TNF-alpha Regulation of Intestinal Paracellular Transport

摘要： 肠上皮细胞之间的肠细胞旁转运或细胞旁渗透受到调节 紧密连接。 克罗恩病（CD）患者存在肠紧密连接（TJ）屏障缺陷，表现为 细胞旁通透性增加。肠TJ屏障功能障碍是肠梗阻的重要致病因素 CD允许毒性管腔抗原的细胞旁渗透增加，导致肠道炎症。 肿瘤坏死因子-<$（TNF-<$），一种促炎细胞因子，已被证明在肿瘤的发生发展中起核心作用。 肠道炎症过程的CD。TNF-α的一个重要的促炎作用是引起功能性炎症， 打开肠TJ屏障，导致有害管腔内毒素的细胞旁渗透增加， 抗原TNF-α诱导的肠道细胞旁通透性增加被认为是一种 在CD和其他炎症性疾病中， 内脏这项资助计划的主要目标是阐明细胞和分子机制 介导TNF-α诱导的肠TJ通透性增加，并确定其可能性。 治疗靶点，以防止TNF-α诱导的TJ屏障缺陷和随后的肠 炎症为了实现这些目标，我们打算使用两种体外（由过滤生长的Caco-2组成） 肠上皮细胞）和体内（小鼠肠灌注系统）模型系统。 我们的初步研究表明，TNF-α诱导的肠上皮TJ通透性增加， 部分受ERK 1/2和p38信号级联诱导的肌球蛋白轻链激酶增加的调节 （MLCK）基因活性和microRNA诱导闭合蛋白基因表达下调。基于我们 初步研究，我们提出了一个新的假设，即TNF-α诱导的肠道TJ增加， 渗透性部分由ERK 1/2和p38信号级联诱导的MLCK基因活化介导， 调节microRNA的表达。提出的具体目标是：1）阐明细胞内和细胞外的 介导TNF-α诱导MLCK基因活性和肠道TJ增加的分子过程 渗透性; 2）描绘介导TNF-α诱导的细胞和分子机制， occludin基因和蛋白质表达的调节; 3）描述TNF-α参与的机制。 诱导体内肠通透性增加，并确定靶向 在TNF-α诱导的肠道炎症中保护TJ屏障功能。

Abstract: Intestinal paracellular transport or paracellular permeation in-between intestinal epithelial cells is regulated by tight junctions. Patients with Crohn's disease (CD) have a defective intestinal tight junction (TJ) barrier, manifested by an increase in paracellular permeability. The defective intestinal TJ barrier is an important pathogenic factor of CD that allows increased paracellular permeation of toxic luminal antigens, leading to intestinal inflammation. Tumor necrosis factor-¿ (TNF-¿), a pro-inflammatory cytokine, has been shown to play a central role in the intestinal inflammation process of CD. An important pro-inflammatory action of TNF-¿ is to cause a functional opening of the intestinal TJ barrier, leading to an increase in paracellular permeation of noxious luminal antigens. The TNF-¿ induced increase in intestinal paracellular permeability has been postulated to be an important mechanism contributing to the intestinal TJ barrier defect in CD and other inflammatory conditions of the gut. The broad objectives of this grant proposal are to elucidate the cellular and molecular mechanisms that mediate the TNF-¿ induced increase in intestinal TJ permeability and to determine the potential therapeutic targets to prevent the TNF-¿ induced defect in TJ barrier and subsequent development of intestinal inflammation. To achieve these objectives, we intend to use both in-vitro (consisting of filter-grown Caco-2 intestinal epithelial cells) and in-vivo (mouse intestinal perfusion system) model systems. Our preliminary studies suggested that the TNF-¿ induced increase in intestinal epithelial TJ permeability was regulated in part by ERK 1/2 and p38 signaling cascade induced increase in myosin light chain kinase (MLCK) gene activity and microRNA induced down-regulation of occludin gene expression. Based on our preliminary studies, we advance a novel hypothesis that the TNF-¿ induced increase in intestinal TJ permeability is mediated in part by ERK1/2 and p38 signaling cascade induced activation of MLCK gene and modulation of microRNA expression. The proposed specific aims are to: 1) elucidate the intracellular and the molecular processes that mediate the TNF-¿ induced increase in MLCK gene activity and intestinal TJ permeability; 2) delineate the cellular and molecular mechanisms that mediate the TNF-¿ induced down- regulation of occludin gene and protein expression; and 3) delineate the mechanisms involved in TNF-¿ induced increase in intestinal permeability in-vivo and determine the therapeutic implications of targeted preservation of TJ barrier function in TNF-¿ induced intestinal inflammation.