Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.
Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.
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DOI：
10.1002/art.39242
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
Jiménez SA
中科院分区：
文献类型：
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作者：
Piera-Velazquez S;Makul A;Jiménez SA
系统性硬化症（SSc）的特征是严重的，往往是渐进的皮肤和多个内部器官纤维化。尽管过度ROS介导的氧化应激已经被牵连，但负责这些改变的机制仍然不清楚。NOX 4是负责ROS产生的七种NADPH氧化酶同工型之一。在这里，我们研究了NOX 4在SSc皮肤和培养的真皮成纤维细胞中的表达以及TGF-β对其的调节。NOX 4在正常和SSc皮肤采用免疫组织学，并在正常和SSc培养的真皮成纤维细胞定量PCR，荧光显微镜和Western印迹进行了评估。通过H2 O2产生的荧光测量来评估ROS水平。使用特异性激酶抑制剂研究了参与N 0X 4刺激的TGF-β信号传导。用选择性N 0X 4小分子抑制剂和N 0X 4 siRNA诱导N 0X 4抑制/下调。与正常皮肤相比，SSc皮肤成纤维细胞显示出强烈的NOX 4染色。培养的SSc成纤维细胞显示增加的NOX 4表达。TGF-β1通过PKC-δ和SMAD 2/3途径在正常和SSc成纤维细胞中引起有效的NOX 4蛋白和mRNA刺激。SSc成纤维细胞中的NOX 4敲低减少了ROS的产生并降低了胶原蛋白I的表达。在SSc皮肤和培养的SSc真皮成纤维细胞中，NOX 4的表达和产生组成性升高。TGF-β1通过PKC-δ和Smad 2/3信号通路刺激正常和SSc成纤维细胞中NOX 4的表达。小分子N 0X 4抑制剂减少成纤维细胞胶原和纤连蛋白的产生，并且N 0X 4 siRNA敲低减少SSc成纤维细胞ROS和胶原的产生。这些结果表明，NOX 4参与了Ssc相关的纤维化，并表明NOX 4抑制剂是SSc的新治疗方法。
Systemic Sclerosis (SSc) is characterized by severe and often progressive skin and multiple internal organ fibrosis. The mechanisms responsible for these alterations remain obscure, although excessive ROS-mediated oxidative stress has been implicated. NOX4 is one of seven NADPH oxidase isoforms responsible for ROS generation. Here, we examined NOX4 expression in SSc skin and cultured dermal fibroblasts and its regulation by TGF-β. NOX4 was assessed in normal and SSc skin employing immunohistology, and in normal and SSc cultured dermal fibroblasts by quantitative PCR, fluorescence microscopy and Western blots. ROS levels were assessed by fluorescence measurements of H2O2 production. The TGF-β signaling involved in NOX4 stimulation was studied employing specific kinase inhibitors. NOX4 inhibition/downregulation was induced with a selective NOX4 small molecule inhibitor and NOX4 siRNA. In contrast with normal skin SSc skin fibroblasts showed intense NOX4 staining. Cultured SSc fibroblasts displayed increased NOX4 expression. TGF-β1 caused potent NOX4 protein and mRNA stimulation in normal and SSc fibroblasts mediated by PKC-δ and SMAD2/3 pathways. NOX4 knockdown in SSc fibroblasts reduced ROS production and lowered collagen I expression. NOX4 expression and production are constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGF-β1 stimulates NOX4 expression in normal and SSc fibroblasts through PKC-δ and Smad2/3 signaling pathways. A small molecule NOX4 inhibitor decreased fibroblast collagen and fibronectin production and NOX4 siRNA knockdown reduced SSc fibroblast ROS and collagen production. These results demonstrate NOX4 involvement in SSc-associated fibrosis and suggest NOX4 inhibitors as novel therapeutic approaches for SSc.