxygenase-2 attenuates abdominal aortic
aneurysm formation in mice. Cardiovasc Res 73(1):227236
35. Samuelsson B, Morgenstern R, Jakobsson PJ (2007) Membrane prostaglandin E synthase-1: a
novel therapeutic target. Pharmacol Rev 59(3):207224
278 U. Yokoyama et al.
36. Wang M et al (2008) Microsomal prostaglandin E synthase-1 deletion suppresses oxidative stress
and angiotensin II-induced abdominal aortic aneurysm formation. Circulation 117(10):13021309
37. Cipollone F et al (2005) Association between prostaglandin E receptor subtype EP4 overex-
pression and unstable phenotype in atherosclerotic plaques in human. Arterioscler Thromb
Vasc Biol 25(9):19251931
38. Linton MF, Fazio S (2008) Cyclooxygenase products and atherosclerosis. Drug Discov Today
Ther Strateg 5(1):2536
39. Yokoyama U et al (2013) The prostanoid EP4 receptor and its signaling pathway. Pharmacol
Rev 65(3):10101052
40. Takayama K et al (2002) Prostaglandin E2 suppresses chemokine production in human macro-
phages through the EP4 receptor. J Biol Chem 277(46):4414744154
41. Yokoyama U et al (2014) Prostaglandin E2 inhibits elastogenesis in the ductus arteriosus via
EP4 signaling. Circulation 129(4):487496
42. Nakashima Y, Sueishi K (1992) Alteration of elastic architecture in the lathyritic rat aorta
implies the pathogenesis of aortic dissecting aneurysm. Am J Pathol 140(4):959969
43. Sibon I et al (2005) Lysyl oxidase deficiency: a new cause of human arterial dissection. Heart
91(5):e33
44. Rao R et al (2007) Prostaglandin E2EP4 receptor promotes endothelial cell migration via
ERK activation and angiogenesis in vivo. J Biol Chem 282(23):1695916968
45. Zhang Y, Daaka Y (2011) PGE2 promotes angiogenesis through EP4 and PKA Cgamma path-
way. Blood 118(19):53555364
46. Yanni SE et al (2009) The role of PGE2 receptor EP4 in pathologic ocular angiogenesis. Invest
Ophthalmol Vis Sci 50(11):54795486
47. Kuwano T et al (2004) Cyclooxygenase 2 is a key enzyme for inflammatory cytokine-induced
angiogenesis. FASEB J 18(2):300310
48. Bayston T et al (2003) Prostaglandin E2 receptors in abdominal aortic aneurysm and human
aortic smooth muscle cells. J Vasc Surg 38(2):354359
49. Chen BC et al (2006) Peptidoglycan-induced IL-6 production in RAW 264.7 macrophages is
mediated by cyclooxygenase-2, PGE2/PGE4 receptors, protein kinase A, I kappa B kinase, and
NF-kappa B. J Immunol 177(1):681693
50. Ray WA et al (2002) COX-2 selective non-steroidal anti-inflammatory drugs and risk of seri-
ous coronary heart disease. Lancet 360(9339):10711073
51. McGettigan P, Henry D (2006) Cardiovascular risk and inhibition of cyclooxygenase: a sys-
tematic review of the observational studies of selective and nonselective inhibitors of cyclo-
oxygenase 2. JAMA 296(13):16331644
52. Peters-Golden M, Henderson WR Jr (2007) Leukotrienes. N Engl J Med 357(18):18411854
53. Funk CD (2005) Leukotriene modifiers as potential therapeutics for cardiovascular disease.
Nat Rev Drug Discov 4(8):664672
54. Spanbroek R et al (2003) Expanding expression of the 5-lipoxygenase pathway within the
arterial wall during human atherogenesis. Proc Natl Acad Sci U S A 100(3):12381243
55. Cipollone F et al (2005) Association between 5-lipoxygenase expression and plaque instability
in humans. Arterioscler Thromb Vasc Biol 25(8):16651670
56. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science
294(5548):18711875
57. Vila L (2004) Cyclooxygenase and 5-lipoxygenase pathways in the vessel wall: role in athero-
sclerosis. Med Res Rev 24(4):399424
58. Berman JP, Farkouh ME, Rosenson RS (2013) Emerging anti-inflammatory drugs for athero-
sclerosis. Expert Opin Emerg Drugs 18(2):193205
59. Revermann M et al (2011) A pirinixic acid derivative (LP105) inhibits murine 5-lipoxygenase
activity and attenuates vascular remodelling in a murine model of aortic aneurysm. Br J
Pharmacol 163(8):17211732
60. Werz O et al (2008) Novel and potent inhibitors of 5-lipoxygenase product synthesis based on
the structure of pirinixic acid. J Med Chem 51(17):54495453
61. Koeberle A et al (2008) Pirinixic acid derivatives as novel dual inhibitors of microsomal pros-
taglandin E2 synthase-1 and 5-lipoxygenase. J Med Chem 51(24):80688076
Chapter 20
Cysteinyl Leukotrienes and Disease
20.1 Introduction
decades after the derivation of their chemical structures, they have fulfilled Kochs
postulates in that regard. Cys-LT biosynthesis is a characteristic feature of asthma
and allergic rhinitis [3, 4] and increases concomitantly with disease activity [5].
Pharmacological inhibitors of 5-LO and the type 1 receptor for cys-LTs (CysLT1R),
respectively, are among the first successful treatments for asthma that were devel-
oped based on specific molecular targets. Their efficacy validates the role of the
cys-LTs in asthma pathogenesis.
This chapter focuses primarily on the role of the cys-LTs in asthma in humans
because of the wealth of pharmacological, functional, immunohistochemical, and
physiological data in the area. Nonetheless, an emerging body of evidence also
implicates their function in cardiovascular disease [610], respiratory viral infec-
tions [11], obstructive sleep apnea [1215], chronic urticaria [16, 17], and Henoch
Schoenlein vasculitis [18]. Confirmatory therapeutic intervention studies are
necessary to firmly implicate cys-LTs in the pathobiology of these conditions.
Additional functions of cys-LTs identified in mouse model systems are covered
elsewhere in this volume, and mouse studies are discussed only in instances that
directly inform observations made in humans.
Fig. 20.1 Leukotriene biosynthesis. 5-Lipoxygenase (5-LO) and 5-LO-activating protein (FLAP)
metabolize arachidonic acid to leukotriene (LT)A4, which can be converted to LTB4 (by LTA4H) or
the cysteinyl leukotrienes (cys-LTs), by leukotriene C4 synthase (LTC4S). LTs then bind to the
indicated G protein-coupled receptors. cPLA2 cytosolic phospholipase A2, BLT leukotriene B
receptor, DiP dipeptidase, g-GL g-glutamyl leukotrienase, g-GT g-glutamyl transpeptidase,
5-HPETE 5-hydroxyperoxyeicosatetraenoic acid. (Reprinted from Annals of Allergy, Asthma, &
Immunology, Volume 111/No. 3. Fanning LB and Boyce JA, Lipid mediators and allergic diseases,
pp. 155162. Copyright 2013, with permission from Elsevier.)
Mast cells [28], basophils [29], eosinophils [30], monocyte/macrophages [31], and
myeloid dendritic cells [32] all possess the complete repertoire of enzymes and
proteins needed to convert endogenous arachidonic acid to LTC4. In allergic dis-
eases, cys-LTs are thought to originate from eosinophils and basophils (which accu-
mulate at sites of inflammation) and activated resident tissue mast cells (which
respond to cross-linkage of their high-affinity receptor for IgE, FcRI, in response
to specific allergens). In bronchial biopsies from atopic asthmatic individuals, mast
cells are the primary site of localization of LTC4S [33], potentially reflecting the
strongly inducible nature of the enzyme in this cell type when exposed to the Th2
cytokine IL-4 [34]. Cys-LT concentrations in the bronchoalveolar lavage fluid
obtained from atopic asthmatic subjects increase sharply following challenge by
inhalation with a specific allergen [3], concomitantly with the release of other mast
cell-derived mediators (histamine, tryptase, and prostaglandin D2 (PGD2)) [3537]
282 L.B. Fanning and J.A. Boyce
that are released or generated de novo following allergen-induced mast cell activa-
tion. Basophils, which also express FcRI and respond to allergen, are recruited to
the bronchial mucosa in the hours after challenge and may contribute to the produc-
tion of cys-LTs during the allergen-induced late-phase response [38]. Increases in
urinary LTE4 occurring following anaphylaxis likely reflect contributions from both
mast cells and basophils [39]. Patients with eosinophilic pneumonia exhibit mark-
edly elevated levels of urinary LTE4 relative to asthmatic and nonasthmatic controls
[40], likely reflecting the large burden of eosinophils capable of LTC4 generation.
Similar to the foregoing myeloid cells, platelets express LTC4S [41]. However,
platelets lack 5-LO and FLAP and therefore cannot generate LTA4 from endogenous
arachidonic acid. However, platelets can convert LTA4 to LTC4 if the unstable pre-
cursor is provided from an adjacent cell source [42]. Activated platelets display
P-selectin on their surfaces, which permits their binding to neutrophils, monocytes,
and eosinophils, all of which have an active 5-LO pathway [43]. Activated neutro-
phils, in particular, generate LTA4 in molar excess of their capacity to convert it to
LTB4, their sole terminal 5-LO pathway product. The excess LTA4, once released,
can be converted to LTC4 by the adherent platelets. Thus, platelet-adherent neutro-
phils comprise a de novo unit potentially capable of amplifying the production of
cys-LTs in circumstances in which platelets become activated by tissue injury or
inflammation. In addition, the presence of adherent platelets primes neutrophils for
augmented 5-LO pathway activity by an unknown mechanism [44]. The bidirec-
tional cooperation between neutrophils and platelets may markedly amplify the pro-
duction of cys-LTs, particularly in aspirin-exacerbated respiratory disease (AERD)
[41], in which plateletleukocyte complexes are detected with aberrantly high
frequencies.
The effects of inhaled cys-LTs on airway physiology were studied in healthy and
asthmatic volunteers subjected to challenges with LTC4, LTD4, or LTE4. The doses
of both LTC4 [49] and LTD4 [50] required to induce bronchoconstriction in healthy
subjects were 1000 to 5000 fold lower than the doses of histamine necessary to
induce an equivalent fall in airflow. The cys-LT-mediated changes in lung function
20 Cysteinyl Leukotrienes and Disease 283
were substantially more prolonged than those in response to histamine and notewor-
thy for the lack of associated cough or hoarseness. The fall in lung function induced
by inhalation of LTD4 occurred more rapidly than that induced by LTC4 [51]. This
difference, in retrospect, might reflect the time required for LTC4 to be converted to
LTD4, or the differences between the two ligands in their receptor-binding proper-
ties. Although LTD4 was also a markedly potent bronchoconstrictor in subjects with
asthma [51], asthmatic subjects were not substantially more responsive to LTD4
than were nonasthmatic controls when corrected for histamine reactivity.
Similar to LTC4 and LTD4, LTE4 also causes bronchoconstriction, but is less
potent than its precursors, inducing reductions in airflow at doses about 1 log lower
than histamine in normal and asthmatic subjects [52]. A separate study, however,
noted that subjects with asthma exhibited selective hyperresponsiveness to LTE4,
but not to LTC4, when compared with nonasthmatic individuals (16 fold more sensi-
tive) [53]. Moreover, individuals with AERD demonstrated even greater sensitivity
(~1 log fold) to LTE4, but identical responsiveness to LTC4, when compared with
aspirin-tolerant asthmatics [54]. These observations fueled speculation that LTE4
acted by receptor-dependent mechanisms distinct from those of LTC4 and LTD4,
and that these mechanisms might be altered as a consequence (and potentially a
pathogenetic factor) of asthma.
Preincubation of guinea pig tracheal smooth muscle with LTE4 shifted the sensitiv-
ity of the organ to constriction in response to the subsequent administration of his-
tamine [45]. This potentiation of histamine sensitivity was not observed in response
to LTC4 or LTD4 and could be blocked by treatment of the tracheal rings with the
cyclooxygenase (COX) inhibitor indomethacin. Pretreatment with LTE4 also poten-
tiated histamine contractility of surgically excised human bronchi. This potentiation
was inhibited by pretreatment with either indomethacin or with a T-prostanoid (TP)
receptor antagonist [55]. In subjects with asthma, inhalation of LTE4 shifted the
doseresponse curve to inhaled histamine challenge at 4 and 7 h [56]. As predicted
from the in vitro studies, this effect was sensitive to inhibition by the oral adminis-
tration of indomethacin. Thus, LTE4 enhances end-organ reactivity to histamine
through induction of COX product(s), potentially thromboxane A2 (TXA2) or PGD2,
both of which mediate their effects on the airway by TP receptors [57, 58].
When inhaled by subjects with mild asthma, LTE4 caused an increase in the
numbers of eosinophils, and to a lesser extent, neutrophils, in bronchial biopsies
obtained 4 h after the inhalation challenge [59]. In a second study, subjects with
asthma underwent sputum induction (as well as bronchial biopsies in a subset) after
inhalations of LTD4, LTE4, and diluent. LTE4, but not LTD4, caused sharp increases
an increase in sputum eosinophils at both 7 and 24 h, and sputum basophils at 7 h
when the dosages of the two cys-LTs were titrated to achieve a comparable degree
284 L.B. Fanning and J.A. Boyce
Although the mechanisms and cell targets that account for LTE4-induced COX acti-
vation and eosinophilic pulmonary inflammation in humans remain to be deter-
mined, in vitro and in vivo models support very different mechanisms of action for
LTE4 than for LTC4 or LTD4. Although the two classical G protein-coupled recep-
tors (GPCRs) for cys-LTs (CysLT1R and the type 2 receptor for cys-LTs, CysLT2R)
are expressed by both hematopoietic and nonhematopoietic cells in human airways
[47, 61], neither of these receptors binds LTE4 with high affinity [46, 47]. One study
demonstrated that LTE4, but not LTC4 or LTD4, could induce the production of PGD2
by a human mast cell line, LAD2 [62]. Importantly, this induction was not altered
by knockdown of either CysLT1R or CysLT2R, and required LTE4-mediated upregu-
lation of COX-2 and signaling through peroxisome proliferator-activated receptor-
(PPAR-). Because PGD2 is both a bronchoconstrictor (acting at TP receptors) [57]
and is chemotactic for eosinophils and basophils [acting at D-prostanoid 2 (DP2)
receptors] [63], the unique properties of LTE4 could reflect, in part, the activation of
pulmonary mast cells and secondary generation of PGD2.
In another study, intrapulmonary administration of LTE4, but not LTD4, potenti-
ated the recruitment of eosinophils to the airways of ovalbumin (OVA)-sensitized
mice if administered with low-dose OVA [64]. This mechanism was independent of
CysLT1R and CysLT2R, but depended exquisitely on platelets and P2Y12 receptors,
which recognize adenosine diphosphate (ADP). The requirement for platelets was
not the result of direct platelet activation by LTE4, suggesting that platelets in this
model may be the target of a mediator released by another cell type that expresses a
true LTE4 receptor. Moreover, P2Y12 receptors do not bind LTE4 directly, but are
involved in certain LTE4-initiated signaling functions [65]. For example, knock-
down of P2Y12 receptors in LAD2 cells markedly suppressed the capacity of LTE4
to induce PGD2 generation and chemokine production [64]. It seems possible that
P2Y12 receptors, which amplify activation signals to other agonists through auto-
crine ADP-mediated circuits in platelets [66], could amplify signaling through a
true LTE4 receptor on mast cells or other cell types. To date, the recently identified
GPR99 is the only GPCR with a preference for LTE4 binding [48]. The distribution
of GPR99, its expression in human airways, and its role in responses to LTE4 in
general and asthma in particular remain to be determined.
20 Cysteinyl Leukotrienes and Disease 285
As noted, a wide range of hematopoietic cells express CysLT1R and CysLT2R [67
70]. Several in vitro studies support potentially important functions of cys-LTs in
activating immune effector cells relevant to asthma. Because primary cells express
various combinations of two or more cys-LT receptors (including yet to be identi-
fied potential receptors), their functional responses to cys-LTs ex vivo often diverge
from the pharmacology predicted from studies of transfected receptors overex-
pressed in isolation. These divergent responses reflect physical or functional inter-
actions between known receptors [71], as well as the potential existence of
previously unidentified receptors. Additionally, the expressions of cys-LT receptors
on cells that also generate cys-LTs (e.g., mast cells, eosinophils) suggest the poten-
tial for autocrine/intracrine signaling functions.
Human mast cells derived in vitro from both cord blood [68, 69] and peripheral
blood [72] express both CysLT1R and CysLT2R. LTC4 and LTD4 both elicit strong
calcium fluxes [68] and phosphorylation of extracellular signal-related kinase
(ERK) [73] in primary cord blood mast cells, as well as the LAD2 cell line [62].
Priming of primary human cord blood mast cells with the Th2 cytokine IL-4 ampli-
fies both calcium flux (particularly in response to LTC4) and ERK activation in
response to the cys-LTs without changing the levels of CysLT1R or CysLT2R expres-
sion [68, 74]. This action differs from that in monocytes, in which IL-4 and IL-13
upregulate the expression of CysLT1R [75] and CysLT2R [76]. LTC4 and LTD4 both
behave as potent agonists for the production of cytokines (IL-5, tumor necrosis fac-
tor, and IL-13) by IL-4-primed human mast cells by a CysLT1R-dependent mecha-
nism [73]. LTD4 also induces proliferation of human mast cells derived from cord
blood or peripheral blood by inducing CysLT1R-dependent transactivation of the
c-kit tyrosine kinase [72, 77]. Interestingly, knockdown of CysLT2 receptors aug-
ments both proliferation and cytokine generation by mast cells in response to stimu-
lation by LTD4, indicating that CysLT2R inhibits CysLT1R functions on this cell
type [71, 77]. CysLT2 forms heterodimers with CysLT1R on mast cells, interfering
with its presentation at the plasma membrane [71]. IL-4, an accessory mitogen for
mast cells, requires both the induced expression of LTC4S and endogenous function
of CysLT1R to drive mast cell proliferation [72]. Of note, both CysLT1R and
CysLT2R proteins localize to the nuclear envelope of human mast cells (the location
of LTC4S), and are thus potentially positioned to induce intracrine signaling in
response to newly synthesized LTC4.
286 L.B. Fanning and J.A. Boyce
20.4.2 Eosinophils
Eosinophils express both CysLT1R and CysLT2R, with the latter receptor being
expressed far more abundantly at the mRNA level [78]. Human peripheral blood
eosinophils secrete IL-4 in response to stimulation with IL-16, eotaxin, or RANTES
by a mechanism that is sensitive to the inhibition of 5-LO, suggesting a requirement
for the synthesis of endogenous cys-LTs [70]. Interestingly, permeabilized eosino-
phils secrete IL-4 in response to LTC4, but not LTD4 or LTE4, by a mechanism that
is resistant to conventional CysLT1R antagonists but sensitive to pertussis toxin
[70]. These findings argue that intracellular cys-LT receptors exhibit a preference
for LTC4, again supporting an intracrine function.
20.4.3 Lymphocytes
Human CD4+ Th2 cells express CysLT1R, as well as the DP2 receptor for PGD2.
Stimulation of human Th2 cells with PGD2 ex vivo induces the release of IL-5 and
IL-13 [79]. Although cys-LTs are comparatively weak as agonists for cytokine gen-
eration by Th2 cells, they synergize markedly with PGD2 for this function, with
LTE4 being more potent than either LTC4 or LTD4 [80]. The effect of LTE4 on T cells
is blocked by treatment of the cells with the CysLT1R antagonist montelukast. The
sensitivity to montelukast is at odds with the rank-order potency of LTE4 over LTD4
for these T-cell-directed effects. Whether a montelukast-sensitive receptor for LTE4
that is not CysLT1R exists is unknown but has been suggested by previous studies of
LAD2 cells [62]. The recently identified LTE4 receptor GPR99 is resistant to block-
ade by CysLT1R antagonists and therefore not likely to account for the effects of
LTE4 observed in T cells [48].
Type 2 innate lymphoid helper cells (ILC2 cells) are robust sources of Th2-type
cytokines that reside constitutively in tissues and are activated directly by epitheli-
ally derived cytokines such as IL-33, IL-25 and thymic stromal lymphopoietin [81].
Similar to Th2 cells, ILC2 cells express DP2 receptors [81], and respond to the
combination of PGD2 and cys-LTs in a synergistic manner by generating IL-13 and
IL-5, as well as IL-9, GM-CSF, and IL-8 [82]. Interestingly, although ILC2 cells do
not produce IL-4 when activated by IL-33 or IL-25, they generate significant
amounts of this cytokine when activated by mast cell supernatants, an activity attrib-
uted to the combination of PGD2 and cys-LTs [82]. The cys-LT receptor(s) expressed
by human ILC2 cells are not yet known, although their counterparts in the mouse
lung strongly express CysLT1R, and generate IL-4, IL-5, and IL-13 when stimulated
with LTD4 or LTE4 [83]. Interestingly, the effects of LTD4 in the mouse lung ILC2
cells are blocked by montelukast, but the effects of LTE4 are montelukast resistant,
suggesting the potential contribution from GPR99 or a different LTE4 receptor.
20 Cysteinyl Leukotrienes and Disease 287
20.4.4 Platelets
Human platelets express both CysLT1R and CysLT2R proteins [84, 85]. Stimulation
of human platelets with LTC4, LTD4, and LTE4 elicits their release of the chemokine
RANTES [84]. A recent mouse study indicated that LTC4 can strongly potentiate
allergen-induced eosinophilic pulmonary inflammation through the activation of
platelets through CysLT2R [85]. Whether platelets have analogous CysLT2R-
dependent functions in humans is presently unknown.
Myeloid dendritic cells in human peripheral blood express CysLT1R [86]. The
administration of montelukast to atopic asthmatic individuals blocked the reduction
in blood dendritic cells elicited by allergen inhalation challenges. Several in vitro
studies also suggest a role for cys-LTs in modulating dendritic cell function. Human
monocyte-derived dendritic cells (moDCs) matured with LPS had a 50 % reduction
in CysLT1R expression but an increase in CysLT2R. In contrast, moDCs treated with
polyI:C had no change in receptor expression. This downregulation of CysLT1R by
LPS was prevented by COX inhibitors. Furthermore, DCs matured with polyI:C
demonstrated chemotaxis in response to LTD4, as well as increased migration in
response to CCL19. This response to LTD4 was seen only weakly in DCs matured
with LPS [87]. Human moDCs cultured with LTC4 release the eosinophil chemoat-
tractant RANTES and induce T-cell proliferation. Both of these effects are blocked
by montelukast [88]. Whether CysLT1R participates in priming for Th2 responses to
dust mite and mold allergens in humans, as it does in mice [89], remains to be seen.
The potency of the cys-LTs as contractile agonists for human airways, and the
observation that urinary levels of LTE4 were elevated in asthmatic subjects present-
ing to the emergency room for spontaneous disease exacerbations [5], suggested
that drugs targeting the synthesis of cys-LTs or blocking their receptors had thera-
peutic potential in asthma. Indeed, the 5-LO inhibitor zileuton and the antagonists
of CysLT1R were among the earliest drugs developed for the treatment of asthma
that were based on a disease-related molecular target.
288 L.B. Fanning and J.A. Boyce
Clinical responses to zileuton and CysLT1R antagonists are not uniform and are at
least partly determined by environmental and genetic controls. Asthmatic children
exposed to cigarette smoke exhibit significantly greater protection from albuterol
usage when treated with montelukast than do children not exposed to smoke [103],
and children with the highest ratios of urinary LTE4 to exhaled nitric oxide are the
most protected [104]. Promoter polymorphisms in the ALOX5 gene (encoding
5-LO) that alter transcription of the enzyme significantly influence the extent to
which treatment with zileuton changes baseline lung function [105107]. The pres-
ence of a common polymorphic variant (A(444)C) of LTC4S, the gene encoding
LTC4S, was associated with a larger increment in baseline FEV1 with administration
20 Cysteinyl Leukotrienes and Disease 289
20.7 Conclusions
Decades after their discovery, the cys-LTs remain a topic of intense interest and to
date are the only successful pharmacotherapeutic mediator target for asthma. The
application of molecular technology and experimental systems have verified the
identity of at least three receptors, suggesting additional potential therapeutic appli-
cations. The astonishing degree of heterogeneity among human subjects in both the
production of cys-LTs and the responsiveness to cys-LTs (as exemplified by studies
20 Cysteinyl Leukotrienes and Disease 293
of AERD), the range of responsiveness to the targeted therapies, and the numbers of
mechanisms that up- or downregulate components of the system hold clues both to
fundamental biology and to underlying genetic and epigenetic modifications that
might serve as more precise guides to therapy in the future.
References
1. Weiss JW, Drazen JM, McFadden ER Jr, Weller PF, Corey EJ, Lewis RA, Austen KF (1982)
Comparative bronchoconstrictor effects of histamine, leukotriene C, and leukotriene D in
normal human volunteers. Trans Assoc Am Physicians 95:3035
2. Soter NA, Lewis RA, Corey EJ, Austen KF (1983) Local effects of synthetic leukotrienes
(LTC4, LTD4, LTE4, and LTB4) in human skin. J Invest Dermatol 80(2):115119
3. Wenzel SE, Larsen GL, Johnston K, Voelkel NF, Westcott JY (1990) Elevated levels of leu-
kotriene C4 in bronchoalveolar lavage fluid from atopic asthmatics after endobronchial aller-
gen challenge. Am Rev Respir Dis 142(1):112119
4. Figueroa DJ, Borish L, Baramki D, Philip G, Austin CP, Evans JF (2003) Expression of cys-
teinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal
allergic rhinitis. Clin Exp Allergy 33(10):13801388
5. Drazen JM, OBrien J, Sparrow D, Weiss ST, Martins MA, Israel E, Fanta CH (1992)
Recovery of leukotriene E4 from the urine of patients with airway obstruction. Am Rev
Respir Dis 146(1):104108
6. Iovannisci DM, Lammer EJ, Steiner L, Cheng S, Mahoney LT, Davis PH, Lauer RM, Burns
TL (2007) Association between a leukotriene C4 synthase gene promoter polymorphism and
coronary artery calcium in young women: the Muscatine Study. Arterioscler Thromb Vasc
Biol 27(2):394399
7. Freiberg JJ, Tybjaerg-Hansen A, Sillesen H, Jensen GB, Nordestgaard BG (2008) Promotor
polymorphisms in leukotriene C4 synthase and risk of ischemic cerebrovascular disease.
Arterioscler Thromb Vasc Biol 28(5):990996
8. Freiberg JJ, Tybjaerg-Hansen A, Nordestgaard BG (2010) Novel mutations in leukotriene C4
synthase and risk of cardiovascular disease based on genotypes from 50,000 individuals. J
Thromb Haemost 8(8):16941701
9. Maznyczka A, Braund P, Mangino M, Samani NJ (2008) Arachidonate 5-lipoxygenase
(5-LO) promoter genotype and risk of myocardial infarction: a case-control study.
Atherosclerosis 199(2):328332
10. Ingelsson E, Yin L, Back M (2012) Nationwide cohort study of the leukotriene receptor
antagonist montelukast and incident or recurrent cardiovascular disease. J Allergy Clin
Immunol 129(3):702707
11. Bisgaard H (2003) A randomized trial of montelukast in respiratory syncytial virus postbron-
chiolitis. Am J Respir Crit Care Med 167(3):379383
12. Goldbart AD, Goldman JL, Veling MC, Gozal D (2005) Leukotriene modifier therapy for
mild sleep-disordered breathing in children. Am J Respir Crit Care Med 172(3):364370
13. Goldbart AD, Krishna J, Li RC, Serpero LD, Gozal D (2006) Inflammatory mediators in
exhaled breath condensate of children with obstructive sleep apnea syndrome. Chest
130(1):143148
14. Stanke-Labesque F, Back M, Lefebvre B, Tamisier R, Baguet JP, Arnol N, Levy P, Pepin JL
(2009) Increased urinary leukotriene E4 excretion in obstructive sleep apnea: effects of obe-
sity and hypoxia. J Allergy Clin Immunol 124(2):364370
15. Shen Y, Xu Z, Shen K (2011) Urinary leukotriene E4, obesity, and adenotonsillar hypertrophy
in Chinese children with sleep disordered breathing. Sleep 34(8):1135041
294 L.B. Fanning and J.A. Boyce
16. Erbagci Z (2002) The leukotriene receptor antagonist montelukast in the treatment of chronic
idiopathic urticaria: a single-blind, placebo-controlled, crossover clinical study. J Allergy
Clin Immunol 110(3):484488
17. Khan S, Lynch N (2012) Efficacy of montelukast as added therapy in patients with chronic
idiopathic urticaria. Inflamm Allergy Drug Targets 11(3):235243
18. Wu SH, Liao PY, Chen XQ, Yin PL, Dong L (2014) Add-on therapy with montelukast in
treatment of Henoch-Schonlein purpura. Pediatr Int 56(3):315322
19. Clark JD, Milona N, Knopf JL (1990) Purification of a 110-kilodalton cytosolic phospholi-
pase A2 from the human monocytic cell line U937. Proc Natl Acad Sci U S A
87(19):77087712
20. Malaviya R, Malaviya R, Jakschik BA (1993) Reversible translocation of 5-lipoxygenase in
mast cells upon IgE/antigen stimulation. J Biol Chem 268(7):49394944
21. Reid GK, Kargman S, Vickers PJ, Mancini JA, Leveille C, Ethier D, Miller DK, Gillard JW,
Dixon RA, Evans JF (1990) Correlation between expression of 5-lipoxygenase-activating
protein, 5-lipoxygenase, and cellular leukotriene synthesis. J Biol Chem
265(32):1981819823
22. Lam BK, Penrose JF, Freeman GJ, Austen KF (1994) Expression cloning of a cDNA for
human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathi-
one to leukotriene A4. Proc Natl Acad Sci U S A 91(16):76637667
23. Leier I, Jedlitschky G, Buchholz U, Cole SP, Deeley RG, Keppler D (1994) The MRP gene
encodes an ATP-dependent export pump for leukotriene C4 and structurally related conju-
gates. J Biol Chem 269(45):2780727810
24. Shi ZZ, Han B, Habib GM, Matzuk MM, Lieberman MW (2001) Disruption of gamma-
glutamyl leukotrienase results in disruption of leukotriene D4 synthesis in vivo and attenua-
tion of the acute inflammatory response. Mol Cell Biol 21(16):53895395
25. Lee CW, Lewis RA, Corey EJ, Austen KF (1983) Conversion of leukotriene D4 to leukotriene
E4 by a dipeptidase released from the specific granule of human polymorphonuclear leuco-
cytes. Immunology 48(1):2735
26. Asano K, Lilly CM, ODonnell WJ, Israel E, Fischer A, Ransil BJ, Drazen JM (1995) Diurnal
variation of urinary leukotriene E4 and histamine excretion rates in normal subjects and
patients with mild-to-moderate asthma. J Allergy Clin Immunol 96(5 pt 1):643651
27. Fanning LB, Boyce JA (2013) Lipid mediators and allergic diseases. Ann Allergy Asthma
Immunol 111(3):155162
28. Murphy RC, Hammarstrom S, Samuelsson B (1979) Leukotriene C: a slow-reacting sub-
stance from murine mastocytoma cells. Proc Natl Acad Sci U S A 76(9):42754279
29. Warner JA, Peters SP, Lichtenstein LM, Hubbard W, Yancey KB, Stevenson HC, Miller PJ,
MacGlashan DW Jr (1989) Differential release of mediators from human basophils: differ-
ences in arachidonic acid metabolism following activation by unrelated stimuli. J Leukoc
Biol 45(6):558571
30. Weller PF, Lee CW, Foster DW, Corey EJ, Austen KF, Lewis RA (1983) Generation and
metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant
production of leukotriene C4. Proc Natl Acad Sci U S A 80(24):76267630
31. Abe M, Hugli TE (1988) Characterization of leukotriene C4 synthetase in mouse peritoneal
exudate cells. Biochim Biophys Acta 959(3):386398
32. Barrett NA, Maekawa A, Rahman OM, Austen KF, Kanaoka Y (2009) Dectin-2 recognition
of house dust mite triggers cysteinyl leukotriene generation by dendritic cells. J Immunol
182(2):11191128
33. Seymour ML, Rak S, Aberg D, Riise GC, Penrose JF, Kanaoka Y, Austen KF, Holgate ST,
Sampson AP (2001) Leukotriene and prostanoid pathway enzymes in bronchial biopsies of
seasonal allergic asthmatics. Am J Respir Crit Care Med 164(11):20512056
20 Cysteinyl Leukotrienes and Disease 295
34. Hsieh FH, Lam BK, Penrose JF, Austen KF, Boyce JA (2001) T helper cell type 2 cytokines
coordinately regulate immunoglobulin E-dependent cysteinyl leukotriene production by
human cord blood-derived mast cells: profound induction of leukotriene C4 synthase expres-
sion by interleukin 4. J Exp Med 193(1):123133
35. Wenzel SE, Westcott JY, Smith HR, Larsen GL (1989) Spectrum of prostanoid release after
bronchoalveolar allergen challenge in atopic asthmatics and in control groups. An alteration
in the ratio of bronchoconstrictive to bronchoprotective mediators. Am Rev Respir Dis
139(2):450457
36. Wenzel SE, Westcott JY, Larsen GL (1991) Bronchoalveolar lavage fluid mediator levels 5
minutes after allergen challenge in atopic subjects with asthma: relationship to the develop-
ment of late asthmatic responses. J Allergy Clin Immunol 87(2):540548
37. Wenzel SE, Fowler AA III, Schwartz LB (1988) Activation of pulmonary mast cells by bron-
choalveolar allergen challenge. In vivo release of histamine and tryptase in atopic subjects
with and without asthma. Am Rev Respir Dis 137(5):10021008
38. Gauvreau GM, Watson RM, OByrne PM (1999) Protective effects of inhaled PGE2 on
allergen-induced airway responses and airway inflammation. Am J Respir Crit Care Med
159(1):3136
39. Taniguchi M, Higashi N, Ono E, Mita H, Akiyama K (2008) Hyperleukotrieneuria in patients
with allergic and inflammatory disease. Allergol Int 57(4):313320
40. Ono E, Taniguchi M, Mita H, Higashi N, Fukutomi Y, Tanimoto H, Sekiya K, Oshikata C,
Tsuburai T, Tsurikisawa N et al (2008) Increased urinary leukotriene E4 concentration in
patients with eosinophilic pneumonia. Eur Respir J 32(2):437442
41. Laidlaw TM, Kidder MS, Bhattacharyya N, Xing W, Shen S, Milne GL, Castells MC, Chhay
H, Boyce JA (2012) Cysteinyl leukotriene overproduction in aspirin-exacerbated respiratory
disease is driven by platelet-adherent leukocytes. Blood 119(16):37903798
42. Maclouf JA, Murphy RC (1988) Transcellular metabolism of neutrophil-derived leukotriene
A4 by human platelets. A potential cellular source of leukotriene C4. J Biol Chem
263(1):174181
43. Maugeri N, Evangelista V, Celardo A, DellElba G, Martelli N, Piccardoni P, de Gaetano G,
Cerletti C (1994) Polymorphonuclear leukocyteplatelet interaction: role of P-selectin in
thromboxane B2 and leukotriene C4 cooperative synthesis. Thromb Haemost 72(3):450456
44. Laidlaw TM, Cutler AJ, Kidder MS, Liu T, Cardet JC, Chhay H, Feng C, Boyce JA (2014)
Prostaglandin E resistance in granulocytes from patients with aspirin-exacerbated respiratory
disease. J Allergy Clin Immunol 133:16921701
45. Lee TH, Austen KF, Corey EJ, Drazen JM (1984) Leukotriene E4-induced airway hyperre-
sponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three sepa-
rate sulfidopeptide leukotriene receptors. Proc Natl Acad Sci U S A 81(15):49224925
46. Lynch KR, ONeill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M,
Figueroa DJ, Zeng Z et al (1999) Characterization of the human cysteinyl leukotriene CysLT1
receptor. Nature 399(6738):789793
47. Heise CE, ODowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN,
Abramovitz M, Cheng R et al (2000) Characterization of the human cysteinyl leukotriene 2
receptor. J Biol Chem 275(39):3053130536
48. Kanaoka Y, Maekawa A, Austen KF (2013) Identification of GPR99 as a potential third cyste-
inyl leukotriene receptor with a preference for leukotriene E4. J Biol Chem 288:1096710972
49. Weiss JW, Drazen JM, Coles N, McFadden ER Jr, Weller PF, Corey EJ, Lewis RA, Austen
KF (1982) Bronchoconstrictor effects of leukotriene C in humans. Science
216(4542):196198
50. Weiss JW, Drazen JM, McFadden ER Jr, Weller P, Corey EJ, Lewis RA, Austen KF (1983)
Airway constriction in normal humans produced by inhalation of leukotriene D. Potency,
time course, and effect of aspirin therapy. JAMA 249(20):28142817
296 L.B. Fanning and J.A. Boyce
51. Griffin M, Weiss JW, Leitch AG, McFadden ER Jr, Corey EJ, Austen KF, Drazen JM (1983)
Effects of leukotriene D on the airways in asthma. N Engl J Med 308(8):436439
52. Davidson AB, Lee TH, Scanlon PD, Solway J, McFadden ER Jr, Ingram RH Jr, Corey EJ,
Austen KF, Drazen JM (1987) Bronchoconstrictor effects of leukotriene E4 in normal and
asthmatic subjects. Am Rev Respir Dis 135(2):333337
53. Arm JP, OHickey SP, Hawksworth RJ, Fong CY, Crea AE, Spur BW, Lee TH (1990)
Asthmatic airways have a disproportionate hyperresponsiveness to LTE4, as compared with
normal airways, but not to LTC4, LTD4, methacholine, and histamine. Am Rev Respir Dis
142(5):11121118
54. Christie PE, Schmitz-Schumann M, Spur BW, Lee TH (1993) Airway responsiveness to leu-
kotriene C4 (LTC4), leukotriene E4 (LTE4) and histamine in aspirin-sensitive asthmatic sub-
jects. Eur Respir J 6(10):14681473
55. Jacques CA, Spur BW, Johnson M, Lee TH (1991) The mechanism of LTE4-induced hista-
mine hyperresponsiveness in guinea-pig tracheal and human bronchial smooth muscle,
in vitro. Br J Pharmacol 104(4):859866. PMCID:PMC1908836
56. Christie PE, Hawksworth R, Spur BW, Lee TH (1992) Effect of indomethacin on leukotriene-
induced histamine hyperresponsiveness in asthmatic subjects. Am Rev Respir Dis
146(6):15061510
57. Beasley RC, Featherstone RL, Church MK, Rafferty P, Varley JG, Harris A, Robinson C,
Holgate ST (1989) Effect of a thromboxane receptor antagonist on PGD2- and allergen-
induced bronchoconstriction. J Appl Physiol (1985) 66(4):16851693
58. Jones GL, Saroea HG, Watson RM, OByrne PM (1992) Effect of an inhaled thromboxane
mimetic (U46619) on airway function in human subjects. Am Rev Respir Dis
145(6):12701274
59. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH (1993) Leukotriene E4 and
granulocytic infiltration into asthmatic airways. Lancet 341(8851):989990
60. Gauvreau GM, Parameswaran KN, Watson RM, OByrne PM (2001) Inhaled leukotriene E4,
but not leukotriene D4, increased airway inflammatory cells in subjects with atopic asthma.
Am J Respir Crit Care Med 164(8 pt 1):14951500
61. Zhu J, Qiu YS, Figueroa DJ, Bandi V, Galczenski H, Hamada K, Guntupalli KK, Evans JF,
Jeffery PK (2005) Localization and upregulation of cysteinyl leukotriene-1 receptor in asth-
matic bronchial mucosa. Am J Respir Cell Mol Biol 33(6):531540
62. Paruchuri S, Jiang Y, Feng C, Francis SA, Plutzky J, Boyce JA (2008) Leukotriene E4 acti-
vates peroxisome proliferator-activated receptor gamma and induces prostaglandin D2 gen-
eration by human mast cells. J Biol Chem 283(24):1647716487
63. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura
K, Nakamura M, Takano S et al (2001) Prostaglandin D2 selectively induces chemotaxis in T
helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2. J
Exp Med 193(2):255261
64. Paruchuri S, Tashimo H, Feng C, Maekawa A, Xing W, Jiang Y, Kanaoka Y, Conley P, Boyce
JA (2009) Leukotriene E4-induced pulmonary inflammation is mediated by the P2Y12 recep-
tor. J Exp Med 206(11):25432555
65. Nonaka Y, Hiramoto T, Fujita N (2005) Identification of endogenous surrogate ligands for
human P2Y12 receptors by in silico and in vitro methods. Biochem Biophys Res Commun
337(1):281288
66. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden
P, Nurden A, Julius D et al (2001) Identification of the platelet ADP receptor targeted by
antithrombotic drugs. Nature 409(6817):202207
67. Bautz F, Denzlinger C, Kanz L, Mohle R (2001) Chemotaxis and transendothelial migration
of CD34+ hematopoietic progenitor cells induced by the inflammatory mediator leukotriene
D4 are mediated by the 7-transmembrane receptor CysLT1. Blood 97(11):34333440
20 Cysteinyl Leukotrienes and Disease 297
68. Mellor EA, Maekawa A, Austen KF, Boyce JA (2001) Cysteinyl leukotriene receptor 1 is also
a pyrimidinergic receptor and is expressed by human mast cells. Proc Natl Acad Sci U S A
98(14):79647969
69. Mellor EA, Frank N, Soler D, Hodge MR, Lora JM, Austen KF, Boyce JA (2003) Expression
of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: functional
distinction from CysLT1R. Proc Natl Acad Sci U S A 100(20):1158911593
70. Bandeira-Melo C, Woods LJ, Phoofolo M, Weller PF (2002) Intracrine cysteinyl leukotriene
receptor-mediated signaling of eosinophil vesicular transport-mediated interleukin-4 secre-
tion. J Exp Med 196(6):841850
71. Jiang Y, Borrelli LA, Kanaoka Y, Bacskai BJ, Boyce JA (2007) CysLT2 receptors interact
with CysLT1 receptors and down-modulate cysteinyl leukotriene dependent mitogenic
responses of mast cells. Blood 110(9):32633270
72. Jiang Y, Kanaoka Y, Feng C, Nocka K, Rao S, Boyce JA (2006) Interleukin 4-dependent mast
cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling. J
Immunol 177(5):27552759
73. Mellor EA, Austen KF, Boyce JA (2002) Cysteinyl leukotrienes and uridine diphosphate
induce cytokine generation by human mast cells through an interleukin 4-regulated pathway
that is inhibited by leukotriene receptor antagonists. J Exp Med 195(5):583592
74. Lin DA, Boyce JA (2005) IL-4 regulates MEK expression required for lysophosphatidic acid-
mediated chemokine generation by human mast cells. J Immunol 175(8):54305438
75. Thivierge M, Stankova J, Rola-Pleszczynski M (2001) IL-13 and IL-4 up-regulate cysteinyl
leukotriene 1 receptor expression in human monocytes and macrophages. J Immunol
167(5):28552860
76. Early SB, Barekzi E, Negri J, Hise K, Borish L, Steinke JW (2007) Concordant modulation
of cysteinyl leukotriene receptor expression by IL-4 and IFN-gamma on peripheral immune
cells. Am J Respir Cell Mol Biol 36(6):715720
77. Jiang Y, Borrelli L, Bacskai BJ, Kanaoka Y, Boyce JA (2009) P2Y6 receptors require an
intact cysteinyl leukotriene synthetic and signaling system to induce survival and activation
of mast cells. J Immunol 182(2):11291137
78. Mita H, Hasegawa M, Saito H, Akiyama K (2001) Levels of cysteinyl leukotriene receptor
mRNA in human peripheral leucocytes: significantly higher expression of cysteinyl leukotri-
ene receptor 2 mRNA in eosinophils. Clin Exp Allergy 31(11):17141723
79. Xue L, Gyles SL, Wettey FR, Gazi L, Townsend E, Hunter MG, Pettipher R (2005)
Prostaglandin D2 causes preferential induction of proinflammatory Th2 cytokine production
through an action on chemoattractant receptor-like molecule expressed on Th2 cells. J
Immunol 175(10):65316536
80. Xue L, Barrow A, Fleming VM, Hunter MG, Ogg G, Klenerman P, Pettipher R (2012)
Leukotriene E4 activates human Th2 cells for exaggerated proinflammatory cytokine produc-
tion in response to prostaglandin D2. J Immunol 188(2):694702
81. Mjosberg JM, Trifari S, Crellin NK, Peters CP, van Drunen CM, Piet B, Fokkens WJ, Cupedo
T, Spits H (2011) Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are
defined by expression of CRTH2 and CD161. Nat Immunol 12(11):10551062
82. Xue L, Salimi M, Panse I, Mjosberg JM, McKenzie AN, Spits H, Klenerman P, Ogg G (2013)
Prostaglandin D activates group 2 innate lymphoid cells through chemoattractant receptor-
homologous molecule expressed on T2 cells. J Allergy Clin Immunol 133:11841194
83. Doherty TA, Khorram N, Lund S, Mehta AK, Croft M, Broide DH (2013) Lung type 2 innate
lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine pro-
duction. J Allergy Clin Immunol 132(1):205213
84. Hasegawa S, Ichiyama T, Hashimoto K, Suzuki Y, Hirano R, Fukano R, Furukawa S (2010)
Functional expression of cysteinyl leukotriene receptors on human platelets. Platelets
21(4):253259
298 L.B. Fanning and J.A. Boyce
85. Cummings HE, Liu T, Feng C, Laidlaw TM, Conley PB, Kanaoka Y, Boyce JA (2013)
Cutting edge: leukotriene C4 activates mouse platelets in plasma exclusively through the type
2 cysteinyl leukotriene receptor. J Immunol 191:58075810
86. Parameswaran K, Liang H, Fanat A, Watson R, Snider DP, OByrne PM (2004) Role for
cysteinyl leukotrienes in allergen-induced change in circulating dendritic cell number in
asthma. J Allergy Clin Immunol 114(1):7379
87. Thivierge M, Stankova J, Rola-Pleszczynski M (2006) Toll-like receptor agonists differen-
tially regulate cysteinyl-leukotriene receptor 1 expression and function in human dendritic
cells. J Allergy Clin Immunol 117(5):11551162
88. Ilarraza R, Wu Y, Adamko DJ (2012) Montelukast inhibits leukotriene stimulation of human
dendritic cells in vitro. Int Arch Allergy Immunol 159(4):422427
89. Barrett NA, Rahman OM, Fernandez JM, Parsons MW, Xing W, Austen KF, Kanaoka Y
(2011) Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes. J
Exp Med 208:593604
90. Liu MC, Dube LM, Lancaster J (1996) Acute and chronic effects of a 5-lipoxygenase inhibi-
tor in asthma: a 6-month randomized multicenter trial. Zileuton Study Group. J Allergy Clin
Immunol 98(5 pt 1):859871
91. Israel E, Rubin P, Kemp JP, Grossman J, Pierson W, Siegel SC, Tinkelman D, Murray JJ,
Busse W, Segal AT et al (1993) The effect of inhibition of 5-lipoxygenase by zileuton in mild-
to-moderate asthma. Ann Intern Med 119(11):10591066
92. Israel E, Chervinsky PS, Friedman B, Van BJ, Skalky CS, Ghannam AF, Bird SR, Edelman
JM (2002) Effects of montelukast and beclomethasone on airway function and asthma con-
trol. J Allergy Clin Immunol 110(6):847854
93. Fitzgerald DA, Mellis CM (2006) Leukotriene receptor antagonists in virus-induced wheez-
ing: evidence to date. Treat Respir Med 5(6):407417
94. Camargo CA Jr, Smithline HA, Malice MP, Green SA, Reiss TF (2003) A randomized con-
trolled trial of intravenous montelukast in acute asthma. Am J Respir Crit Care Med
167(4):528533
95. Adachi M, Taniguchi H, Tohda Y, Sano Y, Ishine T, Smugar SS, Hisada S (2012) The efficacy
and tolerability of intravenous montelukast in acute asthma exacerbations in Japanese
patients. J Asthma 49(6):649656
96. Camargo CA Jr, Gurner DM, Smithline HA, Chapela R, Fabbri LM, Green SA, Malice MP,
Legrand C, Dass SB, Knorr BA et al (2010) A randomized placebo-controlled study of intra-
venous montelukast for the treatment of acute asthma. J Allergy Clin Immunol
125(2):374380
97. Morris CR, Becker AB, Pinieiro A, Massaad R, Green SA, Smugar SS, Gurner DM (2010) A
randomized, placebo-controlled study of intravenous montelukast in children with acute
asthma. Ann Allergy Asthma Immunol 104(2):161171
98. Kikawa Y, Miyanomae T, Inoue Y, Saito M, Nakai A, Shigematsu Y, Hosoi S, Sudo M (1992)
Urinary leukotriene E4 after exercise challenge in children with asthma. J Allergy Clin
Immunol 89(6):11111119
99. Reiss TF, Hill JB, Harman E, Zhang J, Tanaka WK, Bronsky E, Guerreiro D, Hendeles L
(1997) Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced
bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax
52(12):10301035
100. Bancalari L, Conti I, Giannessi D, Lazzerini G, Dente FL, De CR, Paggiaro PL (1999) Early
increase in urinary leukotriene E4 (LTE4) is dependent on allergen dose inhaled during bron-
chial challenge in asthmatic subjects. Allergy 54(12):12781285
101. Rorke S, Jennison S, Jeffs JA, Sampson AP, Arshad H, Holgate ST (2002) Role of cysteinyl
leukotrienes in adenosine 5-monophosphate induced bronchoconstriction in asthma. Thorax
57(4):323327
102. Brannan JD, Gulliksson M, Anderson SD, Chew N, Kumlin M (2003) Evidence of mast cell
activation and leukotriene release after mannitol inhalation. Eur Respir J 22(3):491496
20 Cysteinyl Leukotrienes and Disease 299
120. Sousa A, Pfister R, Christie PE, Lane SJ, Nasser SM, Schmitz-Schumann M, Lee TH (1997)
Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its
cellular distribution in aspirin-sensitive asthma. Thorax 52(11):940945
121. Fischer AR, Rosenberg MA, Lilly CM, Callery JC, Rubin P, Cohn J, White MV, Igarashi Y,
Kaliner MA, Drazen JM et al (1994) Direct evidence for a role of the mast cell in the nasal
response to aspirin in aspirin-sensitive asthma. J Allergy Clin Immunol 94(6 pt
1):10461056
122. Yoshida S, Amayasu H, Sakamoto H, Onuma K, Shoji T, Nakagawa H, Tajima T (1998)
Cromolyn sodium prevents bronchoconstriction and urinary LTE4 excretion in aspirin-
induced asthma. Ann Allergy Asthma Immunol 80(2):171176
123. Liu T, Laidlaw TM, Katz HR, Boyce JA (2013) Prostaglandin E2 deficiency causes a pheno-
type of aspirin sensitivity that depends on platelets and cysteinyl leukotrienes. Proc Natl Acad
Sci U S A 110:1698716992
124. Corrigan CJ, Napoli RL, Meng Q, Fang C, Wu H, Tochiki K, Reay V, Lee TH, Ying S (2012)
Reduced expression of the prostaglandin E2 receptor E-prostanoid 2 on bronchial mucosal
leukocytes in patients with aspirin-sensitive asthma. J Allergy Clin Immunol
129(6):16361646
125. Sousa AR, Parikh A, Scadding G, Corrigan CJ, Lee TH (2002) Leukotriene-receptor expres-
sion on nasal mucosal inflammatory cells in aspirin-sensitive rhinosinusitis. N Engl J Med
347(19):14931499
126. Corrigan C, Mallett K, Ying S, Roberts D, Parikh A, Scadding G, Lee T (2005) Expression
of the cysteinyl leukotriene receptors cysLT1 and cysLT2 in aspirin-sensitive and aspirin-
tolerant chronic rhinosinusitis. J Allergy Clin Immunol 115(2):316322
127. Arm JP, OHickey SP, Spur BW, Lee TH (1989) Airway responsiveness to histamine and
leukotriene E4 in subjects with aspirin-induced asthma. Am Rev Respir Dis 140(1):148153
128. Balzary RW, Cocks TM (2006) Lipopolysaccharide induces epithelium- and prostaglandin
E2-dependent relaxation of mouse isolated trachea through activation of cyclooxygenase
(COX)-1 and COX-2. J Pharmacol Exp Ther 317(2):806812
129. Uematsu S, Matsumoto M, Takeda K, Akira S (2002) Lipopolysaccharide-dependent prosta-
glandin E2 production is regulated by the glutathione-dependent prostaglandin E2 synthase
gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. J Immunol
168(11):58115816
130. Sugimoto Y, Narumiya S (2007) Prostaglandin E receptors. J Biol Chem
282(16):1161311617
131. Luo M, Jones SM, Phare SM, Coffey MJ, Peters-Golden M, Brock TG (2004) Protein kinase
A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523. J Biol
Chem 279(40):4151241520
132. Feng C, Beller EM, Bagga S, Boyce JA (2006) Human mast cells express multiple EP recep-
tors for prostaglandin E2 that differentially modulate activation responses. Blood
107(8):32433250
133. Sestini P, Armetti L, Gambaro G, Pieroni MG, Refini RM, Sala A, Vaghi A, Folco GC,
Bianco S, Robuschi M (1996) Inhaled PGE2 prevents aspirin-induced bronchoconstriction
and urinary LTE4 excretion in aspirin-sensitive asthma. Am J Respir Crit Care Med
153(2):572575
134. Yoshimura T, Yoshikawa M, Otori N, Haruna S, Moriyama H (2008) Correlation between the
prostaglandin D2/E2 ratio in nasal polyps and the recalcitrant pathophysiology of chronic
rhinosinusitis associated with bronchial asthma. Allergol Int 57(4):429436
135. Picado C, Fernandez-Morata JC, Juan M, Roca-Ferrer J, Fuentes M, Xaubet A, Mullol J
(1999) Cyclooxygenase-2 mRNA is downexpressed in nasal polyps from aspirin-sensitive
asthmatics. Am J Respir Crit Care Med 160(1):291296
20 Cysteinyl Leukotrienes and Disease 301
21.1 Introduction
The skin is an organ that serves as an interface between the host and the environ-
ment. The skin provides not only mechanical barrier functions, to restrict water loss
and prevent the entry of harmful environmental substances and microorganisms, but
T. Honda
Department of Dermatology, Kyoto University Graduate School of Medicine,
54 Shogoin-Kawara, Sakyo, Kyoto 606-8507, Japan
Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University
Graduate School of Medicine, Kyoto 606-8501, Japan
K. Kabashima (*)
Department of Dermatology, Kyoto University Graduate School of Medicine,
54 Shogoin-Kawara, Sakyo, Kyoto 606-8507, Japan
e-mail: [email protected]
also an active barrier that provides the first line of immunological defense against
infections [1, 2]. The skin is composed of the epidermis and dermis, and each layer
is composed of several cell types, such as keratinocytes and dendritic cells (DCs),
which are important for maintenance of skin homeostasis and for induction of skin
diseases, such as atopic dermatitis (AD) and psoriasis. Lipid mediators, such as
prostanoids and leukotrienes (LTs), are the candidates for the regulation of its bal-
ance [35].
When tissues are exposed to diverse pathophysiological stimuli, arachidonic acid
(AA) is released from membrane phospholipids and converted to lipid mediators,
such as prostanoids, LTs, and hydroxy-eicosatetraenoic acids (HETEs). Prostanoids
are formed by the cyclooxygenase (COX) pathway, whereas LTs and HETEs are
formed by the 5-, 12-, and 15-lipoxygenase (LO) pathways. COX has two isoforms,
COX-1 and COX-2: COX-1 is constitutively expressed in cells, while COX-2
requires specific stimulation by substances such as acetone and phorbol ester [6].
The COX reaction results in the formation of an unstable endoperoxide intermedi-
ate, prostaglandin (PG) H2, which, in turn, is metabolized to PGD2, PGE2, PGF2,
PGI2, and thromboxane (TX) A2 by their specific synthases. LTs include LTB4 and
cystenyl (Cys) LTs: CysLTs further include LTC4, LTD4, and LTE4.
Prostanoids are released from cells immediately after their formation. Because
they are chemically and metabolically unstable, they usually function only locally
through membrane receptors on target cells [6]. Nine types and subtypes of mem-
brane prostanoid receptors are conserved in mammals from mouse to human: two
subtypes of the PGD receptor (DP and chemoattractant receptor homologous mol-
ecule expressed on Th2 cells, CRTH2), four subtypes of the PGE receptor (EP1,
EP2, EP3, and EP4), the PGF receptor (FP), the PGI receptor (IP), and the TXA
receptor (TP). All are G protein-coupled rhodopsin-type receptors with seven trans-
membrane domains. LTs also exert their functions through their specific G protein-
coupled receptors on the cell surface. LTB4 binds to two kinds of receptors, BLT1
and BLT2, and LTC4 binds to CysLT1 and CysLT2.
Recently, individual prostanoids and LT receptor gene-deficient mice have been
used as models to dissect the respective roles of each receptor in combination with
the use of compounds that selectively bind to the receptors as agonists or antago-
nists [35]. These genetic and pharmacological approaches have revealed new roles
for lipid mediators and their receptors in inflammatory skin diseases. In this review,
we describe the current investigative status of prostanoids and LTs in skin inflam-
matory diseases, focusing on contact dermatitis, atopic dermatitis, and psoriasis,
and discuss the clinical potentials of receptor-selective drugs.
Contact dermatitis, such as metal allergy or plant allergy, is one of the most frequent
skin inflammatory diseases [7, 8]. Most of the chemicals that induce contact derma-
titis are small compound called haptens. The development of contact dermatitis
21 Lipid Mediators and Skin Diseases 305
consists of two phases, the sensitization and elicitation phases. In the sensitization
phase, hapten is captured by cutaneous DCs, which migrate to skin-draining lymph
nodes and present the antigen to nave T cells. Then, the nave T cells differentiate
to the antigen-specific effector T cells. In the elicitation phase, the effector T cells
are recruited to the skin exposed to the haptens and are activated by the skin DCs to
produce inflammatory cytokines. Contact hypersensitivity (CHS) is a frequently
used mouse model of contact dermatitis. By using this model, prostanoids and LTs
have been shown to be essential in each phase of contact dermatitis.
Migration and maturation of cutaneous DCs, such as Langerhans cells (LCs) and
dermal DCs, are the critical steps for sensitization, and several lipid mediators have
been reported to regulate this process [911]. On hapten application to the skin,
PGE2 is produced by keratinocytes and acts at EP4 on LCs to facilitate initiation of
cutaneous immune responses by promoting migration and maturation of cutaneous
DCs [9].
In contrast, PGE2-EP3 signaling suppresses DC migration and maturation after
hapten application and is suggested to function suppressively to prevent excessive
skin inflammation [10]. PGD2 is also reported to inhibit cutaneous DC migration
[12, 13]. PGD2 induced by percutaneous infection with the helminth parasite
Schistosoma mansoni specifically impedes the migration of LCs through the DP
receptor [13]. Administration of a DP agonist, BW245C, inhibits migration of LCs
and attenuates OVA-induced dermatitis [12]. Consistently, DP-deficient mice
exhibit enhanced cutaneous DC migration and exacerbated inflammation in murine
CHS [14]. These activities of lipid mediators are not only limited to prostanoids.
LC migration from the skin to the draining lymph nodes utilizes multidrug
resistance-associated protein 1 as a LTC4 transporter [15]. BLT1-deficient mice
exhibit reduced numbers of migrating cutaneous DCs after hapten application, sug-
gesting that LTB4-BLT1 signaling also promotes DC migration [11].
When cutaneous DCs migrate to draining lymph nodes, DCs present antigens to
nave T cells to prime them. Subsequently, the engagement of the antigen complex
by T-cell receptors triggers clonal expansion and differentiation of T cells. CD4+
helper T (Th) cells are differentiated into at least three subsets: Th1, Th2, and Th17.
Similarly, CD8+ cytotoxic T (Tc) cells undergo differentiation into two subsets: Tc1
cells and Tc17 cells. CHS is mainly mediated by Tc1/Th1 cells and to some extent
by Tc17/Th17 cells [7].
Although the suppressive activity of PGE2 on Th1 differentiation in vitro has
been known since the 1980s, the in vivo role of PGE2 on Th differentiation has only
recently been addressed. In the sensitization phase of CHS, PGE2 produced by DCs
stimulate EP1 receptors on nave CD4+ and CD8+ T cells and promote Th1 and Tc1
306 T. Honda and K. Kabashima
differentiation [16]. Accordingly, EP1-deficient mice exhibit reduced Th1 and Tc1
differentiation and CHS responses [16]. Signaling from EP2 and EP4 receptors also
facilitates Th1 differentiation [17]. In addition to EP receptor signaling, IP signaling
also promotes Th1 and Tc1 differentiation in CHS [18]. Other than Th1/Tc1 dif-
ferentiation, PGE2 promotes Th17 differentiation and expansion through EP2 and
EP4 receptors by increasing interleukin (IL)-23 production from DCs and upregula-
tion of IL-23R on Th17 cells [17]. Consistently, administration of EP4 antagonist
suppresses Th17 differentiation and expansion in CHS [17]. Prostanoids also regu-
late DCT-cell interaction in the priming of nave T cells. Cutaneous DCs produce
abundant TXA2, which acts on nave T cells to impair the DCT-cell interaction
[19]. Predictably, TP-deficient mice or wild-type mice treated with a TP antagonist,
S-145, during the sensitization period exhibit enhanced CHS responses, indicating
that TP signaling negatively regulates the priming of T cells [19].
After establishment of the sensitization phase, antigen re-challenge onto the skin
stimulates keratinocytes (KCs) to produce memory T cell-attracting chemokines,
such as CCL27, and neutrophil-attracting chemokines, such as CXCL1 and CXCL2,
and to evoke inflammation, in a stage called the elicitation phase [7]. It has been
demonstrated that these chemokines are induced by PGE2 [20], and several pros-
tanoid receptors are also involved in this phase. For example, PGD2 promotes neu-
trophil infiltration through CRTH2 and contributes to the progression of inflammation
[21]. Accordingly, administration of a CRTH2 antagonist attenuates the CHS
response [22]. On the other hand, stimulation of the EP3 receptors on KCs inhibits
the chemokine expression in KCs and suppresses the CHS response [23].
The role of LT receptors in the elicitation phase is less clear. As BLT1 is expressed
on effector CD8 or CD4 T cells and mediates the infiltration of those cells into skin
[24, 25] and LTB4 has been reported to enhance CCL27 production from keratino-
cytes [26], LTB4-BLT1-dependent mechanisms may also operate in the elicitation
phase of CHS. The possible roles of individual prostanoid and LT receptors in con-
tact dermatitis are summarized in Figs. 21.1 and 21.2.
Atopic dermatitis (AD) is a common pruritic and chronic inflammatory skin disease
that is regarded as one of the Th2 diseases. In the dermis, a cellular infiltrate is pres-
ent consisting of lymphocytes, monocytes, and mast cells. In biopsy specimens
from patients with AD, PGE2 has been determined in biologically active amounts in
both lesional and perilesional skin [27]. In contrast, normal levels of eicosanoids
21 Lipid Mediators and Skin Diseases 307
PGD2-
CRTH2 PGE2-
Dermal dendritic cell (dDC) Mast cell EP2/EP4
Neutrophil
recruitment Vasodilation
TXA2-TP
T cell activation
: Promotion
: Inhibition
Nave T cell Memory/Effector T cell
Lymph nodes PGE2-EP1/EP2/EP4 Th1 differentiation
PGI2-IP
Fig. 21.1 Roles of prostanoids and leukotrienes (LTs) in the development of contact dermatitis.
Schematic summary of possible roles of prostanoids and LTs in the sensitization phase (left) and
the elicitation phase (right) of contact dermatitis
allergen
PGD2-DP
Pruritus
Neutrophils
vvv asophils
Eosinophils/b
vvv
Blood vessel
: Promotion
: Inhibition
Fig. 21.2 Roles of prostanoids and LTs in the development of atopic dermatitis. Schematic sum-
mary of possible roles of prostanoids and LTs in the development of atopic dermatitis
308 T. Honda and K. Kabashima
were found in the uninvolved skin of these patients [27]. In an OVA-induced mouse
AD model, COX-2-deficient mice exhibited both enhanced eosinophil infiltration
and elevated IL-4 expression in the skin lesion with elevated serum IgE and IgG1
[28]. In in vitro studies, PGE2 drives Ig class switching to IgE by acting at EP2 and
EP4 on B cells under LPS and IL-4 stimulation [29]. These results suggest that
COX-2-derived PGE2 plays both protective and promoting roles in the development
of AD.
PGD2 is the major prostanoid produced by activated mast cells. PGD2 has two
types of receptors, DP and CRTH2. The effect of PGD2 on skin inflammation is not
so simple and is extremely context dependent, because DP and CRHT2 possess
independent, sometimes opposite, functions even in the same pathological condi-
tions [3033]. However, CRTH2 generally seems to be pro-inflammatory and DP
has both pro- and anti-inflammatory effects. CRTH2 induces chemotaxis in Th2
cells, eosinophils, and basophils with enhanced degranulation [34, 35]. In response
to PGD2, CRTH2 also induces Th2 cell and neutrophil migration into inflammatory
skin sites [21]. Virtually all CRTH2+ CD4+ lymphocytes have a pure Th2 phenotype
and occupy not all, but a large proportion, of circulating Th2 cells in both normal
and AD subjects. In AD patients, a preferential increase of CRTH2+ cells was noted
within the disease-related cutaneous lymphocyte-associated antigen-positive CD4+
T-cell compartment [36]. CRTH2-deficient mice exhibit reduced inflammation in
the OVA-induced AD model [30]. These results suggest the importance of CRTH2
on Th2 cells in AD, although there remains a need to clarify the respective roles of
DP and CRTH2 in AD.
As for the role of LTs in the pathogenesis of AD, it has recently been reported
that the LTB4-BLT1 axis of neutrophils is critical to recruit the Th2 cells into skin in
the OVA-induced mice AD model [37].
Pruritus is also an important hallmark of AD. PGE2 is known to evoke pruritus in
AD patients [38]. PGD2, but not a CRTH2 agonist, 13,14-dihydro-15-keto-PGD2,
reduced scratching behavior in NC/Nga AD model mice, suggesting that DP sup-
presses pruritic activity [39]. In addition, TXA2 and LTB4 are known to mediate the
itch sensation [40, 41].
Taken together, these results suggest that appropriate control of lipid mediator
production or signaling in the skin can become a novel drug target for AD
(Fig. 21.3).
LTB4-BLT1
LTB4-BLT1 DCs
Blood vessel
: Promotion
: Inhibition
Fig. 21.3 Roles of prostanoids and LTs in the development of psoriasis. LTB4 and CXCR2 ligand
cooperatively promote neutrophil infiltration into skin. PGE2 may promote IL-23 production from
skin DCs and may contribute to the expansion of Th17 cells
However, the roles of prostanoids and LTs in the development of psoriasis remain
mostly unclear because of the lack of an appropriate animal psoriasis model.
Recently, it has been reported that daily topical application of imiquimod, an
agonist for Toll-like receptor 7 and 8, induced psoriasis-like dermatitis in mice [44].
Using this model, it has recently been shown that BLT1 and CXCR2, a chemokine
receptor, work cooperatively for neutrophil infiltration in the psoriasis lesion [45].
This result is in line with previous reports that a 5-lipoxygenase inhibitor improved
the clinical symptoms of psoriasis whereas indomethacin exacerbated the symp-
toms [43].
In in vitro studies, it has been reported that PGE2 produced by fibroblasts pro-
moted IL-23 production from DCs, which supported the expansion of Th17 cells,
suggesting the possibility that this system may work in psoriasis [46, 47]. We are
currently investigating the role of prostanoids in psoriasis by applying each pros-
tanoid to receptor-deficient mice in this model (manuscript in preparation).
21.6 Conclusions
References
1. Pasparakis M, Haase I, Nestle FO (2014) Mechanisms regulating skin immunity and inflam-
mation. Nat Rev Immunol 14(5):289301. doi:10.1038/nri3646
2. Heath WR, Carbone FR (2013) The skin-resident and migratory immune system in steady
state and memory: innate lymphocytes, dendritic cells and T cells. Nat Immunol 14(10):978
985. doi:10.1038/ni.2680
3. Honda T, Tokura Y, Miyachi Y, Kabashima K (2010) Prostanoid receptors as possible targets
for anti-allergic drugs: recent advances in prostanoids on allergy and immunology. Curr Drug
Targets 11(12):16051613
21 Lipid Mediators and Skin Diseases 311
4. Hirata T, Narumiya S (2012) Prostanoids as regulators of innate and adaptive immunity. Adv
Immunol 116:143174. doi:10.1016/B978-0-12-394300-2.00005-3
5. Yokomizo T (2011) Leukotriene B4 receptors: novel roles in immunological regulations. Adv
Enzyme Regul 51(1):5964. doi:10.1016/j.advenzreg.2010.08.002
6. Narumiya S, Sugimoto Y, Ushikubi F (1999) Prostanoid receptors: structures, properties, and
functions. Physiol Rev 79(4):11931226
7. Honda T, Egawa G, Grabbe S, Kabashima K (2013) Update of immune events in the murine
contact hypersensitivity model: toward the understanding of allergic contact dermatitis. J
Invest Dermatol 133(2):303315. doi:10.1038/jid.2012.284
8. Peiser M, Tralau T, Heidler J, Api AM, Arts JH, Basketter DA, English J, Diepgen TL,
Fuhlbrigge RC, Gaspari AA, Johansen JD, Karlberg AT, Kimber I, Lepoittevin JP, Liebsch M,
Maibach HI, Martin SF, Merk HF, Platzek T, Rustemeyer T, Schnuch A, Vandebriel RJ, White
IR, Luch A (2012) Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro
methods and regulatory aspects. Current knowledge assembled at an international workshop at
BfR, Germany. Cell Mol Life Sci 69(5):763781. doi:10.1007/s00018-011-0846-8
9. Kabashima K, Sakata D, Nagamachi M, Miyachi Y, Inaba K, Narumiya S (2003) Prostaglandin
E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of
Langerhans cells. Nat Med 9(6):744749. doi:10.1038/nm872
10. Shiraishi N, Nomura T, Tanizaki H, Nakajima S, Narumiya S, Miyachi Y, Tokura Y, Kabashima
K (2013) Prostaglandin E2-EP3 axis in fine-tuning excessive skin inflammation by restricting
dendritic cell functions. PLoS One 8(7):e69599. doi:10.1371/journal.pone.0069599
11. Del Prete A, Shao WH, Mitola S, Santoro G, Sozzani S, Haribabu B (2007) Regulation of
dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for
LTB4 in up-regulation of CCR7 expression and function. Blood 109(2):626631. doi:10.1182/
blood-2006-02-003665
12. Angeli V, Staumont D, Charbonnier AS, Hammad H, Gosset P, Pichavant M, Lambrecht BN,
Capron M, Dombrowicz D, Trottein F (2004) Activation of the D prostanoid receptor 1 regu-
lates immune and skin allergic responses. J Immunol 172(6):38223829
13. Angeli V, Faveeuw C, Roye O, Fontaine J, Teissier E, Capron A, Wolowczuk I, Capron M,
Trottein F (2001) Role of the parasite-derived prostaglandin D2 in the inhibition of epidermal
Langerhans cell migration during schistosomiasis infection. J Exp Med 193(10):11351147
14. Yamamoto Y, Otani S, Hirai H, Nagata K, Aritake K, Urade Y, Narumiya S, Yokozeki H,
Nakamura M, Satoh T (2011) Dual functions of prostaglandin D2 in murine contact hypersen-
sitivity via DP and CRTH2. Am J Pathol 179(1):302314. doi:10.1016/j.ajpath.2011.03.047
15. Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ (2000) The leukot-
riene C4 transporter MRP1 regulates CCL19 (MIP-3beta, ELC)-dependent mobilization of
dendritic cells to lymph nodes. Cell 103(5):757768
16. Nagamachi M, Sakata D, Kabashima K, Furuyashiki T, Murata T, Segi-Nishida E, Soontrapa
K, Matsuoka T, Miyachi Y, Narumiya S (2007) Facilitation of Th1-mediated immune response
by prostaglandin E receptor EP1. J Exp Med 204(12):28652874. doi:10.1084/jem.20070773
17. Yao C, Sakata D, Esaki Y, Li Y, Matsuoka T, Kuroiwa K, Sugimoto Y, Narumiya S (2009)
Prostaglandin E2-EP4 signaling promotes immune inflammation through Th1 cell differentia-
tion and Th17 cell expansion. Nat Med 15(6):633640. doi:10.1038/nm.1968
18. Nakajima S, Honda T, Sakata D, Egawa G, Tanizaki H, Otsuka A, Moniaga CS, Watanabe T,
Miyachi Y, Narumiya S, Kabashima K (2010) Prostaglandin I2-IP signaling promotes Th1 dif-
ferentiation in a mouse model of contact hypersensitivity. J Immunol 184(10):55955603.
doi:10.4049/jimmunol.0903260
19. Kabashima K, Murata T, Tanaka H, Matsuoka T, Sakata D, Yoshida N, Katagiri K, Kinashi T,
Tanaka T, Miyasaka M, Nagai H, Ushikubi F, Narumiya S (2003) Thromboxane A2 modulates
interaction of dendritic cells and T cells and regulates acquired immunity. Nat Immunol
4(7):694701. doi:10.1038/ni943
20. Kanda N, Mitsui H, Watanabe S (2004) Prostaglandin E2 suppresses CCL27 production
through EP2 and EP3 receptors in human keratinocytes. J Allergy Clin Immunol 114(6):1403
1409. doi:10.1016/j.jaci.2004.08.041
312 T. Honda and K. Kabashima
36. Iwasaki M, Nagata K, Takano S, Takahashi K, Ishii N, Ikezawa Z (2002) Association of a new-
type prostaglandin D2 receptor CRTH2 with circulating T helper 2 cells in patients with atopic
dermatitis. J Invest Dermatol 119(3):609616. doi:10.1046/j.1523-1747.2002.01862.x
37. Oyoshi MK, He R, Li Y, Mondal S, Yoon J, Afshar R, Chen M, Lee DM, Luo HR, Luster AD,
Cho JS, Miller LS, Larson A, Murphy GF, Geha RS (2012) Leukotriene B4-driven neutrophil
recruitment to the skin is essential for allergic skin inflammation. Immunity 37(4):747758.
doi:10.1016/j.immuni.2012.06.018
38. Neisius U, Olsson R, Rukwied R, Lischetzki G, Schmelz M (2002) Prostaglandin E2 induces
vasodilation and pruritus, but no protein extravasation in atopic dermatitis and controls. J Am
Acad Dermatol 47(1):2832
39. Arai I, Takano N, Hashimoto Y, Futaki N, Sugimoto M, Takahashi N, Inoue T, Nakaike S
(2004) Prostanoid DP1 receptor agonist inhibits the pruritic activity in NC/Nga mice with
atopic dermatitis. Eur J Pharmacol 505(1-3):229235. doi:10.1016/j.ejphar.2004.10.031
40. Andoh T, Haza S, Saito A, Kuraishi Y (2011) Involvement of leukotriene B4 in spontaneous
itch-related behaviour in NC mice with atopic dermatitis-like skin lesions. Exp Dermatol
20(11):894898. doi:10.1111/j.1600-0625.2011.01346.x
41. Andoh T, Nishikawa Y, Yamaguchi-Miyamoto T, Nojima H, Narumiya S, Kuraishi Y (2007)
Thromboxane A2 induces itch-associated responses through TP receptors in the skin in mice.
J Invest Dermatol 127(8):20422047. doi:10.1038/sj.jid.5700810
42. Wagner EF, Schonthaler HB, Guinea-Viniegra J, Tschachler E (2010) Psoriasis: what we have
learned from mouse models. Nat Rev Rheumatol 6(12):704714. doi:10.1038/
nrrheum.2010.157
43. Ikai K (1999) Psoriasis and the arachidonic acid cascade. J Dermatol Sci 21(3):135146
44. van der Fits L, Mourits S, Voerman JS, Kant M, Boon L, Laman JD, Cornelissen F, Mus AM,
Florencia E, Prens EP, Lubberts E (2009) Imiquimod-induced psoriasis-like skin inflammation
in mice is mediated via the IL-23/IL-17 axis. J Immunol 182(9):58365845. doi:10.4049/
jimmunol.0802999
45. Sumida H, Yanagida K, Kita Y, Abe J, Matsushima K, Nakamura M, Ishii S, Sato S, Shimizu
T (2014) Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant
keratinocyte activation in a murine model of imiquimod-induced psoriasis. J Immunol
192(9):43614369. doi:10.4049/jimmunol.1302959
46. Schirmer C, Klein C, von Bergen M, Simon JC, Saalbach A (2010) Human fibroblasts support
the expansion of IL-17-producing T cells via up-regulation of IL-23 production by dendritic
cells. Blood 116(10):17151725. doi:10.1182/blood-2010-01-263509
47. Sheibanie AF, Tadmori I, Jing H, Vassiliou E, Ganea D (2004) Prostaglandin E2 induces IL-23
production in bone marrow-derived dendritic cells. FASEB J 18(11):13181320. doi:10.1096/
fj.03-1367fje
48. Ofuji S, Furukawa F, Miyachi Y, Ohno S (1984) Papuloerythroderma. Dermatologica
169(3):125130
49. Nakahigashi K, Doi H, Otsuka A, Hirabayashi T, Murakami M, Urade Y, Zouboulis CC,
Tanizaki H, Egawa G, Miyachi Y, Kabashima K (2012) PGD2 induces eotaxin-3 via PPAR-
gamma from sebocytes: a possible pathogenesis of eosinophilic pustular folliculitis. J Allergy
Clin Immunol 129(2):536543. doi:10.1016/j.jaci.2011.11.034
50. Sugita K, Ikenouchi-Sugita A, Nakayama Y, Yoshioka H, Nomura T, Sakabe J, Nakahigashi K,
Kuroda E, Uematsu S, Nakamura J, Akira S, Nakamura M, Narumiya S, Miyachi Y, Tokura Y,
Kabashima K (2013) Prostaglandin E2 is critical for the development of niacin-deficiency-
induced photosensitivity via ROS production. Sci Rep 3:2973. doi:10.1038/srep02973
51. Kabashima K, Nagamachi M, Honda T, Nishigori C, Miyachi Y, Tokura Y, Narumiya S (2007)
Prostaglandin E2 is required for ultraviolet B-induced skin inflammation via EP2 and EP4
receptors. Lab Invest J Tech Methods Pathol 87(1):4955. doi:10.1038/labinvest.3700491
314 T. Honda and K. Kabashima
52. Williams TJ, Morley J (1973) Prostaglandins as potentiators of increased vascular permeability
in inflammation. Nature 246(5430):215217
53. Morimoto K, Shirata N, Taketomi Y, Tsuchiya S, Segi-Nishida E, Inazumi T, Kabashima K,
Tanaka S, Murakami M, Narumiya S, Sugimoto Y (2014) Prostaglandin E2-EP3 signaling
induces inflammatory swelling by mast cell activation. J Immunol 192(3):11301137.
doi:10.4049/jimmunol.1300290
54. Goulet JL, Pace AJ, Key ML, Byrum RS, Nguyen M, Tilley SL, Morham SG, Langenbach R,
Stock JL, McNeish JD, Smithies O, Coffman TM, Koller BH (2004) E-prostanoid-3 receptors
mediate the proinflammatory actions of prostaglandin E2 in acute cutaneous inflammation. J
Immunol 173(2):13211326
Chapter 22
Roles and Actions of Arachidonic Acid-
Derived Bioactive Lipids in Stress-Related
Behaviors
22.1 Introduction
Because NSAIDs block PG synthesis, PGE2 synthesized in the brain has been stud-
ied as an inammation-related molecule that mediates pain, febrile, and neuroendo-
crine responses to peripheral inammation [11, 49]. In contrast, the function of
22 Roles and Actions of Arachidonic Acid-Derived Bioactive Lipids 317
Fig. 22.1 Biosynthesis of arachidonic acid (AA)-derived bioactive lipids and their receptors. (a)
Prostaglandin (PG)E2 biosynthesis and its receptors. PGE2 is derived from arachidonic acid (AA)
by sequential actions of cyclooxygenase (COX) and PGE synthase. NSAIDs, such as indometha-
cin and aspirin, suppress PG production by inhibiting COX-1 and COX-2. PGE2 exerts its func-
tions through binding to four G protein-coupled receptors (GPCRs) named EP1, EP2, EP3, and
EP4, each of which is coupled to a distinct signaling pathway. (b) eCB biosynthesis and its recep-
tors. Anandamide (AEA) is synthesized from AA through three distinct pathways.
2-Arachidonoylglycerol (2-AG) is synthesized from AA by sequential actions of phospholipase C
and sn-1 diacylglycerol lipase. AEA and 2-AG act as agonists for the GPCRs CB1 and CB2,
whereas AEA can also bind to TRPV1
effects of antidepressants in depressive patients [2, 31], and the effect of celecoxib
was further conrmed by a meta-analysis based on the results from several clinical
reports [33]. These clinical studies led to the hypothesis that PGE2 may be involved
in the pathophysiology of depression.
To test that hypothesis, a role of PGE2 was examined in repeated social defeat
stress in rodents [45], which has been considered to be a mouse model of depression
[35]. In this stress model, a male mouse of C57BL/6 background is subjected to
agonistic encounters from a male ICR mouse selected based on a high level of
aggression. This social defeat is applied for 10 min daily for 10 consecutive days,
and various behavioral changes are measured. Typically, repeated social defeat
stress induces depression-like behaviors, such as social avoidance and reduced
sucrose preference (anhedonia), and increases anxiety-like behaviors as often mea-
sured by the elevated plus maze test and the lightdark box test [35]. Repeated
social defeat increases PGE2 content in the brain, and EP1-knockout mice failed to
show social avoidance and elevated anxiety [45]. However, EP1-knockout mice
showed submissive posture, an immediate behavioral response to social defeat, to a
normal level. These results showed that the PGE2-EP1 pathway is not critical for
perception of repeated social defeat, but for long-term behavioral changes after
repeated social defeat (Fig. 22.2a).
Fig. 22.2 (continued) release appears to be involved in this 2-AG synthesis, at least in the prefron-
tal cortex. The 2-AG-CB1 pathway in respective brain areas attenuates stress-induced behavioral
changes through distinct mechanisms. Note that red and gray lines indicate active and inactive
processes in respective conditions
Fig. 22.2 Roles of PGE2 and eCB in stress-related behaviors. (a) The PGE2-EP1 pathway facili-
tates stress-related behaviors. A single stress exposure activates the dopaminergic pathway project-
ing to the prefrontal cortex, which suppresses stress-induced social avoidance. After repeated
stress, PGE2 attenuates this dopaminergic pathway through EP1, thereby leading to social avoid-
ance. Because COX-1, a PG synthase enriched in microglia, is critical for PGE2 synthesis and
social avoidance upon stress exposure, microglia are likely to be the cellular source of PGE2 syn-
thesis that contributes to stress-induced social avoidance. (b) A role of eCB-CB1 pathway in atten-
uating stress-related behaviors. Stress exposure increases 2-AG synthesis in multiple brain areas,
such as the prefrontal cortex, hypothalamus, and hippocampus. Stress-evoked corticosterone
320 T. Furuyashiki and S. Kitaoka
[27]. These results suggested that EP1 suppresses dopaminergic activity in multiple
brain structures, and that disinhibited dopaminergic activity caused by EP1 de-
ciency causes aggressive behavior.
EP1 is also critical for dopaminergic changes induced by repeated social defeat
stress [45]. Immunostaining for c-Fos, a marker for neuronal activity, showed that
single exposure to social defeat stress activates dopamine neurons, and that this
stress response of VTA dopamine neurons is attenuated with repetition of social
defeat. A similar change was observed in dopamine turnover in the medial prefron-
tal cortex: Single exposure to social defeat increased dopamine turnover, and this
response was attenuated with stress repetition. In EP1-decient mice, whereas a
single exposure to defeat normally increased c-Fos expression in VTA dopamine
neurons and dopamine turnover in the medial prefrontal cortex, repeated stress-
induced attenuation of both these two indices was abolished. This nding showed
that the PGE2-EP1 pathway mediates suppression, but not facilitation, of dopami-
nergic pathway under stress condition.
In repeated social defeat stress, dopaminergic activity in the medial prefrontal
cortex suppresses induction of social avoidance, thereby leading to stress resilience
[5, 45]. This idea is supported by several ndings. Thus, dopamine turnover in the
medial prefrontal cortex was negatively correlated to the level of social avoidance
induced by repeated social defeat, and pharmacological damage or optogenetic
inhibition of dopaminergic projection to the medial prefrontal cortex facilitated
induction of social avoidance by social defeat stress. Notably, pharmacological
blockade of dopamine D1-like receptors by their antagonist, SCH23390, restored
induction of social avoidance in EP1-decient mice, suggesting that disinhibited
dopaminergic activity in these mice causes the lack of social avoidance.
Collectively, EP1-mediated suppression of dopaminergic projection to the
medial prefrontal cortex appears to underlie at least some of behavioral changes
induced by repeated stress (Fig. 22.2a).
Previous studies showed that PGE2-EP1 can regulate the dopaminergic pathway at
least through two mechanisms.
First, EP1 stimulation can inhibit dopamine neurons through augmenting
GABAergic inhibitory synaptic inputs [46]. EP1 immunostaining showed EP1
localization at GABAergic synaptic terminals formed on SNpc dopamine neurons.
In midbrain slices, pharmacological stimulation of EP1 by ONO-DI-004, an EP1
agonist, increased evoked inhibitory postsynaptic currents in SNpc dopamine neu-
rons, and this EP1 action was abolished by pharmacological blockade or genetic
deletion of EP1. This EP1 action, if extrapolated to VTA dopamine neurons, can
explain the EP1-mediated suppression of dopaminergic activity under stress condi-
tions as described, although this possibility remains to be proven using conditional
EP1-knockout mice.
22 Roles and Actions of Arachidonic Acid-Derived Bioactive Lipids 321
Second, EP1 stimulation can alter the intracellular signaling of dopamine recep-
tors. This mechanism was suggested on the basis of the nding that EP1 deciency
attenuates Thr34 phosphorylation of DARPP-32 induced by dopamine D1 receptor
stimulation [24]. Consistent with this nding, in HEK293T cells overexpressing
both EP1 and D1 receptors, these receptors form a biochemical complex, and phar-
macological stimulation of EP1 by its agonist ONO-DI-004 facilitates cAMP pro-
duction induced by D1 receptor agonists [10]. Originally, this EP1 action appeared
to be opposite to the EP1 action implicated in behavioral regulation under stress.
However, further analysis revealed that dopamine D1 receptor signaling with or
without EP1 stimulation utilizes a distinct molecular pathway [10]. Thus, EP1-
mediated facilitation of D1-induced cAMP production is mediated by G subunits
and adenylyl cyclase 7, a G-sensitive adenylyl cyclase isoform, whereas, without
EP1 stimulation, D1-induced cAMP production was mediated through adenylyl
cyclase 5, a G-insensitive and Ca2+-suppressed adenylyl cyclase isoform. Whether
and how this EP1 action on dopamine receptor signaling could be involved in stress-
induced behavioral changes warrants future investigation.
eCBs are endogenous ligands for receptors that mediate the psychotic and addictive
actions of 9-tetrahydrocannabinol (THC) in cannabis, and include 2-AG and anan-
damide (arachidonoylethanolamide; AEA) [8, 39]. Recent studies have revealed a
critical role for eCBs and their receptors in emotional regulation without or with
stress. Before describing these studies in detail, we briey introduce the synthesis
and metabolism of eCBs and their receptors. The reader can refer to comprehensive
reviews elsewhere [8, 21, 39].
For the synthesis of 2-AG, phospholipase C (PLC) catalyzes the conversion from
sn-2-arachidonate-containing phospholipids to sn-2-arachidonate-containing diac-
ylglycerol (DAG), which is then metabolized to 2-AG by sn-1-diacylglycerol lipase
(DGL). For the synthesis of AEA, multiple enzymatic cascades via phospholipases
of different classes, namely, PLA2, PLC, and PLD, have been proposed, although
the contribution of respective cascades in physiological and pathophysiological
contexts remains to be established (Fig. 22.1b). For inactivation of 2-AG, monoac-
ylglycerol lipase (MGL) metabolizes 2-AG to AA and glycerol. AEA is metabolized
to AA and ethanolamine by fatty acid amide hydrolase (FAAH) for its
inactivation.
2-AG and AEA act as agonists for the G protein-coupled receptors CB1 and
CB2, both of which are primarily coupled to Gi-mediated inhibition of cAMP pro-
duction. CB1 is mainly, but not exclusively, localized at presynaptic terminals of
neurons in the brain. CB1 expression was expressed in multiple brain areas, such as
the striatum, thalamus, hypothalamus, and cerebellar cortex [21]. In addition, CB1
322 T. Furuyashiki and S. Kitaoka
Rodent studies showed roles of CB1 in emotional regulation, although results are
not entirely consistent across studies. For example, systemic injection with the CB1
agonists CP55940 and WIN55212-2 at relatively low doses suppresses anxiety-like
behavior in the elevated plus maze test, whereas systemic injection with the CB1
antagonists AM251 and rimonabant (SR141716) enhances such anxiety-like behav-
ior [38]. Consistently, CB1-decient mice showed elevated anxiety-like behaviors
in elevated or lighted compartments [16, 26]. Because serotonergic neuron-selective
knockout of CB1 also augments the level of anxiety-like behaviors [9], it is plausi-
ble that CB1 reduces the level of anxiety through inhibiting synaptic transmission
from serotonergic neurons. In contrast to these ndings, it was also reported that
systemic treatment with rimonabant rather suppressed anxiety-like behaviors in the
elevated plus maze test [15].
Roles of CB1 in depression-like behaviors are also reported, although results are
again not entirely consistent across studies. CB1-decient mice showed typical
depression-like behaviors, such as increased immobility time in the forced swim
test and reduced motivation for sucrose reward [1, 42]. Consistent with this nding,
9-THC reduced immobility time in the forced swim test, and this effect was
blocked by rimonabant treatment, and thus is likely to be CB1 dependent [17].
However, it was also reported that systemic treatment with rimonabant alone
reduced immobility time in the forced swim test [17], implying the presence of
multiple CB1 actions in depression-like behaviors. Because conditional deletion of
CB1 in GABAergic neurons abolishes rimonabant-induced suppression of
depression-like behaviors [17], this antidepressant-like effect of remonabant
involves disinhibition of GABAergic synaptic transmission, at least in part.
In contrast to CB1, roles of CB2 in emotional regulation have been much less
well characterized, except in a couple of cases. Thus, CB2-decient mice showed
22 Roles and Actions of Arachidonic Acid-Derived Bioactive Lipids 323
Most, if not all, studies in rodents showed that stress exposure of various conditions,
such as single and repeated restraint stress and repeated social defeat stress, aug-
ments 2-AG synthesis in the medial prefrontal cortex [9, 18, 40]. This 2-AG synthe-
sis is blocked by RU486, a GR antagonist, suggesting a role for glucocorticoid
release in this process [18]. On the other hand, 2-AG synthesis in the medial pre-
frontal cortex appears to suppress stress-induced glucocorticoid release, as local
injection of AM251, a CB1 antagonist, to the medial prefrontal cortex augments
corticosterone release upon acute restraint stress [18]. Therefore, the 2-AG-CB1
pathway in the medial prefrontal cortex may provide a negative feedback loop for
stress-induced glucocorticoid release. The eCB-CB1 pathway is also involved in
stress-induced behavioral changes. Thus, local injection of AM251 to the medial
prefrontal cortex facilitates the effect of chronic mild stress on the time of immobil-
ity in the forced swim test [29]. Similarly, systemic injection with URB597, a FAAH
inhibitor, for several consecutive weeks ameliorates chronic mild stress-induced
anhedonia as measured by decreased sucrose preference [3]. Local injection of
URB597 to the medial prefrontal cortex also reduces immobility in the forced swim
324 T. Furuyashiki and S. Kitaoka
test [28]. These behavioral ndings suggest that stress exposure activates the eCB-
CB1 pathway in the medial prefrontal cortex, thereby counteracting neuroendocrine
and behavioral responses to stress.
Besides 2-AG synthesis in the medial prefrontal cortex, repeated social defeat
stress facilitates 2-AG synthesis in other structures, such as the hippocampus and
the hypothalamus [9]. CB1 stimulation in the hypothalamus suppresses excitatory
synaptic inputs to CRH-secreting neurons, thereby inhibiting glucocorticoid release
[7]. CB1 in the hippocampus appears to regulate neurogenesis in the subgranular
zone of the dentate gyrus of the hippocampus, which is critical for the behavioral
effect of antidepressants at least in rodents. Thus, in CB1-decient mice, hippocam-
pal neurogenesis was reduced by half [20], and HU210, a synthetic cannabis, causes
anxiolytic and antidepressant-like actions in a manner dependent on hippocampal
neurogenesis [19].
Collectively, stress activates the eCB-CB1 pathway in multiple brain structures,
including the prefrontal cortex, hippocampus, and hypothalamus, which counteracts
stress responses, perhaps through distinct mechanisms (Fig. 22.2b).
As already described, stress augments synthesis of PGE2 and eCBs in the brain.
COX-1-dependent PGE2 synthesis appears to be critical for stress-induced behav-
ioral changes. Systemic injection with sc-560, a COX-1-selective inhibitor, or
genetic deletion of COX-1 abolishes social avoidance induced by repeated social
defeat, whereas neither pharmacological blockade nor genetic deletion of COX-2
affects the social avoidance [45]. Our preliminary results as well as those of others
[36] showed that COX-1 is critical for PGE2 synthesis in the brain without or with
stress exposure. Combined with the nding that COX-1 is selectively expressed in
microglia in the brain [45], these ndings lead to the hypothesis that COX-1-
expressing microglia may be the source of PGE2 in the brain, especially under stress
conditions. Consistent with this hypothesis, repeated social defeat stress induces
histological changes of microglia, such as enlargement of the cell body and hyper-
trophy of microglial processes, which is reminiscent of microglial activation [45].
Because repeated social defeat stress did not change the expression level of
COX-1 [45], repeated stress may increase the supply of AA for COX-1-mediated
PG production. It has been established that cytosolic PLA2 is critical for the release
of AA from phospholipids of the cell membrane for various physiological and
pathophysiological functions [44]. However, studies using an MGL inhibitor and
MGL-knockout mice recently showed that PGE2 synthesis in brain highly depends
on a pool of AA supplied from MGL-mediated 2-AG metabolism [36]. Therefore,
one can hypothesize that stress-induced increase in PGE2 synthesis in the brain
results from stress-induced 2-AG synthesis, although this hypothesis remains to be
experimentally tested (Fig. 22.3).
22 Roles and Actions of Arachidonic Acid-Derived Bioactive Lipids 325
Fig. 22.3 Crosstalk between eCBs and PGE2. Anandamide (AEA) is metabolized by fatty acid
amide hydrolase (FAAH) into arachidonic acid (AA) and ethanolamine. 2-Arachidonoylglycerol
(2-AG) is metabolized by monoacylglycerol lipase (MGL) into AA and glycerol. It is known that
2-AG is much more abundant than AEA in the brain. It is established that cPLA2-mediated AA
release from the cell membrane is coupled to PGE2 synthesis for various physiological and patho-
physiological functions. However, it was recently shown that PGE2 synthesis in the brain primarily
utilizes a pool of AA derived from 2-AG metabolism. Therefore, it can be hypothesized that stress-
induced 2-AG synthesis leads to concomitant PGE2 synthesis
This review summarizes recent studies in rodents about the roles and actions of the
AA-derived bioactive lipids, PGE2 and eCBs, in regulating emotional behaviors,
especially under stressful conditions. Given historical backgrounds, functions of
326 T. Furuyashiki and S. Kitaoka
PGE2 and eCBs in the brain have originally been studied in different contexts, in
sickness behaviors and addictive behaviors, respectively, and therefore studied by
different groups of researchers. As has been described, most studies so far suggest
distinct, and mostly opposing, actions of PGE2 and eCBs in stress-induced behav-
ioral changes. Based on this idea, the balance between PGE2 and eCBs pathways
appears to be a critical determinant for stress susceptibility as well as the level of
each pathway. An emerging coupling between eCB metabolism and PGE2 synthesis
in the brain all the more highlights the need to integrate research on both of these
bioactive lipids in the same behavioral context. Understanding the roles of PGE2
and eCBs as well as their crosstalk at the level of synthesis, metabolism, and recep-
tor signaling will allow identifying novel targets for pharmaceutical intervention for
stress-related pathophysiology in psychiatric disorders.
References
14. Gobbi G, Bambico FR, Mangieri R et al (2005) Antidepressant-like activity and modulation of
brain monoaminergic transmission by blockade of anandamide hydrolysis. Proc Natl Acad Sci
U S A 102:1862018625
15. Griebel G, Stemmelin J, Scatton B (2005) Effects of the cannabinoid CB1 receptor antagonist
rimonabant in models of emotional reactivity in rodents. Biol Psychiatry 57:261267
16. Haller J, Bakos N, Szirmay M et al (2002) The effects of genetic and pharmacological block-
ade of the CB1 cannabinoid receptor on anxiety. Eur J Neurosci 16:13951398
17. Hring M, Grieb M, Monory K et al (2013) Cannabinoid CB1 receptor in the modulation of
stress coping behavior in mice: the role of serotonin and different forebrain neuronal subpopu-
lations. Neuropharmacology 65:8389
18. Hill MN, McLaughlin RJ, Pan B et al (2011) Recruitment of prefrontal cortical endocannabi-
noid signaling by glucocorticoids contributes to termination of the stress response. J Neurosci
31:1050610515
19. Jiang W, Zhang Y, Xiao L et al (2005) Cannabinoids promote embryonic and adult hippocam-
pus neurogenesis and produce anxiolytic- and antidepressant-like effects. J Clin Invest
115:31043116
20. Jin K, Xie L, Kim SH et al (2004) Defective adult neurogenesis in CB1 cannabinoid receptor
knockout mice. Mol Pharmacol 66:204208
21. Kano M, Ohno-Shosaku T, Hashimotodani Y et al (2009) Endocannabinoid-mediated control
of synaptic transmission. Physiol Rev 89:309380
22. Kathuria S, Gaetani S, Fegley D et al (2003) Modulation of anxiety through blockade of anan-
damide hydrolysis. Nat Med 9:7681
23. Katona I, Freund TF (2012) Multiple functions of endocannabinoid signaling in the brain.
Annu Rev Neurosci 35:529558
24. Kitaoka S, Furuyashiki T, Nishi A et al (2007) Prostaglandin E2 acts on EP1 receptor and
amplies both dopamine D1 and D2 receptor signaling in the striatum. J Neurosci
27:1290012907
25. Lieb J, Karmali R, Horrobin D (1983) Elevated levels of prostaglandin E2 and thromboxane
B2 in depression. Prostaglandins Leukot Med 10:361367
26. Martin M, Ledent C, Parmentier M et al (2002) Involvement of CB1 cannabinoid receptors in
emotional behaviour. Psychopharmacology (Berl) 159:379387
27. Matsuoka Y, Furuyashiki T, Yamada K et al (2005) Prostaglandin E receptor EP1 controls
impulsive behavior under stress. Proc Natl Acad Sci U S A 102:1606616071
28. McLaughlin RJ, Hill MN, Bambico FR et al (2012) Prefrontal cortical anandamide signaling
coordinates coping responses to stress through a serotonergic pathway. Eur
Neuropsychopharmacol 22:664671
29. McLaughlin RJ, Hill MN, Dang SS et al (2013) Upregulation of CB1 receptor binding in the
ventromedial prefrontal cortex promotes proactive stress-coping strategies following chronic
stress exposure. Behav Brain Res 237:333337
30. Mechoulam R, Parker LA (2013) The endocannabinoid system and the brain. Annu Rev
Psychol 64:2147
31. Muller N, Schwarz MJ, Dehning S et al (2006) The cyclooxygenase-2 inhibitor celecoxib has
therapeutic effects in major depression: results of a double-blind, randomized, placebo con-
trolled, add-on pilot study to reboxetine. Mol Psychiatry 11:680684
32. Murakami M, Nakatani Y, Tanioka T, Kudo I (2002) Prostaglandin E synthase. Prostaglandins
Other Lipid Mediat 68-69:383399
33. Na KS, Lee KJ, Lee JS et al (2014) Efcacy of adjunctive celecoxib treatment for patients with
major depressive disorder: a meta-analysis. Prog Neuropsychopharmacol Biol Psychiatry
48:7985
34. Narumiya S, Sugimoto Y, Ushikubi F (1999) Prostanoid receptors: structures, properties, and
functions. Physiol Rev 79:11931226
35. Nestler EJ, Hyman SE (2010) Animal models of neuropsychiatric disorders. Nat Neurosci
13:11611169
328 T. Furuyashiki and S. Kitaoka
36. Nomura DK, Morrison BE, Blankman JL et al (2011) Endocannabinoid hydrolysis generates
brain prostaglandins that promote neuroinammation. Science 334:809813
37. Ortega-Alvaro A, Aracil-Fernndez A, Garca-Gutirrez MS et al (2011) Deletion of CB2 can-
nabinoid receptor induces schizophrenia-related behaviors in mice. Neuropsychopharmacology
36:14891504
38. Patel S, Hillard CJ (2006) Pharmacological evaluation of cannabinoid receptor ligands in a
mouse model of anxiety: further evidence for an anxiolytic role for endogenous cannabinoid
signaling. J Pharmacol Exp Ther 318:304311
39. Piomelli D (2003) The molecular logic of endocannabinoid signaling. Nat Rev Neurosci
4:873884
40. Rademacher DJ, Meier SE, Shi L et al (2008) Effects of acute and repeated restraint stress on
endocannabinoid content in the amygdala, ventral striatum, and medial prefrontal cortex in
mice. Neuropharmacology 54:108116
41. Rubino T, Realini N, Castiglioni C et al (2008) Role in anxiety behavior of the endocannabi-
noid system in the prefrontal cortex. Cereb Cortex 18:12921301
42. Sanchis-Segura C, Cline BH, Marsicano G et al (2004) Reduced sensitivity to reward in CB1
knockout mice. Psychopharmacology (Berl) 176:223232
43. Sciolino NR, Zhou W, Hohmann AG (2011) Enhancement of endocannabinoid signaling with
JZL184, an inhibitor of the 2-arachidonoylglycerol hydrolyzing enzyme monoacylglycerol
lipase, produces anxiolytic effects under conditions of high environmental aversiveness in rats.
Pharmacol Res 64:226234
44. Shimizu T, Ohto T, Kita Y (2006) Cytosolic phospholipase A2: biochemical properties and
physiological roles. IUBMB Life 58:328333
45. Tanaka K, Furuyashiki T, Kitaoka S et al (2012) Prostaglandin E2-mediated attenuation of
mesocortical dopaminergic pathway is critical for susceptibility to repeated social defeat stress
in mice. J Neurosci 32:43194329
46. Tanaka Y, Furuyashiki T, Momiyama T et al (2009) Prostaglandin E receptor EP1 enhances
GABA-mediated inhibition of dopaminergic neurons in the substantia nigra pars compacta and
regulates dopamine level in the dorsal striatum. Eur J Neurosci 30:23382346
47. Vane JR, Bakhle YS, Botting RM (1998) Cyclooxygenases 1 and 2. Annu Rev Pharmacol
Toxicol 38:97120
48. Witting A, Walter L, Wacker J et al (2004) P2X7 receptors control 2-arachidonoylglycerol
production by microglial cells. Proc Natl Acad Sci U S A 101:32143219
49. Zeilhofer HU, Brune K (2006) Analgesic strategies beyond the inhibition of cyclooxygenases.
Trends Pharmacol Sci 27:467474
50. Zorrilla EP, Luborsky L, McKay JR et al (2001) The relationship of depression and stressors
to immunological assays: a meta-analytic review. Brain Behav Immun 15:199226
Part IV
Protocols for Analyzing Lipid Mediators
Chapter 23
Basic Techniques for Lipid Extraction
from Tissues and Cells
Abstract Lipids as well as proteins, nucleic acids, and carbohydrates are essential
components of animals and plants, and their chemical structures and biological
functions are highly variable. However, most lipids are insoluble in aqueous solu-
tions, and careful manipulation using organic solvents is required for the extraction
and purification of lipids. In this chapter, we describe basic techniques for the
extraction and purification of lipids from animal tissues and cells using the Bligh
and Dyer method, solvent fractionation, and column chromatography. General
methods for the extraction and purification of lipids are mentioned here; refer to
other chapters for detailed information on individual lipids.
Lipids are usually defined as molecules that are soluble in organic solvents such as
chloroform, methanol, hexane, and ether. However, some lipids [e.g., lysophospho-
lipids, glycolipids, and phosphoinositides (PIPs)] are readily soluble in aqueous
solutions but are only slightly soluble in organic solvents. Lipid compounds should
be stored in appropriate organic solvents according to their polarity. For example,
polar lipids should be stored in a chloroform:methanol solution with a ratio of 2:1,
whereas nonpolar lipids should be stored in pure chloroform. Organic solvents con-
taining lipids should be stored in glass vials, and plastic lids that have Teflon welded
into their top are recommended. When it is important to completely avoid contami-
nation, such as in mass spectrometric analyses, lipid solutions should be transferred
using glass pipettes, glass syringes, or autopipettors that contain Teflon and have
glass surfaces. Organic solvents should always be prepared in high-quality, fresh
solvents that have a low water content [high performance liquid chromatography
(HPLC) or liquid chromatographymass spectrometry (LC-MS) grade]. In general,
lipids are stored in a deep freezer (below 20 C) and in glass containers at rela-
tively high concentrations. Lipid samples should be stored in containers that have a
blanket of inert gas such as nitrogen or argon in their headspace. In optimal storage
conditions, light and oxygen are absent to avoid isomerization and oxidation of the
double bonds of lipids. When removed from the freezer, lipid samples should be
allowed to reach room temperature before the cap is opened because moisture in the
atmosphere may hydrolyze the ester or amide bonds of lipids.
fluff
organic solvent
Cells in PBS Single-phase Bligh and Dyer Two-phase Bligh and Dyer
(C:M:PBS=1:2:0.8) (C:M:PBS=1:1:0.9)
Fig. 23.1 Lipid extraction by the Bligh and Dyer method. C chloroform, M methanol, PBS
phosphate-buffered saline
1. Collect and suspend cells (~108 cells) in 0.8 ml phosphate-buffered saline (PBS)
and transfer to a glass centrifuge tube with a Teflon cap.
2. Add 3 ml ice-cold chloroform/methanol solution (1:2) and mix to generate a
single-phase Bligh and Dyer mixture (final ratio of chloroform:methanol:PBS =
1:2:0.8). Vortex vigorously and incubate for 5 min.
3. Add 1 ml chloroform and 1 ml PBS and mix to generate a two-phase solution
(final ratio of chloroform:methanol:PBS = 1:1:0.9).
4. Centrifuge at 1000 g for 2 min. The lower phase is composed of
chloroform:methanol:water in a ratio of 86:14:1 (by volume) and contains lipids;
the upper phase consists of the same solvents in the ratio of 3:48:47 (by volume)
and contains the majority of nonlipid contaminants.
5. Recover the lower phase carefully with a Pasteur pipette. Do not remove the
white material at the interface, which contains denatured proteins.
6. (Optional) Add 1 ml chloroform, centrifuge, and recover the lower solution.
7. Concentrate the lower solution under a N2 stream, resuspend the resulting pellets
in chloroform:methanol solution (2:1), and store at 30 C. Generally, about
10 mg dried lipid is recovered from 0.1 g cells or tissues.
The Bligh and Dyer method is also suitable for lipid extraction from animal tissues
(brain, liver, intestine, etc.). The volumes of extraction solvents should be adjusted
to maintain the chloroform:methanol:water ratio at 1:2:0.8 for a single-phase Bligh
and Dyer mixture and at 1:1:0.9 for the formation of a two-phase solution (3,
Fig. 23.1). The exact volumes must be determined empirically for each sample type
when extracting lipids from tissues. The water volume should be considered the
aqueous component of the tissue. Different tissues have different water contents,
and the endogenous water content contributes to the aqueous component in the
334 T. Okuno and T. Yokomizo
Solvent fractionation is a simple method that is sometimes the most efficient means
to crudely separate lipids (Fig. 23.2). This approach depends on the differential
solubility of lipids in various organic solvents.
Acetone precipitation is currently used for one-step separation of polar lipids (e.g.,
phospholipids and glycolipids) from neutral and nonpolar lipids (e.g., triglycerides,
cholesterol, and the majority of fatty acids).
1. Evaporate ~100 mg crude lipid extract in a glass centrifuge tube under a N2
stream and add 2030 volumes (~5 ml) of acetone.
2. Vortex the glass centrifuge tube for 1 min and leave on ice for 1 h.
23 Basic Techniques for Lipid Extraction from Tissues and Cells 335
3. Centrifuge the tube at 2000 rpm for 5 min and carefully collect the supernatant
with a Pasteur pipette.
4. Repeat the procedure (steps 13) and combine the acetone extracts.
5. Dry the pellet, which is rich in phospholipids and glycolipids, under a N2 stream,
redissolve in chloroform/methanol mixture (2:1), and store at 30 C for further
analysis.
6. The acetone extract contains glycerides, sterols, sterol esters, carotenoids, lipid-
soluble vitamins, and fatty acids. Dry the acetone extract under a N2 stream,
redissolve in chloroform:methanol mixture (2:1), and store at 30 C.
1. Equilibrate the column (Oasis HLB, 3 ml/60 mg; Waters) with 3 ml methanol
and 3 ml water containing formic acid (pH 2.03.0).
2. Homogenize ~106 cells or ~100 mg tissue in 1 ml methanol and incubate on ice
for 1 h.
3. Centrifuge at 2000 g for 5 min and recover the supernatant (extract solution).
4. Add 9 ml water containing formic acid (pH 2.03.0) to the extract solution (final
concentration of methanol is 10 %) and apply to a pre-equilibrated reversed-
phase column.
5. Wash the column with 3 ml water.
6. Elute the column with 3 ml hexane. Neutral lipids are eluted.
7. Elute the column with 3 ml methylformate. Fatty acids and oxidized fatty acids
are eluted.
8. Elute the column with 3 ml methanol. Phospholipids and oxidized phospholipids
are eluted.
References
1. Folch J, Ascoli I, Lees M, Meath JA, Le BN (1951) Preparation of lipid extracts from brain tis-
sue. J Biol Chem 191:833841
2. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification
of total lipids from animal tissues. J Biol Chem 226:497509
3. Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J
Biochem Physiol 37:911917
4. Avanti Polar Lipids. Inc. Alabama Technical Support and Lipid Extraction. http://avantilip-
ids.com/
5. LIPID MAPS. University of California, San Diego Protocols http://www.lipidmaps.org/proto-
cols/index.html and Lipidmics methods http://www.lipidmaps.org/resources/tutorials/lipido-
micsmethods.html
6. Brown, HA (ed) (2007) Methods in enzymology, vol 432. Academic, San Diego
Chapter 24
Comprehensive Analysis of Eicosanoids
24.1 Introduction
Y. Kita (*)
Department of Lipidomics, Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Life Sciences Core Facility, Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
e-mail: [email protected]
T. Shimizu
Department of Lipidomics, Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Department of Lipid Signaling, Research Institute, National Center for Global Health and
Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan
Eicosanoids in tissues and biological fluids are extracted and cleaned up before
analysis. Tissue homogenization in aqueous buffer (e.g., saline or phosphate-
buffered saline) can be performed in most biochemical or bioanalytical laboratories
using ordinary homogenizers. Indomethacin (~10 M) and/or butylated hydroxy-
toluene [BHT; 0.1 % (w/v)] can be included in the homogenization buffer to avoid
any ex vivo eicosanoid formation or oxidation. The homogenates are mixed with
approximately 10 volumes of methanol for 1 h at 4 C for extraction and then cen-
trifuged to remove debris and protein precipitates. A more preferable alternative is
to use a cryomill. An SK-mill (Tokken, Chiba, Japan) or similar can be used to
powder frozen tissues in a polypropylene tube without thawing. For example, up to
100 mg frozen tissue can be crushed by a SK-100 mill in a 2-ml Eppendorf Safe-
Lock tube with a stainless steel crusher, to which 1 ml methanol is added directly
for extraction. After extraction, debris and protein precipitates are removed using a
high-speed centrifuge (10,000 g, 10 min, 4 C). The use of a cryomill minimizes the
risk of sample deterioration while providing better throughput. For both methods,
deuterated internal standards for LC-MS quantification are included at this step. For
24 Comprehensive Analysis of Eicosanoids 339
biological fluids, samples are mixed with ten (or more) volumes of methanol and
internal standards, and then centrifuged to obtain extracts. In most cases, methanol
extraction is recommended as the initial extraction procedure. The BlighDyer
method for total lipid extraction [5] is not recommended because of poor recovery
of polar eicosanoids.
The methanol extracts are subjected to solid-phase extraction (SPE). Sep-Pak
C18 (Waters, Milford, MA, USA) or similar reversed-phase SPE cartridges have
been widely used for this purpose, but recent products such as Oasis HLB (Waters)
or similar polymer-based reversed-phase sorbents with polar functional groups pro-
vide better recovery and reproducibility. An Oasis HLB cartridge 1 ml/10 mg
(or 1 ml/30 mg) can be used for 1 ml crude methanol extract (Fig. 24.1). Methanol
extracts are diluted four- or fivefold with 0.03 % formic acid:water and loaded onto
preconditioned cartridges with vacuum. Once diluted, samples are immediately
loaded to avoid losing hydrophobic eicosanoids by adsorption to any contacting
surfaces. The cartridges are then washed serially with 0.1 % formic acid:water,
15 % ethanol:0.03 % formic acid:water, and petroleum ether. Washing with aqueous
solutions removes polar components such as salts, sugars, and polar peptides.
Petroleum ether removes neutral lipids (i.e., triglycerides). After briefly drying the
cartridges by passing air, eicosanoids are eluted with 200 l 0.2 % formic
acid:methanol either by low-speed centrifugation (800 g, 2 min) or by vacuum to
collection vials. If needed, samples can be evaporated and reconstituted to smaller
volumes for analysis.
OH
A
COOH
Intensity
HO O
TXB2 OH
0 1 2 3 4 5 6
Time (min)
OH
B COOH
O
CH2COOH
S N COOH
H
N
H NH2
LTC4 O
Intensity
LTD4
0 1 2 3 4 5 6
Time (min)
Fig. 24.2 Chromatography of TXB2 and LTC4. (a) Resolution of TXB2 by reversed-phase LC
(Capcell Pak MGS3, 1 100 mm, 0.1 % formic acid:37 % acetonitrile/water, isocratic).
Characteristic peak shape is observed. (b) Resolution of LTC4 and LTD4 (Capcell Pak MGS3,
1 100 mm, 0.1 % formic acid:45 % acetonitrile:water, isocratic). Peak tailing of LTC4 is obvious
even under optimized condition (Modified from Kita et al. [6])
24 Comprehensive Analysis of Eicosanoids 341
80 COO- 319
70 257 [M-H]-
155
60
50
40 8(S)-HETE
30 163
20
203 275
10 127
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
m/z
B 100 179
319
[M-H]-
Relative Abundance
90 +H
80 COO-
70
60
50 179
OH
40
257 301
30 12(S)-HETE
20
163 208
10 135
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
m/z
C
SRM (319 > 115) 5-HETE
0 1 2 3 4 5 6
Time (min)
Fig. 24.3 Fragmentation and chromatography of HETE isomers. (a, b) MS/MS spectra for
8-HETE and 12-HETE. (c) Resolution of HETE isomers (Capcell Pak MGS3, 1 100 mm, 0.1 %
formic acid:65 % acetonitrile:water, isocratic). 8-HETE and 12-HETE are not chromatographi-
cally resolved, but all HETE isomers are differentiated by isomer-specific selected reaction moni-
toring (SRM) transitions (Modified from Kita et al. [6])
24 Comprehensive Analysis of Eicosanoids 343
A 315
100 O -H2O
-CO2
Relative Abundance
90
333
80 COO- -H2O
70 351
60 271 [M-H]-
50 HO
OH
40
30 PGE2
20 233
189
10
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
B m/z
100 315
HO
Relative Abundance
90
-CO2 -H2O
80 COO-
70
60 233
50 O 271
OH
40 333 -H2O
30 PGD2
20 351
10 189 251 [M-H]-
0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
C
SRM (351 > 271)
Intensity
PGE2
PGD2
0 1 2 3 4 5 6
Time (min)
Fig. 24.4 Fragmentation and chromatography of PGE2 and PGD2. (a, b) MS/MS spectra for PGE2
(a) and PGD2 (b). Common fragment peak (m/z 271) is used for SRM. (c) Resolution of PGE2 and
PGD2 (Capcell Pak MGS3, 1 100 mm, 0.1 % formic acid:37 % acetonitrile:water, isocratic)
(Modified from Kita et al. [6])
Deuterated eicosanoids are commercially available and can be used for quantifi-
cation by internal standard method. For comprehensive analysis, a mixture of inter-
nal standards is added to the samples before eicosanoid extraction to correct
recoveries throughout the sample preparation and LC-MS procedures. The amount
of internal standards is determined depending on the system sensitivity and calibra-
tion range; generally, the signal of internal standards should be high enough to
achieve good reproducibility in repeated measurements (%CV less than 5 % is rec-
ommended). For example, deuterated PGs can be spiked to the samples at amounts
that give signals corresponding to approximately 100 pg on ordinary triple quadru-
pole MS instruments. Injection of an excess amount of deuterated compound that
contains a small number of deuterium often disturbs the detection of target signals.
For example, injection of PGE2-d4 (m/z 355 > 275) can cause a small, but signifi-
cant, PGE2 signal (m/z 351 > 271), caused by the existence of PGE2 as an impurity
for PGE2-d4 preparation, which becomes more significant when quadrupoles are
operated at low-resolution settings where less deuterated compounds such as
PGE2-d1 or PGE2-d2 also cause PGE2 signals. Thus, the use of excess internal stan-
dards that mask the true signals in the samples should be avoided, although mathe-
matical correction can be performed either manually or by software. For deuterated
compounds containing more numbers of deuterium, such as HETE-d8, such inter-
fering signals are rarely observed.
24 Comprehensive Analysis of Eicosanoids 345
Pump B
6-port
MeCN:Formic acid Trap Column
Valve
Pump C
Water:Formic acid Tee-connector for online-dilution
Fig. 24.5 Online-dilution column-switching LC-MS system. Sample introduced from autosam-
pler is fivefold diluted and concentrated on a trapping column (Opti-guard mini, 1 10 mm;
Optimize Technologies, Oregon City, OR, USA). After valve switches, sample is introduced to
separating column (Capcell Pak MGS3, 1 100 mm) (Modified from Kita et al. [6])
346 Y. Kita and T. Shimizu
6-keto-PGF1
TXB2
PGF2
PGE2 PGD2
LTD4
Relative Abundance
LTC4
LTB4
5-HETE
8-HETE
11-HETE
12-HETE
15-HETE
PAF
0 1 2 3 4 5 6 7 8 9 10
Time (min)
References
1. Samuelsson B (2012) Role of basic science in the development of new medicines: examples
from the eicosanoid field. J Biol Chem 287:1007010080
2. Shimizu T (2009) Lipid mediators in health and disease: enzymes and receptors as therapeutic
targets for the regulation of immunity and inflammation. Annu Rev Pharmacol Toxicol
49:123150
3. Serhan CN (2005) Mediator lipidomics. Prostaglandins Other Lipid Mediat 77:414
4. Rago B, Fu C (2013) Development of a high-throughput ultra performance liquid
chromatography-mass spectrometry assay to profile 18 eicosanoids as exploratory biomarkers
for atherosclerotic diseases. J Chromatogr B Anal Technol Biomed Life Sci 936:2532
5. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 37:911917
6. Kita Y, Takahashi T, Uozumi N, Shimizu T (2005) A multiplex quantitation method for eico-
sanoids and platelet-activating factor using column-switching reversed-phase liquid
chromatography-tandem mass spectrometry. Anal Biochem 342:134143
Chapter 25
Mass Spectrometric Analysis of Phospholipids
by Target Discovery Approach
Kazutaka Ikeda
Abstract Phospholipids (PLs), the most abundant lipids in the cell membrane, are
composed of various types of polar head and fatty acid. Their abundance and distri-
bution vary among organs, and some are oxidized by oxidant stress or inammation.
For this structural diversity, the optimal analytical method for proling is needed.
Lipidomics is an approach well suited to obtain structural information about PL
molecular species and to acquire quantitative proles with a combination of liquid
chromatography (LC) and mass spectrometry (MS). As for the conventional analy-
sis method, a non-target, focusing, or target approach is usually selected, but these
approaches have a disadvantage in sensitivity or completeness. To solve these con-
ventional problems, this chapter introduce the target discovery approach, with
which it is possible to obtain MS/MS data sensitively at high speeds and to combine
a non-target or target approach. This approach has a strong potential as an improved
lipidomics strategy to search new lipids of interest or to identify unknown lipid spe-
cies in a nonbiased manner.
25.1 Introduction
Phospholipids (PLs), the most abundant lipids in the cell membrane, are composed
of a polar head and a nonpolar tail region. The nonpolar tail has two fatty acids of
various lengths and saturation. The polar head region has a phosphate group such as
choline (PC), ethanolamine (PE), inositol (PI), serine (PS), glycerol (PG), or phos-
phatidate (PA). The abundance and distribution of PLs vary among organs, and
K. Ikeda (*)
Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences,
1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and
Technology Agency (JST), 7 Goban-cho, Chiyoda-ku, Tokyo 102-0076, Japan
e-mail: [email protected]
some are oxidized by oxidant stress or inammation. For this structural diversity,
the optimal analytical method for proling is needed.
Lipidomics is an approach well suited to obtain structural information about PL
molecular species and to acquire quantitative proles with a combination of liquid
chromatography (LC) and mass spectrometry (MS). Electrospray ionization (ESI)
MS is considered to be a soft ionization method and is used widely in lipidomics.
As for the conventional analysis method, a non-target, focusing, or target approach
is usually selected [1]. High-resolution MS such as the time-of-ight (TOF) or fou-
rier transform (FT) type is applied for the non-target approach in many cases, and
PL molecular species are globally detected by MS scanning. Precursor ion scanning
or neutral loss scanning of the polar head group or the fatty acid is performed in the
focusing approach, with which it is possible to detect the same class of PL molecu-
lar species widely and sensitively [2, 3]. However, the non-target approach and
focusing approach have a disadvantage in the detection of minor PLs. Multiple reac-
tion monitoring (MRM) (also known as selected reaction monitoring, SRM) is
applied for the target approach, and individual molecular species are detectable
selectively with high sensitively [1, 4], whereas the target approach has a disadvan-
tage in completeness of search range.
To solve these conventional problems, the target discovery approach, with which
it is possible to obtain MS/MS data sensitively at high speeds and to combine with
the non-target or target approach, is effective. The following protocols demonstrate
two types of PL screening methods using the target discovery approach.
25.2.1 Materials
Homogenize tissue
by Shake Master NEO Check each amount of loading
Add 1 mL of MeOH homogenize solvent (A mL)
Tissue 10~50 mg at 1500 rpm for 5min ( 1 5) on prepared Excel data sheet
by liquid-nitrogen cooling
Zr beads (3 mm 5)
for 10 sec for 15 min at R.T. for 10 sec for 15 min at R.T.
Glass tube
1. Pulverize and homogenize mouse skeletal muscle (10 mg) in cold methanol by a
bead pulverizing machine (1500 rpm for 5 min) (Fig. 25.1)
2. Put the homogenized solvent in a 2-ml glass tube [5]
3. Add 250 l chloroform containing internal standard (I.S.) mixture* and remain-
ing methanol (reaching total methanol volume, 500 l)
4. Vortex for 10 s and incubate for 15 min at room temperature
5. Add 50 l Milli-Q water, vortex for 10 s, and incubate for 15 min at room
temperature
6. Centrifuge at 1000 g for 10 min at room temperature
7. Collect the supernatant in an LC-MS vial
8. Analyze the supernatant by LC-MS using the target discovery approach as
follows
1. Analyze PLs by data-dependent MS/MS scanning (Fig. 25.2; Table 25.2) and
identify these molecular species by in-house lipid search
2. Analyze PLs by data in-dependent scanning (Fig. 25.3; Table 25.3)
Table 25.1 Column conditions for reversed-phase liquid chromatography (LC) separation
LC condition Flow rate 0.3 ml/min
Temp 45 C
Solvent A ACN:MeOH:water (=2:2:6) + 5 mM ammonium
formate
B IPA + 5 mM ammonium formate
Gradient (B %) 0 min 0 %
5 min 40 %
7.5 min 64 %
12 min 64 %
12.5 min 82.5 %
19 min 85 %
20 min 95 %
Equilibration time: 5 min
Fig. 25.2 Target discovery approach by data-dependent MS/MS scanning in combination with
in-house lipid search
25 Mass Spectrometric Analysis of Phospholipids by Target Discovery Approach 353
m/z
Q1 isolaon
m/z
(No MS/MS threshold)
m/z (2001250)
MS/MS All data
Each 25 Da
Fig. 25.3 Target discovery approach by data-independent scanning for MS/MS: all analysis
FA(18:1) Choline
derivaon
FA(16:0)
lysoPC(18:0)
lysoPC(16:0)
Q1 Q3 RT
Retro MRM
Fig. 25.4 Retrospective MRM analysis of PL molecular species from MS/MS: all data
25 Mass Spectrometric Analysis of Phospholipids by Target Discovery Approach 355
25.4 Commentary
In the data-dependent scan, precursor ions of PL and lyso-PL molecular species are
globally scanned by high-resolution TOF MS mode, and if some of them are over
the setup threshold, they are secondarily scanned by the sensitive TOF MS/MS
mode, and it is possible then to acquire the structural information (Fig. 25.2). These
MS/MS datasets are applicable to in-house lipid search, and these molecular spe-
cies are identied globally and accurately (manuscript in preparation). The lipid
search is a search engine for the identication of lipid molecular species from MS
raw data that was originally developed by Dr. Taguchi [6] collaborating with Mitsui
Knowledge Industry (MKI).
Our in-house lipid search made some improvements in the accuracy of identi-
cation by new algorithms as compared with conventional searching techniques.
For instance, knockout selection, which sets the order of priority of MS/MS peaks
for high-precision identication of the individual molecular species by reference to
many raw MS/MS data patterns, was adopted in our group and with MKI.
For data in-dependent scan, all precursor ions of PL and lyso-PL molecular spe-
cies are scanned without threshold by sequential window acquisition of all theoreti-
cal fragment ion spectra (SWATH) mode [7]. With this method, it is possible to
obtain all the MS/MS data with high sensitively. In the SWATH mode, the Q1 quad-
rupole is typically stepped at 12.525 amu increments across the 2001250 m/z pre-
cursor range (Fig. 25.3). For data mining, multiple reaction monitoring (MRM)-like
analysis, our termed retro-MRM, is used to accurately identify the molecular spe-
cies. Suitable candidates for fragment ions to identify individual molecular species
of PLs and lyso-PLs are automatically selected from our MS/MS database, and the
356 K. Ikeda
References
1. Taguchi R, Nishijima M, Shimizu T (2007) Basic analytical systems for lipidomics by mass
spectrometry in Japan. Methods Enzymol 432:185211
2. Ekroos K, Chernushevich IV, Simons K, Shevchenko A (2002) Quantitative proling of phos-
pholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-ight mass spec-
trometer. Anal Chem 74:941949
3. Ikeda K, Kubo A, Akahoshi N, Yamada H, Miura N, Hishiki T, Nagahata Y, Matsuura T,
Suematsu M, Taguchi R, Ishii I (2011) Triacylglycerol/phospholipid molecular species proling
of fatty livers and regenerated non-fatty livers in cystathionine beta-synthase-decient mice, an
animal model for homocysteinemia/homocystinuria. Anal Bioanal Chem 400:18531863
4. Nakanishi H, Iida Y, Shimizu T, Taguchi R (2009) Analysis of oxidized phosphatidylcholines as
markers for oxidative stress, using multiple reaction monitoring with theoretically expanded
data sets with reversed-phase liquid chromatography/tandem mass spectrometry. J Chromatogr
B 877:13661374
5. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purication
of total lipids from animal tissues. J Biol Chem 226:497509
6. Taguchi R, Ishikawa M (2010) Precise and global identication of phospholipid molecular spe-
cies by an Orbitrap mass spectrometer and automated search engine Lipid Search. J Chromatogr
A 1217:42294239
7. Gillet LC, Navarro P, Tate S, Rst H, Selevsek N, Reiter L, Bonner R, Aebersold R (2012)
Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a
new concept for consistent and accurate proteome analysis. Mol Cell Proteomics 11:O111.016717
Chapter 26
Determination of Sphingolipids by LC-MS/MS
26.1 Reagents
26.2 Equipments
Siliconized sample tubes and pipette tips are used. For quantitative analysis, stable
isotope analogue internal standards are added to biological samples. The ratio between
the internal standard and the compound of interest can be measured by LC/MS.
26 Determination of Sphingolipids by LC-MS/MS 359
Before analysis, all frozen samples (80 C) should be thawed and allow to equili-
brate at 4 C (or on ice). Do not leave the blood samples on ice before preparing
plasma because red blood cells are a source of the plasma S1P. Thus, it is necessary
360 T. Takahashi et al.
Pass the supernatant through a filter Pass the supernatant through a filter
Centrifuge at 5,100 g Centrifuge at 5,100 g
to be prepared for plasma fraction from whole blood as soon as possible. Sample
preparation for MS is recommended to utilize deproteinization of an organic solvent
such as methanol (Fig. 26.1) as follows:
1. Transfer 10 l plasma into a siliconized sample tube.
2. Add 80 l deproteinization solution (0.1 % formic acid in methanol).
3. Add 10 l IS mixture 1 (final concentration of IS is 100 nmol/l).
Tthe additional volume of IS mixture should be changed to match the stan-
dard calibration curve if you need concentration steps for sample preparation.
4. Close the tube and vortex for 30 s.
5. Then, homogenize for 5 min in an ultrasonic bath on a tube floater.
6. Centrifuge at 16,400 g for 10 min at 4 C and separate supernatant.
7. The supernatant (approximately 100 l) should be passed through a filter (0.2
m pore size, 4 mm inner diameter).
8. Inject 10 l filtered solution into the chromatographic system using an automatic
injector. That is, each internal standard is 1 pmol/on column.
The sample preparation for tissue is also shown in Fig. 26.1 as follows:
1. Tissue (approximately 50 mg) is placed into a siliconized sample tube (2.0 ml).
2. Add 400 l deproteinization solution.
3. Add 50 l IS mixture 1 (final concentration of IS is 100 nmol/l).
The additional volume of IS mixture should be changed to match the standard
calibration curve if you need concentration steps for sample preparation.
26 Determination of Sphingolipids by LC-MS/MS 361
A triple quadrupole mass spectrometer equipped with an ESI source is used for
sphingolipids analysis such as TSQ Quantum Ultra with heated electrospray ioniza-
tion (H-ESI) probe (Thermo Fisher Scientific). Because carryover of samples based
on interactions with metal components and the phosphate group of lipids is a con-
cern, PEEK tubing and connector are useful and are recommended. To optimize the
sensitivity of sphingolipids, several parameters such as spray voltage, sheath gas
pressure, auxiliary gas flow rate, capillary temperature, tube lens offset voltage, and
vaporizer temperature need to be optimized.
1. Infuse a polytyrosine-1,3,6 calibration solution directly into the ESI source with
a syringe pump to tune and calibrate.
2. Save the tune method and calibration files.
3. Fill a clean syringe with 10 g/ml sphingolipid solution and place in the syringe
holder of the syringe pump of MS.
4. Set up the MS detector to tune in H-ESI/MS mode and start infusion. The MS/
MS transitions will be detected in the full scan mode (m/z 50500). Optimal
conditions of tube lens offset and collision energy for SRM analysis employ the
transition of the [M + H]+ precursor ions to their product ions of S1P, dhS1P,
pS1P, Sph, dhSph, and pSph. The parameters of ionization, especially vaporizer
temperature, sheath gas pressure, and auxiliary gas pressure, should be retuned
after LC condition is fixed because they depend on the flow rate of LC. Example
of defined parameters of ionization and MS/MS transitions are described in
Table 26.2.
a) Position A
Analytical
Eluent Pump column
A 1
1 6
Eluent Pump
5
B 2
2 ESI MS / MS
4
3
b) Position B Analytical
Eluent Pump column
A 1
1 6
Eluent Pump
B 2 5
2 ESI MS / MS
4
3
Fig. 26.2 (a) The sample is injected by autosampler and located to atrap column by eluent C at
position A. (b) The traped compounds to a trap column are eluted and loaded to the analytical
column at position B
needed. For low carryover, washing and cleaning of the needle attached to an auto-
matic injector may be effective with more than one washing/cleaning solvent. An
online solid-phase extraction may be valid for ultramicroanalysis. Our LC system
for sphingolipids analysis is described in Fig. 26.2. The sample is injected by an
autosampler and located to a trap column by eluent C for 7 min at position A (16).
After changing from position A to B of a six-port valve, trapped compounds are
eluted from the trap column and loaded to the analytical column by eluent A and B
for 10 min at position B (12).
Isocratic elution or gradient elution may be chosen. The column selection (phase
material, inner diameter, length, etc.) and optimization of the mobile phase are very
important for stable analysis. On sphingolipid analysis using reverse-phase chroma-
tography, pH 4.0 of the mobile phase is especially important (Table 26.3).
Generally, an ODS column is selected for sphingolipids analysis. However, a
peak-tailing problem may be detected because of an interaction between a phos-
phate group of sphingolipids and the residual silanol group (imperfect hydration
state) or a metal ion inside a ODS column. Therefore, column that has as reduced a
residual silanol group as possible to improve the peak-tailing problem should be
selected for sphingolipids analysis. To conduct good analysis, the improvement of
peak shape should be aimed at evaluating a number of theoretical stages and a sym-
metry factor carefully, and this leads to the rise of sensitivity.
364 T. Takahashi et al.
The parameters of ionization using LC change depending on the flow rate, espe-
cially vaporizer temperature, sheath gas pressure, and auxiliary gas pressure. Our
optimal parameters for Nanospace SI 2-Quantum Ultra system are described in
Table 26.4. SRM chromatograms are shown in Fig. 26.3.
0
100 RT: 14.37 4.66e6
50
(b) Sph, m/z 300.3 > 282.2 AA: 25,183,259
0
100 RT: 14.61 1.77e6
(c) dhSph, m/z 302.3 > 284.2
Relative Abundance (%)
AA: 10,977,461
50
0
100 RT: 14.11 3.95e6
50
(d) pSph, m/z 318.3 > 300.2 AA: 2,255,378
100
RT: 14.22 1.71e6
50
(e) C17-S1P, m/z 366.2 > 250.1 AA: 19,961,448
0
100 RT: 14.65 1.89e6
50
(f) S1P, m/z 380.3 > 264.2 AA: 20,081,259
0
100 RT: 14.91 7.52e5
50
(g) dhS1P, m/z 382.3 > 284.1 AA: 8,316,258
0
100 RT: 14.54 6.41e5
50
(h) pS1P, m/z 398.3 > 300.2 AA: 6,424,655
0
0 2 4 6 8 10 12 14 16 18 20 22
Time (min)
a b
6.0 0.15
8.0 0.20
0.15
6.0 0.10
0.05
4.0 0.05
0.00
0 5 10 0.00
S1P concentration (nM) 0 5 10
4.0 Sph concentration (nM)
2.0
2.0
y = 0.01133x + 6.9994e-5 y = 0.01292x + 0.0005889
R2 = 0.9995, W=1/X R2 = 0.9997, W=1/X
0.0 0.0
0 100 200 300 400 500 0 100 200 300 400 500
S1P concentration (nM) Sph concentration (nM)
Fig. 26.4 (a) Caliburation curve for S1P. (b) Caliburation curve for Sph
References
1. Kasumov T, Huang H, Chung YM, Zhang R, McCullough AJ, Kirwan JP (2010) Quantification
of ceramide species in biological samples by liquid chromatography electrospray ionization
tandem mass spectrometry. Anal Biochem 401:154
2. Wewer V, Brands M, Dormann P (2014) Fatty acid synthesis and lipid metabolism in the obli-
gate biotrophic fungus Rhizophagus irregularis during mycorrhization of Lotus japonicus.
Plant J 79:398
3. Liebisch G, Drobnik W, Reil M, Trumbach B, Arnecke R, Olgemoller B, Roscher A, Schmitz
G (1999) Quantitative measurement of different ceramide species from crude cellular extracts
by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J Lipid Res 40:1539
4. Han X (2002) Characterization and direct quantitation of ceramide molecular species from
lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal
Biochem 302:199
5. Gaskell SJ, Edmonds CG, Brooks CJ (1976) Applications of boronate derivatives in the study
of ceramides by gas-liquid chromatography-mass spectrometry. J Chromatogr 126:591
6. Camera E, Picardo M, Presutti C, Catarcini P, Fanali S (2004) Separation and characterisation
of sphingoceramides by high-performance liquid chromatography-electrospray ionisation
mass spectrometry. J Sep Sci 27:971
7. Couch LH, Churchwell MI, Doerge DR, Tolleson WH, Howard PC (1997) Identification of
ceramides in human cells using liquid chromatography with detection by atmospheric pressure
chemical ionization-mass spectrometry. Rapid Commun Mass Spectrom 11:504
8. Hammad SM, Pierce JS, Soodavar F, Smith KJ, Al Gadban MM, Rembiesa B, Klein RL,
Hannun YA, Bielawski J, Bielawska A (2010) Blood sphingolipidomics in healthy humans:
impact of sample collection methodology. J Lipid Res 51:3074
9. Bielawski J, Pierce JS, Snider J, Rembiesa B, Szulc ZM, Bielawska A (2009) Comprehensive
quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-
tandem mass spectrometry. Methods Mol Biol 579:443
10. Shaner RL, Allegood JC, Park H, Wang E, Kelly S, Haynes CA, Sullards MC, Merrill AH Jr
(2009) Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quad-
rupole linear ion trap mass spectrometers. J Lipid Res 50:1692
26 Determination of Sphingolipids by LC-MS/MS 369
30. Jin YX, Cui XH, Paek KY, Yim YH (2012) A strategy for enrichment of the bioactive sphin-
goid base-1-phosphates produced by Hypericum perforatum L. in a balloon type airlift reactor.
Bioresour Technol 123:284
31. Saigusa D, Shiba K, Inoue A, Hama K, Okutani M, Iida N, Saito M, Suzuki K, Kaneko T,
Suzuki N, Yamaguchi H, Mano N, Goto J, Hishinuma T, Aoki J, Tomioka Y (2012)
Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by
liquid chromatography/electrospray ionization tandem mass spectrometry. Anal Bioanal Chem
403:1897
32. Zhao YY, Liu J, Cheng XL, Bai X, Lin RC (2012) Urinary metabonomics study on biochemi-
cal changes in an experimental model of chronic renal failure by adenine based on UPLC
Q-TOF/MS. Clin Chim Acta 413:642
33. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, Bandyopadhyay
S, Jones KN, Kelly S, Shaner RL, Sullards CM, Wang E, Murphy RC, Barkley RM, Leiker TJ,
Raetz CR, Guan Z, Laird GM, Six DA, Russell DW, McDonald JG, Subramaniam S, Fahy E,
Dennis EA (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J
Lipid Res 51:3299
34. Lydic TA, Busik JV, Reid GE (2014) A monophasic extraction strategy for the simultaneous
lipidome analysis of polar and nonpolar retina lipids. J Lipid Res 55:1797
Chapter 27
Lipid Machinery Investigation Using MALDI
Imaging Mass Spectrometry
Abstract Lipids have essential roles in several biological processes, including nor-
mal cell functioning as well as in the onset of diseases. Renewed interest in lipid
research has been largely fueled by emerging evidence linking the augmenting of
corporal adipose tissue in humans with metabolic diseases and cancer. Imaging
mass spectrometry techniques including matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF MS) are analytical methods quite
suitable for the investigation of compounds in biological samples, providing an
enormous amount of information regarding their chemical composition and spatial
distribution with no need for prior labeling. Particularly, MALDI-TOF imaging
mass spectrometry (IMS) has evolved into a very versatile technique capable of
analyzing, at molecular level, a wide range of compounds, including lipids. This
review presents a survey of recent work conducted mainly in our laboratory on full
protocol development for lipid analysis by MALDI-TOF IMS of biological sam-
ples, with emphasis on sample collection and preparation, matrix selection and
deposition, and typical analysis condition settings.
27.1 Introduction
The functions of lipids in energy storage in the body have been well studied and
documented [1]. Nevertheless, although there have been speculations and some evi-
dence about other functions that lipids may have in homeostatic processes and the
onset of disease, until now it has been a greatly neglected area of research. Recently,
scientists have shown renewed interest in lipid research largely fueled by mounting
evidence linking an increase of corporal adipose tissue in humans with metabolic
diseases [2] and cancer [3]. Indeed, new studies are evidencing that a wide range of
lipids, including fatty acids, glycerophospholipids, and sphingolipids, are critical in
a vast number of biological processes [4]. Several mass spectrometry (MS) tech-
niques have been developed for the characterization of biological samples, includ-
ing electrospray ionization (ESI) [5], secondary ion mass spectrometry (SIMS) [6],
and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) [7, 8]. Initially, MALDI-TOF MS was limited to the analysis of
proteins, but since then it has steadily evolved into a very versatile technique capa-
ble of analyzing a wider range of compounds, including lipids.
Lipid analysis by MALDI-TOF MS is potentially a better alternative to more
traditional techniques such as gas chromatography and high performance liquid
chromatography. For example, it requires minimum sample preparation and has
high tolerance to impurities, no cumbersome extractions are needed, and measure-
ments can be completed in a relatively short time. Several factors concur for this
outcome, such as the high abundance of lipids in tissue, inherent ionization of many
lipids forming the bilayer cell membrane, which allows positive or negative ions to
be generated in great number upon laser pulse impact, and efficient ion collection
by modern mass spectrometers for ions having a molecular mass less than 1000 Da
[9]. Nevertheless, as peak intensities of lipids can widely vary during MALDI-TOF
MS analysis, certain identified species may be overestimated, such as in the case of
lipids with ammonia groups, while others are underestimated. Moreover, in the case
of free fatty acids, signals of standard matrices often interfere with those of targeted
fatty acids as they both occupy the same low mass range [10].
To overcome the aforementioned difficulties and to enhance the identification
and characterization of lipids, a number of protocols, including sample collection
and preparation techniques as well as MALDI-TOF MS analysis methods, have
been developed and used worldwide in the past few years to study a wide number of
lipid species at molecular level. Specifically, our laboratory has made a great effort
to develop new methods and modify existing techniques for the study of lipid spe-
cies in tissues showing indication of diseases, including cancer. In the following
sections, we describe some of these achievements in the form of protocols that we
have used for the investigation of the lipid machinery.
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 373
Materials
Anesthetic agents (pentobarbital, ketamine, etc.)
Diethyl ether
Surgical blades and scalpel
Scissors
Dry ice
Liquid nitrogen
Cryogenic protective gloves
Wooden mallet
Sturdy poly-mesh bags
Sieves
Polystyrene foam boxes
374 I. Yao et al.
Postmortem Freezing
1. Pulverize the dry ice with the mallet to produce a very fine powder. If large
chunks still remain, use the sieve to retrieve them. Transfer the powdered dry ice
to a polystyrene foam box. Keep the lid on until further use.
2. To euthanize, deeply anesthesize the animal as required, and cut the head off.
With the scissors, cut open the skin across the top of the head and toward the
nasal region.
3. Insert the tip of a small scissor blade straight into the foramen magnum, lift and
cut the interparietal bone, and push the parietal bones to the sides, like a swing
door.
4. Without damaging the surface of the brain, insert the tip of the scissor blade
underneath the frontal bones and force open the fissure in between them. Do the
same with the nasal bones. Push all bones to the sides as it was done with the
parietal bones.
5. Scoop out the brain with a spatula. It should be removed from the base of the
skull, either from the olfactory nerve side or the cerebellum side.
6. With a gentle but quick motion, place the brain onto the powdered dry ice and
cover completely with it. Let it flash-freeze for approximately 1 min. Remove
the frozen brain and place it inside a precooled Ziploc plastic bag or 50-ml coni-
cal tube. Store the brain at 80 C until further use.
In Situ Freezing
1. In a well-ventilated room, fill a Dewar flask to the brim with liquid nitrogen.
2. Deeply anesthetize the animal with diethyl ether and skin the head.
3. To euthanize the animal and inhibit tissue degradation, dip the top of the head
with care so that the nose is not immersed.
4. Remove the brain and store it as described in Protocol 1.
compound that does not interfere with MS analysis [23]. Other embedding media
may be used for embedding, including gelatine [24] and agarose [25].
With regard to the thickness of sections, thin slices are very frail and difficult to
handle [26]. Conversely, thick slices are not recommended because they take a
longer time to dry and behave as insulating material, which may adversely affect
MS analysis. Thus, slices of 1020 m have been found to be optimal for handling
and analyzing by MS [26]. Once tissues are sectioned, they should be mounted onto
a surface with good electric conductivity. Metal plates are the best conductive mate-
rial for MALDI-TOF MS analysis, but the need to thoroughly clean these plates
after analysis makes their use impractical for multiple measurements. Alternatively,
there are commercially available plastic sheets and glass slides coated with an
excellent conductive layer such as indium tin oxide (ITO) [23], which are also
disposable.
Because lipids are relatively small molecular weight compounds, signals from the
matrix should be sufficiently small to avoid overlap with the target lipids [27]. The
matrix 2,5-dihydroxybenzoic acid (DHB) has been widely used for the analysis of
lipids, as it generally produces few matrix-related ions [28]. Goto-Inoue et al. suc-
cessfully employed DHB to visualize, by MALDI-TOF IMS, the distribution of
ceramides in skin tissue samples from patients suffering from DorfmanChanarin
syndrome [29]. Similarly, He et al. studied the distribution of phophatidylcholines
in Warthin tumor tissues using DHB as matrix [30]. However, the use of traditional
matrices such as DHB and -cyano-4-hydroxycinnamic acid (CHCA) for the
MALDI-TOF MS analysis of lipids such as cholesterol and fatty acids sometimes
results in poor-quality data because an important overlapping occurs between lipid
and matrix ion signals [31], especially in negative-ion mode [28]. The matrix
9-aminoacridine (9AA) is a moderately strong basic compound that has been found
to be a better alternative for the analysis of free fatty acids in negative-ion mode
[32]. Matrix 9AA has also been found to produce better results than newer com-
pounds such as 2,4,6-trihydroxyacetophenone (THAP) [33]. In addition, although
DHB has been used as matrix to detect lipid oxidation products such as peroxides
and reactive oxygen species (occurring in tissues from patients with inflammatory
diseases), chlorinated phosphatidyl ethanolamine species are not easily detected if
analyses are conducted by MALDI-TOF MS with DHB as matrix [31]. Nonetheless,
if 4-chloro--cyanocinnamic acid (Cl-CCA), a chlorinated analogue of cinnamic
acid, is used as matrix, these chloraminic species can be easily detected by MALDI-
TOF MS [34].
376 I. Yao et al.
27.4.1.1 Flavonoids
Two common problems when using DHB as the sole compound in a matrix solution
for MS imaging are (1) the production of nonhomogeneous, needle-shaped crystal-
lization, which appears on the outer rim of the deposited solution, and (2) delocal-
ization of tissue lipids by the solvent used to dissolve this matrix. Ionic matrices are
organic salts used as matrices that are formed by an acidbase reaction, usually
combining a conventional MALDI matrix with an organic base. Ionic matrices may
be either liquid or solid, depending on the molar ratio of components and other
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 377
factors. Liquid ionic matrices based on 2,5-DHB in combination with aniline (ANI),
pyridine (Pyr), and 3-acetylpyridine (3-AP) were investigated to overcome the
drawbacks of crystalline 2,5-DHB [38]. An automatic micro-spotter (Chemical Ink-
Jet Printer CHIP-1000; Shimadzu Biotech, Kyoto, Japan) was used for precise spot-
ting of freshly prepared matrices. Improved imaging of lipids was validated on
human ovarian cancer biopsies. Liquid ionic matrices for r-cyano-4-hydroxycin-
namic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine
(DHBB) were also validated against DHB for enhanced visualization of phospho-
lipids in mouse liver and brain tissue sections [39].
27.4.1.3 Nanomaterials
MALDI matrix layers allow laser energy absorption and energy transfer to analyte
molecules with minimal fragmentation, achieving otherwise unlikely MS measure-
ments. Nonetheless, matrix materials usually have small molecular weights, which
hampers the detection of weak signals of chemical compounds in the small mass
range. Protocols for MS analysis using compounds other than traditional matrices
for enhanced sensitivity in the small mass range rely on nanostructured surfaces, the
techniques being generally called surface-assisted laser desorption/ionization
(SALDI) [40]. Different nanomaterials have been used as substrate for SALDI IMS
lipid measurements, including porous silicon [41], graphene layers [4245],
nanodots [46], and nanometals [47].
Recently, we developed a technique we termed nanoparticle (nano-P)ALDI-IMS
using extremely small (d = 3.7 nm) nanoparticles for the analysis of lipids. Indeed,
this technique employs ferrous nanoparticles as matrix to detect the lipid distribu-
tion in rat brain tissues [48]. In addition, we used alkylcarboxylate- and alkylamine-
modified silver nanoparticles for the lipid analysis of mouse retinal and liver sections
to identify fatty acids such as palmitic, stearic, oleic, linoleic, arachidonic, and
eicosapentaenoic acids, which were not detected with matrix DHB [49]. Similarly,
mapping of the distribution and localization of glycosphingolipids in brain sections
was possible using alkylamine-modified gold nanoparticles as matrix [50]. In this
analysis, detection of glycosphingolipids was 20 fold higher than by DHB.
30, 50, 5260]. Nonetheless, more recently sublimation methods for matrix deposi-
tion have also been applied by our group using an automated deposition device [61].
In the following sections, protocols for both methods are described, but readers are
encouraged to search for information on alternative methods.
An artists airbrush is generally used for matrix spraying. Two minimum features
are required for the airbrush: (1) the operator should be able to adjust the droplet
size and control the mist volume, and (2) the device should be resistant to organic
solvents to avoid contamination of samples with residues derived from parts of the
instrument [51]. In addition, the airbrush should be equipped with a 0.2-mm nozzle.
In our laboratory, the preferred device is Proton Boy FWA Platinum (Mr. Hobby,
Tokyo, Japan) for its simplicity and the ease of handling of its design. Representative
parameters of spraying operation with an airbrush are shown in Fig. 27.1. Important
points to keep in mind to master the airbrushs hand operation follow.
1. Minimize droplet size during spraying.
2. Maintain invariable the distance and angle between the nozzle and the sample
surface.
3. Move the airbrush horizontally in a gradual and smooth manner.
Fig. 27.1 Representative parameters of the spraying operation with an air-brush that must be
maintained between trials for reproducible imaging mass spectrometry (IMS) experiment results
(Reproduced from Sugiura et al. [51])
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 379
The material and protocol for the production and spraying deposition of ferrous
nanoparticle matrix on samples [48] is described next.
General Materials
Alkylcarboxylic acids
Alkylamines
-Aminopropyltriethoxysilane
NaOH
MeOH
380 I. Yao et al.
The protocol for the production and spraying deposition of silver nanoparticle as
matrix onto samples [49, 63] is described next. Please note that some materials are
the same as in the previous section.
Protocol
1. Add an aqueous solution of NaOH (0.150 M) to a 0.150 M suspension of the
required alkylcarboxylic acid in 1.0 l hot water.
2. To the resulting solution, add an aqueous solution of AgNO3 (0.165 M) to obtain
a white precipitate (silver alkylcarboxylate).
3. Collect and dry under reduced pressure at 60 C to produce alkylcarboxylates.
4. Place the silver alkylcarboxylate (1.0 mM) and an equal amount of alkylamines
(1.0 mM) into a three-necked flask. Heat the compounds for 5 h to 120 or 180 C
to cause the reaction mixture to pass from liquid to a brown dispersion with a
metallic luster.
5. Cool the mixture to 80 C and add MeOH. Collect the precipates (silver nanopar-
ticles) by filtration, wash with MeOH, and dry under vacuum.
6. Thaw-mount the tissue section onto an ITO-coated glass slide. Spray 500 l sil-
ver nanoparticle solution (50 mg/ml in 100 % hexane). If spraying with an art-
ists airbrush, ensure 15-cm distance between nozzle and tissue surface. Let dry
at RT.
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 381
The protocol for the production and spraying deposition of gold nanoparticles as
matrix onto samples [50, 64] is described next. Please note that some materials are
the same as in the previous section.
Protocol
1. Place AuCl(SMe2) (295 mg, 1.0 mM) and the alkylamine (10.0 mM) in a 10-ml
flask accessorized with a magnetic stirrer. Depending on the alkylamine, gradu-
ally heat the mixture to 100 or 120 C. Maintain required temperature for 1 h.
Afterward, let solution cool to RT.
2. Add acetone (5 ml) and MeOH (1 ml).
3. Centrifuge at 2000 rpm for 5 min. Decant the solvents; collect and dry the gold
nanoparticles under vacuum.
4. Thaw-mount the tissue section onto an ITO-coated glass slide. Spray 1000 l of
gold nanoparticle solution (50 mg/ml in 100 % hexane). If spraying with artists
airbrush, ensure 15-cm distance between nozzle and tissue surface. Let dry at
RT.
27.5.2 Sublimation
Matrix deposition through sublimation was first proposed by Hankin et al. and
subseuqently has been extensively used [65]. The sublimation process, that is, the
transfer of matrix molecules from solid to gas phase, is a process that depends on
temperature and vacuum conditions. High purity of the matrix material is desirable
for avoiding unknown and unreproducible interactions and ion suppression effects
(described later) with the analytes of the biological sample. The layer quality, that
is, the size of the crystals and thickness uniformity that is suitable for MALDI
measurements, depends on several factors. For example, a deposition time that is
too short may produce unwanted sputtering of matrix grains. In contrast, deposi-
tions at slower speed produce a sublimated layer with small-sized crystals that are
uniformly distributed. Nonetheless, uniformity of the layer thickness across the
sample is also affected by geometric factors such as the distance between the cru-
cible and the substrate and the crucible lid aperture (i.e., its shape and size).
Therefore, ideally the larger the distance between the source and the substrate, and
the aperture size of the crucible lid, the better the uniformity of the deposited layer.
In reality, however, these settings also cause the sublimated matrix to spread more
in space, and thus an optimal layer thickness for a given matrix quantity in the cru-
cible may be difficult to achieve.
382 I. Yao et al.
Fig. 27.2 Picture of iMLayer (Shimadzu, Kyoto, Japan), which is a desktop-size device used for
automatic matrix deposition using sublimation. The vacuum chamber includes the crucible and the
substrate with holders, as well as a laser light source and a detector for measuring the matrix layer
thickness using transmission light signal change and an internal calibration curve. The vacuum
pump and the controlling unit are separate devices and thus not shown in the picture (Photograph
courtesy of Keigo Sano)
Microspatula
Laboratory sieves for sorting the matrix material by grain size (optional)
Protocol
1. Prepare the matrix material by grinding it manually using a mortar and a
pestle.
2. Select the matrix grain size using a sieve with a predetermined hole size
(optional). Only grains smaller than the hole size shall be used for sublimation
(i.e., the distribution in grain size has an upper limit).
3. Weigh the amount of matrix powder for deposition.
4. Place the matrix powder in the crucible.
5. Gently hit the crucible several times with a microspatula to let the powder rear-
range itself at the surface and in volume.
6. Select the lid (cover) for the crucible. The lid may have a round hole or a slit
aperture.
7. Place the lid over the crucible, or alternatively, maintain the crucible
uncovered.
8. Set the desired distance between crucible and substrate.
9. Place the crucible in the special holder electrode.
10. Place the slide with the biological samples in the special slide holder of
iMLayer.
11. Close the device door and set the conditions for temperature and maximal
deposition time, depending on the matrix material used.
12. Start the sublimation.
13. After the matrix sublimation is completed, immediately analyze by MALDI-
TOF IMS.
Typical iMLayer conditions for the sublimation of 9-AA matrix
Matrix grinding time: approximately 5 min
Matrix weight for one sublimation: 500 mg
Temperature: 230 C
Distance from crucible to substrate: 510 cm (better layer uniformity is achieved
when the distance between the crucible and the slide is larger)
Maximum deposition time: 90 min
Expected results: 1-m matrix layer thickness (for MALDI IMS analysis of bio-
logical tissue)
Fig. 27.3 Scheme of our approach for glyco- and lipidomics analyses by TLC-Blot-MALDI-QIT-
TOF MS (Reproduced from 2011 Valdes-Gonzalez T, Goto-Inoue N, Hirano W, Ishiyama H,
Hayasaka T, Setou M, Taki T. Journal of Neurochemistry 2011 International Society for
Neurochemistry)
TLC-Blot
1. Dip the HPTLC plate into a blotting solvent (isopropanol:0.2 % CaCl2:methanol;
40:20:7, v/v). Remove immediately.
2. Mount the plate with the PVDF membrane, the Teflon membrane, and glass fiber
filter paper.
3. Press this assembly for 30 s at 180 C by a thermal blotter. Remove the PVDF
membrane and air dry.
4. Analyze the PVDF membrane with the lipids attached on the reverse side by
MALDI-QIT-TOF MS.
MALDI-QIT-TOF MS
Device: MALDI-QIT-TOF MS (AXIMA-QIT MS; Shimadzu)
Ion mode: positive
Raster scan: automatic
Laser shots: 5 at each spot
Data point interval: 100 m
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 387
27.8 Conclusion
Recent research has been increasingly unveiling the importance of lipids in various
biological processes and the onset of metabolic and degenerative diseases. Upon its
introduction in the late 1980s as a relatively limited tool for the analysis of proteins,
MALDI-TOF MS has become a very versatile technique capable of analyzing a
much wider range of compounds including lipids. Several characteristics including
a low mass range similar to those of matrices commonly used for coating samples,
as well as a preferential identification of certain lipid classes over others, make lipid
analysis by MALDI-TOF MS still a difficult analytical technique to master.
Nonetheless, rapid, breathtaking developments in imaging MS technology are sig-
naling that a breakthrough in subcellular research of the lipid machinery is about to
occur. Thus, we foresee that future work using MALDI-TOF MS as the preferred
tool will be able to further elucidate the importance of individual lipid species and
precisely connect their functions with biological events taking place in tissues, as
well to provide information on new markers for more accurate clinical diagnosis.
References
1. Rosen ED, Spiegelman BM (2006) Adipocytes as regulators of energy balance and glucose
homeostasis. Nature 444(7121):847853. doi:10.1038/nature05483
2. Goto-Inoue N, Manabe Y, Miyatake S, Ogino S, Morishita A, Hayasaka T, Masaki N, Setou M,
Fujii N (2012) Visualization of dynamic change in contraction-induced lipid composition in
mouse skeletal muscle by matrix-assisted laser desorption/ionization imaging mass spectrom-
etry. Anal Bioanal Chem 403(7):18631871. doi:10.1007/s00216-012-5809-x
3. Ide Y, Waki M, Hayasaka T, Nishio T, Morita Y, Tanaka H, Sasaki T, Koizumi K, Matsunuma
R, Hosokawa Y, Ogura H, Shiiya N, Setou M (2013) Human breast cancer tissues contain
abundant phosphatidylcholine (36:1) with high stearoyl-CoA desaturase-1 expression. PLoS
One 8(4):e61204. doi:10.1371/journal.pone.0061204
4. Goto-Inoue N, Hayasaka T, Zaima N, Setou M (2011) Imaging mass spectrometry for lipido-
mics. Biochim Biophys Acta 1811(11):961969. doi:10.1016/j.bbalip.2011.03.004
5. Fenn J, Mann M, Meng C, Wong S, Whitehouse C (1989) Electrospray ionization for mass
spectrometry of large biomolecules. Science 246(4926):6471. doi:10.1126/science.2675315
6. Heeren RMA, McDonnell LA, Amstalden E, Luxembourg SL, Altelaar AFM, Piersma SR
(2006) Why dont biologists use SIMS? A critical evaluation of imaging MS. Appl Surf Sci
252(19):68276835. doi:10.1016/j.apsusc.2006.02.134
7. Karas M, Hillenkamp F (1988) Laser desorption ionization of proteins with molecular masses
exceeding 10,000 daltons. Anal Chem 60(20):22992301. doi:10.1021/ac00171a028
8. Tanaka K, Waki H, Ido Y, Akita S, Yoshida Y, Yoshida T, Matsuo T (1988) Protein and polymer
analyses up to m/z 100 000 by laser ionization time-of-flight mass spectrometry. Rapid
Commun Mass Spectrom 2(8):151153. doi:10.1002/rcm.1290020802
9. Berry KAZ, Hankin JA, Barkley RM, Spraggins JM, Caprioli RM, Murphy RC (2011) MALDI
imaging of lipid biochemistry in tissues by mass spectrometry. Chem Rev 111(10):64916512.
doi:10.1021/cr200280p
10. Fuchs B, S R, Schiller J (2010) An update of MALDI-TOF mass spectrometry in lipid
research. Prog Lipid Res 49(4):450475. doi:10.1016/j.plipres.2010.07.001
388 I. Yao et al.
11. Patterson NH, Thomas A, Chaurand P (2014) Monitoring time-dependent degradation of phos-
pholipids in sectioned tissues by MALDI imaging mass spectrometry. J Mass Spectrom
49(7):622627. doi:10.1002/jms.3382
12. Sugiura Y, Honda K, Kajimura M, Suematsu M (2014) Visualization and quantification of
cerebral metabolic fluxes of glucose in awake mice. Proteomics 14(7-8):829838. doi:10.1002/
pmic.201300047
13. Galli C, Spagnuolo C (1976) The release of brain free fatty acids during ischaemia in essential
fatty acid-deficient rats. J Neurochem 26(2):401404. doi:10.1111/j.1471-4159.1976.
tb04493.x
14. Guidotti A, Cheney DL, Trabucchi M, Doteuchi M, Wang C, Hawkins RA (1974) Focussed
microwave radiation: a technique to minimize post mortem changes of cyclic nucleotides,
dopa and choline and to preserve brain morphology. Neuropharmacology 13(12):11151122.
doi:10.1016/0028-3908(74)90061-6
15. Visioli F, Rodriguez de Turco EB, Kreisman NR, Bazan NG (1994) Membrane lipid degrada-
tion is related to interictal cortical activity in a series of seizures. Metab Brain Dis 9(2):161
170. doi:10.1007/BF01999769
16. Birkle DL, Bazan NG (1988) Cerebral perfusion of metabolic inactivators: a new method for
rapid fixation of labile lipid pools in brain. Neurochem Res 13(9):849852. doi:10.1007/
BF00970752
17. Birkle DL, Kurian P, Braquet P, Bazan NG (1988) Platelet-activating factor antagonist
BN52021 decreases accumulation of free polyunsaturated fatty acid in mouse brain during
ischemia and electroconvulsive shock. J Neurochem 51(6):19001905.
doi:10.1111/j.1471-4159.1988.tb01175.x
18. Reddy TS, Bazan NG (1987) Arachidonic acid, stearic acid, and diacylglycerol accumulation
correlates with the loss of phosphatidylinositol 4,5-bisphosphate in cerebrum 2 seconds after
electroconvulsive shock: complete reversion of changes 5 minutes after stimulation. J Neurosci
Res 18(3):449455. doi:10.1002/jnr.490180311
19. Van Rooijen LAA, Vadnal R, Dobard P, Bazan NG (1986) Enhanced inositide turnover in brain
during bicuculline-induced status epilepticus. Biochem Biophys Res Commun 136(2):827
834. doi:10.1016/0006-291X(86)90515-2
20. Hattori K, Kajimura M, Hishiki T, Nakanishi T, Kubo A, Nagahata Y, Ohmura M, Yachie-
Kinoshita A, Matsuura T, Morikawa T, Nakamura T, Setou M, Suematsu M (2010) Paradoxical
ATP elevation in ischemic penumbra revealed by quantitative imaging mass spectrometry.
Antioxid Redox Signal 13(8):11571167. doi:10.1089/ars.2010.3290
21. Sugiura Y, Zaima N, Setou M, Ito S, Yao I (2012) Visualization of acetylcholine distribution
in central nervous system tissue sections by tandem imaging mass spectrometry. Anal Bioanal
Chem 403(7):18511861. doi:10.1007/s00216-012-5988-5
22. Yao I, Setou M (2010) Animal care and tissue sample extraction for IMS. In: Setou M (ed)
Imaging mass spectrometry. Protocols for mass microscopy. Springer, Tokyo, pp 3339
23. Sugiura Y, Setou M (2010) Preparing biological tissue sections for imaging mass spectrometry.
In: Setou M (ed) Imaging mass spectrometry. Protocol for mass microscopy. Springer, Tokyo,
pp 4154
24. Altelaar AFM, van Minnen J, Jimnez CR, Heeren RMA, Piersma SR (2004) Direct molecular
imaging of Lymnaea stagnalis nervous tissue at subcellular spatial resolution by mass spec-
trometry. Anal Chem 77(3):735741. doi:10.1021/ac048329g
25. Kruse R, Sweedler JV (2003) Spatial profiling invertebrate ganglia using MALDI MS. J Am
Soc Mass Spectrom 14(7):752759. doi:10.1016/S1044-0305(03)00288-5
26. Walch A, Rauser S, Deininger S-O, Hfler H (2008) MALDI imaging mass spectrometry for
direct tissue analysis: a new frontier for molecular histology. Histochem Cell Biol 130(3):421
434. doi:10.1007/s00418-008-0469-9
27. Schiller J, S R, Fuchs B, Mller M, Petkovi M, Zschrnig O, Waschipky H (2007) The
suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of
phospholipids: which isomer for what purpose? Eur Biophys J 36(4-5):517527. doi:10.1007/
s00249-006-0090-6
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 389
45. Qian K, Zhou L, Liu J, Yang J, Xu H, Yu M, Nouwens A, Zou J, Monteiro MJ, Yu C (2013)
Laser engineered graphene paper for mass spectrometry imaging. Sci Rep 3:1415. doi:10.1038/
srep01415
46. Chen W-Y, Chen L-Y, Ou C-M, Huang C-C, Wei S-C, Chang H-T (2013) Synthesis of fluores-
cent gold nanodotliposome hybrids for detection of phospholipase C and its inhibitor. Anal
Chem 85(18):88348840. doi:10.1021/ac402043t
47. Arakawa R, Kawasaki H (2010) Functionalized nanoparticles and nanostructured surfaces for
surface-assisted laser desorption/ionization mass spectrometry. Anal Sci 26(12):12291240.
doi:10.2116/analsci.26.1229
48. Taira S, Sugiura Y, Moritake S, Shimma S, Ichiyanagi Y, Setou M (2008) Nanoparticle-assisted
laser desorption/ionization based mass imaging with cellular resolution. Anal Chem
80(12):47614766. doi:10.1021/ac800081z
49. Hayasaka T, Goto-Inoue N, Zaima N, Shrivas K, Kashiwagi Y, Yamamoto M, Nakamoto M,
Setou M (2010) Imaging mass spectrometry with silver nanoparticles reveals the distribution
of fatty acids in mouse retinal sections. J Am Soc Mass Spectrom 21(8):14461454.
doi:10.1016/j.jasms.2010.04.005
50. Goto-Inoue N, Hayasaka T, Zaima N, Kashiwagi Y, Yamamoto M, Nakamoto M, Setou M
(2010) The detection of glycosphingolipids in brain tissue sections by imaging mass spectrom-
etry using gold nanoparticles. J Am Soc Mass Spectrom 21(11):19401943. doi:10.1016/j.
jasms.2010.08.002
51. Sugiura Y, Setou M, Horigome D (2010) Methods of matrix application. In: Setou M (ed)
Imaging mass spectrometry. Protocols for mass microscopy. Springer, Tokyo, pp 7185
52. Ishikawa S, Tateya I, Hayasaka T, Masaki N, Takizawa Y, Ohno S, Kojima T, Kitani Y, Kitamura
M, Hirano S, Setou M, Ito J (2012) Increased expression of phosphatidylcholine (16:0/18:1)
and (16:0/18:2) in thyroid papillary cancer. PLoS One 7(11):e48873. doi:10.1371/journal.
pone.0048873
53. Kaneko Y, Obata Y, Nishino T, Kakeya H, Miyazaki Y, Hayasaka T, Setou M, Furusu A,
Kohno S (2011) Imaging mass spectrometry analysis reveals an altered lipid distribution pat-
tern in the tubular areas of hyper-IgA murine kidneys. Exp Mol Pathol 91(2):614621.
doi:10.1016/j.yexmp.2011.07.002
54. Kobayashi Y, Hayasaka T, Setou M, Itoh H, Kanayama N (2010) Comparison of phospholipid
molecular species between terminal and stem villi of human term placenta by imaging mass
spectrometry. Placenta 31(3):245248. doi:10.1016/j.placenta.2009.12.026
55. Hayasaka T, Goto-Inoue N, Masaki N, Ikegami K, Setou M (2014) Application of
2,5-dihydroxyacetophenone with sublimation provides more efficient ionization of lipid spe-
cies by atmospheric pressure matrix-assisted laser desorption/ionization imaging mass spec-
trometry. Surf Interface Anal 46(1213):12191222. doi:10.1002/sia.5592
56. Miyamura N, Nakamura T, Goto-Inoue N, Zaima N, Hayasaka T, Yamasaki T, Terai S, Sakaida
I, Setou M, Nishina H (2011) Imaging mass spectrometry reveals characteristic changes in
triglyceride and phospholipid species in regenerating mouse liver. Biochem Biophys Res
Commun 408(1):120125. doi:10.1016/j.bbrc.2011.03.133
57. Nakajima K, Terao M, Takaishi M, Kataoka S, Goto-Inoue N, Setou M, Horie K, Sakamoto F,
Ito M, Azukizawa H, Kitaba S, Murota H, Itami S, Katayama I, Takeda J, Sano S (2013)
Barrier abnormality due to ceramide deficiency leads to psoriasiform inflammation in a mouse
model. J Invest Dermatol 133(11):25552565. doi:10.1038/jid.2013.199
58. Onoue K, Zaima N, Sugiura Y, Isojima T, Okayama S, Horii M, Akai Y, Uemura S, Takemura
G, Sakuraba H, Sakaguchi Y, Setou M, Saito Y (2011) Using imaging mass spectrometry to
accurately diagnose Fabrys disease. Circ J 75(1):221223. doi:10.1253/circj.CJ-10-0767
59. Tanaka H, Zaima N, Yamamoto N, Sagara D, Suzuki M, Nishiyama M, Mano Y, Sano M,
Hayasaka T, Goto-Inoue N, Sasaki T, Konno H, Unno N, Setou M (2010) Imaging mass
spectrometry reveals unique lipid distribution in primary varicose veins. Eur J Vasc Endovasc
Surg 40(5):657663. doi:10.1016/j.ejvs.2010.08.001
27 Lipid Machinery Investigation Using MALDI Imaging Mass Spectrometry 391
Abstract Lipid mediators are intercellular bioactive lipid molecules that induce
various cellular responses mostly through G protein-coupled receptors (GPCRs).
According to the IUPHAR database and recent publications, there are now approxi-
mately 50 lipid-recognizing GPCRs. Monitoring activation of GPCRs by lipid
mediators or synthetic ligands is crucial for studying the actions of bioactive lipids
as well as lipid GPCRs. Because each GPCR differentially couples with heterotri-
meric G proteins (Gs, Gi/o, Gq/11, G12/13), in general it is necessary to prepare multiple
GPCR assays to detect distinct G-protein signaling and individual optimization. In
this protocol section, we describe a recently developed transforming growth factor-
(TGF-) shedding assay, which is a simple and accurate method for measuring acti-
vation of lipid GPCRs. By utilizing coexpression of chimeric G subunits, the
TGF- shedding assay can detect activation of a wide range of GPCRs that are
coupled with any of the four G proteins. Indeed, 39 of 45 (87 %) lipid GPCRs exam-
ined were detectable in the single format of the TGF- shedding assay, demonstrat-
ing, to the best of our knowledge, the greatest coverage of a lipid GPCR assay. Thus,
the TGF- shedding assay provides a useful platform for analyzing lipid GPCRs.
A. Inoue (*)
Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical
Sciences, Tohoku University, Sendai 980-8578, Miyagi, Japan
PRESTO, Japan Science and Technology Agency (JST),
Kawaguchi 332-0012, Saitama, Japan
e-mail: [email protected]
J. Aoki
Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical
Sciences, Tohoku University, Sendai 980-8578, Miyagi, Japan
CREST, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Saitama, Japan
A
Alkaline phosphatase (AP)
Ectodomain
DLLA-VVAA
shedding site
Soluble TGF
(mature form)
Extracellular
Plasma membrane
Membrane-bound TGF
Intracellular
(proform)
B Ectodomain
shedding
Agonist
Soluble
GPCR AP-TGF
TACE
Gq/11 G12/13 Membrane-
bound
AP-TGF
G16
G
s
G
i
q
G
q
G
Fig. 28.1 Mechanism of the transforming growth factor (TGF)- shedding assay. (a) Schematic
structure of the AP-TGF- construct. AP-TGF- is a fusion protein consisting of N-terminal pla-
cental alkaline phosphatase (AP) and C-terminal TGF- with a signal peptide. AP-TGF- is ini-
tially expressed as membrane-bound proform and its juxtamembrane region is cleaved (ectodomain
shedding). In HEK293 cells, TACE is responsible for the ectodomain shedding of AP-TGF-.
Amount of shedding of AP-TGF- is determined by measuring AP activity in conditioned media
(soluble AP-TGF-). (b) Gq/11- and/or G12/13-coupled receptors induce TACE-dependent AP-TGF-
shedding responses upon agonist stimulation. G-protein effectors such as phospholipase C, protein
kinase C, RhoA, and ROCK mediate the process (not shown). (c) In Gs- or Gi-coupled receptors,
chimeric Gq subunits harboring C-terminal Gs-derived sequences (Gq/s; also see Fig. 28.2) or
Gi-derived sequences (Gq/i) and/or promiscuous G16 subunit are coexpressed. When agonist-
bound GPCRs interact with and activate the introduced G subunit(s), TACE-dependent AP-TGF-
shedding responses are induced
396 A. Inoue and J. Aoki
A Six C-terminial
amino acid residues
Gq/s -RQYELL
Gq/i1 -KDCGLF
Gq/i3 -KECGLY
Gq/o -RGCGLY
Gq/z -KYIGLC
B
G16
Fig. 28.2 Schematic structures of the G subunits. (a) Chimeric G subunits used in the TGF-
shedding assay consist of the Gq backbone with substitutions of six C-terminal amino acids.
Among the eight Gi members (Gi1, Gi2, Gi3, Go, Gz, Gt1, Gt2, Gt3), there are four unique
C-terminal sequences because the six amino acid sequences of Gi1, Gi2, Gt1, Gt2, and Gt3 are
identical. (b) G16 belongs to the Gq family, yet is capable of coupling with GPCRs in a relatively
unspecific manner. (Note that C-terminal residues and the backbones of G subunits are critical for
interaction with GPCRs and effectors such as phospholipase C, respectively, and that chimeric G
subunits serve as a converter that switches Gs- or Gi/o-coupled receptor activation to Gq signal-
ing). aa, amino acids
among GPCR assays developed so far. In the following protocol, a standard TGF-
shedding assay (24-well format in a 96-well plate; transfection performed in a
12-well plate) is described.
28.2.1 Materials
174
PAFR
S1P3
S1P1
10
GPR
S 1P
0L
S1
P 2Y
Y1
4
2
S1
P5
R3
P2
18
P4
LP
GP
LP PR
A3
A1 G G R1
LP OX
A2
I2
CB EB
2 LT1
CB1 Cys
2
Detectable CysLT
GPBA
Detectable G2A
GPR84 (with G co-expression) LPA5
GPR119 Not detectable LPA4
FP LPA
1 6
EP GP
R5
TP OX 5
3 FF E
EP A1
2
EP
FF
DP
FF
A2
IP
GP
EP4
A3
DP2
FFA4
BLT1
BLT2
ER
Fig. 28.3 Detection of lipid GPCRs using the TGF- shedding assay. Forty-five lipid-recognizing
GPCRs were subjected to the TGF- shedding assay with or without coexpression of G subunits.
In the absence of G coexpression, 31 GPCRs (69 %) were detectable (shown in red). In the pres-
ence of G coexpression, 8 GPCRs were additionally detectable (yellow). In total, 39 GPCRs
(87 %) were detectable in the TGF- shedding assay. We set 3 % AP-TGF- release as a threshold
to judge detectable or not detectable. A phylogenetic tree is obtained from the Clustal W algo-
rithm and visualized by the FigTree software
24 h
Transfect plasmids
(AP-TGF, GPCR, G subunits)
24 h
Cell
Detach and reseed cells in a 96-well plate
plate (90 L/well)
30 min
60 min
Transfer conditioned media (80 L/well)
CM
plate Dispense p-NPP solution (80 L/well)
Measure OD405 (1st)
60 min
Fig. 28.4 Procedure of the TGF- shedding assay. HEK293 cells are seeded in a 12-well plate and
cultured for 24 h. Cells are transfected with a mixture of plasmids containing the AP-TGF-,
GPCR, and/or G subunit(s) and cultured for 24 h. Transfected cells are detached, suspended in
HBSS, and reseeded in a 96-well plate (cell plate). After 30-min incubation, cells are stimulated
with compounds and incubated for 60 min. Conditioned media are transferred to a blank plate (CM
plate), and p-NPP solution is dispensed in both the cell plate and the CM plate. OD405 is measured
before (first) and after (second) 60-min incubation with p-NPP
8. Remove supernatant.
9. Suspend cells in complete DMEM at a cell density of 2 105 cells/ml.
Option: cells can be seeded 2 days before transfection (1 105 cells/ml) or
same day as transfection (4 105 cells/ml).
10. Seed cell suspension in a 12-well plate (1 ml per well; hereafter, volumes refer
to a single well of a 12-well plate, unless otherwise noted).
If larger amounts of transfected cells are desired, use larger plates, dishes, or
flasks (e.g., 6-well plate, 6-cm dish, 10-cm dish, T75).
11. Place a plate in a CO2 incubator and incubate 24 h until plasmid transfection
A Transfected 1 2 3 4 5 6 7 8 9 10 11 12
cells A AP-TGF AP-TGF AP-TGF AP-TGF
B - - + +
C
D GPCR GPCR GPCR GPCR
E Empty vector H1R Empty vector H1R
F
G
H
1 2 3 4 5 6 1 2 3 4 5 6
Compound A Histamine 0
B (nM) 0
C 1
D 10
E 100
F 1000
G 10000
H TPA (nM) 1000
B C
OD405 1 2 3 4 5 6 7 8 9 10 11 12 OD 405 1 2 3 4 5 6 7 8 9 10 11 12
0h CM A 0.114 0.108 0.109 0.109 0.108 0.108 0.116 0.115 0.116 0.117 0.118 0.118 1h-0h CM A 0.008 0.008 0.007 0.008 0.008 0.008 0.143 0.145 0.142 0.172 0.168 0.178
B 0.110 0.108 0.107 0.109 0.107 0.108 0.121 0.114 0.116 0.119 0.117 0.118 B 0.012 0.008 0.012 0.009 0.009 0.008 0.152 0.149 0.147 0.182 0.179 0.184
C 0.106 0.108 0.107 0.110 0.107 0.106 0.116 0.115 0.117 0.118 0.118 0.119 C 0.008 0.007 0.007 0.008 0.008 0.008 0.149 0.163 0.152 0.203 0.200 0.203
D 0.107 0.133 0.107 0.108 0.105 0.108 0.115 0.114 0.117 0.133 0.132 0.130 D 0.008 -0.019 0.008 0.009 0.009 0.006 0.150 0.150 0.155 0.406 0.385 0.397
E 0.118 0.106 0.105 0.108 0.103 0.120 0.137 0.114 0.115 0.144 0.143 0.144 E -0.003 0.008 0.007 0.009 0.008 0.009 0.146 0.149 0.144 0.640 0.631 0.644
F 0.107 0.111 0.112 0.107 0.114 0.111 0.116 0.115 0.116 0.149 0.150 0.149 F 0.009 0.008 0.008 0.009 0.008 0.007 0.152 0.152 0.147 0.698 0.686 0.692
G 0.107 0.111 0.107 0.106 0.114 0.109 0.116 0.127 0.119 0.154 0.150 0.155 G 0.015 0.002 0.008 0.009 0.008 0.008 0.150 0.154 0.149 0.754 0.717 0.741
H 0.116 0.105 0.107 0.119 0.107 0.113 0.154 0.162 0.155 0.146 0.148 0.148 H 0.005 0.007 0.007 -0.002 0.008 0.002 0.832 0.825 0.839 0.648 0.654 0.661
OD405 1 2 3 4 5 6 7 8 9 10 11 12 OD 405 1 2 3 4 5 6 7 8 9 10 11 12
0h Cell A 0.105 0.105 0.106 0.107 0.105 0.106 0.236 0.276 0.243 0.248 0.256 0.252 1h-0h Cell A 0.020 0.020 0.019 0.018 0.019 0.018 1.660 1.639 1.627 1.428 1.393 1.438
B 0.106 0.106 0.105 0.105 0.103 0.107 0.233 0.240 0.242 0.213 0.235 0.229 B 0.023 0.022 0.020 0.020 0.019 0.021 1.654 1.691 1.682 1.507 1.503 1.502
C 0.108 0.106 0.106 0.108 0.105 0.105 0.207 0.267 0.264 0.223 0.271 0.266 C 0.021 0.022 0.021 0.020 0.020 0.019 1.683 1.717 1.713 1.463 1.429 1.433
D 0.104 0.105 0.105 0.104 0.108 0.109 0.216 0.238 0.247 0.194 0.220 0.248 D 0.022 0.024 0.022 0.022 0.019 0.020 1.670 1.699 1.730 1.276 1.226 1.204
E 0.102 0.101 0.102 0.105 0.103 0.102 0.190 0.199 0.273 0.173 0.196 0.191 E 0.021 0.023 0.019 0.021 0.020 0.020 1.713 1.688 1.713 1.035 0.988 0.966
F 0.105 0.106 0.103 0.105 0.105 0.105 0.199 0.247 0.237 0.179 0.191 0.182 F 0.020 0.024 0.021 0.021 0.021 0.022 1.787 1.690 1.690 0.973 0.911 0.898
G 0.105 0.103 0.104 0.104 0.105 0.103 0.202 0.223 0.247 0.184 0.188 0.175 G 0.019 0.021 0.020 0.022 0.022 0.020 1.706 1.684 1.704 0.943 0.905 0.879
H 0.103 0.100 0.102 0.101 0.099 0.102 0.160 0.170 0.170 0.167 0.177 0.168 H 0.023 0.024 0.022 0.021 0.022 0.020 1.039 0.995 1.014 0.994 0.983 0.987
OD405 1 2 3 4 5 6 7 8 9 10 11 12
1h CM A 0.122 0.116 0.116 0.118 0.116 0.116 0.259 0.260 0.258 0.289 0.286 0.296 AP-TGF in CM (%) 7 8 9 10 11 12
B 0.122 0.116 0.119 0.117 0.116 0.117 0.273 0.264 0.263 0.301 0.296 0.302 A 9.9 10.1 10.0 13.5 13.5 13.8
C 0.114 0.115 0.115 0.118 0.115 0.114 0.264 0.278 0.269 0.321 0.318 0.322 B 10.5 10.1 10.1 13.5 13.3 13.6
D 0.115 0.114 0.114 0.117 0.115 0.114 0.265 0.264 0.271 0.539 0.517 0.527 C 10.1 10.8 10.2 15.2 15.3 15.5
E 0.116 0.113 0.112 0.116 0.111 0.129 0.283 0.263 0.259 0.784 0.774 0.788 D 10.3 10.2 10.3 30.2 29.9 31.0
F 0.116 0.119 0.120 0.116 0.122 0.118 0.267 0.267 0.263 0.847 0.836 0.841 E 9.8 10.1 9.7 47.8 48.7 50.0
G 0.122 0.113 0.115 0.115 0.122 0.116 0.266 0.281 0.267 0.908 0.866 0.895 F 9.8 10.3 10.0 52.2 53.7 54.4
H 0.120 0.112 0.114 0.116 0.115 0.115 0.986 0.987 0.994 0.793 0.802 0.809 G 10.1 10.4 10.0 55.5 55.3 57.2
H 55.6 56.7 56.6 49.3 49.9 50.1
OD405 1 2 3 4 5 6 7 8 9 10 11 12
1h Cell A 0.125 0.126 0.125 0.125 0.124 0.124 1.896 1.916 1.870 1.675 1.649 1.690 Control H1R
B 0.129 0.128 0.125 0.125 0.122 0.127 1.887 1.930 1.924 1.719 1.737 1.731 Mean SD Release Mean SD Release
C 0.129 0.128 0.127 0.128 0.125 0.124 1.890 1.984 1.977 1.685 1.700 1.699 Histamine 0 10.1 0.2 0.0 13.5 0.2 0.0
D 0.126 0.129 0.127 0.126 0.127 0.129 1.885 1.937 1.976 1.470 1.445 1.452 (nM) 1 10.4 0.4 0.3 15.4 0.1 1.8
E 0.122 0.124 0.121 0.125 0.124 0.122 1.902 1.887 1.985 1.207 1.185 1.157 10 10.2 0.1 0.1 30.3 0.6 16.8
F 0.125 0.130 0.124 0.126 0.126 0.126 1.986 1.937 1.926 1.152 1.102 1.080 100 9.9 0.2 -0.2 48.8 1.1 35.3
G 0.124 0.124 0.124 0.126 0.127 0.124 1.907 1.908 1.951 1.127 1.093 1.054 1000 10.0 0.3 -0.1 53.4 1.1 39.9
H 0.126 0.124 0.125 0.122 0.121 0.122 1.199 1.165 1.184 1.161 1.160 1.155 10000 10.2 0.2 0.1 56.0 1.0 42.5
TPA (nM) 1000 56.3 0.6 46.2 49.8 0.4 36.3
D
Control H1R
AP-TGF release (%)
50
40 EC50 15 nM
Emax 42%
30
20
10
0
Fig. 28.5 Calculation processes. (a) A 96-well plate format showing reseeding of transfected cells
and compound treatment. Cells were transfected with the AP-TGF--encoding plasmid (or an
empty vector) and H1R-encoding plasmid (or an empty vector) and treated with vehicle (row A and
B), histamine (row CG, 1 nM to 10 M), or 1 M TPA (row H). (b) Raw OD values. In total, four
measurements for a single 96-well plate assay were performed. (c) Calculation of AP-TGF-
release. (d) Plotting of AP-TGF- release and fitting to four-sigmoidal curves, from which Emax
and EC50 values were obtained
402 A. Inoue and J. Aoki
Conditions (i) and (ii) are negative controls for AP-TGF- expression and AP
activity measurements. Expression of endogenous AP activity in HEK293 cells
should be more than 20 fold as low as AP-TGF--transfected cells. Histamine H1
receptor (H1R) serves as one of excellent positive controls for the following rea-
sons: H1R potently induces shedding response of AP-TGF-; HEK293 do not
endogenously express H1R; variety of H1R ligands including agonists, antagonist,
and inverse agonists are available and testable in the TGF- shedding assay.
13. Dilute 1.25 l Lipofectamine 2000 with 125 l Opti-MEM and incubate 30 min
at room temperature.
14. Add 125 l Lipofectamine 2000-containing Opti-MEM to plasmid-containing
Opti-MEM, mix well, and incubate 20 min at room temperature (transfection
reagent).
15. Gently add 250 l transfection reagent into cell culture plate.
16. Place a plate in a CO2 incubator and incubate 24 h until shedding assay.
32. Suspend cells by pipetting up and down several times. Use a 5-ml liquid pipette
(P5000), if possible.
33. Transfer cell suspension to a reservoir and dispense 90 l cell suspension per
well (hereafter, volume refers to a single well of a 96-well plate, unless other-
wise noted) in a 96-well plate (cell plate). Use 24 wells for each transfected
condition (also see Fig. 28.5).
When using an electric multichannel pipette, select slow dispense speed.
34. Place the cell plate in a CO2 incubator.
35. Incubate 30 min at 37 C.
36. Add 10 l 10 test compounds diluted in HBSS. Use an electric multichannel
pipette, if possible.
If test compounds are not readily soluble in water or have sticky proper-
ties, use bovine serum albumin (BSA) as a carrier. We recommend a concentra-
tion of 0.01 % (w/v) BSA in HBSS as solvent to dilute 10 compounds. In this
condition, a final BSA concentration is 0.001 % (w/v).
37. Incubate 60 min at 37 C in CO2 incubator.
38. Centrifuge the cell plate at 190 g for 2 min at room temperature (optional).
Set slow deceleration speed, if possible. We recommend including this
step to prevent possible contamination of cells in transferred conditioned media
in the following procedure. After centrifugation, quickly start to transfer condi-
tioned media. Do not warm the cell plate as this causes convection within wells
and the cells might be stirred up.
39. Transfer 80 l conditioned media to a new 96-well plate (CM plate) using an
electric multichannel pipette.
Set slow pipetting-up speed. When starting a transfer from row A to B, to
C, using a 12-channel pipette, there is no need to change or wash tips. In
between plates, wash tips once with water. If bubbles appear in the CM plate
(likely to occur in high (0.01 %) concentration of BSA), briefly centrifuge
plates.
40. Dispense 60 l HBSS in cell plate to adjust liquid volumes of 80 l in both cell
plate and CM plate (optional).
41. Leave the cell plate and the CM plate for 10 min at room temperature and cool
them down.
42. Dispense 80 l p-NPP solution to both the cell plate and the CM plate.
Prepare p-NPP solution 1 h before the assay and leave it at room tempera-
ture. Be sure to use room-temperature p-NPP solution as AP reaction depends
on temperature.
43. Measure optical density of a wavelength at 405 nm (OD405) for both the cell
plate and the CM plate.
44. Leave the cell plate and the CM plate in dark for 60 min at room temperature.
404 A. Inoue and J. Aoki
45. Measure OD405 for both the cell plate and the CM plate.
If OD values are small [typically, total OD values (OD405 cell + OD405 CM) 0.5],
additionally incubate the cell plate and the CM plate for 60 min and measure
OD405 thereafter.
46. For each well, calculate AP activity in CM using the following formula.
OD405 Cell = OD405 Cell (at 1 h reaction) OD405 Cell (at 0 h)
OD405 CM = OD405 CM (at 1 h reaction) OD405 CM (at 0 h)
AP-TGF- in CM (% of total AP-TGF-) = OD405 CM/(OD405 Cell
+ OD405 CM) 100 1.25
A factor of 1.25 (100/80) in the formula is used to normalize transferred (mea-
sured) CM volume to the total CM volume (80 l of 100 l in total).
47. Calculate mean and standard deviation (SD) values from multiple
measurements.
48. Subtract mean values of vehicle-treated AP-TGF- in CM from those of
compound-treated ones.
28 Measuring Activation of Lipid G Protein-Coupled Receptors 405
There are a few critical steps to obtain accurate results: (1) uniform resuspension of
cell pellet in HBSS, (2) cautious transfer of CM, and (3) temperature control, espe-
cially during AP reaction.
Development of the TGF- shedding assay for a specific GPCR requires optimi-
zation of the following parameters:
1. G subunit
A mixture of the six G subunits is recommended as a default condition.
However, it is strongly advisable to examine coexpression of G subunits sepa-
rately (seven conditions: no G, Gq/s, Gq/i1, Gq/i3, Gq/o, Gq/z, G16) and to
select one that induces the most potent shedding response.
2. GPCR plasmid
In some GPCRs, GPCR transfection reduces AP-TGF- expression levels
(or total OD405 value OD405 cell + OD405 CM) as compared with mock transfection.
We speculate that the decreased AP-TGF- results from constitutive activity of
GPCRs and/or induction of ER stress by unstable, hydrophobic GPCRs. GPCRs
that have high constitutive activity spontaneously cleave AP-TGF- during 24-h
incubation after transfection, resulting in a smaller amount of AP-TGF- on the
cell surface at the time of the assay.
Use deionized, distilled water or equivalent ultrapure (e.g., Milli-Q from Merck
Millipore) water in all recipes and protocol steps except for p-NPP reagents (deion-
ized water).
Complete DMEM [DMEM supplemented with 10 % fetal calf serum (FCS), 100
U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine].
10 % (w/v) NaHCO3
NaHCO3, 10 g
Water to 100 ml
Close cap tightly, autoclave at 121 C for 15 min, and store at room
temperature
406 A. Inoue and J. Aoki
100 PSG
Penicillin, 1,000,000 U
Streptomycin, 1 g
L-glutamine, 2.92 g
Water to 100 ml
Filtrate through a 0.45-m-pore filter
Make aliquots (10 ml solution in 15-ml tubes) and store at 20 C.
Complete DMEM
DMEM (low glucose, Nissui Pharmaceuticals, cat. no. DMEM2) 4.75 g
Water to 500 ml
Autoclave at 121 C for 15 min; store at 4 C
10 % NaHCO3, 7.5 ml
Heat-inactivated FCS (Life Technologies, cat. no. 26140-079), 50 ml
100 PSG, 5 ml
1 M p-NPP (10 ml)
3.71 g p-nitrophenylphosphate disodium salt hexahydrate (Wako Pure Chemical,
cat. no. 145-02344)
Water to 10 ml (~7.8 ml)
Make aliquots (1 ml solution in 1.5-ml tubes) and store at 20 C.
HBSS (Hanks balanced salt solution) (with Ca2+ and Mg2+ and with 5 mM HEPES
(pH 7.4), without phenol red) (*note 1)
10 HBSS solution 1
KCl, 4 g
KH2PO4, 0.6 g
NaCl, 80 g
Na2HPO412H2O, 1.2 g
D-Glucose, 10 g
Water to 1 l
Autoclave at 121 C for 15 min and store at 4 C
10 HBSS solution 2
CaCl22H2O, 1.85 g
MgCl26H2O, 1 g
MgSO47H2O, 1 g
Water to 1 l
Autoclave at 121 C for 15 min and store at 4 C
1 HBSS
Water, 790 ml
0.5 M HEPES (pH 7.4), 10 ml
1 M KOH (*note 2)
28 Measuring Activation of Lipid G Protein-Coupled Receptors 407
Reference
1. Inoue A et al (2012) TGF shedding assay: an accurate and versatile method for detecting
GPCR activation. Nat Methods 9:1021
Chapter 29
A Novel Anti-FLAG Monoclonal Antibody
Is Useful to Study GPCRs
Common Materials
2H8 monoclonal antibody (mAb) (TransGenic or Gentaur Molecular Products,
available as anti-DYKDDDDK antibody 2H8)
Note: The 2H8 mAb only recognizes an amino-terminal FLAG sequence and does
not recognize carboxy-terminal FLAG or 3 FLAG sequences [8]
Cells or tissues expressing N-terminally FLAG-tagged G protein-coupled receptors
(GPCRs)
Note: We frequently use pcDNA3 or pCXN2 vectors for expression studies of
GPCRs [5, 7]
Materials
Phosphate-buffered saline (PBS) (ice-cold)
PBS/EDTA [PBS, pH 7.4, containing 2 mM ethylenediamine-N,N,N,N-tetraacetic
acid, disodium salt (EDTA-2Na)] (ice-cold)
FACS buffer (PBS, pH 7.4, containing 2 % fetal calf serum and 2 mM EDTA-2Na)
(ice-cold)
Fluorescent dye-labeled anti-mouse IgG antibody [e.g., goat anti-mouse IgG-
phycoerythrin (PE), Becton Dickinson]
7-Amino-actinomycin D (7-AAD, Becton Dickinson)
1. In the case of adherent cells, detach the cells transiently or stably expressing
FLAG-GPCRs using PBS/EDTA after washing with PBS. (Note: Avoid using
trypsin because trypsinization often results in digestion of GPCRs and FLAG
tags.)
2. Transfer the cells to a 96-well V-bottom plate (Nunc) (e.g., 15 105 cells/well).
3. Stain the cells with 1 g/ml 2H8 mAb prepared in FACS buffer for 30 min at
4 C.
4. Wash the cells three times with 150 l PBS/EDTA.
5. Stain the cells with 0.5 g/ml anti-mouse IgG-PE prepared in FACS buffer for
30 min at 4 C.
6. Wash the cells three times with 150 l PBS/EDTA.
7. Suspend the cells in 300 l FACS buffer.
8. To exclude dead cells, stain the cells with 5 l 7-AAD per million cells and incu-
bate for 10 min in the dark.
9. Analyze the cells using a flow cytometer (e.g., FACSCalibur, Becton Dickinson).
Materials
10 mM hydrogen chloride (HCl) (pH 3.0)
Collagen (Cellmatrix Type I-P, Nitta Gelatin)
Fix solution [4 % paraformaldehyde (PFA) prepared in PBS containing 10 mM
glycine] (ice-cold)
PBS-G solution (PBS containing 10 mM glycine)
Blocking solution [PBS containing 3 % bovine serum albumin (BSA)]
Staining solution (PBS containing 1 % BSA)
29 A Novel Anti-FLAG Monoclonal Antibody Is Useful to Study GPCRs 411
Materials
Fix solution (PBS containing 4 % PFA) (ice-cold)
10 mM citrate buffer solution (pH 6.0)
Blocking solution (5 % BSA prepared in PBS containing 0.5 % Triton X-100)
(ice-cold)
Wash solution (PBS containing 1 % BSA and 0.1 % Tween 20)
Horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (e.g., rat anti-
mouse IgG-HRP (Trueblot), Rockland)
Fluorescent dye-labeled tyramide (Tyramide-Alexa Fluor 488, Life Technologies)
DAPI (Sigma)
Mounting medium (Permafluor, Thermo)
1. Prepare the frozen sections of tissues on Matsunami adhesive silane (MAS)-
coated slide glasses (Matsunami).
2. Fix the sections with fix solution for 30 min at room temperature.
412 F. Sasaki and T. Yokomizo
3. Boil the sections in 10 mM citrate buffer solution for 30 min for antigen
retrieval.
4. Incubate the sections in blocking solution for 30 min at room temperature.
5. Stain the sections with 5 g/ml 2H8 mAb prepared in blocking solution over-
night at 4 C.
6. Wash the sections five times with wash solution.
7. Stain the sections with anti-mouse IgG-HRP diluted 100 fold in blocking solu-
tion for 60 min at 4 C.
8. Wash the sections five times with wash solution.
9. Incubate the sections with 1 g/ml DAPI for 30 min at room temperature.
10. Wash the sections twice with PBS.
11. Mount with mounting medium. (Note: Avoid air bubbles.)
12. Visualize the samples using a confocal laser scanning microscope.
29.3.1 Immunoprecipitation
Materials
Lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 % glycerol,
0.3 % sodium deoxycholate, 1 % NP-40, and a protease inhibitor cocktail
(Nacalai)]
Wash buffer (lysis buffer lacking the protease inhibitor cocktail)
Protein A/G-agarose (Santa Cruz)
2 SDS sample buffer (25 mM Tris-HCl, pH 6.8, 0.8 % SDS, 10 % glycerol, 0.1 %
bromophenol blue, 2 % 2-mercaptoethanol)
1. Lyse the cells with 500 l lysis buffer for 15 min at 4 C.
2. Centrifuge at 10,000 g for 30 min at 4 C.
3. Transfer the supernatant to new tubes.
4. Add 10 l protein A/G-agarose and 10100 ng 2H8 mAb.
5. Rotate the tube overnight at 4 C using a rotator.
6. Centrifuge at 1,000 g for 5 min at 4 C.
7. Discard the supernatant.
8. Add 1 ml wash buffer to the pellets.
9. Repeat the procedure (steps 68) four times.
10. Dissolve the immunoprecipitates in 60 l 2 SDS sample buffer.
11. Denature the samples for 30 min at 60 C. (Note: Some GPCRs aggregate after
heat denaturation at 100 C.)
29 A Novel Anti-FLAG Monoclonal Antibody Is Useful to Study GPCRs 413
Materials
30 % Acrylamide (AA)/Bis-acrylamide (Bis) solution (29:1)
1.5 M Tris-HCl, pH 8.8
0.5 M Tris-HCl, pH 6.8
10 % SDS
10 % Ammonium persulfate (AMPS)
N,N,N,N-Tetramethylethylenediamine (TEMED)
12 % SDS-polyacrylamide gel (upper, 6 % stacking gel; lower, 12 % separating gel)
12 % separating gel (8 ml AA/Bis, 5 ml 1.5 M Tris-HCl, 0.2 ml 10 % SDS, 0.2 ml
10 % AMPS, 0.02 ml TEMED, 6.58 ml H2O)
6 % stacking gel (2 ml AA/Bis, 2.5 ml 0.5 M Tris-HCl, 0.1 ml 10 % SDS, 0.1 ml
10 % AMPS, 0.01 ml TEMED, 5.29 ml H2O)
1 running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS, pH 8.3)
1 transfer buffer (25 mM Tris, 192 mM glycine)
TBS-T solution (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 % Tween 20)
Blocking solution (TBS-T containing 5 % skimmed milk)
HRP-labeled anti-mouse IgG polyclonal antibody (PoAb) (e.g., sheep anti-mouse
IgG-HRP, GE Healthcare)
Immobilon Western chemiluminescence HRP substrate solution (Millipore)
1. Separate the proteins (e.g., 1020 l immunoprecipitated samples) by SDS-
PAGE (12 % separating gel).
2. Transfer to a polyvinylidene fluoride (Millipore) membrane in 1 transfer buffer
using a wet transfer system (Bio Craft).
3. Incubate the membrane with blocking solution overnight at 4 C.
4. Incubate the membrane with 0.1 g/ml 2H8 mAb prepared in TBS-T containing
0.5 % skimmed milk for 60 min at room temperature.
5. Wash the membrane five times with TBS-T solution.
6. Incubate the membrane with anti-mouse IgG-HRP diluted 1000 fold in TBS-T
containing 0.5 % skimmed milk for 60 min at room temperature.
7. Wash the membrane five times with TBS-T solution.
8. Incubate the membrane with HRP substrate (e.g., ImmunoStar, Wako) and visu-
alize the signal using an image analyzer (e.g., LAS4000, Fujifilm).
29.4 Results
To evaluate the usefulness of the 2H8 mAb for flow cytometric analysis, we estab-
lished CHO cells expressing various GPCRs with an N-terminal FLAG tag, namely,
mouse and human leukotriene B4 receptor 1 (mBLT1 [3] and hBLT1 [10],
414 F. Sasaki and T. Yokomizo
Fig. 29.1 The 2H8 mAb is sufficiently sensitive to detect N-terminally FLAG-tagged G protein-
coupled receptors (GPCRs). (a) CHO cells stably expressing FLAG-GPCRs and mock cells were
stained with 1 g/ml 2H8 mAb (black) or isotype control (mouse IgG1, white) and then with 1 g/
ml anti-mouse IgG-Alexa Fluor 488 and analyzed with a flow cytometer. (b) CHO cells expressing
FLAG-GPCRs and mock cells were similarly stained in a glass-bottomed dish and visualized by
confocal microscopy. Bar = 50 m
Fig. 29.2 Immunofluorescence staining in the small intestine from villin FLAG-mBLT2-Tg mice.
Frozen sections of the small intestine were stained with 5 g/ml 2H8 mAb followed by anti-mouse
IgG-HRP (upper left panel), or with 10 g/ml rabbit anti-mouse BLT2 PoAb followed by anti-
rabbit IgG-HRP (lower left panel), and then reacted with Alexa Fluor 488-conjugated tyramide.
Right panels show staining using control primary antibodies (mouse IgG1 and rabbit IgG). Nuclei
were visualized by incubation with 1 g/ml DAPI. Bar = 50 m
Some GPCRs are poorly expressed on the plasma membrane, possibly because of
trafficking problems, even when cells overexpress these proteins. Given that it is
generally difficult to generate good mAbs against GPCRs, the 2H8 mAb will be a
powerful tool to detect FLAG-tagged GPCRs with modest or low expression levels
that are currently undetectable using the M2 and M5 mAbs. In addition, we stained
CHO cells expressing FLAG-tagged GPCRs with the 2H8 mAb and detected a
bright signal on the plasma membrane (Fig. 29.1b).
Because of high background staining, especially in the nucleus, conventional
anti-FLAG mAbs (e.g., M2 and M5) are not amenable to immunohistochemical
staining. Therefore, sections of small intestine from villin FLAG-mBLT2 transgenic
mice were stained with the 2H8 mAb or an anti-mBLT2 polyclonal antibodies
(PoAb) (unpublished). We generated villin promoter-driven FLAG-mBLT2 trans-
genic mice, which express FLAG-mBLT2 only in intestinal epithelial cells because
of the specificity of the villin promoter. Both the 2H8 mAb and the anti-mBLT2
PoAb positively stained intestinal epithelial cells (Fig. 29.2, left panels). The signal
generated by the 2H8 mAb was specific because control antibodies did not produce
416 F. Sasaki and T. Yokomizo
Fig. 29.3 The 2H8 mAb efficiently immunoprecipitates FLAG-tagged GPCRs. FLAG-mBLT1
(left) and FLAG-hS1P1 (right) were immunoprecipitated with increasing amounts (0.00110 g)
of the 2H8 mAb. Immunoprecipitates were immunoblotted with 1 g/ml rabbit anti-mBLT1 PoAb
or 1 g/ml mouse anti-hS1P1 mAb and detected with anti-rabbit or anti-mouse PoAb-HRP
(1:1000) as a secondary antibody
any signals (Fig. 29.2, right panels). These data clearly demonstrate that the 2H8
mAb specifically recognizes FLAG sequences fused to GPCRs in vivo and that it is
suitable for immunohistochemical analysis.
Furthermore, we examined whether the 2H8 mAb could immunoprecipitate
FLAG-tagged GPCRs. The 2H8 mAb efficiently immunoprecipitated both FLAG-
mBLT1 and FLAG-hS1P1 (Fig. 29.3). One hundred nanograms of 2H8 mAb was
sufficient for successful immunoprecipitation, and a substantial level of immuno-
precipitation was obtained using only 10 ng 2H8.
29.5 Conclusion
References
1. Feng Y, Broder CC, Kennedy PE, Berger EA (1996) HIV-1 entry cofactor: functional cDNA
cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872877
2. Honda Z, Nakamura M, Miki I, Minami M, Watanabe T, Seyama Y, Okado H, Toh H, Ito K,
Miyamoto T et al (1991) Cloning by functional expression of platelet-activating factor receptor
from guinea-pig lung. Nature 349:342346
29 A Novel Anti-FLAG Monoclonal Antibody Is Useful to Study GPCRs 417
3. Huang WW, Garcia-Zepeda EA, Sauty A, Oettgen HC, Rothenberg ME, Luster AD (1998)
Molecular and biological characterization of the murine leukotriene B4 receptor expressed on
eosinophils. J Exp Med 188:10631074
4. Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T
(1998) Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.
Science 279:15521555
5. Liu M, Saeki K, Matsunobu T, Okuno T, Koga T, Sugimoto Y, Yokoyama C, Nakamizo S,
Kabashima K, Narumiya S, Shimizu T, Yokomizo T (2014) 12-Hydroxyheptadecatrienoic acid
promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2
receptor. J Exp Med 211:10631078
6. Lynch KR, ONeill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M,
Figueroa DJ, Zeng Z, Connolly BM, Bai C, Austin CP, Chateauneuf A, Stocco R, Greig GM,
Kargman S, Hooks SB, Hosfield E, Williams DL Jr, Ford-Hutchinson AW, Caskey CT, Evans
JF (1999) Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature
399:789793
7. Okuno T, Iizuka Y, Okazaki H, Yokomizo T, Taguchi R, Shimizu T (2008) 12(S)-
Hydroxyheptadeca-5Z,8E,10E-trienoic acid is a natural ligand for leukotriene B4 receptor 2. J
Exp Med 205:759766
8. Sasaki F, Okuno T, Saeki K, Min L, Onohara N, Kato H, Shimizu T, Yokomizo T (2012) A
high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled receptor
study. Anal Biochem 425:157165
9. Schweickart VL, Raport CJ, Godiska R, Byers MG, Eddy RL Jr, Shows TB, Gray PW (1994)
Cloning of human and mouse EBI1, a lymphoid-specific G-protein-coupled receptor encoded
on human chromosome 17q12-q21.2. Genomics 23:643650
10. Yokomizo T, Izumi T, Chang K, Takuwa Y, Shimizu T (1997) A G-protein-coupled receptor
for leukotriene B4 that mediates chemotaxis. Nature 387:620624
Index
R Sphingomyelin, 128
Rac, 240, 242, 244, 246 Sphingosine, 128, 357
Raphe nucleus, 63 Sphingosine kinases, 239
Ras-ERK, 240 Sphingosine-1-phosphate (S1P),
Receptor-activator of nuclear factor B ligand 208, 238, 357
(RANKL), 62 phosphatases, 239
Remodeling pathway, 4 receptor, 238
Reproduction, 185 secretion, 215
Resolvin, 31, 72, 157 transporters, 208, 214, 239
Respiratory distress, 241 Sphingosylphosphorylcholine (SPC), 110
Reversed-phase column, 336 SphK1, 239
Reversed-phase liquid chromatography SphK2, 239
(RPLC), 339 S1P lyase (SPL), 133, 239
12R-HETE, 74 S1P metabolic pathway, 132
Rho, 240, 242, 244, 245 Spns2, 204, 210, 239
RhoA, 246 SPP1, 239
RhoA-Rho-kinase (ROCK), 226 s1pr2/mi, 204
Rho kinase, 245 Sprouting angiogenesis, 225, 244
12R-LOX, 7780 Starfish oocytes, 73
Stereo control, 7577
Store-operated Ca2+ entry, 27
S Streptococcus pneumoniae, 99
Saposins, 132 Stress, 316318, 323
Scarlet (ST), 173 Stroma, 253
Schwann cells, 228 Strongyloides venezuelensis, 102
Scramblase, 175176 SUBDUED, 176
Secondary lymphoid organ, 31 Sublimation, 381
Secretory PLA2 group IIA (sPLA2IIA), 120 Substance P (SP), 224
Seizures, 245 Sudden death, 245
Selected reaction monitoring (SRM)/multiple Surface-assisted laser desorption/ionization
reaction monitoring (MRM), 341 (SALDI), 377
Sepsis, 30
Shear stress, 239
Shock, 241 T
SIMS, 372 Target discovery, 350
SjgrenLarsson syndrome (SLS), 133 Testis, 34
Smooth muscle cells (SMCs), 244, 269 12-O-Tetradecanoylphorbol
S-nitrosylation, 243 13-acetate (TPA), 398
Social defeat stress, 318 Th1, 89
Solid phase extraction (SPE), 339 Th2, 89
Soluble epoxide hydrolase (sEH), 231 Th17, 89
Solvent fractionation, 331, 334335 Th1 cytokine, 31
Soybean lipoxygenase (LOX), 70, 73 Th17 immunity., 32
S1P1, 238, 241, 244 Thrombin, 244
S1P2, 238, 244 Thromboxane (TX), 44, 304
S1P3, 238 Thromboxane A2 (TXA2), 26, 53, 283
S1P4, 238, 240 Tip cell, 244
S1P5, 238, 240 TLC-blot, 385, 386
S1P concentration gradient, 239 TMEM16, 175176
Spermatozoa, 34 TMEM16F, 120
Sphingolipid activator proteins, 132 TNF--converting enzyme (TACE), 394
Sphingolipids, 127, 357 TP, 258, 283, 304
Sphingolipid storage diseases (SLSDs), 130 Transacylase, 26
426 Index
V Z
Vascular barrier function, 241, 245 Zebrafish, 200201, 210
Vascular endothelial cells, 239 Zymogen, 33
Vascular endothelial growth factor (VEGF), 245 Zymosan, 32