Rho Regulation of Pulmonary Vascular Permeability

Rho 对肺血管通透性的调节

• 批准号: 6687598
• 负责人: DOLLY MEHTA
• 金额: $ 27.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6687598
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; biological signal transduction; blood proteins; calcium flux; cell line; confocal scanning microscopy; guanine nucleotide binding protein; guanine nucleotides; immunoprecipitation; laboratory mouse; lung; phosphorylation; polymerase chain reaction; protein structure function; thrombin; tissue /cell culture; vascular endothelium permeability; western blottings

DESCRIPTION (provided by applicant): Thrombin by binding to the endothelial cell surface Protease Activated Receptor-1 (PAR-1) activates heterotrimeric proteins Gq and G12/G13, which in turn induces a repertoire of signaling events that mediate increased lung vascular permeability. A primary unresolved question is the nature of the specific signaling pathways emanating from the interaction of PAR-1 with these heterotrimeric G-proteins that induce gaps between endothelial cells. Understanding these specific signals will be critical in defining the mechanisms of increased lung vascular permeability and tissue inflammation. Studies have shown a primary role of RhoA, a monomeric GTPase, in regulating endothelial barrier function. Rho activation is mediated through the tightly regulated GDP/GTP exchange cycle. Both guanine nucleotide dissociation inhibitor (GDI) and guanine nucleotide exchange factors (GEFs) are capable of regulating Rho activation. Studies have shown that the (-subunit of the heterotrimeric G protein family G12/G13 activates Rho via the intermediate p115RhoGEF, a Rho-specific GEF. Interestingly, we have also shown that in endothelial cells, thrombin induces the activation of RhoA via PKC( pathway. Since PKC( is a downstream effector of Gq, our studies suggest that in addition to classical G12/G13 pathway, Gq may play a critical role the activation of Rho. The proposed studies will address the key question that Rho is a point of confluence for PAR-1 initiated signals originating from interactions of PAR-1 with G12/G13 as well as Gq. Studies will also address the potentially important downstream action of RhoA in activating Ca2+ influx pathways in endothelial cells, and thereby in triggering the increase in endothelial permeability. The Specific Aims are: 1. To investigate the specific roles of GDI-1 and p115RhoGEF in triggering the activation of Rho, and thereby in signaling the increase in endothelial permeability. 2. To determine the role of heterotrimeric GTP-binding protein Gq in signaling Rho activation and the loss of endothelial barrier function. 3. To determine the role of RheA in activating store-operated Ca2+ entry and signaling the increase in endothelial permeability. Studies will use genetic approaches in cultured endothelial cells and the intact mouse lung model to establish the roles of GDI-1, p115RhoGEF, and Gq in signaling Rho activation and in regulating endothelial permeability. With the information gained, it is hoped that new lines of therapy could be developed against inflammatory diseases that are characterized by "leaky" pulmonary vessel.

描述（由申请人提供）：凝血酶通过与内皮细胞表面蛋白酶激活受体-1 (PAR-1) 结合来激活异三聚体蛋白 Gq 和 G12/G13，进而诱导介导肺血管通透性增加的一系列信号传导事件。 一个尚未解决的主要问题是 PAR-1 与这些异三聚体 G 蛋白相互作用所产生的特定信号传导途径的性质，这些异三聚体 G 蛋白诱导内皮细胞之间的间隙。 了解这些特定信号对于确定肺血管通透性增加和组织炎症的机制至关重要。 研究表明，RhoA（一种单体 GTP 酶）在调节内皮屏障功能中发挥着主要作用。 Rho 激活是通过严格调控的 GDP/GTP 交换循环介导的。 鸟嘌呤核苷酸解离抑制剂 (GDI) 和鸟嘌呤核苷酸交换因子 (GEF) 都能够调节 Rho 激活。 研究表明，异三聚体 G 蛋白家族 G12/G13 的 (-亚基通过中间体 p115RhoGEF（一种 Rho 特异性 GEF）激活 Rho。有趣的是，我们还发现在内皮细胞中，凝血酶通过 PKC 诱导 RhoA 的激活（由于 PKC( 是 Gq 的下游效应子，我们的研究表明，除了经典的 G12/G13 途径外，Gq 也可能是 Gq 的下游效应子。 Rho 的激活发挥着关键作用。拟议的研究将解决 Rho 是源自 PAR-1 与 G12/G13 相互作用的 PAR-1 启动信号汇合点的关键问题，研究也将解决这一问题。 RhoA 激活内皮细胞中 Ca2+ 流入途径的潜在重要下游作用，从而引发内皮通透性增加。具体目标是： 1. 研究 GDI-1 和 p115RhoGEF 在触发 Rho 激活中的具体作用，从而发出内皮通透性增加的信号。 2. 确定异源三聚体 GTP 结合蛋白 Gq 在信号 Rho 激活和内皮屏障功能丧失中的作用。 3. 确定 RheA 在激活钙池操纵的 Ca2+ 进入和发出内皮通透性增加信号中的作用。 研究将在培养的内皮细胞和完整的小鼠肺模型中使用遗传方法来确定 GDI-1、p115RhoGEF 和 Gq 在信号 Rho 激活和调节内皮通透性中的作用。 根据所获得的信息，希望能够开发出针对以“渗漏”肺血管为特征的炎症性疾病的新疗法。