Excessive extracellular matrix accumulation and tissue contraction, the hallmarks of fibrosis, are mediated by fibroblasts and myofibroblasts activated by TGF-ß 
. Blocking TGF-ß activity therefore represents an appealing approach to fibrosis therapy, and there is intense interest in identifying the intracellular pathways that positively or negatively modulate the fibrotic responses. Recent research findings suggest that PPAR-γ signaling is a novel cell-intrinsic mechanism to negatively regulate profibrotic responses.
PPAR-γ exerts potent antifibrotic effects in vitro and in vivo. We showed previously that PPAR-γ activation by natural or synthetic ligands abrogated collagen synthesis, cell migration and myofibroblast transdifferentiation induced by TGF-ß 
. While the precise mechanisms underlying abrogation of TGF-ß signaling remain to be fully delineated, we found that PPAR-γ blocked Smad-dependent transcriptional responses without interfering with Smad phosphorylation or nuclear accumulation. Instead, PPAR-γ appears to compete with ligand-inducible transcription factors for binding to limiting p300, squelching Smad/p300 complex formation in TGF-ß-stimulated fibroblasts 
. Furthermore, in animal models of TGF-ß-dependent fibrosis, rosiglitazone treatment attenuated the development of dermal thickness and collagen accumulation in vivo 
. Because the biological activities of PPAR-γ ligands depend largely on their interaction with PPAR-γ, the relative levels or biologically availability of PPAR-γ are important factors governing the intensity of ligand-induced cellular responses. Surprisingly, little is known about the expression of PPAR-γ in fibroblasts, and its regulation during inflammation, tissue repair and fibrosis. In the present studies we found that the levels of PPAR-γ protein and mRNA were substantially lower in SSc lesional tissue and explanted skin fibroblasts than in healthy controls. Moreover, genome-wide expression analysis using microarrays demonstrated that SSc skin biopsies with evidence of active TGF-ß signaling had low expression of PPAR-γ-regulated genes, revealing an inverse relationship between TGF-ß activity and PPAR-γ-dependent responses in fibrotic tissues.
We examined the regulation of PPAR-γ by TGF-ß in normal fibroblasts. Despite its importance in determining the intensity of ligand-induced responses, the factors involved in the regulation of PPAR-γ expression are not well understood. Our results demonstrated that TGF-ß caused a potent time- and dose-dependent and cell-type specific down-regulation of PPAR-γ transcription. This inhibitory response was mediated through the canonical Smad signal transduction pathway. A 2.7 kb fragment of the PPAR-γ gene promoter harboring two putative Smad binding elements was sufficient for inhibition by TGF-ß. Taken together with previous findings, the present results indicate that TGF-ß and PPAR-γ are engaged in a mutually antagonistic reciprocal cross-talk, whereby the PPAR-γ pathway abrogates cellular responses induced by TGF-ß, while TGF-ß-Smad inhibits the expression of PPAR-γ and its target genes.
In addition to TGF-ß, IL-13, Wnt, CCN2, as well as hypoxia, have all been shown to inhibit PPAR-γ expression or activity 
. Particularly interesting is lysophosphatidic acid (LPA), a bioactive lipid that potently inhibits the expression of PPAR-γ in a variety of cell types 
, while also acting as a PPAR-γ agonist 
. The signaling mechanisms mediating down-regulation of PPAR-γ in response to these cues are not well understood. It is noteworthy that CCN2 and Wnt are known to be up-regulated in the fibrotic cellular milieu in SSc, and chronic tissue hypoxia due to microvascular insufficiency is prominent 
. Thus, multiple factors implicated in the pathogenesis of fibrosis might contribute to the profound down-regulation of PPAR-γ in SSc. We and others have also observed that PPAR-γ protein and mRNA levels were persistently reduced in explanted SSc fibroblasts 
. At present, it is unclear whether autonomous PPAR-γ down-regulation represents a cell-inherent abnormality in gene regulation, such as epigenetic modifications at the PPAR-γ promoter, or results from constitutive autocrine TGF-ß stimulation. The latter mechanism is plausible in light of the finding that in normal fibroblasts, constitutive basal TGF-ß signaling maintains tonic suppression of PPAR-γ (). Epigenetic modification represents an appealing possibility to account for low level of PPAR-γ gene expression, since methylation-mediated silencing of inhibitory transcription factors such as Fli1 or Smad7 has been implicated as a mechanism for autonomous activation of SSc fibroblasts 
. While we cannot fully exclude the possibility that hypermethylation of the regulatory elements of the PPAR-γ gene result in gene silencing in SSc fibroblasts, in preliminary studies the methylation inhibitor 5-azacytidine failed to normalize PPAR-γ expression (data not shown).
Analysis of microarray data from SSc skin biopsies previously identified a patient subset with a “TGF-ß-responsive gene signature” 
. This pattern of gene expression was only seen in biopsies from patients with dcSSc, and was not found in biopsies from patients with other forms of scleroderma, or from healthy controls. The “TGF-ß responsive signature” identifies a subset of scleroderma patients with extensive skin involvement and propensity to pulmonary fibrosis 
. Remarkably we observed that the expression of a majority of differentially regulated PPAR-γ target genes (80%) was diminished in the “TGF-ß responsive gene signature” subset biopsies. These observations reveal an inverse relationship in the tissue expression of TGF-ß-inducible genes and PPAR-γ inducible genes, a result not unexpected in view of our present findings that TGF-ß suppresses PPAR-γ expression and transcriptional activity in fibroblasts, and our previous demonstration that PPAR-γ directly inhibits Smad-mediated TGF-ß responses. The reciprocal relationship between the expressions of these two sets of genes is consistent with a mutually antagonistic cross-talk of the TGF-ß and PPAR-γ pathways. In this context it is remarkable that diminished PPAP-γ expression or function is a recurring theme in human fibrotic conditions and animal models of fibrosis. For example, intracellular PPAR-γ levels were diminished in alveolar macrophages from patients with pulmonary fibrosis 
, and in the fibrotic lesions in cicatrical alopecia 
. In addition to the association of low PPAR-γ with fibrosis in various human disorders, evidence for a causal relationship comes from animal models. Tissue-specific down-regulation of PPAR-γ, whether due to loss-of-function mutations 
, genetic targeting 
, or overexpression of PPAR-γ inhibitors such as TGF-ß or Wnt 
, is invariably accompanied by exuberant local fibrosis. It is intriguing that in SSc, skin fibrosis is commonly accompanied by a reduction in the subcutaneous fat layer, and in some biopsies, complete adipose tissue atrophy 
. We speculate that in these cases active TGF-ß signaling in the dermis suppresses PPAR-γ in situ with consequent disruption of adipogenesis, loss of adipocytes and lipoatrophy. These observations highlight the importance of a functional antagonism between profibrotic TGF-ß signaling on the one hand, and the anti-fibrotic PPAR-γ pathway on the other, in the pathogenesis of SSc.
One of the critical mechanisms employed by TGF-β in the promotion of fibrosis is epithelial-mesenchymal transition (EMT) 
. In this study, we observed reduced levels of epidermal PPAR-γ in SSc skin biopsies, whereas normal skin biopsies showed strong immunostaining in the basal epidermis (). High level of TGF-ß and ASMA expression in the basal epidermis have been reported previously 
. Because PPAR-γ signaling potently inhibits TGF-β-induced EMT in vitro 
, these observations raise the possibility that an important physiologic role of PPAR-γ in the epidermis is to prevent deregulated EMT, and its loss in SSc may contribute to promotion of EMT and fibrosis.
Since a primary biological function of PPAR-γ is to serve as an intracellular sensor for changes in fatty acid or prostaglandin levels, its abundance and availability are important in determining the intensity of ligand responses. Cellular responses elicited by endogenous ligands, such as adipogenic differentiation of mesenchymal progenitor cells, are dependent on PPAR-γ signaling 
. We have previously shown that augmenting levels of PPAR-γ by means of ectopic overexpression enhanced the cellular responsiveness to a PPAR-γ ligand 
. In light of the potent down-regulation of PPAR-γ observed in TGF-ß-treated cells, we anticipated that TGF-ß might reduce cellular sensitivity to PPAR-γ ligands. We found however that fibroblast activation was blunted by rosiglitazone even when it was added to the cultures 24 h after TGF-ß. Therefore, synthetic PPAR-γ ligands are capable of abrogating profibrotic responses even in the face of low PPAR-γ abundance. Currently approved PPAR-γ ligands such as rosiglitazone have been linked to side effects, including fluid retention, heart failure, and other cardiovascular events, raising questions regarding their safety 
. Novel PPAR-γ agonists with selective activity and potentially improved safety are under active investigation 
In summary, these results reveal that PPAR-γ expression and activity are impaired in SSc, which is correlated with active TGF-ß signaling and fibrosis in lesional tissues. There exists a reciprocal relation between the expression of TGF-ß-activated genes and PPAR-γ-regulated genes in lesional tissue and in explanted SSc fibroblasts. PPAR-γ plays a physiologic role in tissue remodeling as a negative regulator of fibroblast activation and differentiation, which is highlighted by the link between loss of PPAR-γ and spontaneous emergence of fibrosis. Reduced PPAR-γ in SSc, resulting from its suppression by TGF-ß and related cytokines, hypoxia and other mechanisms might contribute to unchecked fibroblast activation and persistent fibrogenesis.