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

Nox 家族 NADPH 氧化酶：在先天免疫和炎症性疾病中的作用

• 批准号: 8555786
• 负责人: THOMAS LETO
• 金额: $ 84.31万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555786
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Affect; Agonist; Anabolism; Animal Model; Antioxidants; Apical; Apoptosis; Arachidonic Acids; Asthma; Bacterial Infections; Biochemical; Blood Vessels; Breast; Cell Aging; Cell Differentiation process; Cell model; Cells; Chronic; Chronic Granulomatous Disease; Cirrhosis; Colitis; Cretinism; Cystic Fibrosis; Data; Defect; Development; Disease; Dominant-Negative Mutation; Enzymes; Epithelial; Epithelial Cells; Exhibits; Exocrine Glands; Extracellular Matrix; Family; Fibronectins; Fibrosis; Gastrointestinal tract structure; Generations; Genetic Polymorphism; Goals; Growth Factor; Heart; Hepatitis C; Hepatitis C virus; Hepatocyte; Hormones; Host Defense; Human; Hydrogen Peroxide; Immune; Infection; Inflammatory; Inflammatory Response; Isoenzymes; Kidney; Lead; Link; Liver; Liver Fibrosis; Lung; Lung diseases; MADH3 gene; Mammary gland; Mediating; Membrane; Messenger RNA; Molecular; Mus; Mutation; NADPH Oxidase; Natural Immunity; Neoplasm Metastasis; Nox enzyme; Oxidants; Oxidases; Oxidation-Reduction; Oxidative Stress; Oxygen; Patients; Pattern; Pattern Recognition; Peroxidases; Phagocytes; Phospholipase A2; Play; Predisposition; Process; Production; Proteins; Pseudomonas Infections; Pseudomonas aeruginosa; Pyocyanine; Reactive Oxygen Species; Role; Salivary; Signal Transduction; Single Nucleotide Polymorphism; Source; Stimulus; Surface; Surveys; System; TGF-beta type I receptor; Therapeutic Intervention; Thyroid Gland; Tissues; Toxin; Transcriptional Regulation; Transforming Growth Factor beta; Viral; Virus Diseases; Wound Healing; antimicrobial; arachidonate; autocrine; base; cell motility; cellular targeting; cytokine; epithelial to mesenchymal transition; feeding; glutathione peroxidase; inhibitor/antagonist; insight; interest; microbial; microbicide; migration; neutrophil cytosol factor 40K; pathogen; patient population; peroxiredoxin; programs; promoter; reconstitution; research study; response; small hairpin RNA; tissue/cell culture; tumor; tumor progression

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).

该计划探讨了先天的抗微生物防御，促炎和其他信号传导机制，涉及NOX家族NADPH氧化酶的故意活性氧（ROS）产生。我们目前的重点研究了调节非肠道NOX家族NADPH氧化酶的细胞机制，主要在上皮细胞中表达（NOX1，NOX4，DUOX1，DUOX2）。研究的组织包括粘膜表面（肺和胃肠道），肝，肾脏，甲状腺和外分泌腺（唾液，乳腺）和血管组织。这些氧化酶产生的ROS会影响对感染，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）和氧气感应的反应。几种非变形细胞NOX酶也用于宿主防御和炎症过程，因为它们主要在上皮细胞的顶端表面表达，并被细胞因子或通过病原体相关的分子模式诱导或激活。最近，我们发现NOX4在丙型肝炎病毒（HCV）感染的肝细胞中诱导，并且是导致肝纤维化（cirrhosis）的过量慢性ROS（氧化应激）的来源。 HCV对NOX4的诱导涉及自分泌TGF-β刺激NOX4表达，这与易感纤维化疾病（肺，肾脏，肝脏，心脏）的几种组织中TGF-β的诱导一致。 在2012年，我们在上皮到间质转变（EMT）的背景下研究了TGF-beta的NOX4诱导，在这种过程中，细胞在正常发育和伤口愈合过程中假设可塑性增加，并且在纤维化和肿瘤转移等病理环境中也起作用。我们的研究表明，NOX4发挥了基于氧化还原的信号传导作用，可促进正常和转移性乳房上皮细胞中EMT和细胞迁移。我们发现TGF-β在正常（MCF10A）和转移性（MDA-MB-231）中诱导NOX4（mRNA和蛋白质）和ROS产生，而人类乳腺上皮细胞，而表达显性阴性（DN）NOX4或NOX4含量的SHRNA的细胞表现出对TGF-BETA的ROS产生较低的ROS产生。组成性活性的TGF-β受体I型显着增加了NOX4启动子活性，mRNA和蛋白质以及ROS的产生。 TGF-beta的NOX4转录调控是基于组成型活性SMAD3的效果的SMAD3依赖性的，而DN SMAD3或SIS3（SMAD3抑制剂）具有相反的效果。 NOX4敲低，DN NOX4或SMAD3或SIS3也钝化的TGF-β诱导伤口愈合和细胞迁移。最后，我们表明NOX4基于DN NOX4在减少TGF-BETA处理的细胞中纤连蛋白mRNA中的影响，在TGF-β调节的纤连蛋白表达中起作用。这些数据表明NOX4有助于NADPH氧化酶依赖性ROS产生对EMT进展和乳腺上皮细胞的迁移至关重要。这些观察结果表明NOX4是治疗干预措施的潜在靶标，以影响伤口愈合，纤维化，癌症进展和转移。 我们对DUOX重新确定的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，其中六个与先天性甲状腺功能减退症有关。在患者人群中筛选出表现出改变DUOX活性的其他常见多态性，以与感染或炎性疾病（哮喘，细菌或病毒感染，囊性纤维化）的敏感性改变有关。我们还对鼠菌株特异性的DUOX多态性进行了调查，在没有谷胱甘肽过氧化物酶1和2的情况下，可能使小鼠易感小鼠结肠炎。相关的实验是探索微生物激动剂的作用，这些实验会激活肠道上皮细胞中Duox2的作用，从而激活肠道上皮细胞，从而刺激炎症反应。 循环吞噬细胞会产生高水平的ROS，这些ROS响应于感染性或炎症刺激，这是NADPH氧化酶活性（NOX2-或基于PHOX的酶）。患有慢性肉芽肿性疾病（CGD）的患者患有NADPH氧化酶缺乏症，导致对微生物感染和异常炎症反应的敏感性增强。在协作研究中，我们正在研究促氧化剂作为吞噬NADPH氧化酶的直接活化剂的作用。我们表明，p40phox的膜易位可以由过氧化氢或花生四烯酸触发，从而导致其他氧化酶成分的组装和激活。其他研究表明，抗氧化酶，过氧蛋白6，伴侣并与胞质phox蛋白共移民，并作为氧化酶活性的阳性调节剂。显示其氧化酶支持功能涉及释放蛛网膜酸的磷脂酶A2活性。最后，还显示出通过细胞内氧化剂的产生，铜绿毒素铜毒素铜绿毒素，铜绿毒素，铜绿假单胞菌毒素。这些研究表明，促氧化剂可以以阳性的馈送信号传导机制作用，以促进过量的ROS产生，从而深入了解在慢性假单胞菌感染（即囊性纤维化）的情况下，发生促炎性肺部疾病发育的机制。