Dual Oxidase in Airway Epithelial Repair and Remodeling

描述（由申请人提供）：最近的研究表明，气道内存在新的NADPH氧化酶（Nox）同源物，与吞噬细胞gp91phox （Nox2）同源，在各种炎症或环境刺激下负责根尖上皮产生H2O2。这些氮氧化物同源物，被称为双氧化酶（Duox），以两种亚型存在，其中Duox1主要在气管支气管上皮中表达，而Duox2在唾液腺或粘膜下腺中被检测到。除了在气道宿主防御中的假设作用外，最近的研究表明气道上皮Duox1的其他功能，包括通过促进生长因子、金属蛋白酶和细胞因子的产生来调节上皮H+运输和参与上皮对损伤的反应，并刺激上皮细胞的迁移和修复。我们最近的研究表明，duox介导的H2O2在气管支气管上皮细胞对机械损伤的反应中产生，这是通过细胞释放ATP和刺激上皮表面的嘌呤能P2受体介导的。此外，上皮细胞的迁移和伤口修复被发现是由ATP介导的丝裂原活化蛋白激酶（MAPK）途径的激活和包括基质金属蛋白酶(MMP)-9在内的金属蛋白酶的激活介导的，其机制涉及Duox1。因此，我们假设Duox1通过刺激损伤后的上皮修复过程、上皮表面的局部氧化事件和/或氧化还原依赖性细胞信号通路的激活，有助于维持气道上皮屏障的完整性。此外，根据最近的观察，Th2细胞因子（IL-13, IL-4）可以诱导上皮Duox1表达，并且在变应性气道炎症小鼠模型中肺Duox1表达显著增加，我们提出过度或持续的Duox1激活可能有助于慢性伤口反应和气道重塑，正如在慢性哮喘中观察到的那样。本研究的主要目的是确定Duox1在体外激活介导上皮细胞迁移和修复的机制，并确定Duox1在体内对气道上皮修复和重塑的贡献。我们将确定细胞外或细胞内H2O2产生和/或局部pH变化和H+转运对duox1介导的上皮细胞迁移（Aim 1）的贡献，确定duox1依赖性MAPK激活和生长因子/MMP激活（Aim 2）的机制，并通过氧化还原蛋白质组学方法表征duox1衍生的H2O2的细胞外和细胞氧化还原敏感靶点（Aim 3）。最后，我们将在萘致上皮损伤模型和变应性气道炎症小鼠模型中研究Duox1对上皮修复的贡献（Aim 4）。总的来说，这些研究将为Duox1的上皮生物学提供重要的新见解，并将确定Duox在哮喘等慢性气道疾病期间气道重塑中的潜在贡献。公共卫生相关性。气道上皮与环境持续接触，在肺部防御吸入的毒素和微生物中起着至关重要的作用。在哮喘等慢性气道疾病中，气道上皮受到损伤，刺激上皮细胞生长和修复的机制被激活。最近的研究发现，气道上皮内存在一种产生氧化剂的酶，Duox1，它可能有助于气道宿主防御，类似于吞噬细胞中的新酶。然而，我们最近的研究也表明，Duox有助于损伤后的上皮修复过程。因此，尽管氧化应激被认为有助于疾病期间的组织损伤，上皮Duox1产生的低水平氧化剂可能有利于维持气道上皮屏障的完整性。该项目的目标是研究Duox1激活介导上皮修复过程的分子机制，以及Duox1产生的细胞氧化剂如何参与这一过程。其次，我们将在上皮损伤和过敏性气道疾病（如哮喘）的体内模型中研究Duox1在上皮修复中的重要性。总的来说，这些研究将进一步加深我们对气道Duox1的生物学作用及其在慢性气道疾病（如哮喘）中的潜在重要性的理解。

DESCRIPTION (provided by applicant): Recent studies have indicated the presence of new NADPH oxidase (Nox) homologs within the airways, with homology to phagocytic gp91phox (Nox2), that are responsible for apical epithelial production of H2O2 in response to various inflammatory or environmental stimuli. These Nox homologs, termed Dual Oxidases (Duox), exist as two isoforms, of which Duox1 is primarily expressed in the tracheobronchial epithelium, whereas Duox2 has been detected in salivary or submucosal glands. In addition to postulated roles in airway host defense, recent studies have suggested alternative functions of airway epithelial Duox1, including regulation of epithelial H+ transport and involvement in epithelial responses to injury by promoting production of growth factors, metalloproteinases, and cytokines, and stimulating epithelial cell migration and repair. Our recent studies have indicated Duox-mediated H2O2 production in tracheobronchial epithelial cells in response to mechanical injury, which is mediated by cellular release of ATP and stimulation of purinergic P2 receptors at the epithelial surface. Moreover, epithelial cell migration and wound repair were found to be mediated by ATP- mediated activation of mitogen-activated protein kinase (MAPK) pathways and activation of metalloproteinases including matrix metalloproteinase (MMP)-9, by mechanisms involving Duox1. Therefore, we hypothesize that Duox1 contributes to maintenance of airway epithelial barrier integrity, by stimulating epithelial repair processes in response to injury, by localized oxidative events at the epithelial surface and/or by activation of redox-dependent cellular signaling pathways. In addition, based on recent observations that Th2 cytokines (IL-13, IL-4) can induce epithelial Duox1 expression, and that lung Duox1 expression is markedly increased in a mouse model of allergic airway inflammation, we propose that exaggerated or persistent Duox1 activation may contribute to a chronic wound response and airway remodeling, as is observed in chronic asthma. The main objectives of this proposal are to identify the mechanisms by which Duox1 activation mediates epithelial cell migration and repair in vitro, and to establish the contribution of Duox1 to airway epithelial repair and remodeling in vivo. We will determine the contribution of extracellular or cellular H2O2 production and/or localized pH changes and H+ transport to Duox1-mediated epithelial cell migration (Aim 1), identify mechanisms involved in Duox1-dependent MAPK activation and growth factor/MMP activation (Aim 2), and characterize extracellular and cellular redox-sensitive targets of Duox1-derived H2O2 by redox proteomics approaches (Aim 3). Finally, we will investigate the contribution of Duox1 to epithelial repair in a model of epithelial injury using naphthalene, and in a mouse model of allergic airway inflammation (Aim 4). Collectively, these studies will provide important new insights into the epithelial biology of Duox1, and will establish the potential contribution of Duox in airway remodeling during chronic airway diseases such as asthma. PUBLIC HEALTH RELEVANCE. The airway epithelium is in continuous contact with the environment and is critical in lung defense against inhaled toxins and microbial organisms. In chronic airway diseases such as asthma, the airway epithelium is injured and mechanisms that stimulate epithelial cell growth and repair are activated. Recent studies have identified the presence of an oxidant-producing enzyme, Duox1, within the airway epithelium, which may contribute to airway host defense, similar to recent enzymes in phagocytes. However, our recent studies have also demonstrated that Duox contributes to epithelial repair processes after injury. Thus, although oxidative stress is believed to contribute to tissue damage during disease, low levels of oxidant production by epithelial Duox1 may be beneficial in maintaining airway epithelial barrier integrity. The goal of this project is to investigate the molecular mechanisms by which Duox1 activation mediates epithelial repair processes, and how cellular oxidants produced by Duox1 contribute to this. Secondly, we will investigate the importance of Duox1 in epithelial repair in in vivo models of epithelial injury and allergic airway disease such as asthma. Collectively, these studies will further our understanding of the biological roles of airway Duox1, and its potential importance in chronic airway diseases such as asthma.