Disruption of transforming growth factor β signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor γ
Disruption of transforming growth factor β signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor γ
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DOI：
10.1002/art.20104
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发表时间：
2004-04-01
影响因子：
--
通讯作者：
Varga, J
中科院分区：
文献类型：
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作者：
Ghosh, AK;Bhattacharyya, S;Varga, J
目标。在成纤维细胞中，转化生长因子β(TGFbeta)通过Smad细胞内信号转导通路刺激胶原合成和肌成纤维细胞转分化。转化生长因子β介导的成纤维细胞活化是硬皮病及相关纤维化疾病的标志，干扰细胞内转化生长因子β/Smad信号可能为控制纤维化提供新的途径。由于其在调节炎症和纤维化反应中的潜在作用，我们检测了核激素受体过氧化体增殖物激活受体γ(PPARGamma)在正常皮肤成纤维细胞中的表达及其对转化生长因子β诱导的细胞反应的影响。通过Northern和Western印迹分析、免疫细胞化学、流式细胞术和报告基因瞬时转染法检测PPARGamma在正常皮肤成纤维细胞中的表达和活性。同样的方法被用来评估自然发生的和合成的配体激活PPARγ对胶原合成和α-平滑肌肌动蛋白(α-SMA)表达的影响。利用野生型和显性负性PPAR-Gamma表达载体进行瞬时转染实验，检测Smad介导的转录反应的调节。PPARGamma受体在静止的正常皮肤成纤维细胞中表达并具有完整的功能。而细胞PPARGamma的配体激活对基础胶原基因的表达有一定的抑制作用，但它以浓度依赖的方式抑制了TGFbeta诱导的刺激。这种反应是通过在成纤维细胞中过表达PPARGamma来模拟的，并被PPARGamma信号的选择性拮抗剂或通过转染显性负PPARGamma结构的成纤维细胞来阻断。此外，PPARγ配体可抑制转化生长因子β诱导的肌成纤维细胞标志物a-SMA的表达。尽管在靶向启动子中缺乏一致的PPARGamma反应元件，但TGFbeta对Smad依赖的转录反应的刺激可被PPARGamma抑制。配体诱导的成纤维细胞Smad3、Smad7蛋白表达无明显变化。通过抑制转化生长因子β对正常成纤维细胞胶原基因表达、肌成纤维细胞转分化和Smad依赖启动子活性的刺激作用，PPARGamma可能在促纤维化反应的调节中发挥生理学作用。此外，我们的结果表明，药物激动剂激活PPARγ可能是控制硬皮病纤维化的一种新方法。
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.