Dosoki Heba, Stegemann Agatha, Taha Muna, Schnittler Hans, Luger Thomas A, Schröder Katrin, Distler Jörg H W, Kerkhoff Claus, Böhm Markus
Department of Dermatology, University of Münster, Münster, Germany.
Department of Botany and Microbiology, Alexandria University, Alexandria, Egypt.
Exp Dermatol. 2017 Jan;26(1):73-81. doi: 10.1111/exd.13180.
Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22 and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-β had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-β -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-β effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.
尽管越来越多的证据表明氧化应激参与胶原蛋白合成和成肌纤维细胞活化，但在人皮肤成纤维细胞（HDFs）和皮肤纤维化的背景下，烟酰胺腺嘌呤二核苷酸磷酸氧化酶（Nox）系统尚未得到充分研究。使用泛Nox抑制剂二苯基碘鎓（DPI）作为初始工具，我们发现HDFs中I型胶原蛋白、α平滑肌肌动蛋白（α-SMA）和纤连蛋白1的基因表达受到抑制。对所有Nox亚型和衔接蛋白的详细表达分析显示，Nox4、p22和Poldip2存在RNA表达和蛋白表达，但未检测到Nox1和Nox2。Nox4可免疫定位到内质网。重要的是，转化生长因子-β（TGF-β）对HDFs中的NADH活性和Nox4基因表达具有剂量和时间依赖性上调作用。在HDFs以及从Nox4基因敲除小鼠建立的鼠成纤维细胞中，小干扰RNA（siRNA）证明Nox4的基因沉默证实了TGF-β介导的I型胶原蛋白基因、α-SMA和纤连蛋白1基因表达依赖于Nox4。如计算机启动子分析、Smad3的药理学抑制和基因沉默所示，这种TGF-β效应由Smad3介导。在博来霉素诱导的硬皮病小鼠模型中突出了这些发现的相关性。DPI治疗减轻了皮肤纤维化和成肌纤维细胞活化。此外，siRNA敲低Nox4可降低体内皮肤胶原蛋白合成、α-SMA和纤连蛋白1表达。最后，对系统性硬化症患者的HDFs分析证实了Nox4及其衔接蛋白的表达，而未检测到Nox1和Nox2。我们的研究结果表明，靶向Nox4是未来治疗纤维化皮肤病的有前景的方法。
