MECHANISMS OF OXIDANT-INDUCED CHRONIC BRONCHITIS

描述（由申请人提供）：该项目的长期目标是了解导致慢性支气管炎相关结构变化的细胞和分子机制。这一应用继续了我们对氧化应激诱导的EGFR激活和粘液高分泌的关注。在目前的资助期内，我们已经证实了最初申请的假设，即活性氧（ROS）诱导透明质酸解聚，释放活性支气管TK，进而导致促egf加工、EGFR激活和下游信号，最终导致粘液细胞增殖和黏液蛋白分泌增加。然而，在气道上皮细胞中，EGFR在拓扑上通过顶端连接复合物（AJP）与其配体分离：配体在顶端膜中表达，而受体主要定位于基底外膜。生长因子与其受体的物理分离表明，必须存在一种受调节的机制，使配体与受体的相互作用仅在AJCs“允许”进入基底外侧的某些条件下发生。我们发现氧化诱导的上皮AJC连接破坏是由RON介导的，RON是一种酪氨酸激酶受体，独特地定位于气道上皮的顶膜。RON的配体是hgf样巨噬细胞刺激蛋白（MSP），它是作为一种非活性前体分泌的，需要通过蛋白水解裂解来刺激RON，在气道中，这种蛋白酶是组织激肽素（TK）。TK存在于气道上皮细胞的顶端表面，在那里它被透明质酸（HA）结合并灭活。ROS诱导的ha解聚导致活性TK的释放，通过这种机制，pro-MSP可以被激活，从而通过其顶膜受体RON发出信号，破坏AJC的完整性，从而允许egfr -配体相互作用和信号传导。我们将验证以下假设：氧化应激过程中，通过激活前msp和RON信号，增加了活性TK的根尖可用性，从而导致AJC破坏，从而允许EGF受体与配体相互作用，最终导致气道粘膜细胞化生和高分泌。目的1将验证以下假设：TK由ROS从上皮结合的HA中释放，将不活跃的pro-MSP加工成活跃的MSP，通过RON结合并发出信号，启动上皮AJC破坏（概念测试）。目的2将验证假设，即msp - ron依赖性AJC破坏允许EGF-EGFR相互作用，最终导致杯状细胞增殖和粘液分泌过多（概念证明）。目的3将测试这些机制在慢性支气管炎患者体内是否有效（气道上皮概念的相关性）。这些研究将提高我们对导致粘液分泌过多的机制的认识，并可能为预防或/和治疗慢性气道疾病，特别是香烟引起的慢性支气管炎提供新的工具。公共卫生相关性。慢性支气管炎是一种以慢性咳嗽和频繁呼吸道感染为特征的致残性疾病，导致频繁住院和生活质量差。慢性支气管炎最常见的原因是暴露于香烟烟雾中。我们建议使用从患有慢性支气管炎的肺供体中获得的细胞，结合最先进的技术来了解为什么发生粘液分泌过多，以及我们如何进行干预以预防疾病或疾病的进展。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the cellular and molecular mechanisms that lead to the structural changes associated with chronic bronchitis. This application continues our focus on oxidative stress-induced EGFR activation and mucus hypersecretion. During the current funding period we have confirmed the hypothesis of the original application, that reactive oxygen species (ROS) induce hyaluronan depolymerization releasing active bronchial TK that, in turn, results in pro-EGF processing, EGFR activation and downstream signaling that ultimately results in mucus cell proliferation and increased mucin secretion. However, in airway epithelial cells, EGFR is topologically separated from its ligands by apical junctional complexes (AJP): ligands are expressed in the apical membrane while receptors are predominantly localized to the basolateral membrane. The physical separation of growth factors from their receptors suggests that a regulated mechanism must exist that allows ligand-receptor interaction to occur only under certain conditions in which AJCs "grant" access to the basolateral side. We have found that oxidant-induced epithelial AJC junction disruption is mediated by RON, a tyrosine kinase receptor that is uniquely localized at the apical membrane of airway epithelia. The ligand for RON is the HGF-like macrophage stimulating protein (MSP), that is secreted as an inactive precursor that needs to be proteolytically cleaved to stimulate RON by a protease that, in the airways, is tissue kallikrein (TK). TK is present at the apical surface of airway epithelial cells, where it is bound and inactivated by hyaluronan (HA). ROS induced-HA depolymerization results in the release of active TK, a mechanism by which pro-MSP could be activated and thereby signal through its apical membrane receptor RON to disrupt AJC integrity allowing EGFR-ligand interaction and signaling. We will test the hypothesis that increased apical availability of active TK during oxidative stress is responsible for AJC disruption by activating pro-MSP and RON signaling, thereby allowing EGF receptor-ligand interaction, which ultimately results in airway mucous cell metaplasia and hypersecretion. This will be tested with the following aims: Aim 1 will test the hypothesis that TK, released from epithelial-bound HA by ROS, processes inactive pro-MSP to active MSP that binds and signals through RON to initiate epithelial AJC disruption (test of concept). Aim 2 will test the hypothesis that MSP-RON-dependent AJC disruption allows EGF-EGFR interaction that ultimately results in goblet cell proliferation and mucus hypersecretion (proof of concept). Aim 3 will test whether these mechanisms are operative in vivo in patients with chronic bronchitis (relevance of concept in airway epithelium). These studies will advance our knowledge on the mechanisms that lead to mucus hypersecretion and likely provide new tools for the prevention or/and treatment of chronic airway diseases, particularly cigarette smoke- induced chronic bronchitis. PUBLIC HEALTH RELEVANCE. Chronic bronchitis is an invalidating disease characterized chronic cough and frequent respiratory infections and results in frequent hospitalizations and poor quality of life. The most common cause of chronic bronchitis is exposure to cigarette smoke. We propose to use cells obtained from lung donors suffering from chronic bronchitis in combination with state of the art techniques to understand why mucous hypersecretion occurs and how can we intervene to prevent the disease or the progression of the disease.