PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

描述(由申请人提供)：我们的长期目标是确定紧密连接(TJ)的生物学及其组成蛋白如何对其脂质环境的变化做出反应。在这一应用中，我们主要关注occludin在肌动蛋白细胞骨架信号传递中的作用。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“击倒”对肌动蛋白介导的细胞功能有何影响？在MTE7b细胞克隆中，siRNA使occludin下降了80%-96%，我们使用一种新的方法来定位细胞内的Rho-GTP，以测试通过全环基金-H1/Rho-GTP/ROCK1途径向肌动蛋白细胞骨架发出的信号的变化。B.细胞凋亡性排泄试验，c.溶质渗透性测量，d.旋转圆盘共聚焦显微镜检查伤口愈合。2)哪些封闭蛋白结构域将信号从质膜传递到肌动蛋白细胞骨架？在MTE7b细胞中，内源性闭塞蛋白被siRNA击倒，并被导入沉默突变的、具有siRNA抗性的闭塞蛋白结构，其中细胞质和胞外区域的特定氨基酸序列被删除或阻断。测试转基因细胞排出凋亡细胞、在胆固醇耗尽后重组皮质肌动蛋白、产生Rho-GTP和改变TJ对大分子有机阳离子的通透性的能力。3)哪些封闭蛋白结构域在维持TJ屏障的同时促进树突状细胞(DC)通过TJ的迁移？利用带有siRNA沉默的内源性occludin的MTE7b细胞和导入siRNA抗性occludin缺失突变体的MTE7b细胞，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TJ的迁移中的作用。4)occludin和gef-H1/RhoA/ROCK I信号通路在吸入性抗原引起的跨上皮树突状细胞迁移中起什么作用？在抗原刺激的封闭小鼠DC迁移模型及其仔鼠对照中，在体内确定了封闭蛋白和全球环境基金-H1/RhoA/ROCK I通路的作用。拟议的研究结果将特别提供有关Occludin最近描述的信号转导功能的新信息，并将为新的战略奠定基础，以调节治疗药物通过肺上皮的通道，促进树突状细胞对肺的监测和/或防止过敏原穿过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.