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 β (TGFβ), and sustained production of TGFβ, connective tissue growth factor, interleukin-1α, and other profibrotic cytokines and growth factors (for review, see ref. 1). Furthermore, lesional fibroblasts show increased expression of the myofibroblast marker α-smooth muscle actin (α-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 TGFβ 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 TGFβ or an exaggerated intensity or duration of the target cell response to TGFβ is primarily responsible for the activated scleroderma fibroblast phenotype.
Recent studies have elucidated the molecular mechanisms underlying the fibrotic responses elicited by TGFβ (3). Receptor-activated Smads (R-Smads) are directly activated by TGFβ/activin (Smad2 and Smad3) or by bone morphogenetic proteins (Smads 1, 5, and 8). Upon ligand binding to the surface TGFβ 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 TGFβ-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 TGFβ/Smad signaling.
We have previously demonstrated that in normal dermal fibroblasts, TGFβ-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 TGFβ/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 TGFβ, indicating intrinsic activation of the TGFβ/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 (α, β, γ) 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-Δ12,14-prostaglandin J2 (15d-PGJ2), 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 PPARγ 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 PPAR-regulated genes linked to glucose homeostasis, apoptosis, and proliferation (15).
Recent discoveries suggest that signaling through PPARγ 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 PPARγ. In these cells, activation of PPARγ is associated with potent antiinflammatory and immunomodulatory effects; these effects are due to suppression of the genes for tumor necrosis factor α, inducible nitric oxide synthase, cyclooxygenase 2, and interleukin-6 (16–20). The immunoregulatory activities of PPARγ involve mechanisms distinct from those that mediate insulin sensitization. Importantly, activation of PPARγ by naturally occurring ligands also appears to have antiinflammatory effects. In the joint, for example, monosodium urate monohydrate crystals have been shown to activate PPARγ on monocytes, presumably via endogenous 15d-PGJ2, and crystal-induced PPARγ 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, PPARγ ligands hold substantial promise as novel immunomodulatory and antiinflammatory agents. These immunoregulatory effects appear to be cell-specific, however, since in monocytes, PPARγ activation resulted in induction, rather than suppression, of cyclooxygenase 2 (22).
The potential involvement of PPARγ in physiologic tissue remodeling, wound healing, and organ fibrosis has thus far received only scant attention. Investigation of PPARγ 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 PPARγ, 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 TGFβ (24). Furthermore, naturally occurring or synthetic ligands of PPARγ 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 PPARγ 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 PPARγ in skin fibroblasts or the role of PPARγ in modulating fibrotic responses in the skin. We report here that PPARγ is constitutively expressed in normal skin fibroblasts, is up-regulated by TGFβ, and can be activated by naturally occurring ligands or by the thiazolidinedione class of antidiabetic drugs. Transient overexpression of PPARγ in fibroblasts dramatically enhanced their sensitivity to PPARγ ligands. Troglitazone and 15d-PGJ2 prevented TGFβ-induced stimulation of type I collagen synthesis at the level of transcription and abrogated α-SMA expression. The inhibitory effects of the PPARγ ligands on these profibrotic responses were specific and PPARγ-dependent. Furthermore, PPARγ activation in fibroblasts had no effect on the level of cellular Smad3 or Smad7 expression. These results indicate that PPARγ inhibits TGFβ-induced profibrotic responses in normal fibroblasts. Together, the findings suggest a novel potential role for PPARγ in the control of skin fibrosis.
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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 TGFβ, 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 TGFβ responses. In addition, various ligands that repress TGFβ 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 PPARγ at the mRNA and protein levels. PPARγ 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 PPARγ expression was found, and the receptor was largely distributed in the cytoplasm. Interestingly, TGFβ induced a significant time-dependent increase in PPARγ protein levels in these fibroblasts. In contrast, a recent study in vascular smooth muscle cells found that TGFβ caused early stimulation and late inhibition of PPARγ expression (49), suggesting that PPARγ regulation by TGFβ may be cell type–specific.
Naturally occurring ligands of PPARγ, such as 15d-PGJ2, as well as the synthetic pharmacologic PPARγ agonist troglitazone, caused activation of the receptor, as indicated by its enhanced nuclear accumulation in the absence of significant change in cellular PPARγ levels and by stimulation of a PPARγ-responsive minimal promoter in transfected fibroblasts. Remarkably, transient overexpression of PPARγ in the fibroblasts dramatically enhanced their sensitivity to stimulation by PPARγ agonists, suggesting that the level of expression is limiting for cellular responsiveness to endogenous PPARγ ligands. It is noteworthy that in diabetic glomerulosclerosis and other forms of fibrosis, PPARγ expression is diminished in affected tissues, potentially rendering them resistant to the potentially protective effects of endogenous PPARγ 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 PPARγ expression or functional activity.
Agonists of the PPARγ 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 TGFβ-induced collagen synthesis and COL1A2 promoter activity. Because 15d-PGJ2 is known to have PPARγ-independent cellular effects (13), it was important to confirm that the repression of TGFβ-induced responses was mediated through PPARγ. By using the potent and selective PPARγ antagonist GW9662, which covalently modifies a cysteine residue in the ligand-binding site of PPARγ (50), we established that the effect of 15d-PGJ2 on repression of TGFβ-induced collagen stimulation could be prevented in a dose-dependent manner. In addition, dominant-negative PPARγ expression vectors blocked the inhibitory effects of 15d-PGJ2 on TGFβ-stimulated COL1A2 promoter activity.
These results further demonstrate that in addition to collagen, the PPARγ ligand also disrupted the induction of α-SMA expression. Because stimulation of α-SMA by TGFβ is one of the key steps in the transdifferentiation of normal fibroblasts into myofibroblasts (40), these results indicate that PPARγ can interfere with multiple cellular events that are important in the pathogenesis of fibrosis. The inhibitory effects of PPARγ on TGFβ-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 TGFβ-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 TGFβ-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 TGFβ (7, 8, 51–56). Furthermore, activation of the p38 and ERK cascades have also been implicated in the stimulation of collagen transcription elicited by TGFβ in fibroblasts (57, 58).
PPARγ functions as a ligand-activated transcription 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 PPARγ-binding elements. This suggests that the inhibitory effect of PPARγ on COL1A2 transcription involves disruption of the cellular transcriptional machinery that mediates the stimulation elicited by TGFβ. Accordingly, we examined the effects of PPARγ 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 PPARγ was able to prevent TGFβ stimulation of these minimal promoter constructs containing the binding sites for only Smad3 and Smad4. These results suggested that PPARγ was able to directly antagonize the activation and/or function of Smad3 in fibroblasts (Figure 8). Furthermore, PPARγ 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 TGFβ was unaffected by PPARγ, indicating that PPARγ did not block all TGFβ responses nonselectively.
Figure 8. Regulation of transforming growth factor β (TGFβ)–induced responses in fibroblasts by peroxisome proliferator–activated receptor γ (PPARγ). Through activation of the intracellular Smad signal transduction pathway, TGFβ stimulates collagen synthesis, α-smooth muscle actin (α-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 PPARγ, resulting in the disruption of TGFβ/Smad signal transduction and the blocking of profibrotic responses. The expression of PPARγ is enhanced on fibroblasts by TGFβ, thereby sensitizing them to the antifibrotic effects of ligands. Activation of PPARγ may represent an effective antifibrotic intervention strategy. 15d-PGJ2 = 15-deoxy-Δ12,14-prostaglandin J2; TGZ = troglitazone.
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The transactivating function of R-Smads may be repressed by PPARγ through cytoplasmic retention of the activated Smad complex. Direct physical interaction of R-Smads has been demonstrated with PPARγ 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 PPARγ and Smads that would account for the repression of the profibrotic TGFβ responses in fibroblasts.
In summary, the results of the present study indicate that normal quiescent skin fibroblasts display constitutive PPARγ expression and activation of PPARγ-dependent transcriptional responses elicited by endogenous and synthetic PPARγ ligands. Up-regulation of PPARγ receptor levels by transient transfection of a PPARγ expression plasmid in fibroblasts markedly enhanced sensitivity to activation by PPARγ agonists. While TGFβ enhanced the expression of endogenous PPARγ in fibroblasts, both endogenous and synthetic ligands of PPARγ caused selective abrogation of TGFβ-induced profibrotic responses. The inhibitory effects of PPARγ on TGFβ-dependent responses involved direct antagonism of Smad signal transduction (Figure 8).
Together, these results suggest that PPARγ represents an important physiologic mechanism in the control of TGFβ responses in normal fibroblasts. Diminished PPARγ expression in affected tissues may be associated with aberrant repair. Accordingly, enhancing fibroblast sensitivity to exogenous PPARγ may represent a novel strategy for the treatment of fibrosis. Furthermore, in light of their potent antiinflammatory properties, pharmacologic PPARγ agonists may be particularly effective in scleroderma and related fibrotic conditions in which both inflammation and fibrosis play prominent roles.
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We are grateful to Drs. Christopher Glass (University of California, San Diego) for the gift of the p(AOx)3-TK-Luc and PPARγ expression vectors, Krishna K. Chatterjee (University 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 PPARγ 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.