MECHANISMS OF OXIDANT-INDUCED CHRONIC BRONCHITIS

描述（由申请人提供）：本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。该申请继续我们对氧化应激诱导的EGFR活化和粘液分泌过多的关注。在当前的资助期内，我们已经证实了原始申请的假设，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pro-EGF加工、EGFR激活和下游信号传导，最终导致粘液细胞增殖和粘蛋白分泌增加。然而，在气道上皮细胞中，EGFR通过顶端连接复合物（AJP）与其配体拓扑分离：配体在顶端膜中表达，而受体主要定位于基底外侧膜。生长因子与其受体的物理分离表明，必须存在一种调节机制，该机制允许配体-受体相互作用仅在特定条件下发生，其中AJCs“授予”进入基底侧。我们已经发现氧化剂诱导的上皮AJC连接破坏是由罗恩介导的，RON是一种酪氨酸激酶受体，其独特地定位于气道上皮的顶膜。罗恩的配体是HGF样巨噬细胞刺激蛋白（MSP），其作为无活性前体分泌，需要通过蛋白酶水解裂解以刺激罗恩，所述蛋白酶在气道中是组织激肽释放酶（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，这是一种可激活MSP原并由此通过其顶端膜受体罗恩发出信号以破坏AJC完整性从而允许EGFR-配体相互作用和信号传导的机制。我们将检验这样的假设：氧化应激期间活性TK的顶端可用性增加是通过激活MSP前体和罗恩信号传导而导致AJC破坏的原因，从而允许EGF受体-配体相互作用，最终导致气道粘液细胞化生和分泌过多。这将按照以下目的进行检验：目的1将检验以下假设：通过ROS从上皮结合HA释放的TK将无活性的pro-MSP加工为活性MSP，活性MSP通过罗恩结合并发出信号以启动上皮AJC破坏（概念检验）。目的2将检验MSP-RON依赖性AJC破坏允许最终导致杯状细胞增殖和粘液分泌过多的EGF-EGFR相互作用的假设（概念证明）。目的3将测试这些机制是否在慢性支气管炎患者体内起作用（与气道上皮概念的相关性）。这些研究将推进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，特别是香烟烟雾诱导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，并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接触香烟烟雾。我们建议使用从患有慢性支气管炎的肺供体获得的细胞与最先进的技术相结合，以了解为什么会发生粘液分泌过多，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.