PERMEABILITY OF THE LUNG TO WATER SOLUBLE SOLUTES

我们的长期目标是确定紧密连接(TJ)的生物学及其组成蛋白如何反应 他们的脂肪环境发生了变化。在本应用中，我们主要关注occludin在肌动蛋白信号转导中的作用 细胞骨架。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？vbl.使用 MTE7b细胞克隆，在其中siRNA将occludin降低80%-96%，我们测试信号的变化 使用一种新的方法定位肌动蛋白细胞内骨架。 细胞Rho-GTP。B.细胞凋亡性排泄试验，c.溶质通透性测定，d.旋转 圆盘共聚焦显微镜检查伤口愈合情况。2.哪些遮挡蛋白结构域传递信号 质膜到肌动蛋白细胞骨架？内源性封闭蛋白被敲除的MTE7b细胞 通过siRNA向下，通过沉默突变的、具有siRNA抗性的阻断素结构来转染，在这些结构中 胞内区和胞外区的氨基酸序列被删除或阻断。被转染者 测试细胞排出凋亡细胞、在胆固醇之后重新组织皮质肌动蛋白的能力。 消耗，产生Rho-GTP，并改变TJ对大的有机阳离子的渗透性。3.哪种遮挡 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？vbl.使用 携带siRNA沉默内源性闭塞蛋白的MTE7b细胞及转导siRNA抗性闭塞蛋白的细胞 缺失突变体，确定特定的闭锁蛋白结构域在促进TJ迁移中的作用 对照表达occludin的DC和从occludin基因缺失小鼠分离的DC。4.遮挡和遮挡起什么作用 全球环境基金-H1/RhoA/ROCK I信号通路在跨上皮树突状细胞迁移中的作用 吸入的抗原？阻滞素和全球环境基金-H1/RhoA/ROCK I通路的作用是在体内确定的 抗原刺激闭塞缺陷型小鼠DC迁移模型及其仔鼠对照。评选结果 拟议的研究将具体提供有关Occludin最近描述的信号的新信息 转导功能，并将成为调节治疗通路的新策略的基础 跨越肺上皮的药物，用于促进树突状细胞对肺的监视和/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.