MECHANISMS OF OXIDANT-INDUCED CHRONIC BRONCHITIS

描述(申请人提供)：这个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。这一应用延续了我们对氧化应激诱导的EGFR激活和粘液高分泌的关注。在目前的资助期间，我们已经证实了最初应用的假设，即活性氧物种(ROS)诱导透明质酸解聚释放活性的支气管TK，进而导致前EGF处理、EGFR激活和下游信号传递，最终导致粘液细胞增殖和粘蛋白分泌增加。然而，在呼吸道上皮细胞中，EGFR与其配体通过顶端连接复合体(AJP)在拓扑上分开：配体表达在顶膜，而受体主要定位于基底膜。生长因子与其受体的物理分离表明，必须存在一种调控机制，允许配体-受体相互作用仅在AJCs“准许”进入基底外侧的特定条件下发生。我们发现，氧化剂诱导的上皮AJC连接断裂是由RON介导的，RON是一种唯一定位于呼吸道上皮细胞顶膜的酪氨酸激酶受体。RON的配体是HGF样巨噬细胞刺激蛋白(MSP)，它是以一种非活性前体的形式分泌出来的，需要被一种在呼吸道中的组织激肽释放酶(TK)蛋白水解酶切割来刺激RON。TK存在于呼吸道上皮细胞的顶端表面，在那里它被透明质酸(HA)结合并失活。ROS诱导的HA解聚导致活性TK的释放，这一机制可以激活原MSP，从而通过其顶膜受体RON发出信号，破坏AJC的完整性，允许EGFR-配体相互作用和信号传递。我们将检验这一假说，即氧化应激期间活性TK的顶端可获得性增加通过激活前MSP和RON信号，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高分泌，从而导致AJC破坏。这将与以下目标进行测试：Aim 1将测试这样的假设，即由ROS从上皮结合的HA释放的TK将非活性的前MSP处理为活性的MSP，后者通过RON结合并发出信号，启动上皮性AJC的破坏(概念测试)。目的2将验证一种假设，即依赖MSP-RON的AJC破坏允许EGF-EGFR相互作用，最终导致杯状细胞增殖和粘液高分泌(概念验证)。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(与呼吸道上皮学概念的相关性)。这些研究将促进我们对导致粘液高分泌的机制的了解，并可能为预防或/和治疗慢性呼吸道疾病，特别是香烟烟雾诱导的慢性支气管炎提供新的工具。与公共卫生相关。慢性支气管炎是一种以慢性咳嗽和频繁的呼吸道感染为特征的致死性疾病，导致频繁住院和生活质量下降。慢性支气管炎最常见的原因是吸烟。我们建议使用从患有慢性支气管炎的肺捐赠者身上获得的细胞，结合最先进的技术来了解为什么会发生粘液高分泌，以及我们如何干预以防止疾病或疾病的进展。

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.