PPARγ downregulation by TGFß in fibroblast and impaired expression and function in systemic sclerosis: a novel mechanism for progressive fibrogenesis

Abstract

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)-dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a "TGF-ß responsive gene signature" in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis.

Figure 1. PPAR-γ expression is reduced in SSc.

A. Expression of PPAR-γ and PAI-1 mRNA was determined by analyzing a previously described skin biopsy microarray dataset from a well-characterized cohort of scleroderma patients (n = 17) . The fold change of mRNA levels was normalized with the average expression level of the samples of whole cohort. Each point represents the relative PPAR-γ and PAI-1 mRNA levels in dcSSc forearm biopsies. B. Skin biopsies from patients with dSSc (n = 3) and normal controls (n = 3) were immunostained with antibodies to PPAR-γ. C. Lung biopsies from patients with SSc-associated advanced pulmonary fibrosis (n = 5) and non-fibrotic controls (n = 5) were immunostained with antibodies to PPAR-γ. Left panels, representative images. Sections incubated with preimmune serum showed absence of staining. Right panel, expression of PPAR-γ in the biopsies was scored from 0 (no immunostaining) -5 (strong immunostaining). The results shown as symbols represent the means from five separate fields per section for each sample at magnification of 630x, and the horizontal lines represent the means.

Figure 2. PPAR-γ expression is reduced in explanted SSc skin fibroblasts.

Dermal fibroblasts explanted from patients with dcSSc (n = 6) or healthy controls (n = 4) were harvested at confluence. A. RNA was subjected to real-time qPCR. The results shown as symbols represent the means of triplicate determinations normalized with ß-actin, and the horizontal lines represent the means. B. Nuclear and cytosolic fractions were subjected to Western analysis. Representative immunoblots.

Figure 3. TGF-ß suppresses PPAR-γ mRNA expression in fibroblasts.

A. Confluent fibroblasts were incubated with 10 ng/ml TGF-ß for indicated period (left panel), or for 24 h with indicated concentrations of TGF-ß (right panel). RNA expression was examined by real-time qPCR. The results, normalized with ß-actin mRNA, represent the means ± SD of triplicate determinations. *p<0.05. B. Total RNA was isolated following 24 h incubation with TGF-ß, and subjected to Northern analysis. C. Fibroblasts and monocyte-derived macrophages were incubated with TGF-ß or IL-4 (both at 10 ng/ml) in parallel for 24 h, and RNA was examined by real-time qPCR. Results represent the means ± SD of triplicate determinations. * p<0.05.

Figure 4. TGF-ß suppresses PPAR-γ expression in fibroblasts.

A. Confluent normal dermal fibroblasts were transiently transfected with PPRE-luc for 18 h. Cultures were preincubated with rosiglitazone (10 µM) for 60 min, followed by TGF-ß for a further 48 h. Whole cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase activity, represent the means ± SD of triplicate determinations. * p<0.05. B. Following incubation with TGF-ß for 48 h, fibroblasts were immunostained using specific antibodies against PPAR-γ (green); DAPI blue. Images were taken under Zeiss UV Meta 510 confocal microscope (original magnification ×400).

Figure 5. Suppression of PPAR-γ via TßRI and Smad3.

A. Confluent foreskin fibroblasts were pre-treated with indicated protein kinase inhibitors for 60 min, followed by incubation with 10 ng/ml TGF-ß. Cultures were harvested 24 h later and mRNA levels were analyzed by real-time qPCR. The results, normalized with ß-actin, represent the means ± SD of triplicate determinations. * p<0.05. B. Fibroblasts were infected with Ad-null or Ad-TßRI^L45 (100 MOI) or indicated concentrations of Ad-TßRI^ca for 48 h, or C. Ad-Smad7 for 24 h (C). RNA subjected to real-time qPCR. The results, normalized with ß-actin, represent the means ± SD of triplicate determinations. * p<0.05. In C, whole cell lysates were subjected to Western analysis. D. Confluent cultures of Smad3^−/− or Smad3^+/+ mouse fibroblasts were incubated with 10 ng/ml TGF-ß for 24 h. RNA was subjected to real-time qPCR. The results, normalized with 18S rRNA, represent the means ± SD of triplicate determinations. * p<0.05. E. Normal dermal fibroblasts were transiently transfected with PPAR-γ-luc along with Smad3 expression vectors. Following a 24 h incubation, cultures were harvested and whole cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase activity, represent the means ± SD of triplicate determinations. ** p<0.005. In A, C, D, open bars, untreated cultures; closed bars, TGF-ß-treated cultures.

Figure 6. TGF-ß inhibits adipogenic differentiation of mesenchymal cell.

Confluent human subcutaneous preadipocytes or adult dermal fibroblasts were induced to undergo adipogenic differentiation in vitro. Subcutaneous preadipocytes were incubated with maintenance media, or differentiation media alone or in the presence of TGF-ß (upper panels). Dermal fibroblasts were incubated with DMEM, or DM-2/AM-1 for three cycles followed by incubation with 10 ng/ml TGF-ß for a further 3 weeks (lower panels), and photographed under a phase contrast microscope. Arrows indicate intracellular accumulation of oil droplets. Original magnification X200.

Figure 7. Rosiglitazione abrogates TGF-ß–induced fibroblast activation.

Confluent dermal fibroblasts were pretreated with TGF-ß for 24 h, followed by rosiglitazone (10 µM) for further 24 h. A. Total RNA was analyzed by real-time qPCR. The results, normalized with 18S, represent the means ± SD of triplicate determinations. * p<0.05. B. Whole cell lysates were subjected to Western analysis. C. Fibroblasts were fixed and stained with antibodies to α-smooth muscle actin (green color). Nuclei were visualized by DAPI (blue). Original magnification X 200.

Figure 8. Reduced PPAR-γ target gene expression in SSc skin.

Gene expression in lesional skin biopsies was examined by analysis of a previously described microarray dataset based on biopsies from patients with SSc (n = 17, red) and healthy controls (n = 6, green) . Samples were ordered based on their TGF-ß-activated signature (red branches, TGF-ß-activated signature; black branches, TGF-ß not activated). Of forty-one PPAR-γ target genes showing significant differential expression in SSc, thirty-three are down-regulated in samples with TGF-ß-activated gene signature, whereas eight genes show the inverse expression pattern.