PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

描述（由申请人提供）：我们的长期目标是定义紧密连接（TJ）的生物学及其组成蛋白如何响应其脂质环境的变化。在这个应用程序中，我们专注于occludin的信号转导的肌动蛋白细胞骨架的作用。使用小鼠气管上皮（MTE7b）细胞系解决了四个中心问题。1)封闭蛋白的"敲低"对肌动蛋白介导的细胞功能有什么影响？使用siRNA敲低闭合蛋白80 - 96%的MTE7b细胞克隆，我们使用a.定位细胞内Rho-GTP的新测定。B.凋亡细胞挤出测定，c.溶质渗透率测量，d。旋转圆盘共聚焦显微镜检查伤口愈合。2)哪一个occludin结构域将信号从质膜传递到肌动蛋白细胞骨架？用沉默突变的siRNA抗性闭合蛋白构建体转染其中内源闭合蛋白被siRNA敲低的MTE7b细胞，其中细胞质和细胞外结构域中的特定氨基酸序列被缺失或阻断。测试转染的细胞挤出凋亡细胞、在胆固醇耗尽后重组皮质肌动蛋白、产生Rho-GTP和改变TJ对大有机阳离子的渗透性的能力。3)哪些闭合蛋白结构域促进树突状细胞（DC）通过TJ迁移，同时维持TJ屏障？使用具有siRNA沉默的内源性闭合蛋白并转染有siRNA抗性闭合蛋白缺失突变体的MTE7b细胞，确定特异性闭合蛋白结构域在促进表达闭合蛋白的对照DC和从闭合蛋白缺失小鼠分离的DC通过TJ的迁移中的作用。4)occludin和GEF-H1/RhoA/ROCK I信号通路在吸入抗原诱导的DC跨上皮迁移中发挥什么作用？在闭合蛋白缺失小鼠及其同窝对照中，在抗原刺激的DC迁移模型中体内确定闭合蛋白和GEF-H1/RhoA/ROCK I途径的作用。拟议的研究结果将提供新的信息，特别是关于occludin的最近描述的信号转导功能，并将形成新的策略的基础，用于调节治疗剂通过肺上皮，促进树突状细胞监测肺和/或防止过敏原渗透通过TJ屏障。

DESCRIPTION (provided by applicant): Our long-term goal is to define the biology of the tight junction (TJ) and how its constituent proteins respond to changes in their lipid environment. In this application we focus on occludin's role in signaling to the actin cytoskeleton. Four central questions are addressed using a mouse tracheal epithelial (MTE7b) cell line. 1) What are the consequences of occludin "knock-down" on actin mediated cell functions? Using MTE7b cell clones in which siRNA knocks down occludin by 80-96%, we test for alterations in signaling to the actin cytoskeleton via the GEF-H1/ Rho-GTP/ ROCK1 pathway using a. A novel assay to localize intra- cellular Rho-GTP. b. An apoptotic cell extrusion assay, c. Solute permeability measurements, d. Spinning disc confocal microscopy to examine wound healing. 2) Which occludin domains transduce signals from the plasma membrane to the actin cytoskeleton? MTE7b cells in which endogenous occludin is knocked down by siRNA, are transfected with silently mutated, siRNA-resistant occludin constructs in which specific amino acid sequences in the cytoplasmic and extra-cellular domains are deleted or blocked. The transfected cells are tested for their ability to extrude apoptotic cells, re-organize cortical actin following cholesterol depletion, generate Rho-GTP and alter TJ permeability to large organic cations. 3) Which occludin domains facilitate dendritic cell (DC) migration through TJs while maintaining the TJ barrier? Using MTE7b cells with siRNA-silenced endogenous occludin and transfected with siRNA-resistant occludin deletional mutants, determine the role of specific occludin domains in facilitating the migration through TJs of control DC that express occludin and DC isolated from occludin null mice. 4) What role does occludin and the GEF-H1/ RhoA/ ROCK I signaling pathway play in trans-epithelial DC migration in response to inhaled antigen? The role of occludin and the GEF-H1/RhoA/ ROCK I pathway is determined in vivo in a model of antigen stimulated DC migration in occludin null mice and their littermate controls. Results of the proposed research will provide new information specifically regarding occludin's recently described signal transducing function and will form the basis for new strategies for regulating the passage of therapeutic agents across the lung epithelium, for facilitating dendritic cell surveillance of the lung and/or for preventing the penetration of allergens across the TJ barrier.