Src Regulation of Lung Endothelial Barrier Function

描述（由申请方提供）：内皮屏障功能丧失在成人呼吸窘迫综合征的发生中很重要。 白蛋白和其他大分子通过内皮小窝的跨细胞转运是调节内皮屏障功能的因素。 使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，可以调节白蛋白的摄取和运输通过小窝之间的特定相互作用。 本研究的主要目的是探讨小窝蛋白-1和Src在细胞内吞和调节内皮屏障功能中的作用。 小窝蛋白-1和Src敲除小鼠将用于研究（1）小窝蛋白-1在肺微血管内皮细胞中小窝的内化和跨内皮转运中的功能和（2）发动蛋白在介导小窝从膜分裂和内皮细胞中跨细胞白蛋白渗透性中的作用。 我们的假设是，Src依赖的小窝蛋白-1和发动蛋白的磷酸化是必不可少的小窝的形成和他们从膜的切断。 我们将测量来自小窝蛋白-1敲除小鼠的小鼠肺制备物中的血管壁白蛋白渗透性。突变小窝蛋白-1，Src和发动蛋白cDNA构建体将用于细胞培养实验中，以检查Src依赖的相互作用在调节小窝蛋白-1和发动蛋白功能中的作用。 霍乱毒素亚单位B和白蛋白的内吞作用和跨细胞转运将通过电子显微镜、共聚焦显微镜和培养细胞中碘化示踪剂的测量来确定。 分子遗传学和生物化学策略将被用来阐明小窝蛋白-1，Src和发动蛋白之间的结构域特异性和磷酸化依赖的相互作用。 因此，这些研究将阐明Src依赖的信号转导机制，调节内吞作用和运输通过肺血管内皮屏障。

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.