Src Regulation of Lung Endothelial Barrier Function

描述（由申请人提供）：内皮屏障功能的丧失在成人呼吸窘迫综合征的发展中很重要。白蛋白等大分子通过内皮小泡的跨细胞转运是调节内皮屏障功能的一个因素。利用肺微血管内皮细胞，我们已经确定了小窝蛋白-1和src家族酪氨酸激酶之间的特定相互作用，这些相互作用可能调节白蛋白的摄取及其通过小窝的运输。该研究的主要目的是研究小洞蛋白-1和Src在细胞内吞作用和内皮屏障功能调节中的作用。研究人员将利用小洞蛋白-1和Src敲除小鼠来研究(1)小洞蛋白-1在肺微血管内皮细胞中内化小洞和跨内皮运输中的功能，以及(2)动力蛋白在介导小洞从膜分裂和内皮细胞跨细胞白蛋白渗透中的作用。我们的假设是src依赖性的小泡蛋白-1和动力蛋白的磷酸化对于小泡的形成和它们从膜上断裂是必不可少的。我们将测量小泡蛋白-1敲除小鼠肺制备中的血管壁白蛋白通透性。突变的caveolin-1、Src和动力蛋白cDNA构建体将用于细胞培养实验，以检验Src依赖的相互作用在调节caveolin-1和动力蛋白功能中的作用。霍乱毒素亚基B和白蛋白的内吞作用和跨细胞运输将通过电子显微镜、共聚焦显微镜和培养细胞中碘示踪剂的测量来确定。分子遗传学和生化策略将用于阐明小洞蛋白-1、Src和动力蛋白之间的结构域特异性和磷酸化依赖性相互作用。因此，这些研究将阐明调节肺血管内皮屏障内吞作用和运输的src依赖的信号机制。

DESCRIPTION (provided by applicant): Loss of endothelial barrier function is important in the development of Adult Respiratory Distress Syndrome. The transcellular transport of albumin and other macromolecules via endothelial caveolae is a factor regulating endothelial barrier function. Using pulmonary microvascular endothelial cells, we have identified specific interactions between caveolin-1 and Src-family tyrosine kinases that may regulate albumin uptake and its transport via caveolae. The primary objective of the proposed investigation is to address the roles of caveolin-1 and Src in endocytosis and regulation of endothelial barrier function. Caveolin-1 and Src knockout mice will be used to investigate (1) the function of caveolin-1 in internalization of caveolae and transendothelial transport in pulmonary microvascular endothelial cells and (2) the role of dynamin in mediating fission of caveolae from the membrane and transcellular albumin permeability in endothelial cells. Our hypothesis is that Src-dependent phosphorylation of caveolin-1 and dynamin is essential for formation of caveolae and their scission from the membrane. We will measure vessel wall albumin permeability in the mouse lung preparation from caveolin-1 knockout mice. Mutant caveolin-1, Src, and dynamin cDNA constructs will be used in cell culture experiments to examine the role of Src-dependent interactions in regulating caveolin-1 and dynamin functions. Cholera toxin subunit B and albumin endocytosis and transcellular transport will be determined by electron microscopy, confocal microscopy, and measurement of iodinated tracers in cultured cells. Molecular-genetic and biochemical strategies will be used to elucidate the domain-specific and phosphorylation-dependent interactions between caveolin-1, Src, and dynamin. Thus, these studies will elucidate the Src-dependent signaling mechanisms that regulate endocytosis and transport across the pulmonary vascular endothelial barrier.