p120 Catenin and Endothelial Monolayer Function

描述(申请人提供)：内皮细胞屏障功能障碍与多种疾病的病因有关，包括动脉粥样硬化、糖尿病微血管高通透性和急性肺损伤后的肺水肿。VE-钙粘蛋白是一种细胞-细胞黏附蛋白，参与调节血管内皮细胞的多种功能。VE-钙粘附素的胞质结构域与一种称为连接素的蛋白质家族结合，形成粘着连接(AJ)。AJ蛋白磷酸化的变化被认为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，这一过程依赖于Src激酶的激活。这项研究的重点是p120，这是一种与VE-钙粘附素的膜旁结合的连接素，在那里它扮演着许多蛋白质的支架角色。有趣的是，p120是Src激酶的靶点，p120的磷酸化状态的变化与炎症介质治疗后内皮屏障功能的降低有关。此外，我们最近的研究表明，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。在此背景下，我们假设VE-钙粘蛋白的膜旁区域及其与p120的相互作用在调节内皮细胞单层的细胞-细胞黏附和屏障功能中起着关键作用。这一假说将通过三个特定的目标来研究：1)确定VE-钙粘蛋白在内皮细胞单层中负责维持VE-钙粘蛋白水平和屏障功能的区域；2)确定p120在从膜释放到细胞质时是否作为信号分子；以及3)确定p120的结构域调控内皮细胞与细胞的黏附和屏障功能。我们将利用VE和N-钙粘蛋白嵌合分子的表达以及p120的缺失突变体来分析p120和VE-钙粘蛋白的结构/功能及其对内皮细胞-细胞黏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附素或p120的情况下进行，这些内源性钙粘附素或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，并可能确定新的干预部位。

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.