Mucin Gene Regulation by Elastase and Oxidants

慢性支气管炎急性加重期肺功能下降的一个主要病理特征是粘液分泌物引起的呼吸道阻塞。在这个应用中，我们提出了一个新的范例，其中一个新的途径将COPD的触发因素、呼吸道炎症和粘液过度生产联系在一起。众所周知，急性加重的触发因素会增加气道中性粒细胞弹性蛋白酶(NE)的浓度。我们之前已经在呼吸道上皮细胞中表明，NE处理会产生细胞内的活性氧物种，这些活性氧物种是NE增加MUC5AC粘蛋白表达所必需的。在这一应用中，我们提出了几个关键的观察结果，将NE与NAD(P)H的激活联系在一起：苯醌氧化还原酶(NQO1)的激活，活性氧物种的增加，以及由于Hur(一种稳定mRNA的RNA结合蛋白)的结合而增强了粘蛋白MUC5AC的mRNA稳定性。根据这些结果，我们提出了以下假设方案：在慢性支气管炎急性加重期间，中性粒细胞弹性蛋白酶(NE)激活NQO1；NQO1产生活性氧(ROS)；ROS改变HUR的表达和/或细胞质定位，从而增强HUR与MUC5AC mRNA的结合；HUR结合增加MUC5AC mRNA的稳定性，导致MUC5AC粘蛋白糖蛋白的产生增加，并增加呼吸道阻塞。为了验证这些假说，我们将使用体外和体内模型系统。具体目的有：1.确定NQO1是否是调节NE诱导的MUC5AC基因表达的信号通路的组成部分。A.确定NE暴露是否影响NQO1的表达或活性。B.确定NE调节的MUC5AC表达是否需要NQO1的表达和活性。具体目的2.确定去甲肾上腺素是否通过HUR结合增加MUC5AC基因的稳定性。A.确定去甲肾上腺素和/或NQO1产生的ROS是否改变了Hur在呼吸道上皮细胞的表达或胞浆定位。B.确定NE和/或NQO1产生的RO是否影响HUR与MUC5AC的结合。C.确定HUR结合是否足以稳定MUC5AC mRNA。

A major pathologic feature contributing to pulmonary function decline in acute exacerbations of chronic bronchitis is airway obstruction by mucus secretions. In this application, we present a new paradigm in which a novel pathway links together COPD triggers, airway inflammation and mucus overproduction. It is known that triggers for acute exacerbations increase airway concentrations of neutrophil elastase (NE). We have previously shown in airway epithelial cells that NE treatment generates intracellular reactive oxygen species and these reactive oxygen species are required for NE-increased MUC5AC mucin expression. In this application, we present several key observations linking NE with activation of NAD(P)H:quinone oxidoreductase (NQO1) activation, increased generation of reactive oxygen species, and increased mucin MUC5AC mRNA stabilization due to binding of HuR, an RNA-binding protein that stabilizes mRNA. From these results, we propose the following hypothetical schema: During acute exacerbations of chronic bronchitis, neutrophil elastase (NE) activates NQO1; NQO1 generates reactive oxygen species (ROS); ROS alter the expression and/or the cytoplasmic localization of HuR, thus enhancing HuR binding to MUC5AC mRNA; HuR binding increases MUC5AC mRNA stability, leading to increased MUC5AC mucin glycoprotein production, and increased airway obstruction. To test these hypotheses, we will use both in vitro and in vivo model systems. The Specific Aims are: Specific Aim 1. To determine whether or not NQO1 is an integral component of the signaling pathway regulating NE-induced MUC5AC mRNA levels. a. To determine whether NE exposure affects NQO1 expression or activity. b. To determine whether NQO1 expression and activity are required for NE-regulated MUC5AC expression. Specific Aim 2. To determine whether or not NE increases MUC5AC mRNA stability via HuR binding. a. To determine whether NE and/or NQO1-generated ROS alter HuR expression or cytoplasmic localization in airway epithelial cells. b. To determine whether NE and/or NQO1-generated ROS affect HuR binding to MUC5AC. c. To determine whether HuR binding is sufficient for MUC5AC mRNA stabilization.