Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis
Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis
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DOI：
10.1111/exd.13180
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发表时间：
2017-01-01
影响因子：
3.6
通讯作者：
Boehm, Markus
中科院分区：
文献类型：
--
作者：
Dosoki, Heba;Stegemann, Agatha;Boehm, Markus
尽管越来越多的证据表明氧化应激参与了胶原合成和肌成纤维细胞的激活，但在人类真皮成纤维细胞(HDFS)和皮肤纤维化的背景下，NADPH氧化酶(NOx)系统的研究还不完全。以PAN-NOx抑制剂二苯基碘(DPI)为初始工具，我们发现HDFS中I型胶原、α-SMA和纤维连接蛋白1的基因表达受到抑制。对所有NOx异构体和接头的详细表达分析表明，NOX4、p22(Phox)和Poldip2都有RNA和蛋白质的表达，但Nox1和NOX2都没有表达。NOX4可免疫定位于内质网。重要的是，转化生长因子-β(1)对HDFS的NADH活性和NOX4基因表达有剂量和时间依赖性的上调作用。在hDFS和从NOX4基因敲除小鼠建立的小鼠成纤维细胞中，通过siRNA证明NOX4的遗传沉默证实了转化生长因子-β(1)介导的I型胶原基因、α-SMA和纤维连接蛋白1基因的表达是依赖于NOX4的。Smad3的电子启动子分析、药理抑制和基因沉默表明，这种转化生长因子-β1的作用是由Smad3介导的。在博莱霉素诱导的硬皮病小鼠模型中，这些发现的相关性得到了强调。DPI治疗可减轻皮肤纤维化和肌成纤维细胞的激活。此外，被siRNA敲除的NOX4在体内减少了皮肤胶原合成、α-SMA和纤维连接蛋白1的表达。最后，对系统性硬化症患者的HDFS的分析证实了NOX4及其接头的表达，而NOX1和NOX2没有被检测到。我们的发现表明，NOX4靶向治疗纤维性皮肤病是一种很有前途的未来治疗方法。
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, alpha-smooth muscle actin (alpha-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(phox) and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-beta(1) 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-beta(1)-mediated collagen type I gene, alpha-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-beta 1 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, alpha-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.