Src Regulation of Lung Endothelial Barrier Function

Src 对肺内皮屏障功能的调节

基本信息

批准号：
7312502
负责人：
RICHARD D MINSHALL
金额：
$ 29.25万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2010-02-28
项目状态：
已结题

项目摘要

Loss of endothelial barrier function is an important characteristic of Acute Lung Injury (ALl). The transcellular transport of albumin and other macromolecules via endothelial caveolae is a factor contributing to endothelial barrier function. We have identified specific interactions between caveolin-1 (a caveolar protein), the heterotrimeric G protein Gi, and Src kinase in the mechanism of caveolae-mediated endocytosis. The goals of Project 4 are to define the role of caveolin-1 as an organizer and regulator of signal transduction cascades essential for plasmatemmal vesicle trafficking and the protein-protein interactions that regulate albumin permeability via transcytosis. The studies in Project 4 will address the following specific aims. Specific Aim #1: To determine the role of the heterotrimeric G protein, Gi, in signaling ceaveolae-mediated endocytosis and transendothelial albumin permeability in endothelial monolayers; Specific Aim #2: To determine the role of Src activation of the GTPase, dynamin-2, in signaling caveolae-mediated endocytosis and transendothelial albumin permeability; Specific Aim #3:To address the component of thrombin/Protease Activated Receptor-1-induced increase in lung vascular permeability resulting from internalization of caveolae and transcelinlar albumin transport. Thus, Project 4 will identify the receptor-coupled signals activating Src, the phosphorylation targets of Src signaling caveolae fission (specifically, caveolin-1 and dynamin-2), and the role of Src activation in regulating transcellular permeability. To address the in vivo relevance and functional significance of these studies in pulmonary microvascular endothelial cells, experiments will also be made, wherever possible, in intact mouse lung models. Studies will employ approaches in both imaging (i.e., using fluorescent probes and electron microscopic assessment) and physiology (i.e., determination of endothelial permeability in monoayers and mouse lung models) to address the role of caveolae-mediated endocytosis in activating increased albumin permeability. Thus, these studies will elucidate the signaling mechanisms that regulate caveolae internalization and plasmalemmal vesicle trafficking, and thus contribute to the mechanism of transendothelial albumin permeability in lungs. The achievement of these objectives will lead to tthe elucidation of the signals regulating caveolae-mediated endocytosis and its role in contributing to the thrombin-induced increase in lung vascular permeability. With the identification of novel signaling pathways, it may be possible to develop therapeutic strategies that specifically target signals leading to inappropriate increase in lung vascular permeability.

内皮屏障功能丧失是急性肺损伤(AL1)的重要特征。白蛋白和其他大分子通过内皮细胞小窝的跨细胞转运是促进内皮屏障功能的一个因素。我们已经确定了caveolin-1（一种caveolar蛋白）、异源三聚体G蛋白Gi和Src激酶在caveolae介导的内吞作用机制中的特异性相互作用。项目 4 的目标是定义以下角色： Caveolin-1 作为信号转导级联的组织者和调节者，对于质膜囊泡运输和通过胞吞作用调节白蛋白通透性的蛋白质-蛋白质相互作用至关重要。项目 4 中的研究将解决以下具体目标。具体目标#1：确定异源三聚体 G 蛋白 Gi 在信号传导 ceaveolae 介导的内吞作用和内皮单层中的跨内皮白蛋白通透性中的作用；具体目标#2：确定 GTP 酶（dynamin-2）的 Src 激活在信号小凹介导的内吞作用和跨内皮白蛋白通透性中的作用；具体目标#3：解决因小窝内化和跨细胞白蛋白转运导致的凝血酶/蛋白酶激活受体 1 诱导的肺血管通透性增加的问题。因此，项目 4 将识别激活 Src 的受体偶联信号，即 Src 信号小凹裂变的磷酸化靶标（具体来说，caveolin-1 和 dynamin-2），以及 Src 激活在调节跨细胞通透性中的作用。为了解决这些研究在肺微血管内皮细胞中的体内相关性和功能意义，只要可能，还将在完整的小鼠肺模型中进行实验。研究将采用成像方法（即使用荧光探针和电子显微镜评估）和生理学方法（即确定内皮渗透性）在单层细胞和小鼠肺模型中），以解决小凹介导的内吞作用在激活白蛋白通透性增加中的作用。因此，这些研究将阐明调节小凹内化和质膜囊泡运输的信号传导机制，从而有助于肺部跨内皮白蛋白通透性的机制。这些目标的实现将导致阐明调节小窝介导的信号内吞作用及其在凝血酶诱导的肺血管通透性增加中的作用。随着新信号通路的识别，有可能开发出专门针对导致肺血管通透性不适当地增加的信号的治疗策略。