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FASEB J. Sep 2009; 23(9): 2968–2977.
PMCID: PMC2735362

Peroxisome proliferator-activated receptor-γ abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator


Ligands of peroxisome proliferator-activated receptor-γ (PPAR-γ) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-β). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-γ in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-β activity by PPAR-γ ligands involves cellular PPAR-γ, since 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) failed to block TGF-β-induced responses in either primary cultures of PPAR-γ-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-γ. Next, we examined the molecular basis underlying the abrogation of TGF-β signaling by PPAR-γ in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-γ without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-β, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-γ. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-β-induced stimulation of COL1A2 in the presence of PPAR-γ ligands. Collectively, these results indicate that PPAR-γ blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-β-induced collagen gene expression. Pharmacological activation of PPAR-γ thus may represent a novel therapeutic approach to target p300-dependent TGF-β profibrotic responses such as stimulation of collagen gene expression.—Ghosh, A. K., Bhattacharyya, S., Wei, J., Kim, S., Barak, Y., Mori, Y., and Varga, J. Peroxisome proliferator-activated receptor-γ abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator.

Keywords: fibrosis, 15d-PGJ2, type I collagen, fibroblast, acetyltransferase

The synthesis of collagen is normally tightly regulated, and excessive accumulation of collagen results in fibrosis (1). Multiple intracellular signaling pathways, transcription factors, and cofactors have been implicated as playing important roles in the regulation of collagen gene expression (2, 3). The multifunctional cytokine transforming growth factor-β (TGF-β) stimulates collagen synthesis and plays a key role in the pathogenesis of scleroderma and related fibrotic disorders (4). Cellular responses induced by TGF-β are mediated intracellularly primarily via the canonical Smad pathway. Upon phosphorylation by the activated type I TGF-β receptor (TβRI), cytoplasmic Smad2 and Smad3 heterodimerize with Smad4 and accumulate within the nucleus, where they recruit cofactors to Smad-binding element (SBE) DNA sequences and activate target gene transcription (5). We showed previously that stimulation of COL1A2 transcription by TGF-β required Smad3 (6, 7), as well as the interaction of activated Smad2/3 with the transcriptional coactivator and histone acetyltransferase p300 (8,9,10). Overexpression of E1A, an inhibitor of p300 function, prevented the stimulation of collagen gene expression by TGF-β (8). In addition to its critical role in mediating profibrotic TGF-β responses, p300 also integrates converging signaling pathways that positively or negatively modulate the expression of collagen genes (11).

The peroxisome proliferator-activated receptors such as PPAR-γ form a family of nuclear hormone receptors that play critical roles in adipogenesis and the regulation of glucose and lipid metabolism. Pathological alterations in PPAR-γ expression or function are implicated in diabetes, obesity, and the metabolic syndrome (12). Recent studies have begun to uncover additional important roles for PPAR-γ in the regulation of inflammation, connective tissue remodeling, and in pathological processes such as glomerulosclerosis, atherosclerosis, cancer, and arthritis (12,13,14,15,16,17,18,19). The biological activity of PPAR-γ is triggered upon its activation by natural ligands, such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), or by synthetic agonists such as rosiglitazone. Activated PPAR-γ acts as an inducible transcription factor that can stimulate, or in some cases, repress, transcription of PPAR-γ target genes (20). Transactivation-mediated by PPAR-γ is generally DNA-dependent, whereas transrepression is DNA independent (21). The full activity of PPAR-γ requires interaction with coactivators such as p300 (22, 23). Indeed, PPAR-γ-driven adipogenic differentiation is completely suppressed in progenitor cells lacking p300 (24).

We recently demonstrated that the PPAR-γ ligands 15d-PGJ2 and troglitazone, as well as transient ectopic expression of PPAR-γ in normal skin fibroblasts, abrogated TGF-β-induced stimulation of collagen synthesis (25). Furthermore, the inhibitory effect could be blocked by pretreatment of cells with a selective PPAR-γ antagonist (25). The molecular mechanisms underlying these important anti-TGF-β activities elicited by PPAR-γ ligands are currently not known. Accordingly, we have undertaken mechanistic studies in normal skin fibroblasts to investigate these mechanisms. We now report that either the transient expression of ectopic PPAR-γ, or ligand-induced activation of endogenous PPAR-γ, suppressed Smad3-dependent transcriptional responses without blocking Smad activation. We further show that TGF-β-induced interaction of p300 with Smad3, as well as recruitment of p300 to the DNA-bound transcriptional complex, and p300-mediated histone H4 hyperacetylation at the COL1A2 locus, were all abrogated. Moreover, forced expression of ectopic p300 rescued the stimulatory TGF-β response in the presence of PPAR-γ ligands. Together, these results delineate a novel mechanism for the anti-TGF-β activities of PPAR-γ, and identify p300 coactivator as an important molecular target.



Transforming growth factor-β2 (Genzyme, Farmingham, MA, USA), troglitazone and 15d-PGJ2 (both from Biomol, Plymouth Meeting, PA, USA), and SB431542 (Glaxo, King of Prussia, PA, USA) were used. Troglitazone and 15d-PGJ2 were used at a concentration of 10 μM unless otherwise indicated.

Cell cultures

Primary cultures of neonatal foreskin fibroblasts were established as described previously (6) and maintained in Eagle’s modified essential medium (EMEM; Biowhittaker, Walkersville, MD, USA) supplemented with 10% FBS, 1% vitamins, 1% penicillin/streptomycin, and 2 mM l-glutamine. Fibroblasts were studied between passages 3 and 7. Cell viability was determined by Trypan blue dye exclusion, and toxicity was measured using EZ4U kit (Alpco Diagnostic, Salem, NH, USA). Murine embryonic fibroblasts (MEFs) null for PPAR-γ (PPAR-γ−/−) and wild-type MEFs were established at embryonic day 13.5 from embryos homozygous for a loxP-flanked PPAR-γ allele (26) and carrying an epiblast-specific Sox2-Cre transgene (27), or matching wild-type controls, respectively. This allelic configuration drives >95% deletion of PPAR-γ in all embryonic tissues outside the placenta (unpublished data). This novel targeting strategy makes it possible to establish PPAR-γ-null MEF donors that survive the placental lethality of standard PPAR-γ-null embryos at E9.5 (28). Primary cultures of PPAR-γ null MEFs were maintained for up to 5 passages in vitro in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemicals, St. Louis, MO, USA) supplemented with 10% FBS, 1% vitamins, 1% penicillin/ streptomycin, and 2 mM l-glutamine.


The 772COL1A2/CAT reporter construct contains the −772 to +58 bp fragment of the human COL1A2 gene in front of CAT gene (29). The expression vector pCMX-mPPAR-γ contains the full-length mouse PPAR-γ cDNA in pCMX vector (13). The pEGFP-Smad3 expression vector contains the full-length human Smad3 cDNA in pEGFPN1 vector (30). The pCI-p300-FLAG and pCI p300ΔHAT-FLAG expression vectors contain wild-type p300 and a HAT-deleted mutant p300 cDNA, respectively, in pCI vector (31). To reduce the cellular levels of PPAR-γ, dermal fibroblasts were transfected with short-hairpin RNA (shRNA) or scrambled control plasmids (SuperArray, Frederick, MD, USA) or lentiviruses (Sigma).

Transient transfection assays

Fibroblasts were transiently transfected with expression vectors for Smad3, PPAR-γ, p300, or p300ΔHAT, along with 772COL1A2/CAT reporter construct and internal standard pRLTK-luc using Superfect reagent (Qiagen, Valencia, CA, USA). Transfected fibroblasts were pretreated with 15d-PGJ2 for 60 min before the addition of TGF-β (12.5 ng/ml). Following further 48-h incubation in medium containing 10% FBS, cultures were harvested, and whole-cell lysates were prepared and assayed for their CAT and luciferase activities (6). Results were normalized for protein concentration in each sample, and transfection efficiency was monitored. To reduce endogenous PPAR-γ, fibroblasts were transfected or infected with PPAR-γ-shRNA plasmids (SuperArray) or lentiviruses (Sigma), or scrambled control oligonucleotides. Twenty four hours following transfection, fresh medium containing 15d-PGJ2 was added to the cultures for 60 min, followed by TGF-β. After an additional 24 h incubation, cultures were harvested and whole-cell lysates, nuclear extracts, or conditioned medium were prepared and subjected to Western blot analysis or electrophoretic mobility shift assays. Efficacy and specificity of suppression by shRNA were evaluated by analysis of protein and RNA levels.

Western and coupled immunoprecipitation-immunoblot analysis

Equal amounts of cellular proteins or aliquots of conditioned culture medium were resolved by electrophoresis in 4–20% Tris-glycine gradient gels (Bio-Rad, Hercules, CA, USA). In other experiments, whole-cell lysates (~200 μg) were immunoprecipitated with indicated antibodies and subjected to immunoblot analysis (10). Membranes were probed with antibodies to human type I collagen (Southern Biotechnology, Birmingham, AL, USA), PPAR-γ (E-8), Smad7 (N-19), Smad4 (B8), Smad1/2/3 (H2), actin (C-2), or p300 (C-20) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), Smad3 (Zymed Lab, San Francisco, CA, USA), phospho-Smad2 or phospho-Smad3 (Cell Signaling, Beverly, MA, USA).

Electrophoretic mobility shift assays

Confluent fibroblasts were preincubated with troglitazone or 15d-PGJ2, followed by TGF-β for 60 min. At the end of the experiments, nuclear extracts were prepared and subjected to electrophoretic mobility shift assays with radiolabeled oligonucleotides harboring the SBE (32) as probes. Antibody supershift assays were performed by preincubating nuclear extracts with antibodies to Smad3 (I-20) (Santa Cruz Biotechnology) or IgG (9).

DNA affinity precipitation assays

Accumulation of DNA binding factors and cofactors on the consensus SBE DNA sequences was determined by DNA affinity precipitation assays (10). Briefly, equal amounts of nuclear proteins (~200 μg) were incubated with biotin-labeled double-stranded deoxyoligonucleotides (3 μg) harboring the SBE sequences (forward: 5′GGAGTATGT CTAGACTGACAATGTAC-3′; reverse: 5′-GTACATTG TCAGTCTAGAC ATACTCC-3′). At the end of a 30-min incubation, a solution of streptavidin-agarose beads (4%) with 50% slurry (Sigma) was added, and mixtures were incubated at 4°C for 45 min on a rotator platform. The streptavidin-agarose beads were precipitated by centrifugation and pellets were washed three times with cold PBS. Bead-bound proteins were resuspended in SDS-loading buffer, boiled, separated in 4–20% denaturing gels, and processed for Western blot analysis.

Chromatin immunoprecipitation (ChIP) assays

In vivo interactions of nuclear factors with the COL1A2 promoter DNA were studied by ChIP assays using the EZ Magna ChIP Assay Kit (Upstate/Millipore, Billerica, MA, USA) following the manufacturer’s instructions. Briefly, fibroblasts were preincubated with 15d-PGJ2 for 60 min (or in selected experiments for 23 h), followed by TGF-β2 (12.5 ng/ml) for 60 min. At the end of the incubation period, 1% formaldehyde was added to cross-link DNA-protein in chromatin, cell lysates were sonicated, and equal aliquots were used for immunoprecipitation with monoclonal antibodies to human Smad1/2/3, p300, and acetylated H4 histone (Upstate/Millipore), or with mouse IgG (Upstate/Millipore). PCR amplification of the captured DNA sequences was performed using primers complementary to COL1A2 sequences flanking the SBE (33), or GAPDH-specific primers. Amplified PCR products were analyzed by electrophoresis in 2% agarose gels. Real-time quantitative PCR of antibody-captured DNA was performed with human COL1A2-specific primers (forward: 5′-AAATTCTGCCCATGTCGGG-3′; reverse: 5′-AAACTCTGGCTCGTTGTCTGC-3) on ABI-Prism 7300 sequence detection PCR machine with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), according to manufacturer’s protocol.

Confocal immunofluorescence microscopy

Fibroblasts were preincubated on coverslips in EMEM with troglitazone or 15d-PGJ2 followed by TGF-β. At the end of the indicated periods, cells were fixed with acetic acid and methanol mixture, and incubated with antibodies to Smad1/2/3 (Santa Cruz) for 60 min. Following washing in PBS, slides were incubated with fluorescein-conjugated anti-mouse IgG (Santa Cruz) for 30 min, and immunofluorescence was evaluated by laser scanning confocal microscopy (9).

Statistical analysis

The data are presented as the means ± sd of multiple determinations. The significance of differences between experimental and control groups was determined by analysis of variance (ANOVA), and the value of P < 0.05 by Student’s t test was considered statistically significant. Student’s t tests were performed using the GraphPad t test calculator (GraphPad, San Diego, CA, USA).


Abrogation of TGF-β stimulation of collagen synthesis by PPAR-γ ligands is PPAR-γ dependent

Brief pretreatment of dermal fibroblasts with 15d-PGJ2 abrogated the stimulation of collagen synthesis and of COL1A2 promoter activity induced by TGF-β (Fig. 1). Ligands of PPAR-γ can elicit cellular responses by binding to and inducing the transcriptional activity of endogenous PPAR-γ, as well as through PPAR-γ-independent signaling pathways (16). Two complementary strategies were pursued to determine whether the inhibitory effect of 15d-PGJ2 on the stimulation of collagen synthesis occurred via PPAR-γ. First, the regulation of collagen gene expression was examined in cells lacking endogenous PPAR-γ. Primary PPAR-γ-null fibroblasts were derived by a novel genetic targeting strategy using midgestation mouse embryos, in which loxP-flanked PPAR-γ was deleted by the epiblast-specific Sox2-Cre in all nonplacental tissues. The MEFs used here are primary fibroblasts, procured directly from mouse embryos in which they have been rendered PPAR-γ-null at the source by germ-line genetic manipulations, and used at early passage. Thus, comparisons to control wild-type MEFs harvested in parallel from wild-type littermates are not complicated by stochastic genetic differences other than deficiency for PPAR-γ. The results shown in Fig. 2A indicated that while 15d-PGJ2 reduced both basal and the TGF-β-induced stimulation of collagen synthesis and secretion in wild-type MEFs (compare lanes 1 and 3, and 2 and 4), PPAR-γ null MEFs were largely refractory to the inhibitory effects of 15d-PGJ2 (compare lanes 5 and 7, and 6 and 8). Levels of both cellular and secreted type I collagen are significantly higher in PPAR-γ-null MEFs compared to levels in wild-type MEFs (compare lanes 8 and 4).

Figure 1.
15d-PGJ2 abrogates stimulation of collagen gene expression. Foreskin fibroblasts transiently transfected with 772COL1A2/CAT reporter construct were preincubated with 15d-PGJ2 (10 μM) for 60 min, followed by TGF-β2 (12.5 ng/ml) for a further ...
Figure 2.
15d-PGJ2 blocks stimulation of collagen gene expression via PPAR-γ. A) Primary cultures of wild-type (PPAR-γ+/+) and PPAR-γ null (PPAR-γ−/−) MEFs, generated as described in Materials and ...

The effect of 15d-PGJ2 on TGF-β stimulation of collagen synthesis was also examined in normal skin fibroblasts where endogenous PPAR-γ was suppressed by RNAi. To achieve PPAR-γ knockdown, confluent fibroblasts were transfected with shRNA constructs. A selective ~50% reduction of PPAR-γ levels was consistently achieved (Fig. 2C and data not shown). In contrast to fibroblasts transfected with scrambled control shRNA (compare lanes 2 and 4), in fibroblasts transfected with PPAR-γ RNAi 15d-PGJ2 caused only a minimal suppression of collagen synthesis and secretion (Fig. 2B, compare lanes 6 and 8). Furthermore, 15d-PGJ2 similarly failed to block TGF-β-induced stimulation of COL1A1 mRNA expression in fibroblasts transfected with PPAR-γ shRNA (Fig. 2D). The effects of 15d-PGJ2 or troglitazone were not a reflection of altered fibroblast growth or viability (Table 1). Together, these results provide evidence that inhibition of TGF-β-stimulated increase in collagen gene expression by 15d-PGJ2 was due to bona fide ligand activation of endogenous PPAR-γ.

Fibroblast viability in the presence and absence of PPAR-γ ligands

PPAR-γ attenuates Smad-dependent transcriptional activity

To explore the molecular mechanisms underlying the abrogation of TGF-β responses by 15d-PGJ2, we first focused on the canonical Smad signaling pathway. First, the regulation of Smad-dependent transcription by 15d-PGJ2 was examined in fibroblasts transfected with Smad3 expression vectors and 772COL1A2/ CAT. The results showed that while ectopic Smad3 induced an >6-fold increase in COL1A2 promoter activity, even in the absence of TGF-β, 15d-PGJ2 abrogated this response in a dose-dependent manner (Fig. 3A). Furthermore, enhanced Smad3 transactivation in the presence of TGF-β was also blocked by 15d-PGJ2. The high-affinity PPAR-γ ligand troglitazone exerted comparable inhibition of Smad3-mediated transactivation (data not shown). Expression of ectopic PPAR-γ in the fibroblasts was sufficient to mimic the effect of 15d-PGJ2 and abrogate the stimulation of COL1A2 promoter activity (Fig. 3B, top panel), and type I collagen synthesis (Fig. 3B, bottom panel) induced by Smad3. Taken together, these results indicate that PPAR-γ targets the transcriptional activity of Smad3, accounting for inhibition of TGF-β responses in fibroblasts. Coupled immunoprecipitation/immunoblot assays revealed a low level constitutive interaction of Smad3 with endogenous PPAR-γ in unstimulated fibroblasts that was modestly enhanced by TGF-β treatment, but unaffected by 15d-PGJ2 or by troglitazone (Fig. 3C and data not shown).

Figure 3.
Both 15d-PGJ2 and ectopic PPAR-γ abrogate Smad3-dependent transcription. A, B) Foreskin fibroblasts transiently transfected with Smad3 expression vector (A) or PPAR-γ expression vector (B) along with 772COL1A2/CAT reporter were preincubated ...

Ligand-dependent Smad activation in the presence of PPAR-γ

In some studies, the antagonistic modulation of TGF-β responses by PPAR-γ ligands was associated with interference with Smad2 or Smad3 phosphorylation in hepatic stellate cells and vascular smooth muscle cells (34,35,36,37,38). To investigate the effect of PPAR-γ ligands on Smad activation in fibroblasts, a series of experiments were performed. Western blot analysis indicated that TGF-β-induced rapid phosphorylation of Smad2/3 was unaffected by preincubation of fibroblasts with 15d-PGJ2 (Fig. 4A, top panel). Moreover, a comparable increase in nuclear accumulation of Smad2/3 and phospho-Smad2/3 was seen in the presence or absence of 15d-PGJ2 (Fig. 4A bottom panel, B) or troglitazone (data not shown). In contrast, the ALK5 inhibitor SB431542 completely blocked these TGF-β-induced responses, as expected (Fig. 4B).

Figure 4.
15d-PGJ2 fails to prevent Smad activation. Confluent fibroblasts pretreated with 15d-PGJ2 for 23 h (A) or for 60 min (B) were incubated with TGF-β2 for a further 60 min. A) Cultures were harvested and whole-cell lysates (top panel) or nuclear ...

To examine the effect of 15d-PGJ2 on Smad2/3 DNA binding, nuclear extracts were prepared from fibroblasts stimulated with TGF-β in the presence or absence of 15d-PGJ2 and incubated with radiolabeled oligonucleotide probes harboring SBE sequence, and subjected to electrophoretic mobility shift analysis. The results showed that TGF-β induced the formation of a protein-DNA complex that was identified as Smad3 by antibody supershift assays (Fig. 4C). Notably, the relative intensity of the Smad3-DNA complex was comparable in fibroblasts incubated in the presence or absence of 15d-PGJ2. Collectively, these results indicate that PPAR-γ did not interfere with TGF-β-induced activation of the Smad pathway, but rather abrogated Smad transcriptional activity.

PPAR-γ blocks the recruitment of the p300 coactivator

The transcriptional coactivator p300 is intimately involved in intracellular TGF-β signal transduction (11). Furthermore, by virtue of its intrinsic histone acetyltransferase activity, p300 plays a fundamental role in the induction of fibrotic responses (11). We had shown previously that Smad-dependent transactivation of COL1A2 in TGF-β-stimulated fibroblasts required the physical and functional interaction of p300 with activated Smad2/3 (9, 10). The transcriptional activities of PPAR-γ similarly require interaction with p300 (22). In light of the requirement for p300 as a shared cofactor that is indispensable for both Smad2/3-dependent and PPAR-γ-dependent transcription, we hypothesized that the antagonistic regulation of TGF-β signaling by PPAR-γ may be associated with disrupted p300 recruitment or function. This notion was examined by three complementary approaches. First, coupled immunoprecipitation-immunoblot assays revealed that while TGF-β stimulation enhanced the interaction between endogenous Smad3 and p300, as expected (Fig. 5), preincubation of the cultures with 15d-PGJ2 blocked this response, while at the same time enhancing the interaction of PPAR-γ with p300 (Fig. 5). Second, the effect of PPAR-γ on p300 recruitment was examined by DNA affinity precipitation assay using biotin-labeled oligonucleotides harboring SBE. The results demonstrated that while Smad2/3 binding to SBE sequences in TGF-β-treated fibroblasts was unaffected by 15d-PGJ2, recruitment of p300 was completely blocked (Fig. 6A). Similar results were obtained with troglitazone as the PPAR-γ ligand (data not shown). To extend these observations to the chromatin context in live cells, ChIP assays were performed. Fibroblasts were stimulated with TGF-β in the presence of absence of 15d-PGJ2, and formaldehyde crosslinked DNA-protein complexes were immunoprecipitated with antibodies to Smad2/3, p300 or acetylated histone H4. The results revealed that, whereas 15d-PGJ2 had no detectable effect on the increased levels of Smad2/3 accumulation on the COL1A2 promoter, in fibroblasts treated with TGF-β, both the increased recruitment of p300, as well as histone H4 hyperacetylation at the COL1A2 locus, were reduced to near-basal levels in the presence of 15d-PGJ2 (Figs. 6B, C).

Figure 5.
15d-PGJ2 abrogates induction of p300-Smad2/3 interaction. Following pretreatment with 15d-PGJ2 for 60 min, fibroblasts were incubated with TGF-β for 60 min. Whole-cell lysates were immunoprecipitated with antibodies to p300 and subjected to Western ...
Figure 6.
15d-PGJ2 abrogates p300 recruitment to SBE. A) DNA affinity precipitation assays. Nuclear extracts from fibroblasts treated with TGF-β in the presence and absence of 15d-PGJ2 were incubated with biotin-labeled oligonucleotide probes harboring ...

Forced expression of p300 rescues collagen stimulation in the presence of PPAR-γ

These findings led us to hypothesize that interference with the recruitment of p300 to the Smad2/3-containing transcriptional complex in 15d-PGJ2-treated fibroblasts resulted in loss of histone H4 hyperacetylation at target gene promoters, accounting for abrogation of Smad-dependent TGF-β responses. If the hypothesis is correct, then forced expression of p300 would be expected to rescue TGF-β stimulation in the presence of 15d-PGJ2 in a histone acetyltransferase-dependent manner. To test this notion, fibroblasts were cotransfected with expression vectors for wild-type p300 or a mutant form of p300 that lacks HAT domain (31). Transient transfection assays revealed that while 15d-PGJ2 almost completely prevented the stimulation of COL1A2 promoter activity in TGF-β-treated fibroblasts, ectopic p300 rescued the stimulatory response in a dose-dependent manner (Fig. 7). By itself, ectopic p300 caused a significant increase in basal, as well as TGF-β-stimulated, COL1A2 promoter activity (data not shown). In contrast, the HAT-deficient mutant p300 failed to reverse the suppression of TGF-β responses. These results indicate that p300 reversed the suppressive effects of 15d-PGJ2 on TGF-β responses, and the intrinsic acetyltransferase activity of the coactivator was required for this effect.

Figure 7.
Ectopic p300 rescues stimulation of COL1A2 activity in the presence of 15d-PGJ2. Confluent fibroblasts transiently cotransfected with increasing concentration of p300 or HAT-deleted mutant p300 (p300ΔHAT) expression vectors along with 772COL1A2/CAT ...


Aberrant TGF-β expression or activity of TGF-β is implicated in the pathogenesis of fibrosis in a broad range of conditions (1). In light of its critical role in fibrosis, there is substantial interest in understanding the physiological modulation of TGF-β signaling intensity, and in blocking the expression or function of TGF-β as potential therapeutic strategies (39). We previously demonstrated that ligands of PPAR-γ blocked the stimulation of collagen synthesis induced by TGF-β in dermal fibroblasts (25). Both natural and synthetic ligands exerted comparable inhibitory effects (25). Suppression of the stimulatory response occurred at the level of transcription. Other studies have reported similar effects of PPAR-γ in lung fibroblasts, mesangial cells and hepatic stellate cells (17, 40,41,42). In addition, PPAR-γ ligands have been shown to ameliorate fibrosis in vivo in rodent models of fibrosis of the kidney (43), liver (18), lung (44, 45), and dermis (46). Taken together, these observations point to a potentially important novel facet of PPAR-γ biology that is related to connective tissue homeostasis and the control of tissue remodeling. There is growing recognition of the potential roles of PPAR-γ in regulating physiological and pathological matrix remodeling (45). The molecular mechanisms underlying these effects are incompletely understood. The present studies demonstrate that the antagonistic regulation of profibrotic TGF-β signaling in normal fibroblasts by ligand agonists of PPAR-γ is a bona fide PPAR-γ response and involves suppression of Smad2/3 transcriptional activity. The inhibitory effect was not associated with disruption of Smad2/3 activation or nuclear accumulation or Smad-SBE interaction, but rather involved in suppression of the recruitment of p300, a coactivator and histone acetyltransferase that is indispensable for stimulation of collagen gene expression (11).

Ligands of PPAR-γ are known to exert their effect on target gene expression in both PPAR-γ-dependent and PPAR-γ-independent manner (47). We demonstrate here that PPAR-γ ligands blocked TGF-β-induced stimulation of collagen synthesis via PPAR-γ. That endogenous PPAR-γ was a bona fide mediator of the inhibitory effect of PPAR-γ ligands on TGF-β responses was established by several lines of observation (25, 48). First, we showed that 15d-PGJ2 was unable to abrogate TGF-β-induced transcription in MEFs lacking endogenous PPAR-γ. Second, the inhibitory effect was abrogated by pretreatment of normal fibroblasts with a selective and irreversible PPAR-γ antagonist. A potential mechanism for the antifibrotic effects of PPAR-γ is the ability to antagonize Smad-mediated profibrotic responses elicited by TGF-β. Blockade of TGF-β signaling by PPAR-γ has been shown to involve disruption of Smad nuclear translocation in NRK fibroblasts (37) or phosphorylation in hepatic stellate cells (35). However, in other studies with vascular smooth muscle cells, PPAR-γ ligands enhanced, or had no effect on, Smad phosphorylation and nuclear translocation (34, 37, 38). The present results indicate that PPAR-γ targeted the transcriptional activity of Smads in skin fibroblasts. We found that 15d-PGJ2 blocked Smad-mediated transcriptional responses without disrupting TGF-β-induced Smad2/3 activation. The effects of PPAR-γ on Smad signal transduction may therefore depend on the cell type. The antifibrotic activities of PPAR-γ may be mediated via the induction of endogenous repressor molecules. For instance, in mesangial cells, PPAR-γ ligands stimulated the expression of hepatocyte growth factor, which, in turn, induced the transcriptional corepressor TGIF (37). Recent studies implicate induction of the tumor suppressor PTEN as responsible for the attenuation of bleomycin-induced pulmonary fibrosis by PPAR-γ (49,50,51).

The dual-function coactivator and histone acetyltransferase p300 is essential for Smad-dependent TGF-β responses. Full transcriptional stimulation of TGF-β target genes requires recruitment of p300 to DNA and the accompanying locus-specific histone hyperacetylation and chromatin remodeling (52). Ligand-dependent Smad2/3 interaction with p300 is implicated in TGF-β stimulation of collagen synthesis (10, 53). As a promiscuous cofactor, p300 interacts with and is required for the transcriptional activity of multiple DNA-binding factors (54). Furthermore, p300, as well as its orthologue CBP, also serves as an essential cofactor for nuclear receptors, including PPAR-γ (22). Because p300 is available in cells in limiting amounts, it has been suggested that PPAR-γ competes in a cell-type-specific manner with ligand-inducible transcription factors for binding to limiting p300, a mechanism called squelching (55). We believe that this may represent a generalized mechanism for regulation of other genes. For example, a strong correlation between PPAR-γ interaction with p300 and repression of inflammation induced-iNOS promoter activity has been found (56). A similar mechanism underlies PPAR-γ suppression of TGF-β-induced CTGF gene expression (57). Squelching is also implicated as a mechanism for PPAR-γ abrogation of inflammatory responses such as Cox-2 production (58). Our results identify a similar mechanism in TGF-β-stimulated fibroblasts treated with PPAR-γ ligands. Competition by activated PPAR-γ for limiting amounts of p300 in activated fibroblasts could interfere with the recruitment of p300 to the COL1A2 promoter, thereby reducing local histone modification and inhibiting collagen transcription. In the present studies, we found that treatment with 15d-PGJ2 reduced p300 accumulation and H4 histone hyperacetylation at the COL1A2 locus in TGF-β-treated fibroblasts. Ligand activation of cellular PPAR-γ disrupted the interaction of Smad2/3 with p300 induced by TGF-β. Taken together, these observations implicate competition for limiting amounts of p300 as a potential mechanism accounting for the antifibrotic effects of PPAR-γ ligands, and point to mechanistic parallels between the anti-inflammatory and the antifibrotic effects of PPAR-γ, both of which appear to involve squelching as the mechanism for transrepression (59). Alternate mechanisms to explain the inhibitory activities of PPAR-γ have also been identified. For instance, an inducible interaction of PPAR-γ with an inhibitory transcriptional complex containing CIITA and RFX-5 in IMR-90 lung fibroblasts has been implicated in repression of collagen synthesis by interferon-γ (60).

The present results now allow us to propose a novel mechanistic model to explain how PPAR-γ causes suppression of Smad-dependent transcriptional responses in the context of fibrogenesis. In normal fibroblasts, TGF-β stimulated the recruitment of p300 histone acetyltransferase to the COL1A2 promoter, which was associated with hyperacetylation of histone H4 at the COL1A2 locus. Recruitment of p300 to the Smad complex on the COL1A2 promoter SBE sequences is required for maximal transactivation induced by TGF-β. PPAR-γ inhibited the recruitment of p300 to the promoter in TGF-β-treated fibroblasts, and prevented histone H4 hyperacetylation. Inhibition of p300 recruitment is likely to account for the suppression of histone acetylation. We, therefore, propose that PPAR-γ-mediated abrogation of the stimulatory TGF-β response was due, at least in part, to decreased histone H4 acetylation at the COL1A2 promoter, possibly due to the reduced recruitment of p300. This observation may be relevant for the development of targeted antifibrotic therapies. Scleroderma fibroblasts are characterized by elevated p300 expression, constitutive Smad2/3 activation and constitutive interaction of Smad2/3 with p300 (53, 61, 62). By disrupting recruitment and function of p300, PPAR-γ might normalize altered fibroblast function in scleroderma and control TGF-β-dependent fibrotic processes.


We are grateful to Christopher K. Glass (University of California, San Diego, CA, USA) and Joan Boyes (Institute of Cancer Research, London, UK) for PPAR-γ and p300 expression vectors. This work was supported by grants from the Scleroderma Foundation to A.K.G. and the National Institutes of Health (AR-49025) to J.V.


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