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

项目摘要 慢性阻塞性肺疾病（COPD）是美国肺气肿的第四大死亡原因 进展和肺功能下降，有证据表明粘液纤毛清除不良和气道上皮改变 以及肺泡破坏。我们发现，在COPD中，粘附连接显著减少， 蛋白质E-钙粘蛋白，其是决定细胞-细胞粘附的主要成分，这一发现已被证实， 几个研究者和大的队列研究。在这个提议中，我们将破译E-cadherin的作用， 调节促进气道上皮功能的变化和实质建模，并确定 增加的E-钙粘蛋白保护上皮细胞和肺免受香烟烟雾（CS）的影响。我们建议 减少的肺上皮E-钙粘蛋白导致上皮细胞极性丧失，破坏上皮屏障， 并降低纤毛搏动频率以促进气道功能障碍。此外，我们建议，E- 钙粘蛋白促进实质重塑。我们将剖析两个具体的机制， 蛋白质水平下降。在目标1中，我们将确定细胞、离体气管和气管中的E-钙粘蛋白敲低是否是一种有效的方法。 小鼠模型导致上皮功能障碍和改变的组织完整性，表明E-钙粘蛋白的丢失与 慢性烟雾暴露或COPD患者在COPD的发展中起因果作用。 具体来说，我们将确定是否肺E-钙粘蛋白丢失改变上皮极性和减少纤毛搏动 频率、粘膜纤毛清除和维持空气表面液体。此外，我们将确定是否E- 使用小鼠模型，钙粘蛋白损失促进实质重塑和炎症。在目标2中，我们将 研究CS暴露和COPD背景下E-cadherin降低的新机制。 具体来说，我们将研究启动子的表观遗传调控是否有助于mRNA和蛋白质的减少， 丰饶。我们的数据显示，CDH 1在富含调控元件的区域被甲基化，因此我们将研究 这些元素，以确定什么是改变与CS曝光。此外，我们还发现了一个职位- 在群体研究中与E-钙粘蛋白功能相关的翻译修饰。具体来说，我们将 确定E-钙粘蛋白的末端岩藻糖基化是否增加其粘附强度以增强功能。我们有 确定了一个特定的多态性或与功能变化相关的单倍型， 将其遗传工程化到肺细胞系中以确定这些是否影响基于E-钙粘蛋白的细胞-细胞粘附， 功能此外，我们将确定缺乏这种岩藻糖基化的小鼠是否会增加对CS的易感性 exposure.在目标3中，我们将进行概念验证研究，以确定E-钙粘蛋白的上调是否作为 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， 操纵Aim 2中鉴定的途径上调E-钙粘蛋白并改善CS背景下的功能 exposure.

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.