Nox family NADPH oxidases: roles in innate immunity and inflammatory disease

该计划探讨了先天性抗微生物防御，促炎和其他信号机制，涉及Nox家族NADPH氧化酶的故意活性氧（ROS）产生。我们目前的重点是研究细胞机制调节非吞噬细胞的Nox家族NADPH氧化酶主要在上皮细胞（Nox 1，Nox 4，Duox 1，Duox 2）。研究的组织包括粘膜表面（肺和胃肠道）、肝、肾、甲状腺和外分泌腺（唾液腺、乳腺）以及血管组织。由这些氧化酶产生的ROS影响对感染、生长因子、激素、细胞因子、细胞分化、细胞衰老、程序性细胞死亡（凋亡）和氧感测的响应。几种非吞噬性Nox酶也在宿主防御和炎症过程中起作用，因为它们主要在上皮细胞的顶端表面上表达，并且由细胞因子或通过识别病原体相关的分子模式诱导或激活。最近，我们发现Nox 4在丙型肝炎病毒（HCV）感染的肝细胞中被诱导，并且是过量慢性ROS（氧化应激）的来源，可导致肝纤维化（肝硬化）。HCV对Nox 4的诱导涉及Nox 4表达的自分泌TGF-β刺激，这与在易患纤维化疾病的几种组织（肺、肾、肝、心脏）中TGF-β对其的诱导一致。 在2012年，我们研究了在上皮-间充质转化（EMT）的背景下TGF-β对Nox 4的诱导，EMT是一个细胞在正常发育和伤口愈合过程中表现出可塑性增加的过程，在病理环境中也起作用，如纤维化和肿瘤转移。我们的研究表明，Nox 4在正常和转移性乳腺上皮细胞中起着基于氧化还原的信号传导作用，促进EMT和细胞迁移。我们发现TGF-β诱导正常（MCF 10A）和转移性（MDA-MB-231）人乳腺上皮细胞中的Nox 4（mRNA和蛋白质）和ROS生成，而表达显性阴性（DN）Nox 4或Nox 4靶向shRNA的细胞显示对TGF-β的反应较低的ROS生成。组成型活性TGF-β受体I型显着增加Nox 4启动子活性，mRNA和蛋白质，和ROS的产生。Nox 4转录调节TGF-β是SMAD 3依赖性的，基于组成型活性SMAD 3的作用，而DN SMAD 3或SIS 3，SMAD 3抑制剂，具有相反的作用。Nox 4敲低、DN Nox 4或SMAD 3或SIS 3也减弱TGF-β诱导的伤口愈合和细胞迁移。最后，基于DN Nox 4在TGF-β处理的细胞中降低纤连蛋白mRNA的作用，我们表明Nox 4在TGF-β调节的纤连蛋白表达中起作用。这些数据表明，Nox 4有助于NADPH氧化酶依赖性ROS的产生，这对乳腺上皮细胞的EMT进展和迁移至关重要。这些观察结果表明，Nox 4是影响伤口愈合、纤维化、癌症进展和转移的治疗干预的潜在靶标。 我们对Duox重建细胞模型的研究已用于鉴定几种Duox单核苷酸多态性（SNP）和改变氧化酶功能或细胞靶向的突变，其中6种与先天性甲状腺功能减退症有关。在患者人群中筛选其他表现出Duox活性改变的常见多态性，以确定其与感染性或炎性疾病（哮喘、细菌或病毒感染、囊性纤维化）易感性改变的联系。我们还进行了一项调查，小鼠品系特异性Duox多态性，可能使小鼠结肠炎的谷胱甘肽过氧化物酶1和2的情况下。相关实验正在探索微生物激动剂的作用，这些微生物激动剂激活肠道上皮细胞中的Duox 2，从而刺激炎症反应。 循环吞噬细胞产生高水平的ROS，其作为重要的杀微生物剂响应于感染或炎症刺激，这归因于NADPH氧化酶活化（基于Nox 2或phox的酶）。患有慢性肉芽肿病（CGD）的患者患有NADPH氧化酶缺陷，导致对微生物感染和异常炎症反应的敏感性增强。在合作研究中，我们正在研究促氧化剂作为吞噬细胞NADPH氧化酶的直接激活剂的作用。我们发现，过氧化氢或花生四烯酸可以触发p40 phox的膜转位，导致其他氧化酶组分的组装和激活。其他研究表明，抗氧化酶，peroxiredoxin 6，协会和共同迁移与胞质phox蛋白，并作为一个积极的调节氧化酶活性。其氧化酶支持功能被证明涉及释放花生四烯酸的磷脂酶A2活性。最后，氧化还原活性铜绿假单胞菌毒素，绿脓菌素，也被证明可以通过产生细胞内氧化剂来触发氧化酶激活。这些研究表明，促氧化剂可以在正前馈信号传导机制中起作用，以促进过量ROS的产生，这提供了对慢性假单胞菌感染情况下发生的促炎性肺病发展机制的了解（即，囊性纤维化）。

This program explores innate anti-microbial defense, pro-inflammatory and other signaling mechanisms involving deliberate reactive oxygen species (ROS) production by Nox family NADPH oxidases. Our current focus investigates cellular mechanisms regulating nonphagocytic Nox family NADPH oxidases expressed primarily in epithelial cells (Nox1, Nox4, Duox1, Duox2). Tissues studied include mucosal surfaces (lung and gastrointestinal tract), liver, kidney, thyroid and exocrine glands (salivary, mammary), and vascular tissues. ROS produced by these oxidases affect responses to infection, growth factors, hormones, cytokines, cell differentiation, cellular senescence, programmed cell death (apoptosis) and oxygen sensing. Several non-phagocytic Nox enzymes also serve in host defense and inflammatory processes, as they are expressed predominately on apical surfaces of epithelial cells and are induced or activated by cytokines or by recognition of pathogen-associated molecular patterns. Recently, we found Nox4 is induced in Hepatitis C virus (HCV)-infected hepatocytes and is a source of excess chronic ROS (oxidative stress) that can lead to liver fibrosis (cirrhosis). The induction of Nox4 by HCV involves autocrine TGF-beta stimulation of Nox4 expression, consistent with its induction by TGF-beta in several tissues susceptible to fibrotic disease (lung, kidney, liver, heart). In 2012, we studied Nox4 induction by TGF-beta in the context of the epithelial-to-mesenchymal transition (EMT), a process in which cells assume increased plasticity during normal development and wound healing that also functions in pathological settings such as fibrosis and tumor metastasis. Our studies show that Nox4 serves a redox-based signaling role promoting the EMT and cell migration in normal and metastatic breast epithelial cells. We found TGF-beta induces Nox4 (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells, whereas cells expressing dominant-negative (DN) Nox4 or Nox4-targeted shRNA show lower ROS production in response to TGF-beta. Constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3-dependent, based on effects of constitutively active SMAD3, whereas DN SMAD3 or SIS3, a SMAD3 inhibitor, had the opposite effects. Nox4 knockdown, DN Nox4 or SMAD3, or SIS3 also blunted TGF-beta induced wound healing and cell migration. Finally, we showed that Nox4 plays a role in TGF-beta regulated fibronectin expression, based on the effects of DN Nox4 in reducing fibronectin mRNA in TGF-beta treated cells. These data indicate Nox4 contributes to NADPH oxidase-dependent ROS production critical for EMT progression and migration of breast epithelial cells. These observations suggest Nox4 as a potential target for therapeutic intervention to affect wound healing, fibrosis, cancer progression and metastasis. Our studies on Duox-reconstituted cell models have been used to identify several Duox single nucleotide polymorphisms (SNPs) and mutations that alter oxidase function or cellular targeting, six of which have been linked to congenital hypothyroidism. Other common polymorphisms exhibiting altered Duox activity are being screened in patient populations for links to altered susceptibilities to infectious or inflammatory disease (asthma, bacterial or viral infection, cystic fibrosis). We have also undertaken a survey of murine strain-specific Duox polymorphisms that may predispose mice to colitis in the absence of glutathione peroxidase 1 and 2. Related experiments are exploring effects of microbial agonists that activate Duox2 in gut epithelial cells that would thereby stimulate inflammatory responses. Circulating phagocytes generate high levels of ROS that serve as important microbicidal agents in response to infectious or inflammatory stimuli, which is attributed to NADPH oxidase activation (Nox2- or phox-based enzyme). Patients with chronic granulomatous disease (CGD) suffer from NADPH oxidase deficiencies resulting in enhanced susceptibility to microbial infections and aberrant inflammatory responses. In collaborative studies, we are examining roles of pro-oxidants as direct activators the phagocytic NADPH oxidase. We showed that membrane translocation of p40phox can be triggered by hydrogen peroxide or arachidonic acid, leading to assembly and activation of other oxidase components. Other studies showed that the antioxidant enzyme, peroxiredoxin 6, associates and co-migrates with cytosolic phox proteins and serves as a positive regulator of oxidase activity. Its oxidase-supportive function was shown to involve phospholipase A2 activity that releases arachidonate. Finally, the redox-active Pseudomonas aeruginosa toxin, pyocyanin, was also shown to trigger oxidase activation through generation of intracellular oxidants. These studies demonstrating that pro-oxidants can act in a positive feed-forward signaling mechanism to promote excess ROS generation provide insight on mechanisms of pro-inflammatory lung disease development occurring in cases of chronic Pseudomonas infection (i.e., cystic fibrosis).