The NAD(P)H Oxidase in Airways Remodeling and Reactivity

描述(申请人提供)：目标：本项目的目标是 内源性NAD(P)H氧化酶在增生性鼻咽癌中的作用 反应和增强的伸缩性导致呼吸道平滑肌(AWSM) 哮喘的重塑和高反应性。假说：包容性假说 有待检验的是，气道平滑肌NAD(P)H氧化酶受到高度调控 ROS产生酶在启动AWSM增殖中扮演不同的角色 和收缩。具体施舍：第一个具体目标将是 呼吸道平滑肌细胞NAD(P)H氧化酶(S)及其活性的测定 对ROS的生成做出了贡献。这个目标将解决这一假设 NAD(P)H氧化酶4(NOX4)与内质网膜的结合 是AWSM增殖过程中NAD(P)H氧化酶的催化亚基。这个 第二个具体目标将确定NAD(P)H氧化酶(S)在AWSM中的作用 扩散。这一目标将检验特定增长因素 通过激活NOX4氧化酶及其产物诱导AWSM增殖 核因子-kB的反式激活。第三个具体目标是 明确NAD(P)H氧化酶(S)在AWSM收缩功能中的作用。这一目标 将检验由NAD(P)H氧化酶产生的ROS诱导AWSM的假设 收缩，但收缩表型中氧化物酶成分 与增殖性表型不同。研究计划：针对 第一个目标是，我们的战略是定义和充分描述 负责AWSM NAD(P)H氧化酶活性的组分并确定其 亚细胞定位和相互作用。我们还将描述该活动的特征， AWSM NAD(P)H氧化酶的氧化还原中点电位及个体的作用 在ROS的生成中的组件。对于第二个目标，我们将利用 NAD(P)H氧化酶组分缺乏的纯化细胞直接 确定氧化酶在AWSM增殖中的重要性。重点将是 致力于确定氧化酶在核因子-kB反式激活中的作用 对平滑肌增殖至关重要的因子。为了第三个目标，我们将 再利用NAD(P)H基因缺陷的纯化细胞和动物 直接确定氧化物组分的重要性和特点 调节呼吸道收缩功能的氧化酶。重点将是 放在研究是否有一个表型转换的氧化酶 组件。意义：这项工作将使我们更好地了解 AWSM NAD(P)H氧化酶在介导呼吸道平滑肌中的重要性 哮喘时的重塑和哮喘状态下的收缩能力 ROS在呼吸生理学和疾病中作用的重要性。

DESCRIPTION (provided by applicant): OBJECTIVE: The goals of this project are to identify the roles of endogenous NAD(P)H oxidases in the proliferative responses and enhanced contractility leading to airways smooth muscle (AWSM) remodeling and hyperreactivity in asthma. HYPOTHESIS: The inclusive hypothesis to be tested is that airway smooth muscle NAD(P)H oxidases are highly regulated ROS producing enzymes that play distinct roles in initiating AWSM proliferation and contraction. SPECIFIC AlMS: The first specific aim will characterize the NAD(P)H oxidase(s) of airway smooth muscle (AWSM) cells and determine its contribution to the generation of ROS. This aim will address the hypothesis that NAD(P)H Oxidase 4 (Nox4) bound to membranes in the endoplasmic reticulum is the catalytic subunit of the NAD(P)H oxidase in proliferating AWSM. The second specific aim will define the role of NAD(P)H oxidase(s) in AWSM proliferation. This aim will test the hypotheses that specific growth factors induce AWSM proliferation via activation of the Nox4 oxidase with resultant transactivation of nuclear factor-kappa B (NF-kB). The third specific aim will define the role of NAD(P)H oxidase(s) in AWSM contractile function. This aim will test the hypothesis that ROS generated by an NAD(P)H oxidase induce AWSM contraction, but that components of the oxidase in the contractile phenotype are distinct from those of the proliferative phenotype. RESEARCH PLAN: For the first aim, our strategy is to define and fully characterize the structure of the components responsible for AWSM NAD(P)H oxidase activity and pinpoint their subcellular location and interaction. We will also characterize the activity, the redox midpoint potential of AWSM NAD(P)H oxidase and the role of individual components in the generation of ROS. For the second aim we will utilize purified cells with deficiencies of NAD(P)H oxidase components to directly establish the importance of the oxidase in AWSM proliferation. Emphasis will be placed on establishing the role of the oxidase in transactivation of NF-kB, a factor essential for smooth muscle proliferation. For the third aim we will again utilize purified cells and animals with genetic deficiences of NAD(P)H oxidase components to directly establish the importance and characteristics of the oxidase that regulates airways contractile function. Emphasis will be placed upon investigating whether there is a phenotype switch in the oxidase components. SIGNIFICANCE: The work will provide a better understanding of the importance of AWSM NAD(P)H oxidase in mediating airways smooth muscle remodeling in asthma and contractility in the asthmatic state, with broad importance in the role of ROS in respiratory physiology and disease.