Role of E-cadherin in modulating airway epithelial function and parenchymal remodeling

项目总结 慢性阻塞性肺疾病(COPD)是美国肺气肿患者的第四大死因 病情进展和肺功能下降，有粘液纤毛清除不良和呼吸道上皮病变的证据 以及肺泡破坏。我们发现，在COPD患者中，粘附素连接显著减少 蛋白质E-钙粘附素是决定细胞间黏附的主要成分，这一发现已经得到了 几名调查人员和一大群人进行了研究。在这项提案中，我们将破译E-钙粘附素的作用 调节促进呼吸道和实质模型上皮功能的改变，并确定 增加的E-钙粘附素保护上皮细胞和肺免受香烟烟雾(CS)的影响。我们建议 肺上皮细胞E-钙粘附素降低导致上皮细胞极性丧失，破坏上皮屏障 减少纤毛跳动频率，促进呼吸道功能障碍。此外，我们建议E-的损失- 钙粘附素促进实质重塑。我们将剖析两个具体的机制，它们可能有助于 蛋白质水平降低。在目标1中，我们将确定E-钙粘素在细胞、体外气管和 小鼠模型导致上皮功能障碍和组织完整性改变，表明E-钙粘附素与 慢性吸烟暴露或慢性阻塞性肺病患者在慢性阻塞性肺疾病的发展中起因果作用。 具体地说，我们将确定肺E-钙粘附素缺失是否会改变上皮细胞的极性并减少纤毛搏动 空气表面液体的频率、粘液纤毛清除和维护。此外，我们将确定E- 在小鼠模型中，钙粘附素丢失促进实质重塑和炎症。在目标2中，我们将 研究在CS暴露和COPD背景下E-钙粘附素降低的新机制。 具体地说，我们将研究启动子的表观遗传调控是否会导致mrna和蛋白质的减少。 富足。我们的数据显示，cdh1在一个富含调控元件的区域发生甲基化，因此我们将研究 这些元素可以确定CS暴露后会发生什么变化。此外，我们已经确定了一个职位- 人口研究中与E-钙粘附素功能相关的翻译修饰。具体来说，我们将 确定E-钙粘附素的末端岩藻糖基化是否增加其黏附强度以增强功能。我们有 确定了与功能变化相关的特定多态或单倍型，并将 通过基因工程将其转化为肺细胞系，以确定它们是否影响基于E-钙粘附素的细胞-细胞黏附和 功能。此外，我们将确定缺乏这种岩藻糖基化的小鼠是否增加了CS的易感性 曝光。在目标3中，我们将进行概念验证研究，以确定E-钙粘附素的上调是否起到 在原代人类细胞和小鼠模型中的治疗策略。此外，我们将确定是否 操纵Aim 2中确定的通路上调E-钙粘素并改善CS的功能 曝光。

PROJECT SUMMARY Chronic Obstructive Pulmonary Disease (COPD) is the 4th leading cause of death in the US with emphysema progression and lung function decline with evidence of poor mucociliary clearance and airway epithelial changes as well as alveolar destruction. We have found that in COPD there is a significant decrease in adherens junction protein E-cadherin, which is a primary component dictating cell-cell adhesion a finding which has been verified by several investigators and in large cohort studied. In this proposal, we will decipher the effects of E-cadherin regulation in promoting changes in epithelial function in the airways and parenchymal modeling and determine if increased E-cadherin protects the epithelia and the lungs from cigarette smoke (CS). We propose that decreased lung epithelial E-cadherin leads to loss of polarity of the epithelial cell, disrupts the epithelial barrier f and decreases ciliary beat frequency to promote airway dysfunction. Furthermore, we propose that loss of E- cadherin promotes parenchymal remodeling. We will dissect two specific mechanisms which could contribute to decreased protein levels. In Aim 1, we will determine if E-cadherin knockdown in cells, ex vivo trachea and mouse models lead to epithelial dysfunction and altered tissue integrity indicating that loss of E-cadherin with chronic smoke exposure or in patients with COPD serves a causal role in the development of COPD. Specifically, we will determine if lung E-cadherin loss alters epithelial polarity and decreases ciliary beat frequency, mucociliary clearance, and maintenance of the air surface liquid. In addition, we will determine if E- cadherin loss promotes parenchymal remodeling and inflammation using mouse models. In Aim 2, we will study novel mechanisms by which E-cadherin is decreased in the context of CS exposure and COPD. Specifically, we will study if epigenetic regulation of the promoter contributes to decrease in mRNA and protein abundance. Our data shows that CDH1 is methylated in an area rich with regulatory elements, so we will study these elements to determine what is altered with CS exposure. In addition, we have identified a post- translational modification which correlates with E-cadherin function in population studies. Specifically, we will determine if terminal fucosylation of E-cadherin increases its adhesion strength to enhance function. We have identified a specific identified polymorphism or a haplotype that is associated with functional changes and will genetically engineer this into a lung cell line to determine if these affect E-cadherin based cell-cell adhesion and function. In addition, we will determine if mice lacking this fucosylation have increased susceptibility to CS exposure. In Aim 3, we will perform proof of concept studies to determine if upregulation of E-cadherin serves as a therapeutic strategy in primary human cells and mouse models. In addition, we will determine if manipulating the pathways identified in Aim 2 upregulate E-cadherin and improve function in the context of CS exposure.