THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 19,Issue of July 5,pp.

9397-9403, 1987
0 1987 by The American Society of Biological Chemists, Inc. Printed in U.S. A.

Reduction of Collagen Production inKeloid Fibroblast Culturesby

(Received for publication, September 18, 1986)

Tetsuo Sasaki, KariMajamaaS, and Jouni Uittog

Excessive accumulation of collagen is the hallmark pathologic condition resulting from excessive accumulation of
of several clinical conditions characterized by tissue type I collagen in tissue. Several different mechanisms could
fibrosis. Previously, 3,4-dihydroxybenzoic acid, a explain the excessive deposition of collagen in the lesional
structural analog of a-ketoglutarate and ascorbate, has areas of skin. Previous studies have indicated that many, but
been shown to inhibit the activityof purified prolyl 4- not all, keloid fibroblast cultures are characterized by en-
hydroxylase, theenzyme catalyzing the synthesis of 4- hanced collagen production in uitro (3-6). This overproduc-
hydroxyprolineduringintracellular biosynthesis of tion of collagen in cultures, up to 4-fold higher than in the
procollagen. In this studya hydrophobic modification, control cultures, has been directed predominantly toward the
an ethyl ester, of 3,4-dihydroxybenzoic acid was tested
for itseffects on collagen synthesis andprolyl hydrox- synthesis of type I procollagen (3).
ylase activity in human skin fibroblast cultures. The The increase in the rate of procollagen synthesis is also
results indicatedthat 0.4 mM ethyl-3,4-dihydroxyben- accompanied by increased activity of prolyl 4-hydroxylase
zoate markedly inhibited the synthesis of 4-hydroxy- (prolyl hydroxylase), the enzyme which catalyzes the conver-
proline in normal cell cultures apparently as a result sion of selected prolyl residues to 4-hydroxyproline during
of reduced prolyl4-hydroxylase activity, and the syn-intracellular elaboration of procollagen polypeptides (2, 3, 7,
thesis and secretionof both type I and type I11 procol- 8). The presence of a critical number (approximately 100) of
lagens were markedly reduced. Control experiments 4-hydroxyprolyl residues/pro-u chain is required for these
indicated that the test compound did not affect the polypeptides to fold into the triple-helical conformation char-
viability,proliferation, or plating efficiency of the acteristic of collagenous molecules (9-11). The triple-helical
cells, and ithad little, if any, effect on the synthesisof conformation, in turn, is required for secretion of procollagen
noncollagenous proteins. Furthermore, determinationsto the extracellular milieu at normal rate (lo), and in the
of type I and typeI11 procollagen mRNA steady-state absence of 4-hydroxyproline the nonhelical polypeptides are
levels by slot-blot hybridizations suggested that the
inhibition of procollagen production did not occur on subject to rapid degradation both in the intracellular and
the pretranslational level. Thus, ethyl-3,4-dihydroxy- extracellular space (12). Thus, inhibition of the synthesis of
benzoate selectively reduced procollagen production in 4-hydroxyproline would be expected to result in reduced col-
fibroblast culturesby inhibiting the post-translational lagen deposition, and apharmacological reduction in collagen
synthesis of 4-hydroxyproline. Similar inhibitionwas accumulation in tissues could be thought to be beneficial to
also observed in keloid fibroblast cultures, demonstrat- the patientswith keloids (13).
ing the potential applicability of ethyl-3,4-dihydroxy- Prolyl hydroxylase belongs to a group of enzymes which
benzoate, or other structural a-ketoglutarate ascor- or requires ferrous ion, molecular oxygen, ascorbate, and u-
bate analogs, for treatment of fibrotic diseases. ketoglutarate for its activity (14, 15). The latter compound
serves as a cosubstrate, which is stoichiometrically decarbox-
ylated to yield succinate and COZ, in a reaction coupled to the
hydroxylation. Recent biochemical studies have demonstrated
Keloids are relatively common cutaneous lesions histologi- that certain structuralanalogs of a-ketoglutarate canserve as
cally characterized by an abundance of the extracellular ma- potent competitive inhibitors of prolyl hydroxylase (16).
trix of connective tissue (1, 2). Recent biochemical studies These analogs compete with a-ketoglutarate for the binding
have concluded that the major extracellular component of in the active site of the enzyme, and thus they inhibit the
keloids is collagen, type I being the predominant genetically activity of prolyl hydroxylase (17). Recently, another group
distinct collagen (3). Thus, keloids can be described asa of compounds was described which are analogous both to a-
ketoglutarate and ascorbate, and the inhibitory kinetics also
* This work was supported in part by United States Public Health
Service-National Institutes of Health Grants GM-28833, AR-28450, suggested partial identityof the u-ketoglutarate and theascor-
AR-38923, and AR-35297. The costs of publication of this article bate binding sites of the enzyme (18).The most potent inhib-
were defrayed in part by the payment of page charges. This article itor of this group was 3,4-dihydroxybenzoic acid which had
must therefore be hereby marked “advertisement” in accordance with the K; of approximately 5 p M when tested with purified prolyl
18 U.S.C. Section 1734 solely to indicate this fact. hydroxylase (18).This compound, however, was found to be
$ Fellow of the American Heart Association, Greater Los Angeles
Affiliate.
a poor inhibitor of colIagen production under cell culture
3 To whom correspondence should be addressed: Dept. of Derma- conditions, probably because this molecule is relatively polar
tology, Jefferson Medical College of Thomas Jefferson University, and may cross the cell membranes only with difficulty (19).
1020 Locust St., “46, Philadelphia, PA 19107. In the present study we have examined a hydrophobic modi-

9397
9398 Inhibition of Collagen Production
fication, an ethyl ester, of 3,4-dihydroxybenzoic acid for its or 0.4 mM ethyl-3,4-dihydroxybenzoateand 0.1 mM ascorbic acid for
effect on prolyl hydroxylation in keloid fibroblast cultures. 16 h. The medium wasthen removed, and thecells were homogenized
The results indicate that ethyl-3,4-dihydroxybenzoatemark- in buffer containing4 M guanidinium thiocyanate, 5 mM sodium
citrate, 0.5% sarkosyl, 0.1 M 2-mercaptoethanol, and 0.1% Antifoam
edly inhibits collagen production in keloid fibroblast cultures.A (Sigma) (25). Total RNA was isolated by cesium chloride density
gradient centrifugation using 5.7 M Cscl cushion in SW 40.1 rotor
EXPERIMENTALPROCEDURES (Beckman Instruments) for 16 h at 35,000 rpm (25). The pellet
Materials-The two test compounds examined in this study, ethyl- containing total RNA was rinsed with absolute ethanol, dissolved in
3,4-dihydroxybenzoate and 3,4-dihydroxyhenzoic acid, were pur- distilled water, and reprecipitated with 70% (v/v) ethanol containing
chased fromAldrich. The compounds were initially dissolved in 0.4 M NaCl; the precipitate was collected by centrifugation at 18,000
absolute ethanol in 200 mM final concentration. The incubations with X g for 30 min at -10'C. The total RNA in the final pellet was
cells or partially purified prolyl hydroxylase contained ethanol in dissolved in sterilized water, and theRNA concentration was assessed
0.2% final concentration, and theconcentration of ethanol incontrols by absorbance at 260/280 nm.
as well as in all samples incubated with intermediate concentrations For specific mRNA level determinations, varying concentrations
of the test compounds was adjusted to 0.2%. of total RNA were dotted on nitrocellulose filters using a commercial
Fibroblast Cultures-Skin fibroblast primary cultures were estab- vacuum manifold (Schleicher & Schuell) (3). RNA was immobilized
lished either from normal human skin obtained from cosmetic surgery on the filters by heating at 78 "C under vacuum for 90 min, and the
after informed consentor from biopsy specimens obtained from filters were first prehybridized and then hybridized (26, 27) with
patients with keloids. The primary cultures were passed by trypsini- human pro-al(1) collagen (28), pro-al(II1) collagen (29), or fibronec-
zation, and the subcultures were maintained in Dulbecco's modified tin (30) specific cDNA probes. The cDNA probes were radioactively
Eagle's medium plus glutamine and supplemented with 10% fetal calf labeled by nick translation with 32P-deoxyribonucleotides(31). Fol-
serum under 5% CO,, 95% air atmosphere in a humidified tissue lowing hybridizations, the filters were washed, with the stringency of
culture incubator at 37 "C. The fibroblast cultures were studied in the final wash consisting of 37.5 mM NaCl, 3.75 mM sodium citrate,
passages 3-9. pH 6.8, and containing 0.1% sodium dodecyl sulfate (SDS)' (27).The
Collagen Assays-For determination of collagen production in fi- [azP]cDNA-mRNAhybrids were visualized by autoradiography in x-
broblast cultures, 3.0-4.5 x lo4cells/well were plated in 24-well tissue ray cassettes equipped with intensifying screens, and the mRNA
culture plates. At early visual confluency the incubation medium was levels were quantitated by scanning densitometry at 700 nm.
Assay of Prolyl Hydroxylase-For prolyl hydroxylase assay, fibro-
replaced by fresh medium containing 0.1 mM ascorbic acid, 25 rg/ml
8-aminopropionitrile, 20% dialyzed fetal calf serum, and supple- blasts were cultured with or without
ethyl-3,4-dihydroxybenzoateon
75-cm2tissue culture flasks under conditions described above. A t the
mented with the test compound (19). Following a 4-h preincubation,
end of a 16-h incubation, medium was removed, and the cells were
30 pCi of radioactive proline (~-[2,3,4,5-~H]proline, specific activity
rinsed with Hanks' balanced salt solution and scraped with a rubber
107 Ci/mmol; Amersham Corp.) was added, and theincubations were policeman in 1.0mlof20 mM Tris-HC1, pH 7.5, containing 0.2 M
continued for 16 h. At the end of the incubation, the medium was NaCl, 50 p~ dithiothreitol, 10 pg/ml soybean trypsin inhibitor, and
removed, and stock solutions of proteinase inhibitors were added to 0.01% Triton X-100 (32). The cell suspension was homogenizedwith
yield the final concentrations of 10 mM N-ethylmaleimide, 0.3 mM a Teflon-glass tissue homogenizer, stirred gently at 4 "C for 60 min,
phenylmethylsulfonyl fluoride, and 20 mM disodium ethylenedia- and centrifuged a t 4 "C a t 30,000 X g for 30 min. Aliquots of the
minetetraacetate. Cells were sonicated in 1.2 ml of 50 mM Tris-HC1, supernatant were dialyzed for 3 h with two changes against 20 mM
pH 7.5, containing 0.4 M NaCl and the same proteinase inhibitors as Tris-HC1, pH 7.5, containing 0.2 M NaC1. Prolyl hydroxylase activity
above. Aliquots of the media and the cell homogenates were dialyzed was then determined by incubation with [3H]proline-labeled proto-
against running tap water. The samples were then hydrolyzed with collagen (unhydroxylated collagen), prepared from 17-day-old chick
an equal volume of 12 N HCl for 20 h a t 120 'C. The total incorpo- embryo tendons, as described elsewhere (9).The enzyme preparations
ration of 3H radioactivity and the synthesis of [3H]hydroxyproline were tested infive different concentrations to ensure that theactivity
were then determined, as described elsewhere (20). Aliquots of the determinations were performed on the linear range of the assay. The
cell homogenate were also used for protein determinationusing a dye- enzyme activity was assayed in the presence of the following cofactors
binding assay (21). in final concentrations: 2 mM ascorbic acid, 0.08 mM FeSO,, and 0.5
To study the synthesis of type I and type III procollagens, fibro- mM a-ketoglutaric acid (33). The enzyme incubations were performed
blasts in 75"' tissue culture flasks were preincubated for 4 h with at 37 "C for 180 min, and thereaction was terminated by the addition
0, 0.2, or 0.4 mM ethyl-3,4-dihydroxybenzoate,and 100 NCiof [3H] of an equal volume of 12 N HC1. The tubes were sealed, hydrolyzed,
proline was added. After 16 h of incubation, protease inhibitors were and assayed for [3H]hydroxyproline (20).
added to the medium fraction, and newly synthesized proteins were In some experiments, prolyl hydroxylase was extracted from fibro-
precipitated by the addition of 176 mg/mlof ammonium sulfate (30% blasts incubated on 150-cmZtissue culture flasks, and the effects of
of saturation). The precipitate was collected by centrifugation for 60 0.4 mM ethyl-3,4-dihydroxybenzoateor 3,4-dihydroxybenzoicacid on
min at 18,000 X g at 4 "C. The precipitates were dissolved in 25 mM the enzyme activity in uitro were examined.
Tris-HC1, pH 7.5, containing 2 M urea. The samples were then Other Assays-For assay of the synthesis of noncollagenous pro-
chromatographed on a 1.5 X 20-cm column of DEAE-cellulose as teins, fibroblasts were cultured on 24-well tissue culture plates, as
described elsewhere (22, 23). The proteins were eluted with a linear described above. After a 4-h preincubation with or without 0.4 mM
gradient from 0 to 0.22 M NaCl in 25 mM Tris-HC1, pH 7.5, containing ethyl-3,4-dihydroxybenzoate, radioactive tryptophan ( ~ - [ G - ~ H ] t r y p -
2 M urea (23). The recovery of 3H-labeledproteins in DEAE-cellulose tophan, specific activity 3.7 Ci/mmol; Amersham Corp.), 10 &/well,
chromatography varied from 76 to 94%. The radioactive protein peaks was added; the incubation was then continued for 16 h. The incor-
corresponding to type I and type I11 procollagens were pooled, and poration of [3H)tryptophan into macromolecules precipitable with
their [3H]hydroxyproline content was determined after dialysis and 10% trichloroacetic acid was then determined. The amount of cell
hydrolysis in 6 N HCl (20). protein in the same cultures was determined as above (21), and the
To test the helical stability of the newly synthesized collagens, incorporation of 3H radioactivity was expressed as cpm/pg cell pro-
fibroblasts were cultured on 25-cm2tissue culture flasks with 0, 0.2, tein.
or 0.4 mM ethyl-3,4-dihydroxybenzoatefor 4 h as described above. To assay the effect of the test compounds on cell proliferation,
Radioactive proline, 50 &i/flask, was added, and theincubation was fibroblasts in 96-well microtiter plates were incubated with or without
continued for 16 h. The medium was then removed, mixedwith stock 0.4 mM ethyl-3,4-dihydroxybenzoatefor 72 h. Radioactive thymidine
solutions of protease inhibitors (see above), andthe radioactive ([methyL3H]thymidine, specific activity 84 Ci/mmol; Amersham
procollagens were recovered by precipitation with 176 mg/ml of Corp.), 10 &i/well, was added, and the incubations were continued
ammonium sulfate. The precipitate, recovered by centrifugation, was for 6 h at 37 'C. The cells were harvested with an automated multi-
dissolved in 0.1 N acetic acid, pepsin (in a final concentration of 300 sample cell harvester (PHD, Cambridge Technology, Inc., Boston).
pg/ml) was added, and the samples were digested for 6 h a t 15 "C The radioactive macromolecules were collected on glass-fiber filters
(23). The digests were then examined by SDS-polyacrylamide gel and counted by liquid scintillation counting.
electrophoresis using 8%polyacrylamide gels, as described previously. To assay the plating efficiency of fibroblasts, 2 X lo5 fibroblasts
The radioactive polypeptides were visualized by fluorography (24). were plated on 25-cm2 tissue culture flasks in medium containing
For determination of collagen- or fibronectin-specific mRNA lev-
els, the cells were cultured in 75-cn-1' tissue culture flasks with 0, 0.2, The abbreviation used is: SDS, sodium dodecyl sulfate.
 Inhibition of Collagen Production 9399
10% fetal calf serum and with or without 0.4 mM ethyl-3,4-dihydrox- KELOIDS
ybenzoate (34). After a 4-h incubation, the medium was removed, the
cells were rinsed with Hanks’ balanced salt solution, and detached by
brief trypsinization. The number of cells attached to the flasks was
then determined by counting in a hemocytometer.
To determine the viability of the cells incubated in the presence of
0.4 mM ethyl-3,4-dihydrosybenzoate, a trypan blue exclusion test was
performed as described previously (35).
The statistical significance of the differences was calculated by
Student’s t test.

-
RESULTS
Inhibition of Prolyl Hydroxylation in Keloid Fibroblast Cul-
tures-In the first setof experiments, a keloid fibroblast cell
line was incubated with [3H]proline in the presence of 0.2 or ~ 0 0.4
0.4 mM ethyl-3,4-dihydroxybenzoate,andthe synthesis of
[3H]hydroxyproline was assayed as a marker of collagen pro- E - 3.4 - DHB (mM)
duction. The results indicated a marked inhibition in the
FIG. 1. Inhibition of collagen production by 0.4 m M ethyl-
synthesis of [3H]hydroxyproline (Table I). In the same exper- 3.4-dihydroxybenzoate in control and keloid fibroblast cul-
iment, the cells were also incubated with the parent com- tures. Seven keloid and six normal control fibroblast cultures in 24-
pound, 3,4-dihydroxybenzoic acid, in the corresponding con- well tissue culture plates were incubated with and without ethyl-3,4-
centrations. Theparent compound did notinhibit prolyl dihydroxybenzoate, as indicated under “Experimental Procedures.”
hydroxylation at 0.2 mM concentration and showed only an After a 4-h preincubation, the cultures were labeled with [3H]proline,
11%inhibition at 0.4 mM concentration, similar to findings and the synthesis of [3H]hydroxyproline (PHIHYPRO)both in cell
and medium fractions as well as theincorporation of total 3H radio-
in a previous report (19) (Table I). activity were determined. The individual values represent mean8 of
In subsequent studies, seven different keloid fibroblast lines three parallel determinations.
were incubated in thepresence of 0.4 mM ethyl-3,4-dihydrox-
ybenzoate, and theeffect on collagen production was assessed tures incubated without the inhibitor. This variability prob-
as the ratio of newly synthesized [3H]hydroxyproline/total ably reflects the known heterogeneity of fibroblast popula-
incorporation of 3H radloactivity into protein. Incubation of tions with respect to their procollagen synthesis (see Ref. 2).
keloid cell cultures with the test compound resulted in a Demonstration of Reduced Prolyl Hydroxylase Activity-To
marked inhibition of the synthesis of [3H]hydroxyproline(Fig. examine the mechanisms leading to reduced synthesis of
I). Specifically, the relative collagen synthesisincultures hydroxyproline in cultures incubated with ethyl-3,4-dihydrox-
incubated with 0.4 mM ethyl-3,4-dihydroxybenzoatewas re-
ybenzoate, prolyl hydroxylase was extracted from the cells
duced to 26.1 & 10.5% (mean & S.D.) of the controls incubated
and the activity determined by in vitro incubation with bio-
without inhibitor. At the same time, a similar reduction in
logically prepared protocollagen, unhydroxylated [3H]proline-
the synthesis of [3H]hydroxyproline was observed in normal
fibroblast cultures, the values in cultures incubated with 0.4 labeled collagen, as substrate. The results indicated that the
mM ethyl-3,4-dihydroxybenzoatebeing 28.5 k 8.8% (mean & activity of prolyl hydroxylase in cultures incubated with 0.4
S.D.; n = 6) of the controls (Fig. 1).As noted in Fig. 1, there mM ethyl-3,4-dihydroxybenzoatewas markedly reduced, and
is considerable variability in the relative synthesis of [3H] intermediate values were observed in cultures incubated in
hydroxyproline both in the control and keloid fibroblast cul- the presence of 0.2 mM concentration of the test compound
(Fig. 2). Theseresultsthen suggest inactivation of prolyl
TABLE I hydroxylase, and the reduction in prolyl hydroxylation by
Effects of 3,4-dihydroxybenzoicacid and its ethyl ester derivative ethyl-3,4-dihydroxybenzoatenoted in cell culture is appar-
ethyl-3,4-dihydroxybenzoateon the synthesis of hydroxyproline in ently a result of reduced activity of this enzyme.
keloid fibroblast cultures To examine the mechanism of the inhibition of prolyl
Keloid fibroblasts were cultured in 24-well tissue culture plates a t hydroxylase activity in further detail, the enzyme from keloid
early confluency. The cultures were incubated with 30 pCi of [3H] fibroblast cultures was extracted, and its activity was deter-
proline for 16 h, and the synthesis of [3H]hydroxyproline in medium
and cell fraction was determined separately. E-3.4-DHB.. ethvl-3.4- mined in vitro in the presence of varying concentrations of
- .
dihydroxybenzoate 3,4-DHB, 3,4-dihydroxybenzoic acid. the inhibitor using protocollagen as substrate. Incubation of
the enzyme in the presence of 3,4-dihydrobenzoic acid or
.“”
TmQt [3H]Hydroxyprolinesynthesized
ethyl-3,4-dihydroxybenzoate, both in 0.4 mM concentration,
compound Medium Cells resulted in a marked inhibition of the prolyl hydroxylase
mM dpm x Sb dpm X %b activity in vitro (Table 11). Thus, ethyl-3,4-dihydroxybenzoate
Control 8.17 & 0.06 100.0 0.65 2 0.05 100.0 appears to be an inhibitor of prolyl hydroxylase in uitro, but
E-3,4-DHB 0.24.97 & 0.40’ 60.9 0.43 f 0.02d 66.6 it is unclear from these experiments whether its conversion
E-3,4-DHB 0.4 1.60 f0.14‘ 19.5 0.24 +0.02f 37.8
3,4-DHB 0.28.34 k 0.7Y 102.0 0.69 k 0.01’ 107.1 into the parent compound by cleavage of the ester bond is
3,4-DHB 0.4 7.28 f 0.468 89.1 0.81 f 0.06B 124.8 required for the activity, since 3,4-dihydroxybenzoicacid was
Values are dpm of [3H]hydroxyproline/pg of cell protein (mean & an equally effective inhibitor.
S.E. of three parallel determinations). Consequences of Reduced Hydroxyproline Synthesis-As in-
* Calculated as percent of the control incubated with 0.2% ethanol dicated above, the presence of 4-hydroxyproline is critical for
used as solvent for the inhibitors. normal synthesis of triple-helical procollagen molecules. It
The values are significantly different (p < 0.02) from the control. wasof interest, therefore, to measure the stability of the
The values are significantly different (p < 0.05) from the control.
e The values are significantly different (p < 0.001) from the control.
triple-helical conformation of procollagen synthesized in the
’The values are significantly different (p < 0.01) from the control. presence of ethyl-3,4-dihydroxybenzoate.For this purpose,
#The values are not significantly different (p > 0.1) from the newly synthesized [3H]proline-labeled macromolecules were
control. subjected to limited proteolytic digestion with pepsin utilizing
9400 Inhibition of Collagen Production
.”

-
30-
I
n
J
a
e
0
t-
;20-
01
a
->.
I
I
R
dye
front -
Y

x
0 ‘0-
P 0 0.2 0.4
0
E-3.4-DHB (mM1
FIG.3. Demonstration of reduced stability of newly synthe-
sized type I procollagen synthesized in the presence of ethyl-
3,4-dihydroxybenzoate. Normal control fibroblasts in 25-cm’ tis-
sue culture flasks were preincubated for 4 h with 0, 0.2, or 0.4 mM
E-3,4-DHB (mM) ethyl-3,4-dihydroxybenzoate.[‘HIProlinewas then added, incuba-
FIG.2. Inhibition of prolyl hydroxylase activity in cell cul- tions were continued for 16 h, and the stability of the newly synthe-
tures incubated with ethyl-3,4-dihydroxybenmate.Two keloid sized procollagen molecules was determined by limited pepsin prote-
and one normal control fibroblast lineswere incubated in 75-cm2 olysis, as indicated under “Experimental Procedures.” The samples
tissue culture flasks with varying concentrations of ethyl-3,lt-dihy- were electrophoresed on SDS-polyacrylamide gel electrophoresis us-
droxybenzoate for 16 h. At the end of the incubation, prolyl hydrox- ing 8%polyacrylamide gels. The figure represents fluorograms of [3H]
ylase was isolated from the cells and its activity determined by in proline-labeled polypeptides, the electrophoretic mobilities of al(1)
vitro incubation with biologically prepared protocollagen (unhydrox- and a2(I) of type I collagen polypeptides as well as of bromphenol
ylated type I collagen) as substrate. The values are expressed as[’HI blue (dye front) being indicated.
hydroxyproline (p’H/HYPRO) synthesized with [’Hlproline-labeled
protocollagen (percent of total radioactivity; mean f S.E. of three
parallel cultures). procollagen in the presence of 0.4 mM ethyl-3,4-dihydroxy-
benzoate was markedly reduced (see below), essentially no
TABLE I1 procollagen molecules with stabletriple-helical conformation,
Inhibition of prolyl hydroxylase activity in vitro by 3,4-DHB and i t s which would resist pepsin proteolysis, were present in these
ethyl ester derivative ethyl-3,4-dihydroxybenzoate cultures. In cultures incubated with 0.2 mM ethyl-3,4-dihy-
Prolylhydroxylase was extracted from three 150-cm2 flasks of droxybenzoate, an intermediate quantityof radioactive poly-
keloid fibroblasts in early confluency, as indicated under “Experi- peptides in the migration position of a-chains was noted,
mental Procedures.” Aliquots of the enzyme preparation were assayed suggesting that a critical number of prolyl residues in some
for prolyl hydroxylaseactivity by incubation with [’Hlproline-labeled of the molecules were sufficientlyhydroxylated to permit
unhydroxylated collagen as substrate, with or without the inhibitors. pro-a chains tofold into a stable triple helix (Fig. 3).
Amount [3H]Hydroxyproline Since the synthesis of triple-helicaltype I collagen was
Test compound enzymeOf synthesized
added markedly inhibited by ethyl-3,4-dihydroxybenzoate,as judged
by theSDS-polyacrylamide gel electrophoresis of pepsin-
Pl dpm’ %b

None 0 177 f 33
resistant a-chains (see Fig. 3), it was of interest to study
None 10.2% ethanol) 400 13.268 f 329 100.0 whether the synthesis and secretion of intact procollagens
E-3.4-DHB’ 400 2I148 f 25d 15.1 type I and type I11 were equally inhibited. For this purpose,
3,4-DHB’ 400 3,265 f23.6
404d cells were incubated with 0, 0.2, or 0.4 mM ethyl-3,4-dihy-
Values are expressed as dpm of [1H]hydroxyproline synthesized droxybenzoate, and the newly synthesized procollagen mole-
with 1 x lo5 cpm of radioactive substrate during a 3-h incubation in cules were recovered by precipitation with 30% ammonium
vitro (mean f S.E. of three parallel determinations). sulfate. The radioactive proteins were then subjected to
Calculated as percent of the controls incubated with 0.2% ethanol DEAE-cellulose chromatography utilizing conditions which
used as solvent for the inhibitors after subtraction of 177 dpm from
the values. allow separation of intact typeI and typeI11 procollagens, as
‘Test compounds were added in 0.4 mM final concentration. E- shown in control cell cultures (Fig. 4). In contrast, very little
3,4-DHB, ethyl-3,4-dihydroxybenzoate3,4-DHB, 3,4-dihydroxyben- radioactivity was detected in the corresponding peaks in sam-
zoic acid. ples incubated with either 0.2 or 0.4 mM ethyl-3,4-dihydrox-
The values are significantly different(p < 0.001) from the control ybenzoate. For quantitation of type I and type I11 procolla-
incubated with ethanol alone but not different from each other.
gens, the radioactivity in the corresponding peaks was pooled,
and [”Hlhydroxyproline was determined as a specific marker
conditions under which the triple-helical collagen domain of of procollagen. Quantitation of [‘H]hydroxyproline indicated
procollagen molecules resists proteolysis while nonhelical a- that the synthesis and secretion of both type I and type I11
chains aredigested into small fragments. Examinationof the procollagens were markedly inhibited by 0.2 or 0.4 mM ethyl-
digests by SDS-polyacrylamidegel electrophoresis revealed in 3,4-dihydroxybenzoate (Table 111). Thus, the results indicated
control cultures thepresence of intact al(1) and a2(I)chains that a consequence of the inhibition of prolyl hydroxylation
of type I collagen (Fig. 3). In cultures incubated in the pres- during intracellular biosynthesis of procollagens is a marked
ence of 0.4 mM ethyl-3,4-dihydroxybenzoate,very little radio- reduction in the amount of both type I and type I11 procolla-
activity was detected in the position of a-chains. Although gens recovered in the extracellularspace.
the total synthesis of [‘H]proline-labeled type I and type 111 Demonstration That Ethyl-3,4-dihydroxybenzoateDoes Not
 Inhibition of Collagen Production 9401
Amount of

. RNA ( y g )
TYPE I
PROCOLLAGEN

yl

b 3.0 TYPE
PROCOLLAGEN
m - 4.0
I
- 2.0
- 1.0
- 0.5
- 0.25
IO 20 30 40 50 60
FRACTION NO.
- 0.125
FIG. 4. DEAE-cellulose chromatography of ['Hlproline-la-
beled medium proteins synthesized with or without 0.4 mM 0 0.2 0.4
(ethyl-3.4-dihydroxybenzoate). Normalcontrolfibroblastcul-
tures in 75-cm2 tissue culture flasks were incubated with or without E-3.4-DHB (mM)
the test compound, as indicated under "Experimental Procedures." FIG.5. Demonstration that E-3.4-DHB does not affect the
The newly synthesized procollagens were recovered from the medium steady-state abundance of type I and I11 procollagen or fibro-
fraction by precipitation with 30% ammonium sulfate and chromat- nectin mRNAs. Normal control fibroblast cultures were incubated
ographed on DEAE-cellulose chromatography. The proteins initially in 75-cm' tissue culture flasks with varying concentrations of ethyl-
bound to the column were eluted with a linear gradient of 0-0.22 M 3,4-dihydroxybenzoate (E-3,4-DHB) for 16 h. Total RNA wasisolated
NaCl between fractions 11 and 56. The remaining radioactivity was by cesium chloride density gradient centrifugation, and typeI and 111
eluted by a stepwise addition of 1 M NaC1. Fractions of 3 ml were procollagen and fibronectin mRNA levels were determined by slot
collected, and the radioactivity in the fractions was determined. The blot hybridizations with humansequence specific cDNA probes. The
recovery of total radioactivity in control cultures was 76%, whereas figure represents autoradiograms of [32P]cDNA-mRNAhybrids with
the correspondingvalue in the cultures incubated with 0.4 mM ethyl- type I11 procollagen cDNA probe; similar results were obtained with
3,4-dihydroxybenzoate was 94%. The elution positions of type I and type I procollagen and fibronectin cDNA probes. The steady-state
111 procollagens synthesized in control fibroblast cultures are indi- levels of mRNA were quantitated by scanning densitometry at 700
cated by arrows. nm (see Table IV).
TABLE 111
enous glycoprotein synthesized by the fibroblasts, was also
Inhibition of the synthesi of type I and type III procollngens by
ethyl-3,I-dihydroxybenzoate determined. Again, no difference in fibronectin mRNA
Fibroblast cultures in 75-cmZ tissue culture flasks were preincu- steady-state level was noted in cultures incubated withvary-
bated with varying concentrations of E-3,4-DHB and then labeled ing concentrations of the test compound (Table IV). These
with 100 pCi of [3H]proline for 16 h. The 3H-labeled proteins in the observations suggest that ethyl-3,4-dihydroxybenzoatedoes
medium fractions were precipitated with ammonium sulfate, and type not affect procollagen gene expression on pretranslational
I and type 111 procollagens were separated by DEAE-cellulose chro- level and further support the notion that the inhibition is a
matography, as shown in Fig. 4. E-3,4-DHB, ethyl-3,4-dihydroxyben-
zoate. post-translational eventat thelevel of prolyl hydroxylation.
ControlExperiments-To examinethe possibility that
Test [SH]Hydroxyprolinesynthesized ethyl-3,4-dihydroxybenzoatemight exert itseffect on collagen
compound 'On'
Type I" Type 111" Totalb production throughgeneralized inhibition of protein synthesis
mM proteindpm/pg or by being toxic to the fibroblasts, several control experi-
Control 69.44 1.41 70.85 ments were performed. First, the incorporation of [3H]thy-
E-3,4-DHB 0.2 0.72 0.07 0.78 midine into the cells cultured with or without 0.4 mM ethyl-
0.12
E-3,4-DHB 0.4 0.15 0.27 3,4-dihydroxybenzoate was determined. In these experiments,
a Values are expressed as dpmof [3H]hydroxyproline in the corre- both keloid and normal fibroblast cultures were tested. The

sponding peaks recovered by DEAE-cellulose chromatography and results indicated that there was no inhibition of ['Hlthymi-
corrected for the cell protein content of the corresponding cultures.
*Values are dpm of [3H]hydroxyproline in type I plus type I11 dine incorporation in cultures incubated with 0.4 mM ethyl-
procollagen peaks. 3,4-dihydroxybenzoate. Specifically, the incorporationof ['HI
thymidine in normal fibroblast cultures incubated with 0 or
Affect Type I and Type III Procollagen or Fibronectin mRNA 0.4 mM ethyl-3,4-dihydroxybenzoate was 1.77 +. 0.46 and 1.89
Steady-state Levels-To examine the possibility that ethyl- f 0.20 x lo3cpm/2 x lo3 cells, respectively (mean f S.D. of
3,4-dihydroxybenzoate might affect procollagen production 3-4 parallel cultures). The corresponding valuesin keloid
on the transcriptional level, the steady-state abundance of cultures without and with the test compound were 1.66 f 0.12
al(1) and al(1II) procollagen mRNAs was determinedin and 2.12 f 0.34 X IO3 cpm, respectively.
fibroblast cultures incubated with and without the testcom- Second, the viability of the cells incubated with and without
pound. Slot-blot hybridizations with human sequence specific the test compound in 0.4 mM concentration was examined by
cDNA probes under hybridization and washing conditions trypan blue exclusiontest. Resultsindicated that theviability
which exclude cross-hybridizations between type I and type of normal fibroblasts incubated with and without the test
111 procollagen cDNA probes and the corresponding mRNAs compound was 95.5 & 2.3 and 94.6 +. 1.2%, respectively (mean
(27) were utilized in these studies. The results indicated thatf S.D. of four parallel determinations). The corresponding
there was no difference in al(1) andal(II1) procollagen values inkeloid fibroblast cultures incubated with and without
mRNA levels in cultures incubated in the presenceof 0, 0.2, the test compound were 98.1 +. 0.5 and 96.0 f 3.6%, respec-
or 0.4 mM ethyl-3,4-dihydroxybenzoate(Fig. 5 and TableIV). tively.
For comparison, the levels of fibronectin mRNA,a noncollag- Third, theeffect of ethyl-3,4-dihydroxybenzoateon plating
9402 Inhibition of Collagen Production
TABLEIV
Steady-statelevels of al(I) of type Zprocohgen, al(III) of type IIIprocollugen, and fibronectin messenger RNAs in
cultured human skin fibroblasts incubated with ethyl-3,4-dihydroxybenzoate(E-3,4-DHB)
Concentration of Messenger RNA levels*
E-3,4-DHBE al(I) al(II1) Fibronectin
mM units/& % of controld units/& % of controld units/& % of controt‘
0 556 f 54 100.0 677 f 12 100.0 210 f 9 100.0
5890.2 f 29 105.9 681 f 44 100.6 213 f 23 101.4
0.4 569 f 56 102.3 630 f 21 93.1 209 f 12 99.5
“Fibroblasts, cultured in 75-cm2 tissueculture flasks, were incubatedfor 16 h at 37 “C with ethyl-3,4-
dihydroxybenzoate in final concentrations indicated.All cultures, including the controls, contained 0.2% ethanol
used as solvent for ethyl-3,4-dihydroxybenzoate.
Total RNA was isolated by CsCl density gradient centrifugation, andmessenger RNA levels were determined
by molecular hybridizations withhuman al(I), al(III),and fibronectin specific cDNA probes as indicated under
“Experimental Procedures.”
e The values are expressed as densitometric absorbance units obtainedby scanning densitometryat 700 nm/pg
total RNA (mean f S.E. of four to six parallel determinations) and
do not reflect absolute levels of type I and type
I11 procollagen and fibronectin mRNAs.
Calculated as percent of the control cultures incubated without
ethyl-3,4-dihydroxybenzoate.

efficiency of the fibroblasts was tested. No difference in the in cell cultures. Unfortunately, many of the compounds are
presence and absence of the testcompound in 0.4 mM concen- not specific for collagen, and their clinical efficacy is fre-
tration was noted, the plating efficiency being 65.9 f 0.1 and quently compromised by side effects. Consequently, there is
69.9 f 0.1% in normal fibroblast cultures and 79.5 f 0.1 and a further need for pharmacologic agents which would display
76.5 f 0.1% in keloid cell cultures, respectively (mean f S.D.; action properties specific for collagen metabolism.
n = 4). Collagens are a family of closely related connective tissue
Fourth, the effects of ethyl-3,4-dihydroxybenzoateon the proteins which have several features in common (37, 38). All
synthesis of noncollagenous proteins were tested by incubat- collagen polypeptides undergo extensive post-translational
ing the cells with [3H]tryptophan, an amino acid which is not modifications, many of which are catalyzed by specific en-
present in any significant quantities in collagenous proteins; zymes with stringent cofactor and cosubstrate requirements
the incorporation of [3H]tryptophan was determined in pro- (14, 15, 37). Thus, the enzymatically mediated modification
teins precipitable with 10% trichloroacetic acid. The results reactions, many of them unique to collagen, could serve as
indicated that 0.4 mM ethyl-3,4-dihydroxybenzoatehad little, targets for pharmacologic modulation. Of particular interest
if any, effect on the synthesis of noncollagenous proteins. is the reaction leading to formation of 4-hydroxyproline, an
Specifically, the values for [3H]tryptophan incorporation in imino acid necessary for normal synthesis of triple-helical
normal and keloid fibroblast cultures in the absence of the procollagen molecules (9-11,39). Inhibition of prolyl hydrox-
test compound was 7.0 -+ 1.6 and 6.0 f 0.6 x 10‘ cpm/pg cell ylation leads to reduced secretion of newly synthesized pro-
protein(mean f S.D. of three parallel incubations). The collagen polypeptides, and the nonhelical pro-a-chains are
corresponding values in the same cell cultures incubated with subject to rapid both intra- andextracellular degradation.
0.4 mM ethyl-3,4-dihydroxybenzoatewere 6.3 f 0.8 and 5.9 & In thepresent study, we have demonstrated that ethyl-3,4-
0.7 X 10’ cpm/pg, respectively (statistically not significant; p dihydroxybenzoate is a potent inhibitor of prolyl hydroxyla-
> 0.1). Thus,the control experiments demonstrated that tion during intracellular biosynthesis of procollagen in human
ethyl-3,4-dihydroxybenzoatein 0.4 mM concentration had no skin fibroblast cultures. As a consequence of the inhibition of
effect on cell proliferation, viability, or plating efficiency. prolyl hydroxylation, the secretion of type I and type I11
Also, the test compound did not affect the synthesis of non- procollagens was markedly reduced, apparently due to com-
collagenous proteins, suggesting a specific effect on collagen promised stability of the procollagen triple helix. The inhibi-
production. tion of procollagen production was shown not to result from
generalized inhibition of protein synthesis orfrom toxicity of
DISCUSSION the test compound to the cells. Furthermore, type I and type
Excessive deposition of collagen is a major pathologic fea- I11 procollagen mRNA steady-state level determinations sug-
ture in several fibrotic diseases which can affect either skin gested that the inhibition was a post-transcriptional event.
or a variety of internal organs (2, 13, 36). In many of these Further studies specifically demonstrated that theactivity of
conditions, increase in collagen synthesis may not be the prolyl hydroxylase, the enzyme catalyzing the conversion of
primary event of the disease process; nevertheless, the exces- prolyl residues to 4-hydroxyproline, was inhibited by the test
sive accumulation of collagen has major consequences in compound.
terms of structure and function of the affected organs. Thus, Previous studies (18)have demonstrated that 3,4-dihydrox-
a pharmacologic approach which could arrest collagen depo- ybenzoic acid, the parent molecule for the ethylester deriva-
sition in the tissues would be thought to be beneficial to the tive tested in this study, is a competitive analog of a-ketoglu-
patients suffering from clinical fibrotic diseases. tarate and ascorbate, a cosubstrate and a cofactor for prolyl
Previously, several pharmacologic agents have been tested hydroxylase. 3,4-Dihydroxybenzoic acid is able to compete
for their effects on collagen metabolism (see Refs. 13, 17, and with a-ketoglutarate and ascorbate for the binding to the
36). Many of these compounds have been shown to be effective active site of the enzyme (18).Thus, ethyl-3,4-dihydroxyben-
ininhibiting collagen production, and some of themare zoate would appear to be a relatively specific inhibitor for
currently in clinical use for treatment of fibrotic diseases. The prolyl hydroxylation with limited effects on other enzymes or
rationale for the clinical use in most cases has been derived on the synthesis of noncollagenous proteins.
from studies on collagen metabolism in isolated tissues and To demonstrate the potential feasibility of ethyl-3,4-dihy-
 Inhibition of Collagen Production 9403
droxybenzoate for inhibition of collagen production in fibrotic Disease (Jayson, M. I. V., and Weiss, J. B., eds) pp. 101-120,
skin diseases, several keloid cell lines were examined in this Churchill Livingstone, New York
15. Kivirikko. K. I.. and Mvllvla,
" . R. (1985) Ann. N . Y. Acad. Sci.
study for their response to ethyl-3,4-dihydroxybenzoate.As 460,187-201'
indicated above, keloids are cutaneous lesions characterized 16. Maiamaa. K.. Hanauske-Abel. H. M.. Giinzler. V.. and Kivirikko.
by excessive accumulation of type I collagen, and this accu- K. I. (1984) Eur. J. Biochen. 138,' 239-245 '
mulation appears to result from enhanced synthesis of colla- 17. Kivirikko, K. I., and Majamaa, K. (1985) in Ciba Foundation
gen by lesional fibroblasts. The inhibition of collagen produc- Symposium 114,Fibrosis, pp. 34-64, Pitman Books, Turnbridge
Wells, UK
tion in keloid fibroblast cultures demonstrated in this study 18. Majamaa, K., Giinzler, V., Hanauske-Abel, H. M., Myllyla, R.,
suggests that ethyl-3,4-dihydroxybenzoate,or other structural and Kivirikko, K. I. (1986) J. Biol. Chem. 261, 7819-7823
analogs of a-ketoglutarate and ascorbate, might serve as use- 19. Majamaa, K., and Uitto, J. (1986) Clin. Res. 34, 417A (abstr.)
ful agents to limit collagen production in uiuo. 20. Juva, K., and Prockop, D. J. (1966) Anal. Biochem. 15,77783
21. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254
22. Lichtenstein, J. R., Byers, P. H., Smith, B. D., and Martin, G. R.
Acknowledgments-Human sequence specific cDNA probes were (1975) Biochemistry 14, 1589-1594
kindly provided by Drs. Mon-Li Chu, Francesco Ramirez, and Darwin 23. Uitto, J., Booth, B. A., and Polak, K. L. (1980) Biochim. Biophys.
J . Prockop, Department of Biochemistry, University of Medicine and Acta 6 2 4 , 545-561
Dentistry of New Jersey, Rutgers Medical School. We thank Helmi 24. Bonner, W.M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,
Konola for skillful technical help and Charlene D. Aranda for expert 83-92
secretarial assistance. 25. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,
W. J . (1979) Biochemistry 18, 5294-5299
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