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: 6760007
• 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/6760007
• 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 在诸如胚胎发育、伤口愈合、肿瘤生长和 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 已经建立了一个在双层纤维蛋白中形成毛细管的模型 体外 在纤维蛋白中，这种功能的关键结构是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，p120 ctn结合 高度保守的八肽VE-钙粘蛋白胞浆跨膜区 该地区应存在。 VE-钙粘蛋白与 p120 ctn似乎调节细胞生长和增殖。 我们建议 进一步表征VE-钙粘蛋白-p120 ctn相互作用在 调节细胞生长。 我们将分析CHO的生长特征 细胞，缺乏钙粘蛋白，转染野生型或突变型VE- 钙粘蛋白不能结合p120 ctn。 我们将确定基因是否 已知调节细胞生长的是p120 ctn的靶点， 我们知道p120 ctn是自由的还是结合的。 我们也将尝试 使用微阵列技术来鉴定是否有基因 当p120 ctn与VE-钙粘蛋白结合或不结合时表达不同。 我们 还提出了表征VE-钙粘蛋白之间的相互作用的作用， 和p120 ctn在毛细血管形成的双层纤维蛋白中的作用 体外 我们认为VE-钙粘蛋白和p120 ctn之间的相互作用是 p120 ctn对血管生成过程很重要，p120 ctn可能参与血管生成过程。 在纤维蛋白接合VE的细胞外结构域后的信号转导- 钙粘蛋白 我们的假设是VE-钙粘蛋白和 p120 ctn在毛细管形成过程中是动态的。 我们将研究他们的合作- 在毛细管形成过程中的定位和磷酸化状态， 免疫组化法和免疫沉淀法。 我们还将 测定p120 ctn的下调是否消除管形成。 最后我们 将决定野生型钙粘蛋白是否能恢复形成 毛细血管内皮细胞不表达VE-钙粘蛋白， 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 不能结合p120 ctn赋予相同的性质。 我们的假设是 毛细血管需要VE-钙粘蛋白和p120 ctn之间的相互作用 因此，我们预测我们的突变VE-钙粘蛋白不能结合 p120 ctn不能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-cadherin与p120 ctn之间的相互作用可以调节 血管生成