PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

我们的长期目标是确定紧密连接（TJ）的生物学特性及其组成蛋白如何响应 脂质环境的变化。在这个应用程序中，我们专注于occludin的作用，在信号的肌动蛋白 细胞骨架使用小鼠气管上皮（MTE 7 b）细胞系解决了四个核心问题。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 在siRNA敲低闭合蛋白80- 96%的MTE 7 b细胞克隆中，我们测试了信号转导的改变， 使用a.通过GEF-H1/ Rho-GTP/ROCK 1途径的肌动蛋白细胞骨架。一种新的检测方法， 细胞Rho-GTP。B.凋亡细胞挤出测定，c.溶质渗透率测量，d。纺丝 圆盘共聚焦显微镜检查伤口愈合。2.封闭蛋白的哪些结构域从 细胞膜和肌动蛋白骨架之间的联系敲除内源性闭合蛋白的MTE 7 b细胞 用沉默突变的、siRNA抗性闭合蛋白构建体转染，其中特异性 细胞质和细胞外结构域中的氨基酸序列被缺失或封闭。转染的 测试细胞挤出凋亡细胞的能力，重组皮质肌动蛋白， 耗尽，产生Rho-GTP并改变TJ对大有机阳离子的渗透性。3.这些阻塞 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和用siRNA抗性闭合蛋白转染的MTE 7 b细胞 缺失突变体，决定了特定的闭合蛋白结构域在促进通过TJ的迁移中的作用， 表达闭合蛋白的对照DC和从闭合蛋白缺失小鼠分离的DC。4.封闭蛋白和 GEF-H1/ RhoA/ ROCK I信号通路在DC跨上皮迁移中起作用 吸入性抗原闭合蛋白和GEF-H1/RhoA/ ROCK I通路的作用在体内在一个实验中确定。 在occludin敲除小鼠及其同窝对照中抗原刺激的DC迁移的模型。结果 拟议的研究将提供新的信息，特别是关于occludin最近描述的信号， 转导功能，并将形成新的策略，用于调节通过治疗的基础上， 用于促进肺的树突状细胞监视和/或用于预防 过敏原穿过TJ屏障的渗透。

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 DCmigration 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.