Disruption of transforming growth factor signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferatoractivated receptor ╨Ю╤Ц.код для вставкиСкачать
ARTHRITIS & RHEUMATISM 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 Smad7. 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 constructs. 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: firstname.lastname@example.org. 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, 1305 1306 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). GHOSH ET AL 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- REGULATION OF PROFIBROTIC RESPONSES BY PPAR␥ 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. MATERIALS AND METHODS 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 1307 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 1308 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 GHOSH ET AL 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 significant. RESULTS 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 probes. REGULATION OF PROFIBROTIC RESPONSES BY PPAR␥ 1309 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- 1310 GHOSH ET AL 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 REGULATION OF PROFIBROTIC RESPONSES BY PPAR␥ 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 expression. 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 1311 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 1312 GHOSH ET AL 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 REGULATION OF PROFIBROTIC RESPONSES BY PPAR␥ 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. 1313 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 1314 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 GHOSH ET AL 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. DISCUSSION 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 REGULATION OF PROFIBROTIC RESPONSES BY PPAR␥ 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– specific. 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 1315 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 activity. 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 toxicity. 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- 1316 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- GHOSH ET AL 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. 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