p120 Catenin and Endothelial Monolayer Function

描述（由申请人提供）：内皮细胞屏障功能障碍与多种疾病状态的病因学有关，包括动脉粥样硬化、糖尿病微血管通透性过高和急性肺损伤后的肺水肿。VE-钙粘蛋白是一种细胞-细胞粘附蛋白，参与许多内皮细胞功能的调节。VE-钙粘蛋白的胞质结构域与称为连环蛋白的蛋白质的犰狳家族结合以形成粘附连接（AJ）。AJ蛋白磷酸化的变化与调节屏障功能有关。例如，AJ蛋白的磷酸化发生在中风和心肌梗死后水肿的形成过程中，这是一个依赖于Src激酶活化的过程。这个建议集中在p120上，这是一种与VE-钙粘蛋白的质膜结合的连环蛋白，在那里它对许多蛋白质起支架作用。有趣的是，p120是Src激酶的靶点，p120磷酸化状态的变化与炎症介质治疗后内皮屏障功能降低相关。此外，我们最近的出版物表明，p120与VE钙粘蛋白的相互作用是维持内皮细胞中VE钙粘蛋白水平所必需的。在这种情况下，我们假设，血管内皮细胞-钙粘蛋白及其与p120的相互作用的内膜区域在调节内皮细胞单层的细胞-细胞粘附和屏障功能中起着至关重要的作用。该假说将通过执行三个特定目标来研究：1）鉴定负责维持内皮细胞单层中VE-钙粘蛋白水平和屏障功能的VE-钙粘蛋白区域; 2）确定当p120从膜释放到细胞质中时是否充当信号分子;和3）鉴定调节内皮细胞-细胞粘附和屏障功能所需的p120的结构域。我们将利用VE和N-cadherin嵌合分子以及p120缺失突变体的表达来进行p120和VE-cadherin的结构/功能分析以及它们对内皮细胞-细胞粘附和屏障功能的影响。这些分子的表达将在不存在内源性钙粘蛋白或p120的情况下进行，所述内源性钙粘蛋白或p120将使用siRNA耗尽。这些研究将为控制内皮屏障功能的细胞和分子机制提供新的见解，并可能确定新的干预位点。

DESCRIPTION (provided by applicant): Dysfunction of the endothelial cell barrier has been implicated in the etiology of a variety of disease states including atherosclerosis, microvascular hyperpermeability with diabetes and pulmonary edema following acute lung injury. VE-cadherin is a cell-cell adhesion protein that participates in the regulation of a number of endothelial cell functions. The cytoplasmic domain of VE-cadherin binds to the armadillo family of proteins called catenins to form the adherens junction (AJ). Changes in the phosphorylation of AJ proteins have been implicated in regulating barrier function. For example, phosphorylation of AJ proteins occurs during the formation of edema following stroke and myocardial infarction, a process that is dependent on activation of Src kinase. This proposal focuses on p120, a catenin that binds to the juxtamembrane of VE- cadherin where it serves a scaffolding role for a number of proteins. Interestingly, p120 is a target for Src kinase and changes in the phosphorylation state of p120 are associated with decreased endothelial barrier function following treatment with inflammatory mediators. In addition, our recent publications have shown that the interaction of p120 with VE-cadherin is required for maintaining VE-cadherin levels in endothelial cells. Within this context, we hypothesize that the juxtamembrane region of VE-cadherin and its interaction with p120 plays a critical role in regulating cell-cell adhesion and barrier function in endothelial cell monolayers. This hypothesis will be investigated by performing three specific aims: 1) identify the regions of VE-cadherin that are responsible for maintaining VE-cadherin levels and barrier function in endothelial cell monolayers; 2) determine if p120 serves as a signaling molecule when released from the membrane into the cytoplasm; and 3) identify the domains of p120 required for the regulation of endothelial cell-cell adhesion and barrier function. We will use expression of chimeric molecules of VE and N-cadherin as well as deletion mutants of p120 to perform structure/function analysis of p120 and VE-cadherin and their effects on endothelial cell-cell adhesion and barrier function. Expression of these molecules will be performed in the absence of endogenous cadherin or p120 that will be depleted using siRNA. These studies will provide new insights into the cellular and molecular mechanisms controlling endothelial barrier function and potentially identify new sites for intervention.