Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.
OBJECTIVE
In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/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 (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses.


METHODS
The expression and activity of PPAR gamma 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 gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs.


RESULTS
The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7.


CONCLUSIONS
By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.
Ghosh
Asish K
AK
University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60607, USA.
Bhattacharyya
Swati
S
Lakos
Gabriella
G
Chen
Shu-Jen
SJ
Mori
Yasuji
Y
Varga
John
J
D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D024042:Collagen Type I; D004268:DNA-Binding Proteins / Q000378:metabolism; D020405:Dermis / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005355:Fibrosis; D015870:Gene Expression; D006801:Humans; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D012595:Scleroderma, Systemic / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D000199:Actins; D024042:Collagen Type I; D004268:DNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D051785:Smad Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D003094:Collagen