Disruption of transforming growth factor β signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor γ

Objective. In fibroblasts, transforming growth factor beta (TGFbeta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGFbeta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGFbeta/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 gamma (PPARgamma) in normal skin fibroblasts and its effect on TGFbeta-induced cellular responses. Methods. The expression and activity of PPARgamma 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 PPARgamma activation by naturally occurring and synthetic ligands on collagen synthesis and a-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPARgamma expression constructs. Results. The PPARgamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPARgamma resulted in modest suppression of basal collagen gene expression, it abrogated TGFbeta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPARgamma in fibroblasts, and was blocked by a selective antagonist of PPARgamma signaling or by transfection of fibroblasts with dominant-negative PPARgamma constructs. Furthermore, PPARgamma ligands abrogated TGFbeta-induced expression of a-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGFbeta was suppressed by PPARgamma despite the absence of a consensus PPARgamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPARgamma had no effect on protein expression of cellular Smad3 or Smad7. Conclusion. By abrogating of TGFbeta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPARgamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPARgamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.