Mechanism of the paracellular molecular transport

细胞旁分子转运机制

基本信息

批准号：
12144208
负责人：
HORIGUCHI Yasuhiko
金额：
$ 32.77万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12144208/
关键词：
tight iunction claudin 13 aracellular pathway nephron enteropathogenic E. coli ウェルシュ菌上皮細胞上皮細胞バリアー 3型分泌機構インチミン Tir 水分蒸散 EPEC Caco2 上皮細胞間電気抵抗

项目摘要

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.

为了理解紧密连接（TJ）作为细胞旁空间分子传递和生理屏障的装置，我们分析了组成TJ的紧密连接蛋白的功能以及细菌毒力因子影响TJ.1细胞旁屏障功能的作用方式。 (1)我们分别制备了claudin-1缺陷型和claudin-5缺陷型小鼠，发现claudin-1构成了首先在表皮中证实的TJ，而claudin-5本质上在表皮中形成了TJ。 (2)我们利用表达GFP标记的密蛋白的Eph4上皮细胞分析了TJ链的动态行为。研究发现，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，多余的紧密蛋白被内吞到相邻细胞之一中。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是造成这种情况的原因。此外，EPEC 和上皮细胞之间的密切联系被发现会触发除 Map 之外的毒力因子的分泌，这对于屏障破坏活性至关重要(2)我们分析了claudins和产气梭菌肠毒素(CPE)之间的结合性质，已知CPE能够特异性地将claudins识别为受体。结果表明，除了迄今为止报道的紧密蛋白3和4之外，CPE还与紧密蛋白6、7、8和14结合，并且CPE识别紧密蛋白的第二细胞外环。体外和体内实验均表明，CPE 的密蛋白结合片段打开了上皮细胞的细胞旁通路。