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Disruption of transforming growth factor signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferatoractivated receptor ╨Ю╤Ц.

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Vol. 50, No. 4, April 2004, pp 1305–1318
DOI 10.1002/art.20104
© 2004, American College of Rheumatology
Disruption of Transforming Growth Factor ␤ Signaling and
Profibrotic Responses in Normal Skin Fibroblasts by
Peroxisome Proliferator–Activated Receptor ␥
Asish K. Ghosh, Swati Bhattacharyya, Gabriella Lakos, Shu-Jen Chen,
Yasuji Mori, and John Varga
Results. The PPAR␥ receptor was expressed and
fully functional in quiescent normal skin fibroblasts.
Whereas ligand activation of cellular PPAR␥ resulted
in modest suppression of basal collagen gene expression, it abrogated TGF␤-induced stimulation in a
concentration-dependent manner. This response was
mimicked by overexpressing PPAR␥ in fibroblasts, and
was blocked by a selective antagonist of PPAR␥ signaling or by transfection of fibroblasts with dominantnegative PPAR␥ constructs. Furthermore, PPAR␥ ligands abrogated TGF␤-induced expression of ␣-SMA, a
marker of myofibroblasts. Stimulation of Smaddependent transcriptional responses by TGF␤ was suppressed by PPAR␥ despite the absence of a consensus
PPAR␥-response element in the targeted promoters.
Ligand-induced activation of fibroblast PPAR␥ had no
effect on protein expression of cellular Smad3 or
Conclusion. By abrogating of TGF␤-induced
stimulation of collagen gene expression, myofibroblast
transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR␥ may play a physiologic role in the regulation of the profibrotic response.
Furthermore, our results suggest that PPAR␥ activation
by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.
Objective. In fibroblasts, transforming growth
factor ␤ (TGF␤) stimulates collagen synthesis and
myofibroblast transdifferentiation through the Smad
intracellular signal transduction pathway. TGF␤mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting
the intracellular TGF␤/Smad signaling may provide a
novel approach to controlling fibrosis. Because of its
potential role in modulating inflammatory and fibrotic
responses, we examined the expression of the nuclear
hormone receptor peroxisome proliferator–activated receptor ␥ (PPAR␥) in normal skin fibroblasts and its
effect on TGF␤-induced cellular responses.
Methods. The expression and activity of PPAR␥
in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry,
flow cytometry, and transient transfections with reporter constructs. The same approaches were used to
evaluate the effects of PPAR␥ activation by naturally
occurring and synthetic ligands on collagen synthesis
and ␣-smooth muscle actin (␣-SMA) expression. Modulation of Smad-mediated transcriptional responses
was examined by transient transfection assays using
wild-type and dominant-negative PPAR␥ expression
Supported by grants from the NIH (AR-46390 and AR42309) and the Scleroderma Foundation.
Asish K. Ghosh, PhD, Swati Bhattacharyya, PhD, Gabriella
Lakos, MD, PhD, Shu-Jen Chen, PhD, Yasuji Mori, MD, PhD, John
Varga, MD: University of Illinois at Chicago, College of Medicine,
Chicago, Illinois.
Address correspondence and reprint requests to John Varga,
MD, Section of Rheumatology, M/C 733, Department of Medicine,
University of Illinois at Chicago, 1158 Molecular Biology Research
Building, 900 South Ashland Avenue, Chicago, IL 60607. E-mail:
Submitted for publication May 2, 2003; accepted in revised
form December 3, 2003.
Abnormal synthesis and tissue accumulation of
collagen are hallmarks of scleroderma and are responsible for the damage and failure of affected organs.
Lesional scleroderma fibroblasts display an activated
phenotype characterized by accelerated transcription of
genes coding for collagen and other extracellular matrix
proteins, increased expression of cell surface receptors
for transforming growth factor ␤ (TGF␤), and sustained
production of TGF␤, connective tissue growth factor,
interleukin-1␣, and other profibrotic cytokines and
growth factors (for review, see ref. 1). Furthermore,
lesional fibroblasts show increased expression of the
myofibroblast marker ␣-smooth muscle actin (␣-SMA)
and resistance to apoptosis (2). Although the nature of
the stimulus that triggers fibroblast activation in scleroderma remains unknown, the close topographic association between activated tissue fibroblasts and infiltrating
inflammatory cells suggests that mononuclear cell–
derived signals may be responsible. Because TGF␤ is
consistently detected in lesional tissue and is known to
be a potent inducer of extracellular matrix synthesis,
growth factor production, and fibroblast proliferation,
chemotaxis, and terminal differentiation, it is widely
regarded as the pivotal mediator in pathologic fibrosis. It
remains unclear, however, whether an excess of TGF␤
or an exaggerated intensity or duration of the target cell
response to TGF␤ is primarily responsible for the activated scleroderma fibroblast phenotype.
Recent studies have elucidated the molecular
mechanisms underlying the fibrotic responses elicited by
TGF␤ (3). Receptor-activated Smads (R-Smads) are
directly activated by TGF␤/activin (Smad2 and Smad3)
or by bone morphogenetic proteins (Smads 1, 5, and 8).
Upon ligand binding to the surface TGF␤ receptors,
R-Smads are phosphorylated and then heterodimerize
with Smad4 and translocate from the cytoplasm into
nucleus. Once inside the nucleus, the Smad complex
recognizes specific DNA sequences in TGF␤-regulated
target genes, stimulating or repressing their transcription (3,4). In contrast to R-Smads, Smad7 is an inhibitory member of the Smad family that abrogates TGF␤/
Smad signaling.
We have previously demonstrated that in normal
dermal fibroblasts, TGF␤-induced stimulation of type I
collagen gene (COL1A2) transcription requires cellular
Smad3 and is abrogated by Smad7 (5,6). We also
demonstrated that interaction of the DNA-bound Smad
complex with transcription coactivators and histone
acetyltransferases p300/CREB binding protein is required for maximal TGF␤/Smad3-induced type I collagen synthesis in normal dermal fibroblasts (7,8). In
scleroderma lesional fibroblasts, R-Smads display increased phosphorylation and nuclear accumulation in
the absence of exogenous TGF␤, indicating intrinsic
activation of the TGF␤/Smad signal transduction pathway (9). Substantial evidence indicates that altered
regulation of Smad signaling is also implicated in the
pathogenesis of lung, liver, and kidney fibrosis in humans and in experimentally induced fibrosis in animal
models (for review, see ref. 10).
Peroxisome proliferator–activated receptors
(PPARs) represent a family of nuclear hormone receptors that are expressed at high levels in adipose tissues
and were originally identified as key regulators of adipocyte differentiation and insulin sensitivity (11). Three
PPAR isoforms (␣, ␤, ␥) have been identified and have
been shown to be encoded by separate genes (for review,
see ref. 12). The PPARs function as ligand-dependent
transcription factors that regulate the expression of
target genes. Fatty acids, eicosanoids, and prostaglandins, such as 15-deoxy-⌬12,14-prostaglandin J2 (15dPGJ2), have been proposed to function as naturally
occurring ligands for PPARs (12,13). The thiazolidinedione class of drugs used in diabetes and dyslipidemias
have also been shown to activate PPAR␥ and, thus, are
synthetic ligands.
Like other nuclear hormone receptors, PPARs
are modular in structure, with N-terminal transcriptional
activation domain, C-terminal ligand-binding domain,
and the activation function 2 domain required for interaction with coactivators/corepressors (12,14). The middle region contains the DNA binding domain, consisting
of a zinc finger that specifically recognizes conserved
DNA sequences called PPAR-response elements
(PPREs) that are present in the promoters of PPARregulated genes linked to glucose homeostasis, apoptosis, and proliferation (15).
Recent discoveries suggest that signaling through
PPAR␥ influences a wide range of cellular responses
that are entirely unrelated to adipogenesis and insulin
homeostasis. Macrophages, microglia, chondrocytes, T
cells, and synovial fibroblast-like cells all express
PPAR␥. In these cells, activation of PPAR␥ is associated
with potent antiinflammatory and immunomodulatory
effects; these effects are due to suppression of the genes
for tumor necrosis factor ␣, inducible nitric oxide synthase, cyclooxygenase 2, and interleukin-6 (16–20). The
immunoregulatory activities of PPAR␥ involve mechanisms distinct from those that mediate insulin sensitization. Importantly, activation of PPAR␥ by naturally
occurring ligands also appears to have antiinflammatory
effects. In the joint, for example, monosodium urate
monohydrate crystals have been shown to activate
PPAR␥ on monocytes, presumably via endogenous 15dPGJ2, and crystal-induced PPAR␥ activation was implicated as a potential mechanism to explain the spontaneous resolution of acute inflammation associated with
gouty arthritis (21). In light of its potent antiinflammatory effects, PPAR␥ ligands hold substantial promise as
novel immunomodulatory and antiinflammatory agents.
These immunoregulatory effects appear to be cell-
specific, however, since in monocytes, PPAR␥ activation
resulted in induction, rather than suppression, of cyclooxygenase 2 (22).
The potential involvement of PPAR␥ in physiologic tissue remodeling, wound healing, and organ fibrosis has thus far received only scant attention. Investigation of PPAR␥ in these processes has focused primarily
on the pancreas, liver, and kidney, the target organs in
diabetes. It has been shown that through activation of
cellular PPAR␥, the thiazolidinedione antidiabetic drug
troglitazone inhibited collagen synthesis in mesangial
cells from diabetic rats (23) and in mesangial cells
activated in vitro by glucose or TGF␤ (24). Furthermore,
naturally occurring or synthetic ligands of PPAR␥ have
been shown to inhibit proliferation (25), myofibroblast
transdifferentiation (26), and collagen synthesis (27,28)
in hepatic and pancreatic stellate cells. Long-term troglitazone administration prevented the development of
glomerulosclerosis (29) and pancreatic fibrosis (30) in
rodent models of diabetes. Together, these findings
indicate that activation of PPAR␥ by naturally occurring
ligands or synthetic agonists causes repression of profibrotic responses in vitro and is associated with reduction
or prevention of organ fibrosis.
Virtually nothing is known about the expression,
function, or mechanism of action of PPAR␥ in skin
fibroblasts or the role of PPAR␥ in modulating fibrotic
responses in the skin. We report here that PPAR␥ is
constitutively expressed in normal skin fibroblasts, is
up-regulated by TGF␤, and can be activated by naturally
occurring ligands or by the thiazolidinedione class of
antidiabetic drugs. Transient overexpression of PPAR␥
in fibroblasts dramatically enhanced their sensitivity to
PPAR␥ ligands. Troglitazone and 15d-PGJ2 prevented
TGF␤-induced stimulation of type I collagen synthesis
at the level of transcription and abrogated ␣-SMA
expression. The inhibitory effects of the PPAR␥ ligands
on these profibrotic responses were specific and
PPAR␥-dependent. Furthermore, PPAR␥ activation in
fibroblasts had no effect on the level of cellular Smad3
or Smad7 expression. These results indicate that PPAR␥
inhibits TGF␤-induced profibrotic responses in normal
fibroblasts. Together, the findings suggest a novel potential role for PPAR␥ in the control of skin fibrosis.
Fibroblast culture. Primary cultures of human dermal
fibroblasts were established from newborn foreskins obtained
from the delivery suite, as described previously (31). Fibroblasts were maintained at 37°C in an atmosphere of 5% CO2 in
Eagle’s minimum essential medium (EMEM) containing 5 mM
glucose supplemented with 10% fetal bovine serum (FBS), 1%
vitamins, 1% penicillin/streptomycin, and 2 mM L-glutamine
(all from BioWhittaker, Walkersville, MD). For all experiments, fibroblasts were studied between passages 3 and 7. Cell
viability was determined by trypan blue dye exclusion.
Plasmids. The 772COL1A2/CAT plasmid contains sequences from ⫺772 to ⫹58 of the human COL1A2 gene linked
to the chloramphenicol acetyltransferase (CAT) reporter gene
(32). The p(AOx)3-TK-Luc plasmid (obtained from Dr. Christopher Glass, University of California, San Diego) contains 3
copies of the PPRE from the acyl-coenzyme A (acyl-CoA)
oxidase gene linked to thymidine kinase and luciferase genes.
The expression vector pCMX-mPPAR␥ contains the fulllength PPAR␥ complementary DNA under the cytomegalovirus (CMV) promoter in the pCMX vector. Dominant-negative
PPAR␥ mutant L466A was generated by polymerase chain
reaction–based site-directed mutagenesis (obtained from Dr.
J. Larry Jameson, Northwestern University Medical School,
Chicago, IL) (33). Dominant-negative PPAR␥ (L468A/
E471A) was constructed by mutation of 2 highly conserved
residues in the ligand-binding domain (obtained from Dr.
Krishna K. Chatterjee, University of Cambridge, Cambridge,
UK) (34). SBE4-TK-Luc contains 4 copies of the consensus
Smad-binding element linked to thymidine kinase and luciferase genes (35). The p3637-TK-Luc construct contains 6
copies of the COL1A2 Smad-binding element (5⬘ATGCAGACA-3⬘) sequences linked to thymidine kinase promoter in pGL3-basic vector (36). The pRL-TK renilla luciferase (pRL-TK-Luc) construct served as an internal control.
Transient transfection. Fibroblasts were seeded in
6-well clusters at 105 cells/well. At confluence, fibroblasts were
transiently transfected with reporter constructs along with
PPAR␥ expression vectors or appropriate empty vector using
Superfect reagent (Qiagen, Valencia, CA). Transfected fibroblasts were pretreated with troglitazone or 15d-PGJ2 (both
from Biomol, Plymouth Meeting, PA) or vehicle (0.1%
DMSO) for 1 hour, followed by TGF␤2 (Genzyme, Framingham, MA). To test the PPAR␥ dependence of ligand-mediated
effects on fibroblasts, the PPAR␥ antagonist GW9662 (Cayman Chemical, Ann Arbor, MI) was added to cultures 30
minutes prior to the PPAR␥ ligand. Following a further
48-hour incubation, fibroblasts were harvested and CAT or
luciferase activities were determined in triplicate samples
containing equal amounts of proteins. For determination of
CAT activities, a phase extraction procedure was used. Luciferase activities were determined by scintillation counting.
Transfection efficiency was monitored by measuring renilla
luciferase activity in each sample. Each experiment was repeated 2–3 times, and the results were consistent.
Western blot analysis. At the end of the incubation
period, confluent fibroblasts were harvested and whole cell
lysates or cell fractions were prepared, as described previously
(5). The amount of protein in the cell lysates or nuclear
fractions was determined using the Bio-Rad protein assay kit
(Bio-Rad, Hercules, CA). Samples containing equal amounts
of proteins (10–15 ␮g) were subjected to electrophoresis in
4–20% Tris–glycine gradient gels. Proteins were then transferred to polyvinylidene difluoride membranes, blocked with
10% fat-free milk in TBST buffer (20 mM Tris HCl, 137 mM
NaCl, and 0.05% Tween 20). The membranes were incubated
with antibodies against PPAR␥ (1:200 dilution), Smads 1, 2,
and 3 (1:200 dilution), actin (1:200 dilution), or histone H3
(1:3,000 dilution) (all from Santa Cruz Biotechnology, Santa
Cruz, CA), Smad7 (1:500 dilution; Novus Biologicals, Littleton, CO), type I collagen (1:250 dilution; Southern Biotechnology, Birmingham, AL), or ␣-SMA (1:500 dilution; Sigma,
St. Louis, MO) in TBST buffer overnight. Membranes were
then washed 3 times and incubated with appropriate secondary
antibodies for 45 minutes.
Antigen–antibody complexes were visualized using the
enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. Membranes were exposed to autoradiography, and signals were scanned for quantitation. The results
were normalized against the intensity of the actin signal.
Northern blot analysis. At the end of the incubation
period, total RNA was isolated from confluent fibroblasts by a
1-step extraction procedure using TRIzol reagent (Life Technologies, Grand Island, NY), and analyzed by Northern blotting as described previously (37). Nitrocellulose filters were
sequentially hybridized with 32P-dCTP–labeled probes for human COL1A2, PPAR␥, or GAPDH. Membranes were exposed to autoradiography, and signals were scanned for messenger RNA (mRNA) quantitation. The results were
normalized against the intensity of the 18S ribosomal RNA or
GAPDH probe.
Immunocytochemistry. Fibroblasts were cultured on
8-well chamber slides in EMEM with 0.1% FBS in the
presence or absence of troglitazone or 15d-PGJ2 (10 ␮M). At
the end of the incubation period, cells were fixed in methanol,
washed in phosphate buffered saline (PBS), and incubated
with primary antibodies against PPAR␥ (Santa Cruz Biotechnology) for 30–60 minutes, as described previously (9). Slides
were then washed with PBS and treated with fluoresceinconjugated anti-mouse IgG (Santa Cruz Biotechnology) for 30
minutes. To stain the nuclei, chambers were mounted with
Vectashield (Vector, Burlingame, CA). Nonimmunized IgG
was used as a negative control.
Following stringent washing of the slides, the subcellular distribution of fluorescence was evaluated by immunofluorescence or confocal laser scanning microscopy using a Zeiss
LSM 510 microscope (Zeiss, Wetzlar, Germany). Each experiment was repeated at least 3 times, and the results were
consistent. Quantitative analysis was performed by scoring 100
individual fibroblasts from different microscopic fields as
showing predominantly nuclear or predominantly cytoplasmic
distribution of immunofluorescence. The observer was blinded
to the identity of each section.
Flow cytometry. Fibroblasts were incubated with 10
␮M troglitazone or 15d-PGJ2 in the presence or absence of
TGF␤. After 48 hours, fibroblasts were harvested by gentle
trypsinization, washed in ice-cold buffer, and fixed with 1%
paraformaldehyde, followed by permeabilization with 0.2%
saponin (Sigma) for 10 minutes. Direct immunofluorescence
staining was performed using fluorescein isothiocyanate–
conjugated monoclonal antibody to ␣-SMA (1 ␮g) or an
isotype control mouse IgG (BD Biosciences, San Diego, CA).
Aliquots of equal numbers of cells (104) were analyzed using a
FACSCalibur instrument (Becton Dickinson, Mountain View,
CA) equipped with CellQuest software. The percentage of
positive cells was measured from a cutoff set determined by
using isotype-matched control, and the mean channel fluorescence was measured over the entire distribution. Data are
expressed as the percentage of ␣-SMA–positive fibroblasts
from 3 separate experiments.
Statistical analysis. Values are expressed as the
mean ⫾ SD. Statistical differences between experimental and
control groups were determined by analysis of variance. P
values less than 0.05 by Student’s t-test were considered
Expression and activation of PPAR␥ in normal
skin fibroblasts. The nuclear hormone receptor PPAR␥
was originally detected in adipocytes. Subsequent studies demonstrated that PPAR␥ is also expressed in endothelial cells, monocytes, macrophages, chondrocytes,
and synovial fibroblast-like cells (for review, see ref. 38).
In order to examine PPAR␥ protein expression in skin
fibroblasts, low-passage confluent dermal fibroblasts
were incubated for 48 hours in EMEM, harvested, and
whole-cell lysates were analyzed by Western immunoblotting. In unstimulated fibroblasts, a single ⬃50-kd
band corresponding to PPAR␥ was detected (Figure
1A). Incubation with TGF␤ resulted in a timedependent increase in cellular PPAR␥ levels, with a
maximal 6-fold increase at 18 hours. Next, Northern
blot analysis was used to examine fibroblast PPAR␥
mRNA expression. The results revealed the presence of
a single 1.8-kb band corresponding to PPAR␥ mRNA
(Figure 1B).
Having demonstrated that normal dermal fibroblasts express PPAR␥ mRNA and protein, we used 2
complementary approaches to determine whether the
PPAR␥ signaling pathway was functional in these cells in
Figure 1. Expression of peroxisome proliferator–activated receptor ␥
(PPAR␥) in human skin fibroblasts. Confluent cultures of foreskin
fibroblasts were incubated in media with 10% fetal bovine serum in the
absence or presence of transforming growth factor ␤2 (TGF␤2; 12.5
ng/ml) for the indicated periods. A, Whole cell lysates were analyzed by
Western blotting with antibodies against PPAR␥ or actin. A representative autoradiogram is shown. B, Total RNA was extracted from
quiescent fibroblasts and analyzed by Northern blotting using radiolabeled PPAR␥ polymerase chain reaction products or GAPDH as
Figure 2. Peroxisome proliferator–activated receptor ␥ (PPAR␥) is functional in human skin fibroblasts.
A, Confluent fibroblasts were incubated with vehicle (DMSO) or the PPAR␥ ligands troglitazone (TGZ)
or 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2) (both at 10 ␮M) for 24 hours. Fibroblasts were then fixed
and processed for immunocytochemistry using specific antibodies. Left, Confocal microscopy images
representative of 3 separate experiments. Green indicates PPAR␥; blue indicates nucleus (original
magnification ⫻ 250). DAPI ⫽ 4⬘,6-diamidino-2-phenylindole. Right, Quantitation of the data from the
control and PPAR␥ ligand–treated fibroblasts. Values are the mean ⫾ SEM of 3 separate experiments.
B, Fibroblasts were incubated with DMSO or 15d-PGJ2 (10 ␮M) for 24 hours. Nuclear extracts were
prepared, and equal amounts of proteins were subjected to Western blot analysis for PPAR␥ or for histone
H3 to confirm that the proteins were nuclear. C, Fibroblasts transiently transfected with the PPRE-TKLuc construct (PPAR␥ response element [PPRE]; 200 ng/well) were incubated in media containing
15d-PGJ2 (10 ␮M) and/or GW9662 (1 ␮M). Following 48 hours of incubation, cells were harvested and
luciferase activity was measured. Values are the mean ⫾ SD of triplicate determinations from a single
experiment normalized against protein concentrations and are expressed in arbitrary units. Transfection
efficiency was monitored using renilla luciferase assay of each sample. Results were consistent in 3
separate experiments. ⴱ ⫽ P ⬍ 0.05 versus control.
vitro. First, confluent fibroblasts were incubated with the
naturally occurring PPAR␥ ligand 15d-PGJ2 or with the
synthetic PPAR␥ ligand troglitazone (both at 10 ␮M
concentration), stained with specific antibody against
PPAR␥, and examined by confocal immunofluorescence
microscopy. The results showed the presence of low
levels of PPAR␥ distributed throughout the cytosol in
unstimulated fibroblasts (Figure 2A). Treatment with
troglitazone or 15d-PGJ2 resulted in substantial nuclear
accumulation of cellular PPAR␥. Increased nuclear accumulation of PPAR␥ was detectable as early as 30
minutes (data not shown), and was maximal after 24
hours of exposure to the ligand. In the absence of
primary antibody, no staining was detected (data not
shown). There was no increase in cellular trypan blue
staining in ligand-treated fibroblasts compared with
fibroblasts treated with DMSO (data not shown).
To further document the stimulation of nuclear
accumulation of cellular PPAR␥, Western immunoblotting was performed. Exposure of fibroblasts to 15dPGJ2 for 24 hours resulted in substantial increase in
nuclear levels of PPAR␥ (Figure 2B). In contrast,
PPAR␥ ligands failed to induce a change in the
total levels of cellular PPAR␥ in these fibroblasts
(Figure 3A).
To further confirm that the PPAR␥ axis was
functional in dermal fibroblasts, cells were transiently
transfected with reporter vector p(AOX)3-TK-Luc con-
Figure 3. Abrogation of transforming growth factor ␤ (TGF␤)–stimulated type I collagen synthesis in skin
fibroblasts by peroxisome proliferator–activated receptor ␥ (PPAR␥) activation. Confluent fibroblasts were
pretreated with DMSO or troglitazone (10 ␮M) or with 15-deoxy-⌬12,14-prostaglandin J2 (15d-PGJ2; 10
␮M), followed by incubation in media with or without TGF␤ (12.5 ng/ml) for 48 hours. A, Whole cell lysates
were prepared, and collagen and PPAR␥ levels were analyzed by Western immunoblotting. B, Total RNA
was extracted and subjected to Northern blot analysis using a COL1A2 probe. 18S ribosomal RNA (18S
rRNA) was used as loading control. A representative autoradiogram is shown. Bottom, The autoradiogram
was scanned by laser densitometry to determine the intensity of bands. Results were normalized against 18S
rRNA in each lane. Open bars show untreated fibroblasts; closed bars show TGF␤-treated fibroblasts.
taining the minimal PPRE from the acyl-CoA oxidase
gene and incubated with 15d-PGJ2 for 48 hours. The
results showed that 15d-PGJ2 induced a ⬎2-fold increase in PPRE-driven luciferase activity (Figure 2C).
To confirm that the observed 15d-PGJ2 response in
fibroblasts was due to activation of cellular PPAR␥, a
selective inhibitor was used. GW9662 is an irreversible
PPAR␥ ligand that works as an effective and nontoxic
PPAR␥ antagonist (39). Transfected fibroblasts were
pretreated for 30 minutes with GW9662 (1 ␮M) and
then stimulated with 15d-PGJ2 for 48 hours. Luciferase
assays showed that whereas GW9662 by itself had no
effect on PPRE-driven promoter activity, it completely
abrogated ligand-induced stimulation, indicating that
the response was mediated through activation of endogenous PPAR␥ (Figure 2C). Together, these results
provide the first direct evidence of functional PPAR␥
expression in normal skin fibroblasts.
Abrogation of TGF␤-stimulated collagen synthesis by PPAR␥ ligands. The presence of a functional
PPAR␥ pathway in fibroblasts raised the possibility that
it may be involved in regulation of collagen gene expres-
sion in these cells. We therefore sought to determine the
effect of PPAR␥ activation on the rate of basal and
TGF␤-stimulated synthesis of collagen. For this purpose, confluent skin fibroblasts were incubated with
15d-PGJ2 or troglitazone (both at 10 ␮M concentration)
in the presence or absence of TGF␤2 (12.5 ng/ml). At
the end of the 48-hour incubation period, whole cell
lysates were prepared from fibroblasts and examined by
Western immunoblot analysis using anti–type I collagen
antibodies. The results revealed that while TGF␤ caused
a ⬎2-fold increase in the levels of cellular type I
collagen, pretreatment with 15d-PGJ2 or troglitazone
reduced this stimulation by 55% and 73%, respectively
(Figure 3A). No change in cellular PPAR␥ levels was
induced by either PPAR␥ ligand.
Decreased levels of intracellular collagen may
result from decreased synthesis, increased secretion, or
turnover of newly synthesized collagen. In order to
directly determine the effects of PPAR␥ ligands on
collagen gene expression, total RNA from ligand-treated
fibroblasts was subjected to Northern blot analysis.
While, as expected, TGF␤ induced a ⬎2-fold increase in
COL1A2 mRNA levels incubation of fibroblasts with
15d-PGJ2 or troglitazone caused a significant decrease
in collagen mRNA expression (Figure 3B). Importantly,
short preincubation with either ligand almost completely
abrogated the stimulation of collagen mRNA expression
induced by TGF␤. These results indicate that naturally
occurring or synthetic ligands of PPAR␥ inhibited
TGF␤-stimulated collagen synthesis in normal dermal
fibroblasts. Significantly, PPAR␥ activation had only a
relatively modest effect on basal levels of collagen in
unstimulated fibroblasts, suggesting that the major function of PPAR␥ is repression of TGF␤-induced responses.
Prevention of the stimulation of ␣-SMA expression in fibroblasts by PPAR␥. In fibrosis, a subgroup of
resident fibroblasts transdifferentiate into myofibroblasts that express high levels of ␣-SMA normally found
only in smooth muscle cells. Because these specialized
fibroblasts show accelerated synthesis of extracellular
matrix proteins, are resistant to apoptosis, and have
contractile properties, they have a significant functional
role in pathologic fibrosis. A major fibrogenic effect of
TGF␤ is stimulation of fibroblast–myofibroblast transdifferentiation (for review, see ref. 40).
In order to investigate the effect of PPAR␥
activation on the myofibroblast response, quiescent fibroblasts were incubated with TGF␤2 (12.5 ng/ml) in the
presence or absence of 10 ␮M troglitazone or 15d-PGJ2
for 48 hours. Western blot analysis showed that TGF␤
induced a significant increase in cellular levels of
␣-SMA, detected as a single 42-kd band (Figure 4A).
Preincubation of the fibroblasts with either PPAR␥
ligand had no effect by itself, but in both cases resulted
in substantial suppression of TGF␤-stimulated ␣-SMA
To further characterize the effect of PPAR␥ on
myofibroblast transdifferentiation and to determine the
proportion of ␣-SMA-positive fibroblasts, flow cytometry was used. Approximately 20% of unstimulated
fibroblasts were strongly positive for ␣-SMA. Stimulation with TGF␤ resulted in a ⬎2-fold increase in the
proportion of fibroblasts with high levels of ␣-SMA
expression (Figure 4B) and a similar increase in the
mean intensity of ␣-SMA staining (data not shown).
Consistent with the results of Western blot analysis,
pretreatment with troglitazone caused a 30% reduction
in the proportion of fibroblasts with high levels of
␣-SMA expression and decreased the mean intensity of
staining as well. Thus, these findings demonstrate that in
normal dermal fibroblasts, activation of PPAR␥ by
either naturally occurring or synthetic ligands inhibits
Figure 4. Prevention of TGF␤-stimulated expression of ␣-smooth
muscle actin (␣-SMA) by PPAR␥ ligands. Confluent fibroblasts were
incubated in media containing DMSO or troglitazone (10 ␮M) or
containing 15d-PGJ2 (10 ␮M) in the presence or absence of TGF␤
(12.5 ng/ml) for 48 hours. A, Whole cell lysates were analyzed by
Western blotting using specific antibodies against ␣-SMA or actin. B,
Fibroblasts were stained for ␣-SMA, and 104 cells were subjected to
flow cytometric analysis, and the proportion of cells with high-level
␣-SMA expression was determined. Isotype staining has been subtracted from each set of data. Results from a representative experiment are shown. Open bars show untreated fibroblasts; closed bars
show TGF␤-treated fibroblasts. See Figure 3 for other definitions.
the induction of critical profibrotic responses (stimulation of collagen synthesis and of ␣-SMA expression)
induced by TGF␤.
Prevention of COL1A2 stimulation by PPAR␥
activation. The expression of collagen mRNA is regulated predominantly at the level of transcription. To
determine whether PPAR␥-induced inhibition of collagen gene expression involved transcriptional repression,
fibroblasts were transiently transfected with a COL1A2
Figure 5. Prevention of TGF␤ stimulation of the COL1A2 promoter by PPAR␥ ligands. Confluent
fibroblasts were transiently transfected with the 772COL1A2/CAT construct (2.5 ␮g/well). A, Fibroblasts were
pretreated with the indicated concentrations of troglitazone or 15d-PGJ2 for 1 hour, followed by 12.5 ng/ml of
TGF␤2. After a further 48-hour incubation, fibroblasts were harvested and chloramphenicol acetyltransferase
(CAT) activities were determined. Values are the mean ⫾ SD of triplicate determinations from an experiment
representative of 3 separate experiments. Open bars show untreated fibroblasts; closed bars show TGF␤treated fibroblasts. B, Fibroblasts were pretreated with GW9662 for 30 minutes followed by 15d-PGJ2 (5 ␮M)
and TGF␤2 for 48 hours. ⴱ ⫽ P ⬍ 0.005 versus control. C, Fibroblasts were transfected with wild-type or
dominant-negative mutant PPAR␥ expression vectors (L468A/E471A or L466A) or empty vector, along with
772COL1A2/CAT reporter constructs. Cultures were incubated with DMSO or 15d-PGJ2 (10 ␮M) for 1 hour,
followed by 12.5 ng/ml of TGF␤2. After a further 48-hour incubation, fibroblasts were harvested and CAT
activities were determined. See Figure 3 for other definitions.
reporter construct. The 772COL1A2/CAT construct
harbors a Smad-response element between ⫺258 and
⫺263 bp of the human COL1A2 5⬘-flanking region, but
lacks a consensus PPRE sequence. Consistent with
previous results (5–8), exposure to TGF␤ caused a
⬎2-fold increase in COL1A2 promoter activity in transiently transfected fibroblasts (Figure 5A). Treatment
with either 15d-PGJ2 or troglitazone alone for 48 hours
had little or no effect on basal levels of COL1A2
promoter activity, but caused a dose-dependent suppression of stimulation induced by TGF␤.
We next sought to determine whether suppression of TGF␤-induced stimulation was mediated via the
PPAR␥ pathway by use of a selective PPAR␥ antagonist.
For this purpose, fibroblasts were pretreated with
GW9662, followed by treatment with 15d-PGJ2 and
stimulation with TGF␤. Transient transfection analysis
showed that suppression of TGF␤-induced COL1A2
activity by 15d-PGJ2 was abrogated by GW9662 in a
dose-dependent manner, whereas GW9662 by itself had
no effect on either basal promoter activity or its TGF␤induced stimulation (Figure 5B and data not shown).
To further confirm that ligand-induced PPAR␥
signaling in fibroblasts was directly responsible for the
suppressive effects of 15d-PGJ2, dominant-negative mutants of PPAR␥ were used. Both PPAR␥ mutant constructs L468A/E471A and L466A abrogated the inhibitory effects of 15d-PGJ2 on TGF␤-induced stimulation
of COL1A2 promoter activity in transiently transfected
fibroblasts (Figure 5C). Together, these findings suggest
that the inhibitory effect of PPAR␥ on collagen transcription in transfected fibroblasts was mediated through
cellular PPAR␥.
Abrogation of COL1A2 stimulation by overexpression of PPAR␥. To further characterize the role of
cellular PPAR␥ in the repression of TGF␤-induced
collagen stimulation, fibroblasts transiently cotransfected with PPAR␥ expression vector along with
772COL1A2/CAT or PPRE-TK-Luc were incubated in
the presence or absence of TGF␤ for 48 hours. Transfection of PPAR␥ resulted in increased cellular PPAR␥
protein levels (Figure 6B, top). The stimulation of
COL1A2 promoter activity induced by TGF␤ was completely abrogated in the presence of overexpressed
PPAR␥ (Figure 6A). As expected, the activity of the
PPRE reporter construct was increased in PPAR␥transfected fibroblasts, even in the absence of ligand,
and was dramatically further enhanced when the PPAR␥
pathway was activated by incubation of the fibroblasts
with 15d-PGJ2 (Figure 6B). Together, these results
suggest that overexpression of PPAR␥ results in complete suppression of collagen gene transcription stimulated by TGF␤, even in the absence of activating ligand.
Overexpression of the PPAR␥ receptor by itself results
in increased PPAR␥-dependent transcription and causes
dramatic sensitization of fibroblasts to the activating
ligands. These observations suggest that the relatively
low level of PPAR␥ expression in normal fibroblasts may
be limiting for negative regulation of TGF␤ responses.
Figure 6. Prevention of TGF␤ stimulation of the COL1A2 promoter
by PPAR␥. Fibroblasts were cotransfected with PPAR␥ expression
vector or empty vector along with A, 772COL1A2/CAT (COL1A2) or
B, PPRE-TK-Luc (PPAR␥ response element [PPRE]) reporter constructs. Following incubation in the presence (closed bars) or absence
(open bars) of A, TGF␤ or B, 15d-PGJ2 for 48 hours, fibroblasts were
harvested and chloramphenicol acetyltransferase (CAT) or luciferase
activities were determined. The blot at the top of B shows PPAR␥
protein expression in whole cell lysates prepared from empty vector–
transfected and PPAR␥ expression vector–transfected fibroblasts. See
Figure 3 for other definitions.
Disruption of intracellular TGF␤/Smad signal
transduction by activation of PPAR␥. We showed that
troglitazone or 15d-PGJ2 abrogated multiple TGF␤induced responses in normal fibroblasts and that this
inhibition involved activation of the endogenous PPAR␥
pathway. Transcriptional responses in fibroblasts elicited
by TGF␤ are known to be mediated in large part
through the Smad intracellular signal transduction pathway (10). In particular, we have recently demonstrated
that in dermal fibroblasts, Smad3 was both necessary
and sufficient to mediate TGF␤-induced stimulation of
collagen gene expression (5). Transactivation of
COL1A2 by Smad3 is mediated through a Smad-binding
regulatory element containing the canonical CAGACA
sequence (6). Stimulation of the myofibroblast marker
␣-SMA induced by TGF␤ was also shown to be dependent on Smad-mediated signal transduction (41,42).
To determine if the repression of TGF␤-induced
responses by PPAR␥ was due to disruption of intracellular Smad signaling, we used a minimal reporter construct containing 4 copies of the consensus binding sites
for Smad3/Smad4. Fibroblasts transiently transfected
with SBE4-TK-Luc plasmid were incubated with 15dPGJ2 (10 ␮M) or vehicle in the presence and absence of
TGF␤2 for 48 hours, followed by determination of
reporter activity. Only relatively low levels of luciferase
Figure 7. Prevention of Smad2/3-driven transcriptional responses by
PPAR␥ activation. A, Fibroblasts were transiently transected with
SBE4-TK-Luc (Smad-binding element [SBE]) reporter construct along
with pRL-TK-Luc. Fibroblasts were pretreated with 15d-PGJ2 (10 ␮M)
for 1 hour, followed by 12.5 ng/ml of TGF␤2. After a further 48-hour
incubation, fibroblasts were harvested and luciferase activities were
determined. Values are the mean ⫾ SD of triplicate determinations.
Open bars show untreated fibroblasts; closed bars show TGF␤-treated
fibroblasts. ⴱ ⫽ P ⬍ 0.05 versus control. B, Fibroblasts cotransfected
with SBE4-TK-Luc reporter plasmid along with PPAR␥ expression
vector or empty vector and pRL-TK-Luc reporter constructs were
incubated with or without TGF␤2 for 48 hours. Cells were harvested,
and cell lysates were subjected to luciferase assays. ⴱ ⫽ P ⬍ 0.01 versus
control. C, Skin fibroblasts were incubated with 15d-PGJ2 (10 ␮M) and
or GW9662 in the presence and absence of TGF␤2 for 48 hours.
Whole cell lysates were then prepared and analyzed by Western
blotting using antibodies against Smad3, Smad7, or actin. See Figure 3
for other definitions.
activity were detected in unstimulated fibroblasts,
whereas TGF␤ caused marked up-regulation (Figure
7A). However, the TGF␤ response was almost completely abrogated when fibroblasts were pretreated with
15d-PGJ2. Essentially identical results were obtained
when p3637-TK, which contains 6 copies of the COL1A2
promoter SBE sequence, was used as the reporter
construct in transient transfections (data not shown).
Together, these results strongly suggest that ligand activation of the PPAR␥ pathway disrupted the Smaddependent intracellular TGF␤ signaling.
To confirm the direct effect of PPAR␥ on Smaddependent TGF␤-signaling, fibroblasts cotransfected
with PPAR␥ expression vectors along with the SBE4TK-Luc reporter construct were incubated for 48 hours
in the presence or absence of TGF␤. The results showed
that overexpression of PPAR␥ resulted in abrogation of
TGF␤ stimulation of the minimal Smad-responsive promoter (Figure 7B), indicating that PPAR␥ signaling can
repress Smad-dependent transcriptional responses in
the absence of ligand. Because there are no consensus
PPRE sequences in either 772COL1A2 or SBE4-TK,
these results further suggest that PPAR␥-mediated suppression of TGF␤ signaling in fibroblasts is independent
of PPAR␥ interaction with its cognate DNA elements.
In normal fibroblasts, TGF␤ causes rapid phosphorylation and nuclear translocalization of cellular
Smad3 and increased levels of inhibitory Smad7 (37).
The repression of Smad-dependent transcriptional responses observed in PPAR␥-treated fibroblasts could be
the result of decreased steady-state levels of cellular
Smad3, interference with TGF␤-induced nuclear import
of Smad3, or alternately, diminished induction of Smad7
in response to TGF␤. In order to determine if PPAR␥
activation in the fibroblasts modulates the expression
level or activation state of cellular Smads, fibroblasts
incubated for 48 hours with TGF␤ in the presence or
absence of 15d-PGJ2 were analyzed by Western immunoblotting.
The results showed that in whole cell lysates, the
cellular levels of Smad3 and Smad7 proteins were comparable in vehicle-treated and 15d-PGJ2–treated fibroblasts (Figure 7C, compare the first and fifth lanes).
Furthermore, at this late time point, TGF␤ treatment
resulted in a marked down-regulation of Smad3 both in
the presence and in the absence of 15d-PGJ2 (compare
the second and sixth lanes). Therefore, inhibition of
Smad-mediated TGF␤ responses by PPAR␥ was not
associated with alterations in the expression levels of
Smad3 or Smad7 in these fibroblasts. These results also
indicate that PPAR␥ is not simply a nonspecific inhibitor
of all TGF␤ responses (such as repression of Smad3),
which suggests that PPAR␥ antagonistic effects on
TGF␤ signaling are likely to involve interference with
nuclear Smad activation or function.
In scleroderma, lesional fibroblasts are responsible for the development of tissue fibrosis. In contrast to
the signals and pathways implicated in the activation of
fibroblasts, the endogenous mechanisms that limit this
response have thus far received scant attention. Yet,
such mechanisms must clearly be important in order to
regulate fibroblast function and to prevent unopposed
activation. For example, during physiologic processes of
tissue remodeling, such as organogenesis or wound
healing, the synthesis and accumulation of collagen must
be terminated precisely at the appropriate stage. Failure
of the inhibitory mechanisms would result in an exaggerated magnitude, or prolonged duration, of profibrotic
responses, culminating in aberrant repair and pathologic
fibrosis. In the case of TGF␤, the most potent of the
profibrotic mediators, several mechanisms that repress
intracellular signaling have been identified. One such
mechanism is the induction of Smad7, the inhibitory
member of the Smad family that blocks Smad phosphorylation and functions as an endogenous negative feedback to terminate TGF␤ responses. In addition, various
ligands that repress TGF␤ responses also can induce
Smad7 expression (43–45). It is not surprising that
defective expression and/or function of endogenous inhibitors of fibroblast activation is linked to deregulated
tissue remodeling and pathologic fibrosis (46).
The findings of the present study indicate that
quiescent normal dermal fibroblasts express PPAR␥ at
the mRNA and protein levels. PPAR␥ was originally
identified in adipocytes and was more recently shown to
also be expressed in the liver, pancreas, kidney, and
vascular tissues, as well as in a variety of inflammatory
cells (11,27,28,47,48). In unstimulated fibroblasts, only
relatively low levels of PPAR␥ expression was found,
and the receptor was largely distributed in the cytoplasm. Interestingly, TGF␤ induced a significant timedependent increase in PPAR␥ protein levels in these
fibroblasts. In contrast, a recent study in vascular smooth
muscle cells found that TGF␤ caused early stimulation
and late inhibition of PPAR␥ expression (49), suggesting
that PPAR␥ regulation by TGF␤ may be cell type–
Naturally occurring ligands of PPAR␥, such as
15d-PGJ2, as well as the synthetic pharmacologic
PPAR␥ agonist troglitazone, caused activation of the
receptor, as indicated by its enhanced nuclear accumulation in the absence of significant change in cellular
PPAR ␥ levels and by stimulation of a PPAR ␥ responsive minimal promoter in transfected fibroblasts.
Remarkably, transient overexpression of PPAR␥ in the
fibroblasts dramatically enhanced their sensitivity to
stimulation by PPAR␥ agonists, suggesting that the level
of expression is limiting for cellular responsiveness to
endogenous PPAR␥ ligands. It is noteworthy that in
diabetic glomerulosclerosis and other forms of fibrosis,
PPAR␥ expression is diminished in affected tissues,
potentially rendering them resistant to the potentially
protective effects of endogenous PPAR␥ ligands (24,27).
This loss of responsiveness to an antifibrotic mechanism
may play a significant role in the pathogenesis of fibrosis
in these conditions. It will be of great interest to
determine whether scleroderma fibroblasts show altered
PPAR␥ expression or functional activity.
Agonists of the PPAR␥ receptor in normal fibroblasts caused suppression of collagen gene expression.
Both 15d-PGJ2, an endogenously produced prostanoid,
and synthetic thiazolidinedione drugs were effective
inhibitors of TGF␤-induced collagen synthesis and
COL1A2 promoter activity. Because 15d-PGJ2 is known
to have PPAR␥-independent cellular effects (13), it was
important to confirm that the repression of TGF␤induced responses was mediated through PPAR␥. By
using the potent and selective PPAR␥ antagonist
GW9662, which covalently modifies a cysteine residue in
the ligand-binding site of PPAR␥ (50), we established
that the effect of 15d-PGJ2 on repression of TGF␤induced collagen stimulation could be prevented in a
dose-dependent manner. In addition, dominant-negative
PPAR␥ expression vectors blocked the inhibitory effects
of 15d-PGJ2 on TGF␤-stimulated COL1A2 promoter
These results further demonstrate that in addition to collagen, the PPAR␥ ligand also disrupted the
induction of ␣-SMA expression. Because stimulation of
␣-SMA by TGF␤ is one of the key steps in the transdifferentiation of normal fibroblasts into myofibroblasts
(40), these results indicate that PPAR␥ can interfere
with multiple cellular events that are important in the
pathogenesis of fibrosis. The inhibitory effects of
PPAR␥ on TGF␤-induced fibroblast activation were
selective (see below), and were not attributed to cellular
The results of the transient transfection studies
indicated that inhibition of TGF␤-stimulated collagen
synthesis was mediated at least in part through a transcriptional mechanism. The regulation of COL1A2 transcription in normal fibroblasts has been investigated
extensively (for review, see ref. 51). Such studies indicate
that TGF␤-induced activation of the Smad signal transduction pathway results in rapid nuclear accumulation of
the Smad2/3/4 complex and its binding to a CAGACA
sequence in the COL1A2 promoter region (5,6). In
addition, interaction of the Smad complex with multiple
coactivators and cofactors is also required for optimal
transcriptional stimulation of COL1A2 by TGF␤
(7,8,51–56). Furthermore, activation of the p38 and
ERK cascades have also been implicated in the stimulation of collagen transcription elicited by TGF␤ in
fibroblasts (57,58).
PPAR␥ functions as a ligand-activated transcrip-
tion factor that modulates target gene transcription
through direct binding to its cognate PPRE element
(12). Sequence analysis of the COL1A2 promoter reveals the presence of a consensus SBE, but there are no
putative PPAR␥-binding elements. This suggests that
the inhibitory effect of PPAR␥ on COL1A2 transcription involves disruption of the cellular transcriptional
machinery that mediates the stimulation elicited by
TGF␤. Accordingly, we examined the effects of PPAR␥
on Smad-mediated signaling using reporter constructs
containing either a consensus SBE or tandem repeats of
the Smad-binding element of the COL1A2 promoter.
The results indicated that PPAR␥ was able to prevent
TGF␤ stimulation of these minimal promoter constructs
containing the binding sites for only Smad3 and Smad4.
These results suggested that PPAR␥ was able to directly
antagonize the activation and/or function of Smad3 in
fibroblasts (Figure 8). Furthermore, PPAR␥ did not
decrease the protein expression of stimulatory Smad3 or
increase the expression of inhibitory Smad7. The results
also demonstrated that the suppression of Smad3 induced by TGF␤ was unaffected by PPAR␥, indicating
that PPAR␥ did not block all TGF␤ responses nonselectively.
The transactivating function of R-Smads may be
repressed by PPAR␥ through cytoplasmic retention of
the activated Smad complex. Direct physical interaction
of R-Smads has been demonstrated with PPAR␥ in
vascular smooth muscle cells (48) and with the estrogen
receptor, another nuclear hormone receptor, in kidney
carcinoma cells (59). We are pursuing further studies in
order to precisely delineate the level of antagonistic
interaction between PPAR␥ and Smads that would
account for the repression of the profibrotic TGF␤
responses in fibroblasts.
In summary, the results of the present study
indicate that normal quiescent skin fibroblasts display
constitutive PPAR␥ expression and activation of
PPAR␥-dependent transcriptional responses elicited by
endogenous and synthetic PPAR ␥ ligands. Upregulation of PPAR␥ receptor levels by transient transfection of a PPAR␥ expression plasmid in fibroblasts
markedly enhanced sensitivity to activation by PPAR␥
agonists. While TGF␤ enhanced the expression of endogenous PPAR␥ in fibroblasts, both endogenous and
synthetic ligands of PPAR␥ caused selective abrogation
of TGF␤-induced profibrotic responses. The inhibitory
effects of PPAR␥ on TGF␤-dependent responses involved direct antagonism of Smad signal transduction
(Figure 8).
Together, these results suggest that PPAR␥ rep-
Figure 8. Regulation of transforming growth factor ␤ (TGF␤)–
induced responses in fibroblasts by peroxisome proliferator–activated
receptor ␥ (PPAR␥). Through activation of the intracellular Smad
signal transduction pathway, TGF␤ stimulates collagen synthesis,
␣-smooth muscle actin (␣-SMA) expression, and myofibroblast transdifferentiation of resident fibroblasts in the skin. These responses
contribute to tissue fibrosis. Naturally occurring endogenous ligands or
synthetic pharmacologic agonists activate cellular PPAR␥, resulting in
the disruption of TGF␤/Smad signal transduction and the blocking of
profibrotic responses. The expression of PPAR␥ is enhanced on
fibroblasts by TGF␤, thereby sensitizing them to the antifibrotic effects
of ligands. Activation of PPAR␥ may represent an effective antifibrotic
intervention strategy. 15d-PGJ2 ⫽ 15-deoxy-⌬12,14-prostaglandin J2;
TGZ ⫽ troglitazone.
resents an important physiologic mechanism in the
control of TGF␤ responses in normal fibroblasts. Diminished PPAR␥ expression in affected tissues may be
associated with aberrant repair. Accordingly, enhancing
fibroblast sensitivity to exogenous PPAR␥ may represent a novel strategy for the treatment of fibrosis.
Furthermore, in light of their potent antiinflammatory
properties, pharmacologic PPAR␥ agonists may be particularly effective in scleroderma and related fibrotic
conditions in which both inflammation and fibrosis play
prominent roles.
We are grateful to Drs. Christopher Glass (University
of California, San Diego) for the gift of the p(AOx)3-TK-Luc
and PPAR␥ expression vectors, Krishna K. Chatterjee (Uni-
versity of Cambridge, Cambridge, UK), Thomas P. Burris
(Lilly Corporate Center, Indianapolis, IN), and J. Larry Jameson (Northwestern University, Chicago, IL) for the wild-type
and mutant PPAR␥ expression vectors, Jean-Michel Gauthier
(Glaxo Wellcome, Les Ulis, France) for the p3637-TK-Luc
plasmid, and Leigh Zawel (Johns Hopkins University, Baltimore, MD) for the SBE4-TK-Luc plasmid.
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disruption, growth, skin, norman, transforming, factors, proliferatoractivated, response, profibrotic, peroxisomal, receptov, signaling, fibroblasts
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