Mechanism of the paracellular molecular transport

为了了解紧密连接（TJ）作为细胞旁空间的分子传递和生理屏障的装置，我们分析了组成紧密连接的claudins的功能以及细菌毒力因子影响紧密连接的细胞旁屏障功能的作用方式。claudins的功能分析。(1)我们分别产生了claudin-1缺陷型和claudin-5缺陷型小鼠，并发现claudin-1构成了首次在表皮中证明的TJ，而claudin-5基本上在血脑屏障处形成TJ。(2)我们通过使用表达GFP标记的紧密连接蛋白的Eph 4上皮细胞来分析TJ链的动态行为。结果发现，TJ链在伴随细胞运动的重塑过程中保持其连续性。当TJ链缩短时，多余的claudin被内吞到相邻的细胞中。这种动态行为被认为是TJ.2的本构屏障功能的基础。细菌毒力因子影响TJ屏障功能的机制分析（1）我们在上皮细胞模型中研究了肠致病性大肠杆菌（EPEC）如何降低细胞旁屏障功能，发现EPEC毒力因子之一的Map是导致这种功能降低的原因。发现EPEC和上皮细胞之间的紧密结合触发了（a）除Map以外的毒力因子的分泌，其对于EPEC的屏障破坏活性是必需的。(2)我们分析了claudins和产气荚膜梭菌肠毒素（CPE）之间的结合性质，已知CPE特异性识别claudins作为受体。结果表明，CPE与claudin 6、7、8和14结合，而与claudin 3和4结合，CPE识别claudin的第二个胞外环。体外和体内实验均表明，CPE的claudin结合片段打开了上皮细胞的细胞旁通路。

To understand the tight junction (TJ) as the apparatus for molecular transfer and physiological barrier at the paracellular space, we analyzed function of claudins composing TJ and the mode of action by which bacterial virulence factors affect the paracellular barrier function of TJ.1. Functional analyses of claudins.(1) We generated claudin-1-deficient and claudin-5-deficient mice, respectively, and found that claudin-1 constitutes TJ that was first demonstrated in the epidermis, and claudin-5 essentially forms TJ at the blood-brain barrier.(2) We analyzed dynamic behavior of the TJ strand by using Eph4 epithelial cells expressing GFP-tagged claudin. It was found that the TJ strands retain their continuities during their remodeling accompanied by the cell movement When the TJ strands shortened, the superfluous claudins were endocytosed into one of the adjacent cells. This dynamic behavior is considered to underlie the constitutive barrier function of TJ.2. Analyses for mechanisms by which bacterial virulence factors affect the barrier function of TJ(1) We examined how enteropathogenic Escherichia coli (EPEC) reduces the paracellular barrier function in the epithelial cell model, and found that Map, one of EPEC virulence factors, is responsible for it Moreover, the intimate association between EPEC and epithelial cells was found to trigger the secretion of (a) virulence factor(s) other than Map, which are essential for the barrier-disrupting activity of EPEC.(2) We analyzed the binding nature between claudins and Clostridium perfiivens enterotoxin (CPE), which is known to specifically recognize claudins as receptors. The results demonstrated that CPE binds to claudin 6,7,8, and 14 besides claudin 3 and 4, which had been reported so far, and that CPE recognizes the second extracellular loop of the claudins. Both in vitro and in vivo experiments revealed that a claudin-binding fragment of CPE opened the paracellular pathways of epithelial cells.