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有关，包括动脉粥样硬化、微血管高渗透性伴糖尿病和急性肺损伤后的肺水肿。VE-cadherin是一种参与调节多种内皮细胞功能的细胞-细胞粘附蛋白。ve -钙粘蛋白的细胞质区域与犰狳家族的连环蛋白结合，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卒中和心肌梗死后水肿的形成过程中，这一过程依赖于Src激酶的激活。这一建议的重点是p120，一种结合到VE-钙粘蛋白近膜上的连环蛋白，在那里它为许多蛋白质提供支架作用。有趣的是，p120是Src激酶的靶标，p120磷酸化状态的变化与炎症介质治疗后内皮屏障功能下降有关。此外，我们最近的出版物表明，p120与VE-cadherin的相互作用是维持内皮细胞中VE-cadherin水平所必需的。在这种情况下，我们假设VE-cadherin的近膜区域及其与p120的相互作用在调节内皮细胞单层细胞的细胞粘附和屏障功能中起关键作用。这一假设将通过三个具体目标进行研究：1)确定ve -钙粘蛋白负责维持内皮细胞单层中ve -钙粘蛋白水平和屏障功能的区域；2)确定p120从膜释放到细胞质时是否作为信号分子；3)确定调节内皮细胞-细胞粘附和屏障功能所需的p120结构域。我们将利用VE和N-cadherin嵌合分子的表达以及p120的缺失突变体进行p120和VE-cadherin的结构/功能分析及其对内皮细胞-细胞粘附和屏障功能的影响。这些分子的表达将在缺乏内源性钙粘蛋白或p120的情况下进行，p120将被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，并有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.