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）的生物学特性及其组成蛋白如何响应脂质环境的变化。在这个应用中，我们关注闭塞蛋白在肌动蛋白细胞骨架信号传导中的作用。使用小鼠气管上皮（MTE7b）细胞系解决了四个核心问题。1) occludin“敲低”对肌动蛋白介导的细胞功能有什么影响？使用siRNA敲除闭塞蛋白80-96%的MTE7b细胞克隆，我们使用一种定位细胞内Rho-GTP的新方法，通过GEF-H1/ Rho-GTP/ ROCK1途径检测肌动蛋白细胞骨架信号的变化。b.凋亡细胞挤压实验，c.溶质渗透性测量，d.旋转盘共聚焦显微镜检查伤口愈合。2)哪些occludin结构域将信号从质膜传递到肌动蛋白细胞骨架？内源性occludin被siRNA敲低的MTE7b细胞，转染了沉默突变的siRNA抗性occludin构建物，其中细胞质和细胞外结构域的特定氨基酸序列被删除或阻断。检测转染细胞挤压凋亡细胞的能力，胆固醇消耗后重组皮质肌动蛋白，产生Rho-GTP和改变TJ对大有机阳离子的通透性。3)哪些occludin结构域在维持TJ屏障的同时促进树突状细胞（DC）通过TJ的迁移？利用sirna沉默内源性occludin的MTE7b细胞，转染sirna耐药的occludin缺失突变体，确定特定的occludin结构域在促进表达occludin的对照DC和从occludin缺失小鼠分离的DC通过TJs迁移中的作用。4) occludin和GEF-H1/ RhoA/ ROCK I信号通路在吸入抗原的跨上皮DC迁移中起什么作用？occludin和GEF-H1/RhoA/ ROCK I通路在体内的作用是在occludin缺失小鼠及其窝鼠对照中抗原刺激DC迁移的模型中确定的。拟议的研究结果将提供关于occludin最近描述的信号转导功能的新信息，并将为调节治疗药物通过肺上皮，促进肺树突状细胞监测和/或防止过敏原穿过TJ屏障的渗透的新策略奠定基础。

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.