ORIGINAL RESEARCH

Glucocorticoids Retain Bipotent Fibroblast Progenitors during

Alveolar Septation in Mice
Stephen E. McGowan and Diann M. McCoy
Department of Veterans Affairs Research Service and Department of Internal Medicine, University of Iowa Carver College of Medicine,
Iowa City, Iowa
ORCID ID: 0000-0003-3084-1962 (S.E.M.).

Abstract signaling through the Smad1/5 pathway, which increased uncoupling

protein-1 in a lung fibroblast progenitor cell line. We conclude that
Glucocorticoids have been widely used and exert pleiotropic effects on glucocorticoid receptor manipulation can sustain fibroblast plasticity,
alveolar structure and function, but do not improve the long-term and posit that targeting downstream glucocorticoid responsive
clinical outcomes for patients with bronchopulmonary dysplasia, pathways could steer fibroblast progenitors along more desirable
emphysema, or interstitial lung diseases. Treatments that foster regenerative pathways.
alveolar regeneration could substantially improve the long-term
outcomes for such patients. One approach to alveolar regeneration is to Keywords: progenitor cells; pulmonary alveolar development;
stimulate and guide intrinsic alveolar progenitors along developmental platelet-derived growth factor receptor-a; myofibroblast;
pathways used during secondary septation. Other investigators and we lipofibroblast
have identified platelet-derived growth factor receptor-a–expressing
fibroblast subpopulations that are alternatively skewed toward
myofibroblast or lipofibroblast phenotypes. In this study, we Clinical Relevance
administered either the glucocorticoid receptor agonist dexamethasone
(Dex) or the antagonist mifepristone to mice during the first postnatal Current treatments for bronchopulmonary dysplasia,
week and evaluated their effects on cellular proliferation and adoption emphysema, and interstitial fibrosis do not restore damaged
of a-smooth muscle actin and lipid droplets (markers of the alveoli. This study shows how glucocorticoids regulate
myofibroblast and lipofibroblast phenotypes, respectively). We fibroblast progenitors, which contribute to alveolar septal
observed that Dex increased the relative abundance of fibroblasts with formation during postnatal development. The findings identify
progenitor characteristics, i.e., containing both a-smooth muscle actin glucocorticoid-responsive pathways that could be used to
and lipid droplets, uncoupling protein-1 (a marker of brown and beige stimulate alveolar regeneration.
adipocytes), delta-like ligand-1, and stem cell antigen-1. Dex enhanced

The unique contributions of individual rats (1) or fibrotic injury in adult mice (2) storage subpopulation (referred to as
pulmonary alveolar fibroblast have shown that structural abnormalities lipofibroblasts [LIFs]) (3). Recent
subpopulations to air-space development are ameliorated by steering fibroblasts studies using lineage tracing indicate
and regeneration remain incompletely toward a lipid-storage phenotype. that the fate of LIF progenitors is defined
understood. Studies of hyperoxia-induced Conversely, adoption of myofibroblast later than that of MFs, which arise
bronchopulmonary dysplasia in neonatal (MF) characteristics reduces the lipid- from a prenatally defined population of

( Received in original form November 17, 2016; accepted in final form February 14, 2017 )
This study was funded by a Merit Review award from the Department of Veterans Affairs research service. Flow cytometry was performed at the Flow
Cytometry Facility, which is a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility at the University of Iowa. The Facility is
funded through user fees and the generous financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City
Veteran’s Administration Medical Center. The Aria flow cytometer was funded by a grant from the National Center for Research Resources of the National
Institutes of Health under award number 1S10 RR027219.
Author Contributions: S.E.M. designed studies, acquired and analyzed data, and wrote the manuscript. D.M.M. acquired data and edited the manuscript.
Correspondence and requests for reprints should be addressed to Stephen E. McGowan, M.D., Division of Pulmonary, Critical Care, and Occupational Medicine,
C33B GH, Department of Internal Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242. E-mail: [email protected]
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Am J Respir Cell Mol Biol Vol 57, Iss 1, pp 111–120, Jul 2017
Copyright © 2017 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2016-0376OC on May 21, 2017
Internet address: www.atsjournals.org

McGowan and McCoy: Glucocorticoids Retain Alveolar Fibroblast Progenitors 111

 ORIGINAL RESEARCH

sonic-hedgehog–responsive, Gli1-expressing proteins, whereas GFPlow lung fibroblasts Production and nuclear localization of GFP
progenitors (3). MF differentiation is showed higher expression of cell surface are under the control of the endogenous
dependent on transforming growth factor-b proteins involved in migration and pdgfra promoter, and spatially and
(TGF-b) signaling through TGF-b receptor cytoskeletal rearrangement (15). It remains temporally recapitulate endogenous
1 (Alk5), and disruption of Alk5 results in unclear whether these differences in pdgfr-a expression (10). The mice were
expansion of the LIF subpopulation (4). ontogeny and gene expression profiles heterozygous for the pdgfra-GFP allele and
Manipulating the differentiation of one result from divergent terminal differentiation were phenotypically identical to wild-type
subpopulation influences that of the other, along predefined pathways, adaptation to (GFP2) mice. Members within 4 separate
but it remains unclear whether this only their current environment, or both. litters of mice were allocated to receive Dex
occurs before birth or LIFs and MFs To address this issue, we manipulated or phosphate-buffered saline (in one litter,
maintain postnatal plasticity. This is an glucocorticoid signaling in mice and fibroblasts were isolated separately from
important consideration when developing examined the effects on LIF and MF two Dex-treated mice, yielding a total of
regenerative strategies for the treatment phenotypes. Glucocorticoids are used five). In the Dex-treated mice, Dex was
of destructive alveolar diseases such as clinically but have shown limited efficacy in delivered subcutaneously at 100 ng/g body
bronchopulmonary dysplasia, emphysema, treating bronchopulmonary dysplasia and weight on P1 and at 50 ng/g on subsequent
and interstitial fibrosis (5). pulmonary fibrosis. A better understanding days. Mifepristone (Mfp) was dissolved in
Mesenchymal progenitor cells with of the effects of glucocorticoids on fibroblast propylene glycol and 50 mg/g body weight
the potential to differentiate into adipocytes subpopulations may enable us to identify was delivered subcutaneously to male
or another specialized cell (myocyte, signaling pathways that could be specifically mice from five separate litters, using
oligodendrocyte, or adipocyte) express targeted to circumvent some of the other littermates as controls. To analyze
platelet-derived growth factor receptor-a undesirable effects of long-term proliferation, the pups were treated
(PDGFRa), which influences progenitor corticosteroid administration. with 100 ng/g body weight of 5-ethynyl-
proliferation and differentiation (6–8). Both augmentation and suppression of 29deoxyuridine (EdU) 3 h before they were
Furthermore, PDGFRa-expressing signaling have been used to define the effects killed and lung fibroblasts were isolated
adipocyte precursors can assume of glucocorticoids on alveolar septation. (13). The protocols for animal use were
characteristics of brown adipocytes and However, the findings are enigmatic because approved by the Iowa City Veterans
participate in the development of brown they vary with the timing of the stimuli, the Affairs Medical Center Animal Use
fat within deposits of white fat (beige fat) cell populations that were targeted, and Committee (10).
induced by cold exposure or b3-adrenergic which cellular properties were studied. The
stimulation (9). Using mice of different glucocorticoid agonist dexamethasone Lung Inflation and Fixation
perinatal ages, investigators observed that (Dex) delays capillary maturation and Lungs from mice bearing the PDGFRa-GFP
PDGFRa is expressed in both LIFs (which produces transient alveolar wall thinning, insert at P8 and P12 were uniformly inflated
stain for adipocyte-related differentiation but enhances the expression of elastin (50 ml of fixative per gram body weight),
protein [ADRP, also known as perilipin 2] (16–18). Targeted deletion of the fixed, and sectioned, and the Ki67antigen
and contain lipid droplets) and MFs (a glucocorticoid receptor (GR) in was analyzed by laser scanning confocal
larger proportion contain a-smooth muscle mesenchymal cells was shown to increase the microscopy and enumerated using the
actin [a-SMA], and few contain ADRP) volume density (VD) and proliferation of the optical fractionator stereological probe (14).
(10–12). Studies using flow cytometry to airspace mesenchyme, diminish elastic fiber Tissues used to analyze the gas-exchange
classify fibroblasts isolated from mice formation and epithelial maturation, and surface area and alveolar wall thickness
bearing green fluorescent protein (GFP) halt progression beyond the saccular stage, were embedded in London Resin (LR)
driven by the endogenous PDGFRa such that few mice survived after P1 (19). White. They were analyzed using the
promoter (PDGFRa-GFP) identified We hypothesized that the differential effects Cycloids for Sv stereological probe to
fibroblasts with two levels of GFP intensity, of glucocorticoids on the LIF and MF determine the gas-exchange surface area,
which during alveolar development fibroblast subpopulations could explain and the Mertz probe to determine the
correlated with the abundance of PDGFRa why GR deletion increases alveolar arithmetic mean barrier thickness.
molecules on the cell surface (13). During mesenchymal cells but diminishes elastin,
Postnatal Day 4 (P4) through P14, a major product of these cells. Isolation of Primary Mouse
PDGFRa-GFPlow cells predominantly Fibroblasts and Flow Cytometry
reside at the base of elongating secondary Lung fibroblasts were isolated on P8 using
septa and store neutral lipids (14). PDGFR- Materials and Methods a previously reported method involving
GFPhigh fibroblasts congregate at the digestion with collagenase and adherence to
alveolar entry ring (secondary septal tip) A complete list of the materials used in this tissue-culture plastic for 1 h (10). Epithelial
and exhibit more a-SMA. Flow cytometry work and a more detailed explanation of the and endothelial cells comprised z 2.5% and
has also been used to sort lung fibroblasts methods employed are provided in the 1.6%, respectively, whereas macrophages
from adult mice based on their intensity online supplement. were only detected in the PDGFRa-GFP2
of GFP fluorescence. In adult mice, population (10). Fibroblasts stained for
PDGFRa-GFPhigh cells demonstrated a Mice ADRP, a-SMA, phosphorylated Smad1/5
gene expression profile for abundant Mice bearing the PDGFRa-GFP construct (pSmad1/5), or uncoupling protein-1
production of extracellular structural have been described previously (10). (UCP1) were permeabilized before

112 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017
ORIGINAL RESEARCH

immunostaining (10). When staining for A Postnatal day C control Mfp Dex
LipidTOX red (LTR) and stem cell antigen-1

alveolar surface (cm2)

01 8 12 100
(Sca1), the cells were not permeabilized.
80
CD451 cells, which had adhered to the EdU * Euthanize
culture plastic after 1 h, were excluded from inflation, 60
* fixation
analysis. Virtually all of the PDGFRa- 40 * *
Dex or Mfp
P8 FACS
expressing fibroblasts were in the CD452 * 20 *
fraction. Forward scatter and side scatter 0
were used to exclude small (presumably
B control Mfp Dex P8 P12
apoptotic) cells and aggregates. The
background fluorescence from the D
corresponding immunoglobulin G isotype
P8
control Mfp Dex
controls was subtracted to quantify the

barrier thickness (μm)

8
different fibroblast populations. *

arithmetic mean
6 *
Isolation of Mitochondria for UCP1 4 *
Immunoblotting
P12

Neonatal mouse lung fibroblasts (MLg) 2

2908 cells were subjected to adipogenic 0
induction, and after a 16 h period without P8 P12
Dex, they were treated with 5 ng/ml TGF-b
Figure 1. Perturbing glucocorticoid signaling disrupts alveolar structure. Dexamethasone (Dex) or
or 100 nM Dex, or remained untreated, and mifepristone (Mfp) was administered, whereas controls were untreated, during the postnatal days shown in
the mitochondria were isolated (20). After (A) (horizontal lines) and mice were killed (*) at Postnatal Day 8 (P8) or P12. (B) Lungs were uniformly inflated
incubation with 0.5% n-octyl-B-D- with low-melting-point agarose, fixed, embedded in London Resin White, sectioned, stained, and
glucopyranoside to solubilize UCP1, the imaged (scale bars are 50 µm). Insets are lower magnification to show larger fields of view. (C) Combined
samples were subjected to SDS-PAGE and alveolar surface (including both alveoli and alveolar ducts) for both lungs. Mean 6 SEM, n = 4 mice per
immunoblotting. MLg 2908 cells were treatment. (D) Alveolar wall (arithmetic mean barrier) thickness was altered by both Mfp and Dex at P8,
induced and similarly treated with TGF-b1 but only by Mfp at P12. Mean 6 SEM, n = 4 mice for each treatment group, *P , 0.05 for Dex-
or Dex before immunoblotting for pSmad2, or Mfp-treated mice compared with controls at the respective ages. EdU, ethinyl deoxyuridine; FACS,
Smad2 pSmad1/5, Smad1/5, and b-tubulin. fluorescence-activated cell sorter.

P8, after which the GFPlow LIFs diminish proportion of EdU1 GFPlow fibroblasts at
Results (10, 16). The observation that both Dex and P8 (Figure 2C). Our prior studies showed
Mfp reduced the alveolar surface area indicates that proliferation is sustained in PDGFRa-
Glucocorticoid Signaling Regulates that septal outgrowth requires properly GFP fibroblasts, which bear the progenitor
Alveolar Wall Thickness and Surface balanced glucocorticoid signaling, and that marker Sca1, and Dex increases the
Area both excessive Dex and insufficient Mfp are proportion of Sca11 LFs within the GFPhigh
Dex, a potent GR agonist, or Mfp, a GR as detrimental. population (Figure 2D) (10). However,
well as a progesterone receptor antagonist, Dex reduced the size of the entire GFPhigh
was administered during P1–P7 or P1–P11 Increased Proliferation Contributes to population when expressed as a percentage
and the mice were killed on P8 or P12 Increased Alveolar Wall Thickness in of all CD452 fibroblasts (Figure 3A). These
(Figure 1A). Images of lungs that had been Mfp-Treated Mice observations suggest that Dex retains
uniformly inflated with agarose during The increase in alveolar wall thickness after GFPhigh fibroblasts as progenitors.
fixation are shown in the figure. Residual Mfp treatment could represent enhanced
agarose accounts for the gray background mesenchymal cell proliferation, as others PDGFRa GFPhigh Fibroblasts Exhibit
in the air spaces. Casual inspection showed have observed in GR-deleted mice (19). We LIF Characteristics after Treatment
that Dex increased the VD of the airspace at examined proliferation in situ using the with Mfp or Dex
P8 and P12, and that the GR antagonist marker Ki67 or by pulse labeling with PDGFRa1 mesenchymal progenitors retain
Mfp had the same effect at P8 (Figure 1B). EdU before isolating fibroblasts at P8. a less differentiated, progenitor state
Stereological analysis confirmed that the Although the volume-corrected number of in other organs; for example, both
alveolar surface was smaller in both Dex- nonproliferating cells (GFP2 cells included preadipocytes and myoblasts can assume
and Mfp-treated mice at P8 (Figure 1C). resident cells in the alveolar wall that either a lipid-storage or muscle-like
Mfp increased the mean barrier (alveolar were not fibroblasts) was not altered, we phenotype (21). Although Mfp is a GR
wall) thickness at both ages, whereas wall observed an increase in Ki671 GFPlow, antagonist, its functional effects are not
thinning was only observed at P8 in Dex- but not GFPhigh, fibroblasts after exposure always converse to those of Dex (22). Our
treated mice (Figure 1D). Others have to Mfp (Figures 2A and 2B). When we readouts were sensitive to the size (only
observed that pharmacological manipulation analyzed isolated fibroblasts using LTR) and the abundance (both LTR and
of the GR transiently alters the surface area, fluorescence-activated cell sorting, ADRP) of lipid droplets. These readouts
indicating that critical effects are manifest by treatment with Mfp increased the reflect not only the immediate effects on

McGowan and McCoy: Glucocorticoids Retain Alveolar Fibroblast Progenitors 113

 ORIGINAL RESEARCH

A B Ki67–
Control Dex
400

(cells/2 lungs) X 10–9

control
300 Mfp

200

100

0
all (GFPneg) GFPlow GFPhigh

60 Ki67+

(cells/2 lungs) X 10–9

control
Mfp
40

20
C *
50 GFP neg

GFP low 0
% of CD45- cells

40
GFP high all (GFPneg) GFPlow GFPhigh
30
* D
20
80 GFP neg

Sca1+ % of population
10
GFP low
60 GFP high
0
control mifepristone
40

50 GFP neg 20
% of CD45- cells

40
GFP low *
GFP high 0
30 control dexamethasone
Mfp
20

10

0
control dexamethasone
Figure 2. Glucocorticoids differentially alter proliferation in PDGFRa-GFP–expressing subpopulations. Lung fibroblast proliferation was assessed by the
abundance of Ki67 antigen or by flow cytometry at P8, using mice that were treated as shown in Fig. 1A. (A) Representative confocal images of lungs stained for
Ki67 (blue) or the nuclear counterstain PoPo3 (red). PDGFRa-expressing cells exhibited GFP fluorescence (green, scale bar: 20 µm). (B) Ki671 nuclei were
enumerated within the GFPhigh and GFPlow populations in control or Mfp-treated mice on P8. GFP2 includes alveolar cells other than fibroblasts, whereas only
fibroblasts contained GFP. Mean 6 SEM, n = 4 mice for each treatment group. *P , 0.05 for Mfp- or Dex-treated mice compared with controls. (C) EdU
was administered to untreated (control) or Dex- or Mfp-treated mice on P8. Fibroblasts were isolated and subjected to flow cytometry, gating out CD451 cells
(macrophages or fibrocytes), so that the only GFP2 cells analyzed were fibroblasts. Mean 6 SEM of the EdU1 fibroblasts as percent of CD452 cells, n = 5 sets
of 1 or 2 mice from 4 separate litters for each glucocorticoid treatment group. *P , 0.05 for Mfp-treated mice compared with controls. (D) Percent Sca11,
CD452 within each population stratified by GFP intensity. Mean 6 SEM, n = 4 control and Dex-treated littermates derived from 4 separate litters. *P , 0.05 for
Dex-treated mice compared with controls. GFP, green fluorescent protein; PDGFRa, PDGFR, platelet-derived growth factor receptor-a.

GR but also differentiation (lipid droplets Mfp modestly increased the proportion of fibroblasts that contained a-SMA
were observed both in GFPhigh progenitors LTR1 GFPhigh fibroblasts (P = 0.045, a (Figure 4A). This indicates that
and in mature GFPlow fibroblasts), the small but statistically significant difference). glucocorticoid signaling influences the
balance between fibroblast lipogenesis and Gating on LTR fluorescence intensity, relative abundance of lipid droplets and
lipolysis, and the response to hormones Dex increased the per-cell abundance of a-SMA in PDGFRa-expressing fibroblasts.
such as insulin (22). These factors are lipids in both the GFPlow and GFPhigh Whereas Mfp did not alter the proportions
addressed in more detail in the DISCUSSION. populations (Figure 3C). Mfp increased the of ADRP and a-SMA double-positive cells
Mfp increased lipid storage in the proportion of GFPlow fibroblasts, whereas compared with controls (data not shown),
GFPhigh population (Figures 3B and 3D). Dex decreased the proportion of GFPhigh Dex increased the proportion of GFPhigh

114 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017
ORIGINAL RESEARCH

A B preadipocyte differentiation toward brown

GFP neg GFP low GFP high adipocytes. Flow-cytometric analysis
100 100 GFP neg
showed that Mfp did not alter the
GFP low
proportions of UCP11 cells within the

% of CD45- cells
% of CD45- cells

80 80 three populations (data not shown),

% of LTR+ CD45- LF
100 GFP high
60 80 whereas Dex decreased the proportion
60 * of UCP11 fibroblasts in the GFPlow GFPhigh
60
40 40 population (Figure 5B). Both UCP1 mRNA
40 (Figure 5A) and protein (Figure 5B) were
20 20
20 highest in the GFPlow population of control
0 0 0 fibroblasts. Both Mfp and Dex increased
Control Mfp Control Dex control mifepristone the proportion of ADRP, UCP1 double-
positive cells in the GFPhigh population
C LTR low
GFP neg (Figure 5C), which resulted in part from the
100
LTR high
GFP low higher prevalence of ADRP1 cells in the
GFPhigh population (Figure 3D). Dex
% of LTR+ CD45- LF 100 GFP high
% CD45- GFP+

80
80 diminished the proportion of ADRP,
60
60 UCP1 double-positive cells in the GFPlow
*
40 * * population (Figure 5C). We also observed
40
that Mfp increased the proportion of
20 * 20 GFP low fibroblasts that contained both
0 0 a-SMA1 and UCP11 (Figure 5D) and
GFP low GFP high GFP low GFP high control dexamethasone
increased the proportion of GFPhigh
Control Dexamethasone fibroblasts that contained a-SMA, ADRP,
D GFP neg and UCP1 (Figure 5E). Therefore, as with
GFP low GFP neg beige adipocytes (24, 25), manipulation
100 GFP high 100 GFP low of glucocorticoids lessened the distinction
GFP high between ADRP1 GFPlow and a-SMA1
% of population

% of population

80 80
GFPhigh fibroblasts, and conferred a
ADRP+

*
ADRP+

60 60 * more adipocytic phenotype to PDGFRa-

40 40 expressing fibroblasts, which also
20 20 contain a-SMA.
0 0
control mifepristone control dexamethasone Dex Stimulates Phosphorylation
Figure 3. Perturbing glucocorticoid signaling alters lipid storage in the GFP population. (A) Flow high of Smad1/5 in the Context of
cytometry showed that Dex, but not Mfp, altered the relative abundance of the three lung fibroblast Increased UCP1
(LF) populations defined by PDGFRa-GFP intensity: GFP2, GFPlow, and GFPhigh. Mean 6 SEM, n = 5 Dex augments Smad1/5 phosphorylation in
control and treated littermates from 4 litters. *P , 0.05, size of GFPlow or GFPhigh component NIH3T3 cells and adult human primary
compared with control. *P , 0.05 for GFPhigh, comparing Dex-treated mice with controls. (B) Effects of lung fibroblasts (26). Others have shown
Mfp and Dex on the proportions of neutral lipid–containing fibroblasts (stained with LipidTox red [LTR]) that that the TGF-b homolog BMP7
within each subpopulation stratified by intensity of GFP-fluorescence. Mean 6 SEM, n = 5 mice, for each enhances phosphorylation of pSmad1/5,
treatment, from 4 separate litters. *P , 0.05 comparing Mfp-treated mice with controls. (C) Using the
inducing C3H10T1/2 cells to acquire
cells shown in B, the LTR1 CD452 fibroblasts were gated into two populations based on the intensity of
LTR fluorescence. The distribution of GFPlow and GFPhigh cells in LTRlow and LTRhigh populations is
characteristics of brown adipocytes,
shown. Mean 6 SEM, n = 5, same mice as in B. (D) Analysis similar to that shown in B, except that lipid including increased UCP1 (23). Therefore,
droplets were identified by perilipin-2 (ADRP). Mean 6 SEM, n = 5 mice, for each treatment, from we examined whether Dex augments
4 separate litters. *P , 0.05 comparing Mfp- or Dex-treated mice with littermate controls. Smad1/5 phosphorylation and induces
UCP1 in the progenitor-like (bipotent
differentiation to MF or adipocyte
fibroblasts that contained both ADRP and differentiate to smooth-muscle– and characteristics) MLg 2908 lung fibroblast
a-SMA (Figure 4B). This is consistent with brown-adipocyte–like phenotypes (23). The cell line. Dex increased Smad1/5
GFPhigh fibroblasts retaining their bipotent observation of a similar relationship in phosphorylation (Figures 6A and 6B) and
progenitor status and delaying commitment PDGFRa1 alveolar fibroblasts (10) UCP1 protein in isolated mitochondria
to differentiated MFs. prompted us to examine their similarity to (Figure 6C). To determine whether Dex
brown adipocytes using the marker UCP1. alters Smad1/5 signaling in vivo, we
PDGFRa-Expressing Fibroblasts In controls without manipulation of the administered Dex to mice during P1–P8,
Exhibit Characteristics of Brown GR, UCP1 gene expression was higher in and using immunostaining and flow
Adipocytes the GFPlow population, which also cytometry, we observed that the proportion
Others have shown that C3H10T1/2 exhibited higher expression of PRDM16 of pSmad1/5 1 GFP high fibroblasts
mesenchymal progenitor cells alternatively (Figure 5A), an important regulator of (73.6 6 3.9, mean 6 SEM, n = 5) was

McGowan and McCoy: Glucocorticoids Retain Alveolar Fibroblast Progenitors 115

 ORIGINAL RESEARCH

A Dex Retains Lung Fibroblast increased the proportions of ADRP1

100
GFP low
Progenitors (Figure 3D) and UCP11, ADRP1 double-
GFP high
Dex increased the proportion of GFPhigh LF positive fibroblasts (Figure 5C) in the
% of population

80
that contained both ADRP and a-SMA GFPhigh population. Dex increased the
* intensity of LTR fluorescence per cell
αSMA+

60 (Figure 4B), and a larger proportion were

40
Sca11 (Figure 2D), suggesting that GFPhigh (Figure 3C), which is consistent with an
cells retained progenitor characteristics increased aggregate volume of intracellular
20 with markers of both MF and LIF. lipid droplets. Dex also increased the
0 We posited that Dex may restrain the proportion of LTR1 cells in the GFPhigh
control mifepristone divergence into LIF and MF phenotypes by population (Figure 3B). Mfp increased
holding more fibroblasts in a bipotent state. the proportion of ADRP1 fibroblasts
100 GFP low Therefore, we examined the expression of (dependent on increased surface area
GFP high sterol receptor binding protein-1 (SREBP1), and not droplet volume) in the GFPhigh
% of population

80 which indicates a transition to an population (Figure 3B). Mfp decreased the

αSMA+

60 * adipocyte-like phenotype, and delta-like lipid droplet size (with a corresponding

ligand-1, splice product-C (Dlk1C), a increase in the surface/volume ratio) in
40
progenitor marker that is regulated adipocytes of mice receiving a high-fat diet
20 by PDGFRa during mouse alveolar (29, 30). If Mfp similarly affects fibroblast
0 development (10, 27). Treatment with Dex lipid droplets, one would expect an
control dexamethasone increased Dlk1C and decreased SRBP1 increased proportion of ADRP1, but not
mRNA relative to untreated controls, LTR1, fibroblasts, which is what we
B consistent with retention of progenitor observed. Further complexity arises from
GFP neg
80 characteristics (Figure 7). differential effects of Dex on preadipocytes
αSMA+ and ADRP+

GFP low
Our findings illustrate how versus adipocytes. Dex increased
% of population

60 GFP high
glucocorticoids influence the balance proliferation, accelerated differentiation,
40 between LIFs and MFs, and sustain and increased the UCP1 gene expression
progenitors, which share characteristics of of brown preadipocytes (31). However,
20
* both phenotypes. Lung fibroblasts contain Dex antagonized the cyclic adenosine
the beige adipocyte progenitor marker monophosphate (cAMP)-mediated
0
control dexamethasone UCP1, which is augmented by Dex in the stimulation of oxygen consumption and
PDGFRa-GFPhigh population. They also UCP1 expression in differentiated brown
Figure 4. Perturbing glucocorticoid signaling
illustrate how the effects of glucocorticoids adipocytes (32), and increased lipolysis in
alters the proportions of a-smooth muscle actin
(a-SMA)-containing fibroblasts. Fibroblasts were depend on the differentiation status, and mature white adipocytes (33). Although Dex
isolated from mice treated with Mfp or Dex that timing and context are essential for and Mfp exert opposing effects on the GR, this
during P1–P7, or untreated littermate controls targeting their effects on alveolar does not consistently translate into obverse
(see Fig. 1), and subjected to flow cytometry. (A) development or regeneration. findings at the cellular and organ levels.
The proportions of CD452 GFPlow and GFPhigh
fibroblasts that contained a-SMA are shown. (B) Glucocorticoids Have Time-Sensitive
A minority of fibroblasts contained both a-SMA Discussion and Context-Dependent Effects on
and ADRP, and Dex increased the proportion of Alveolar Septation
double-positive cells in the GFPhigh population.
Glucocorticoids Complexly Regulate The effects of glucocorticoids on alveolar
Mean 6 SEM, n = 5 treated mice from
Lipid-Droplet Metabolism development are influenced by the age at
4 litters each for Mfp and Dex with 5 littermate
controls, which differed for Mfp and Dex We used two different markers to identify which they are administered. Massaro and
exposures. *P , 0.05 comparing Dex- or lipid-storage cells by flow cytometry. LTR coworkers (34) showed that administering
Mfp-treated mice with the respective control accumulates inside lipid droplets, where it 0.1 mg of Dex (which reduced body weight
populations stratified by GFP fluorescence binds to triglycerides and cholesterol esters. by 10%) to rats between P4 and P13
intensity. ADRP (perilipin 2) is located on the outer diminished the alveolar surface area at P14,
phospholipid coating rather than in the and it remained lower than in saline-treated
interior of lipid droplets. Therefore, the LTR controls at P60. The alveolar wall interstitial
significantly higher than in controls intensity depends on the droplet volume, thickness, the VD of lipid-laden and
(63.8 6 3.0, P , 0.05). Gating on the whereas the anti-ADRP fluorescence non–lipid-laden interstitial cells, and the
CD452 fibroblasts, which were also intensity depends on the droplet surface VD of fibroblast lipid droplets were all
ADRP1, demonstrated a more substantial area. Like other cells, LIFs contain multiple reduced at P14 (18). Tschanz and
effect of Dex on the proportion of droplets of various sizes (28). The size of the colleagues (35), and later Roth-Kleiner and
GFPhigh fibroblasts containing pSmad1/5 lipid droplets is controlled by the balance colleagues (17), showed that administering
(Figure 6D). Therefore, as in preadipocytes, between lipid esterification and lipolysis, Dex from P1 through P4 produced septal
Smad1/5 signaling was accompanied by a and therefore is sensitive to agents such as thinning and diminished alveolar
shift of differentiation toward the LIF Mfp and Dex, which modulate lipolysis (28). capillaries, both of which normalized after
phenotype. We observed that both Dex and Mfp P13. This abbreviated administration of

116 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017
ORIGINAL RESEARCH

A B Dex only transiently interrupted the

GFP neg
UCP1 PRDM16 100 proliferation and apoptosis of septal cells at P4

UCP1+ % of population
5 0.20 GFP low
2-(ΔΔCT) (fold GFPneg )

2-(ΔΔCT) (fold GFPneg )

GFP high
and diminished elastin and tenascin-C at P6
4 80 (36). These studies identified a critical time
0.15
3 60 during which Dex reversibly interrupts
* 0.10 mesenchymal cell functions, and coincides
2 40 *
with the postnatal period in which LIFs
1 0.05 20 maximally proliferate and accumulate neutral
* lipids (14, 37). The study presented here
0 0.00 0
PDGFRα-GFP low high low high control dexamethasone explains some of these time-sensitive effects of
glucocorticoids during secondary septation.
C D First, proliferation of the GFPlow population
GFP neg GFP neg was lower in control than in Mfp-exposed
100 100
GFP low GFP low fibroblasts at P8 (Figure 2B). Therefore, the
UCP1+ and ADRP+

UCP1+ and αSMA+

80 GFP high GFPlow population is particularly susceptible
% of population

% of population
80 GFP high

60 60 to manipulation of GR signaling before P5.

* Second, although GFPhigh fibroblasts
40 40 * diminished as a proportion of CD452 cells
20 20 (Figure 3A), the GFPhigh, ADRP1, a-SMA1,
and UCP11 progenitor populations increased
0 0
control mifepristone control mifepristone (Figure 5E). Retained progenitors may
contribute to the “catch-up” septal formation
neg neg
100
GFP
100
GFP that is observed when Dex is withdrawn
GFP low GFP low before P5 (17, 35). Ntokou and coworkers
UCP1+ and αSMA+
UCP1+ and ADRP+

GFP high
% of population

GFP high 80 (12) reported that only fibroblasts that had

% of population

80
60 60 been lineage marked on or before P2 were
observed in both lipid-laden and soma-
40 * * 40
expressing populations at P7. This suggests
20 20 that by preserving PDGFRa-expressing
* progenitors, transient (P1–P4) glucocorticoid
0 0
control dexamethasone control dexamethasone administration may enable alveolarization to
recover.
E
Significance of UCP1 in Lung
αSMA+, ADRP+, and UCP1+

GFP neg
80 Fibroblast Progenitors
GFP low
GFP high UCP1-expressing adipocytes arise from
% of population

60 at least two progenitor populations.

Classical brown adipocytes are defined
40
embryonically and arise along myotomes
20 from Pax71, Myf51 precursors, which
* may also differentiate into myocytes (38).
0 Beige (sometimes termed brite) adipocytes
control dexamethasone arise within white adipose tissue from
progenitors with characteristics of smooth
Figure 5. GFP high
fibroblasts that acquire more lipid droplets exhibit characteristics of brown
adipocytes. (A) Fibroblasts were isolated from PDGFRa-GFP mice (control, not treated) and
muscle cells (i.e., they are PDGFRa1 and
subjected to flow-cytometric sorting, gating on CD452 cells, and their GFP fluorescence intensity. a-SMA1, and some are Myh111). Whereas
Using quantitative RT-PCR, uncoupling protein-1 (UCP1, a marker of brown adipocytes) and PR UCP1 is constitutively expressed by brown
domain containing 16 (Prdm16), mRNA from the GFPlow and GFPhigh populations was normalized to adipocytes, expression by beige adipocytes
mRNA from the CD452, GFP2 population from the same fibroblast isolation. Mean 6 SEM, n = 5 requires induction from cold exposure or
mice, all from separate litters. *P , 0.05 comparing GFPlow and GFPhigh. (B) Fibroblasts were isolated b3-adrenergic stimulation (25). Adrenergic
from mice treated with Dex during P1–P7 and subjected to flow cytometry after staining for UCP1, induction of UCP1 in beige adipocytes
ADRP, and a-SMA. Mean 6 SEM, n = 6 mice for each treatment group from 4 separate litters. *P , 0.05 follows expansion of the adipose stromal
for GFPhigh, comparing Dex-treated mice with untreated controls. (C) Dex and Mfp altered the vascular cell population and is dependent
proportions of fibroblasts that stained positively for ADRP as well as UCP1. Mean 6 SEM, n = 5 mice
on vascular endothelial growth factor
for each treatment group from 4 separate litters. *P , 0.05 for GFPlow or GFPhigh, comparing Dex- or
Mfp-treated mice with controls. (D) Dex and Mfp altered the proportions of fibroblasts that stained
(VEGF)-A and vascular endothelial growth
positively for both a-SMA and UCP1. Mean 6 SEM, n = 5. Control and Mfp- or Dex-treated mice are factor receptor-2 (VEGF receptor-2 or
the same as those shown in C. (E) Proportions of cells that stained positively for a-SMA, ADRP, VEGFR2) (39). Relevant to our findings is
and UCP1 within the GFP2, GFPlow, and GFPhigh populations in fibroblasts isolated from control and the novel observation of Seki and associates
Dex-treated mice. Mean 6 SEM, n = 5 using the same mice shown in D. (40) that the adipose stromal vascular

McGowan and McCoy: Glucocorticoids Retain Alveolar Fibroblast Progenitors 117

 ORIGINAL RESEARCH

A B now observed that Dex increases Dlk1C,

Fβ consistent with their progenitor state.

Fβ
ex 8

ex
Therefore, like beige adipocyte precursors,
TG

TG
control

SMAD (fold control)

IC

IC
C

D
*
6
TGFβ1
PDGFRa-expressing lung fibroblast

pSMAD density
pSmad2 pSmad 1/5 Dex
progenitors retain characteristics of both
4
Smad2 Smad1/5 smooth-muscle and lipid-storage cells (41).
2 Precisely what drives PDGFRa-expressing
βTubulin βTubulin fibroblasts to express UCP1 remains
0
pSMAD2 pSMAD1/5 unclear, because they are in a warm
C D environment and b3-adrenergic receptors
have not been observed in lung fibroblasts

pSMAD 1/5+ and ADRP+

100
2.0 GFP neg (42). UCP1 more likely marks progenitor
*

% of population
80 GFP low
Fβ

cells, which have not fully committed to

UCP1 density
(fold control)
ex

1.5
TG

GFP high
IC

60
*
either an MF or a lipid-storage phenotype.
UCP1 1.0
40
0.5 20 Contributions of Fibroblast Plasticity
HSP60
0.0 0 to Alveolar Repair and Regeneration
Control TGFβ Dex control dexamethasone In mice and rats, neutral lipid droplets
Figure 6. Dex increases UCP1 in cultured MLg 2908 cells and pSMAD1/5 both in culture and in vivo. (A) appear during the late canalicular stage and
MLg 2908 neonatal mouse lung fibroblasts were induced to assume a lipid-storage phenotype and then remain abundant through the first two
exposed to medium alone (control) or supplemented with either transforming growth factor b 1 (TGF-b1) or postnatal weeks (14, 37, 43). Although their
Dex and then harvested for Western immunoblotting. The blots were probed for either pSmad2 and presence in the lungs of human newborns
Smad2 or pSmad1/5 and Smad1/5, followed by b-tubulin. C, uninduced control; IC, induced control. (B) The remains controversial, lipid-laden fibroblasts
density of pSmads in immunoblots is expressed relative to the abundance of the corresponding Smad contribute to alveolar repair and regeneration
for each sample and normalized to the ratio for the control. Mean 6 SEM, n = 4 separate experiments, *P ,
in models of human disease (44).
0.05 comparing Smad1/5 for Dex-treated MLg with controls. (C) MLg cells were induced and stimulated
as in A before mitochondrial isolation and Western immunoblotting. The density of UCP1 relative to the
Other investigators have studied murine
respective induced control (set to a density of one) is shown for three separate experiments. Mean 6 SEM, fibroblast subpopulations during
*P , 0.05 comparing Dex-treated mice with controls. (D) On P8, fibroblasts were isolated from a compensatory right lung growth (CLG)
separate cohort of controls or mice that had been treated with Dex as shown in Fig. 1A. (A) After staining for after a left pneumonectomy. It was found
pSmad1/5 and ADRP, flow cytometry was performed, gating on CD452 cells. Mean 6 SEM, n = 5 mice that in adult mice, a larger proportion of
for each treatment group from four different litters. *P , 0.05 comparing the GFPhigh population from Dex- PDGFRa-GFPlow fibroblasts contained
treated mice with controls. HSP60, heat shock protein 60; pSMAD, phosphorylated form of a homolog of the a-SMA, and this population increased after
Drosophila family of proteins including SMA (small body size) and mothers against decapentaplegia (MAD). a pneumonectomy (11). Administration
of rosiglitazone, a peroxisome proliferator-
endothelium produces PDGF-CC (a (10), and have now shown that Dex activated recptor-g (PPARg) agonist that
PDGFRa ligand), which stimulates UCP1 expands the Sca11, GFPhigh subpopulation promotes lipid accumulation in LIFs,
gene expression in PDGFRa1, CD341, and and increases the proportion of these cells increased the abundance of GFPhigh,
Sca11 perivascular mesenchymal cells. We that express UCP1. Likewise, we previously a-SMAlow fibroblasts. In a follow-up study,
previously found that some PDGFRa1 lung showed that targeted pdgfra deletion the same group explored differences
fibroblasts express CD34 and Sca1 at P8 increased Dlk1C mRNA (10), and have between the GFP low and GFP high
populations in sham-operated and
pneumonectomized mice (15). In the sham-
A B operated mice, the lipid-laden cells also
SREBP1 Dlk1C
2.0 2.0 expressed CD34. Pneumonectomy reduced
2-(ΔΔCT) (fold normalizer)

2-(ΔΔCT) (fold normalizer)

low
Control GFP
the proportion of lipid-laden cells and
GFP high *
1.5 Dex 1.5 increased the proportion of a-SMA1
cells within the CD341 population.
1.0 * 1.0 Adrenalectomy influenced CLG much
like targeted GR deletion did during
0.5 0.5 the saccular stage of development.
Adrenalectomized rats exhibited a
0.0 0.0
GFP neg GFPlow GFPhigh Control Dex thickened interstitium with more
fibroblasts during CLG compared with
Figure 7. Dex increases the retention of fibroblasts as progenitors. Fibroblasts were isolated from control
pneumonectomized controls with
and Dex-treated PDGFRa-GFP mice and subjected to flow-cytometric sorting, gating on CD452 cells and
their GFP fluorescence intensity. Using quantitative RT-PCR, (A) sterol receptor binding protein-1 (SREBP1)
intact adrenal glands (45). Therefore,
and (B) delta-like ligand-1, splice product-C (Dlk1C) mRNA was analyzed and the cycle threshold (CT) pneumonectomy modifies the CD341
values were normalized to the CT value for a single normalizing RNA sample, which was used in all fibroblast population by reducing the
analyses. n = 5 separate cell isolations from control and Dex-treated mice, which were obtained from proportion that contains lipid droplets,
different litters. Mean 6 SEM, *P , 0.05 comparing Dex-treated GFPhigh mice with controls. increasing the proportion that contains

118 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017
ORIGINAL RESEARCH

a-SMA, and increasing the expression of fibroblast phenotype. In murine neonates PDGFRa function in alveolar fibroblasts (47).
the extracellular matrix proteins tenascin C and adults, GFP intensity correlates Additional experimentation is required to
and periostin, consistent with a more with quantitative rather than qualitative understand the impact of these various
synthetic phenotype. Recently, El Agha and differences in lipid storage or a-SMA, as pathways on alveolar fibroblasts.
associates (46) lineage traced the lipid- both the GFPlow and GFPhigh populations
storage and myofibroblastic phenotypes in contain cells exhibiting both lipid droplets Potential Clinical Application
adult mice. They demonstrated that after and a-SMA (15). Gene expression profiling Lineage-tracing studies have demonstrated
bleomycin administration, fibroblasts that in adult mice characterized which genes are the importance of Smad2 signaling for
were lineage labeled for a-SMA acquired more highly expressed in GFPlow (vinculin the differentiation of Shh-responsive,
lipid droplets, suggesting that adult lung and integrin a8, consistent with a PDGFRa1, mesenchymal progenitors
fibroblasts retain plasticity and may have contractile phenotype) and GFPhigh (the destined to be MFs (3). A second
salutary effects after lung injury. extracellular matrix proteins periostin, population of PDGFRa1 cells, from
collagen 3a1, fibrillin 1, consistent with a the same mesenchymal lineage, can
PDGFRa Marks Progenitors, but synthetic phenotype) populations (15). differentiate postnatally into a lipid-storage
Does Not Exclusively Control their However, PDGFRa signaling is also regulated phenotype in the absence of Smad2
Differentiation post-transcriptionally. Studies using signaling (4). Our studies suggest that a
Our study also clarifies how PDGFRa oligodendrocyte precursor cells (which are portion of this second population maintains
signaling contributes to the balance similar to cancer stem cells) from the bipotency at least through P8, combining
between progenitors and differentiated periphery of glioblastoma multiforme characteristics of both MFs (a-SMA) and
alveolar septal MFs. Although alveolar tumors showed that PDGFRa signal LIFs (ADRP, UCP1). These less committed
fibroblast subpopulations can be transduction preserves “stemness” manifested cells more likely retain the progenitor
distinguished by their apparent level of as self-renewal, retention of markers of markers Dlk1C and Sca1, and contain
PDGFRa gene transcription (with GFP as a multipotency, and invasiveness (47). both a-SMA and lipid droplets, if Dex
reporter of endogenous PDGFRa gene Other examples of post-transcriptional is administered during this early
expression), it remains unclear how well regulation include (1) the abundance of postnatal time window. Administering
GFP intensity correlates with PDGFRa PDGFRa on the cell surface, which is glucocorticoids to newborns with
signaling activity. During secondary regulated by endosomal recycling and bronchopulmonary dysplasia or adults with
septation, the GFP intensity correlates with exosomal shedding (48, 49); (b) the location pulmonary fibrosis has not improved their
the abundance of CD140a (PDGFRa) on on the cell surface (i.e., whether PDGFRa clinical outcome. However, our studies
the cell surface and the abundance of localizes to membrane lipid rafts) (48); and suggest that Dex sustains plasticity, and
PDGFRa mRNA (10), and the PDGFR- (3) the proximity to phosphatase inhibitors, that targeting additional downstream
kinase inhibitor imatinib primarily which are also regulated by recycling pathways could steer fibroblasts away from
suppresses the proliferation of GFPhigh and membrane location (50). These fibrogenesis toward a more salutary
fibroblasts (10). However, factors in factors influence how PDGFRa impacts phenotype. Further investigation is
addition to gene expression may regulate oligodendrocyte precursor cell stemness and required to identify and modify these
PDGFRa signaling and impact the differentiation, and could also influence incompletely defined pathways. n

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120 American Journal of Respiratory Cell and Molecular Biology Volume 57 Number 1 | July 2017