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).

本项目探索先天抗微生物防御、促炎和其他信号机制，包括Nox家族NADPH氧化酶产生活性氧（ROS）。我们目前的重点是研究主要在上皮细胞中表达的非吞噬性Nox家族NADPH氧化酶（Nox1, Nox4, Duox1, Duox2）的细胞机制。研究的组织包括粘膜表面（肺和胃肠道）、肝脏、肾脏、甲状腺和外分泌腺（唾液腺、乳腺）和血管组织。这些氧化酶产生的活性氧影响对感染、生长因子、激素、细胞因子、细胞分化、细胞衰老、细胞程序性死亡（凋亡）和氧感知的反应。几种非吞噬性Nox酶也在宿主防御和炎症过程中发挥作用，因为它们主要在上皮细胞的顶端表面表达，并由细胞因子或病原体相关分子模式的识别诱导或激活。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诱导Nox4涉及自分泌tgf - β刺激Nox4的表达，这与tgf - β在一些纤维化疾病易感组织（肺、肾、肝、心）中的诱导一致。