p120 catenin/VE-cadherin interaction in angiogenesis

p120 连环蛋白/VE-钙粘蛋白在血管生成中的相互作用

• 批准号: 6638796
• 负责人: ANDRES FERBER
• 金额: $ 13.28万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638796
• 关键词: CHO cells; angiogenesis; biological signal transduction; cadherins; capillary; cell adhesion; cell growth regulation; fibrin; immunocytochemistry; immunoprecipitation; microarray technology; protein localization; protein protein interaction

DESCRIPTION (Applicant's abstract) The formation of new vessels plays a fundamental role in processes such as embryonic development, wound healing, tumor growth and ischemia-reperfusion. In those processes the interaction between the endothelial cells and proteins of the extracellular matrix is essential. We have developed a model of capillary tube formation in a double layer of fibrin in vitro. In fibrin the critical structure for this function is the N terminal 15-42 aminoacids of the beta chain. This action is mediated by associating with VE-cadherin. We found that for p120ctn to bind the cytoplasmic juxtamembrane domain of VE-cadherin a highly conserved octapeptide of that region should be present. The interaction between VE-cadherin and p120ctn seems to modulate cell growth and adhesiveness. We proposed to further characterize the role of the VE-cadherin -p120ctn interaction in regulating cellular growth. We will analyze the growth characteristics of CHO cells, devoid of cadherin, transfected with either wild type or mutant VE- cadherin unable to bind p120ctn. We will determine whether genes that are known to regulate cellular growth are targets of p120ctn taking advantage of our system where we know whether p120ctn is free or bound. We will also try to identify using microarrays techniques whether there are genes that are expressed differently when p120ctn is bound or unbound to VE-cadherin. We also propose to characterize the role of the interaction between VE-cadherin and p120ctn in capillary tube formation in the double layer of fibrin in vitro. We propose that the interaction between VE-cadherin and p120ctn is important for the angiogenesis process and that p120ctn may be involved in the transduction of a signal upon fibrin engaging the extracellular domain of VE- cadherin. Our hypothesis is that the relationship between VE-cadherin and p120ctn is dynamic during capillary tube formation. We will study their co- localization and phosphorylation state during capillary tube formation by immunohistochemical methods and immunoprecipitation methods. We will also assay whether downregulation of p120ctn abrogates tube formation. Finally we will determine whether wild type cadherin can restore the ability to form capillary tubes to endothelial cells that do not expressed VE-cadherin and do not form capillaries. If so we will determine whether our mutant VE-cadherin unable to bind p120ctn confers the same properties. Our hypothesis is that the interaction between VE-cadherin and p120ctn is needed for capillary tubes to form and therefore we predict that our mutant VE-cadherin unable to bind p120ctn will not restore the ability on those cells to form tubes. These studies will help elucidate the basic biochemical and biological responses by which the interaction between VE-cadherin and p120ctn may modulate angiogenesis.

描述 （申请人摘要）新血管的形成起着基础性作用 在胚胎发育、伤口愈合、肿瘤生长等过程中 缺血再灌注。 在这些过程中， 内皮细胞和细胞外基质的蛋白质是必不可少的。 我们 开发了双层纤维蛋白中毛细管形成的模型 体外。 在纤维蛋白中，该功能的关键结构是 N β链末端15-42个氨基酸。 这一行动是由 与 VE-钙粘蛋白结合。 我们发现 p120ctn 结合 高度保守的八肽 VE-钙粘蛋白的细胞质近膜结构域 该区域的应该存在。 VE-钙粘蛋白与 p120ctn 似乎可以调节细胞生长和粘附性。 我们提议 进一步表征 VE-钙粘蛋白 -p120ctn 相互作用在 调节细胞生长。 我们将分析CHO的生长特性 细胞，缺乏钙粘蛋白，用野生型或突变型VE-转染 钙粘蛋白无法结合 p120ctn。 我们将确定基因是否 已知调节细胞生长是 p120ctn 的目标，利用 在我们的系统中，我们知道 p120ctn 是游离的还是结合的。 我们也会尝试 使用微阵列技术来鉴定是否有基因 当 p120ctn 与 VE-钙粘蛋白结合或未结合时，表达有所不同。 我们 还建议描述 VE-钙粘蛋白之间相互作用的作用 和 p120ctn 在纤维蛋白双层毛细管形成中的作用 体外。 我们认为 VE-cadherin 和 p120ctn 之间的相互作用是 对于血管生成过程很重要，p120ctn 可能参与 纤维蛋白与 VE- 胞外域结合时信号的转导 钙粘蛋白。 我们的假设是 VE-钙粘蛋白和 p120ctn 在毛细管形成过程中是动态的。 我们将研究他们的合作 毛细管形成过程中的定位和磷酸化状态 免疫组织化学方法和免疫沉淀方法。 我们还将 测定 p120ctn 的下调是否会消除管形成。 最后我们 将确定野生型钙粘蛋白是否可以恢复形成能力 毛细管连接到不表达 VE-钙粘蛋白但不表达 VE-钙粘蛋白的内皮细胞 不形成毛细血管。 如果是这样，我们将确定我们的突变 VE-钙粘蛋白是否 无法结合 p120ctn 赋予相同的特性。 我们的假设是 毛细管需要 VE-钙粘蛋白和 p120ctn 之间的相互作用 形成，因此我们预测我们的突变 VE-钙粘蛋白无法结合 p120ctn 不会恢复这些细胞形成管的能力。 这些 研究将有助于阐明基本的生化和生物反应 VE-钙粘蛋白和 p120ctn 之间的相互作用可能会调节 血管生成。