Mucin Gene Regulation by Elastase and Oxidants

慢性支气管炎急性发作期肺功能下降的主要病理特征是粘液分泌物阻塞气道。在本申请中，我们提出了一种新的范例，其中一种新的途径将COPD触发因素、气道炎症和粘液过度产生联系在一起。已知急性加重的触发因素增加嗜中性粒细胞弹性蛋白酶（NE）的气道浓度。我们先前已经在气道上皮细胞中显示NE处理产生细胞内活性氧，并且这些活性氧是NE增加的MUC 5AC粘蛋白表达所需的。在本申请中，我们提出了几个关键的观察与NAD（P）H的激活：醌氧化还原酶（NQO 1）的激活，活性氧的产生增加，并增加粘蛋白MUC 5AC mRNA的稳定性，由于结合的HuR，RNA结合蛋白，稳定mRNA。根据这些结果，我们提出以下假设：在慢性支气管炎急性加重期，中性粒细胞弹性蛋白酶（NE）激活NQO 1，NQO 1产生活性氧（ROS），ROS改变HuR的表达和/或胞浆定位，从而增强HuR与MUC 5AC mRNA的结合; HuR结合增加MUC 5AC mRNA稳定性，导致MUC 5AC粘蛋白糖蛋白产生增加和气道阻塞增加。为了检验这些假设，我们将使用体外和体内模型系统。具体目标是：具体目标1。确定NQO 1是否是调节NE诱导的MUC 5AC mRNA水平的信号通路的组成部分。a.确定NE暴露是否影响NQO 1表达或活性。B.确定NE调节的MUC 5AC表达是否需要NQO 1表达和活性。具体目标2。确定NE是否通过HuR结合增加MUC 5AC mRNA稳定性。a.确定NE和/或NQO 1产生的ROS是否改变气道上皮细胞中HuR的表达或细胞质定位。B.确定NE和/或NQO 1产生的ROS是否影响HuR与MUC 5AC的结合。C.确定HuR结合是否足以稳定MUC 5AC 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.