Mechanisms and consequences of cytokine-induced tight junction barrier regulation

描述（由申请人提供）：肠屏障功能在炎症、感染、缺血和免疫介导的肠道疾病中受损。在前一个周期中，我们使用体外和体内模型研究了肿瘤坏死因子（TNF-）诱导的屏障丧失的机制和影响，以：i）定义TNF-诱导的肌球蛋白轻链激酶（MLCK-）依赖性紧密连接调节; ii）显示MLCK是TNF-诱导的腹泻所需的; iii）鉴定和表征人MLCK启动子; iv）证实了MLCK在人炎症性肠病（IBD）中活化;和v）表明慢性上皮MLCK活化刺激粘膜免疫细胞并使小鼠对实验性IBD敏感。我们现在已经开始定义TNF特异性激活MLCK 1（一种长MLCK剪接变体）以触发紧密连接蛋白occludin的内吞作用的机制。这产生了紧密连接“泄漏”，允许大的大分子的细胞旁通量而不影响离子选择性。我们的初步数据表明，在体内的组成型活性MLCK的表达增加了通过'泄漏'途径的流量。此外，与体外MLCK激活不同，慢性体内MLCK激活改变了细胞旁屏障的离子选择性。这是由于粘膜IL-13产生增加，其诱导上皮claudin-2表达以产生小的阳离子选择性“孔”，其允许Na+而不是大的大分子穿过细胞旁途径。该体内结果强调了穿过紧密连接的两种不同途径的存在以及它们之间的相互作用;允许小的不带电溶质和特定离子通过的高容量“孔”途径和可渗透较大的不带电大分子但不具有离子选择性的低容量“泄漏”途径。“孔”和“漏”途径通过粘膜免疫系统（即IL-13产生）相互作用的事实表明，这些途径可能对疾病发病机制有不同的贡献。这些和其他数据导致了中心假设，即至少有两种调节机制调节独特的细胞旁途径，即“孔”和“泄漏”，以差异化地影响粘膜稳态和疾病发病机制。本申请的目的是i）定义TNF触发MLCK 1连接周围运输和酶激活以增强穿过紧密连接“泄漏”途径的通量的机制; ii）鉴定调节密蛋白-2表达和“孔”途径通量的机制;和iii）确定MLCK、闭合蛋白和密蛋白-2表达对“孔”和“漏”途径功能以及慢性肠道疾病的起始和进展的影响。作为一个整体，这些研究将确定特定的上皮调控过程和免疫信号在调节“孔”和“漏”途径旁细胞流量和疾病发病机制的相对作用。这些数据将为肠道屏障功能促进人类健康和疾病的机制提供新的理解，为开发纠正屏障功能障碍的新治疗策略奠定必要的基础。 公共卫生相关性：肠道内层（上皮）必须保持一个屏障，使肠道内容物与身体的其余部分分开。这一功能在肠道疾病中经常受到损害，并被认为是疾病发展的早期步骤。拟议的研究将促进对屏障调节和失调的机制理解，因此，导致开发新的治疗方法来改善人类健康。

DESCRIPTION (provided by applicant): Intestinal barrier function is compromised in inflammatory, infectious, ischemic, and immune-mediated intestinal disease. In the previous cycle we studied mechanisms and impact of tumor necrosis factor- (TNF-) induced barrier loss using in vitro and in vivo models to: i) define TNF-induced, myosin light chain kinase- (MLCK-) dependent tight junction regulation; ii) show that MLCK is required for TNF-induced diarrhea; iii) identify and characterize the human MLCK promoter; iv) document MLCK activation in human inflammatory bowel disease (IBD); and v) show that chronic epithelial MLCK activation stimulates mucosal immune cells and sensitizes mice to experimental IBD. We have now begun to define the mechanisms by which TNF specifically activates MLCK1, a long MLCK splice variant, to trigger endocytosis of the tight junction protein occludin. This creates tight junction 'leaks' that allow paracellular flux of large macromolecules without affecting ion selectivity. Our preliminary data show that expression of constitutively-active MLCK in vivo increases flux across the 'leak' pathway. Moreover, unlike in vitro MLCK activation, chronic in vivo MLCK activation alters ion selectivity of the paracellular barrier. This is due to increased mucosal IL-13 production, which induces epithelial claudin-2 expression to create small, cation-selective 'pores' that allow Na+, but not large macromolecules, to traverse the paracellular pathway. This in vivo result emphasizes the presence of, as well as interactions between, two distinct pathways across the tight junction; a high capacity 'pore' pathway that allows small uncharged solutes and specific ions to pass and a low capacity 'leak' pathway that is permeable to larger, uncharged macromolecules but is not ion selective. The fact that 'pore' and 'leak' pathways interact through the mucosal immune system, i.e. IL-13 production, suggests that these pathways may make distinct contributions to disease pathogenesis. These and other data have led to the central hypothesis that at least two mechanisms of regulation modulate unique paracellular pathways, i.e. 'pore' and 'leak,' to differentially impact mucosal homeostasis and disease pathogenesis. The aims of this application are to i) define the mechanisms by which TNF triggers MLCK1 perijunctional trafficking and enzymatic activation to enhance flux across the tight junction 'leak' pathway; ii) identify the mechanisms that regulate claudin-2 expression and 'pore' pathway flux; and iii) determine the impact of MLCK, occludin, and claudin-2 expression on 'pore' and 'leak' pathway function as well as initiation and progression of chronic intestinal disease. As a whole, these studies will define the relative roles of specific epithelial regulatory processes and immune signaling in modulation of 'pore' and 'leak' pathway paracellular flux and disease pathogenesis. The data, which will provide new understanding of the mechanisms by which intestinal barrier function contributes to human health and disease, will lay the foundation necessary for development of novel therapeutic strategies to correct barrier dysfunction. PUBLIC HEALTH RELEVANCE: The intestinal lining (epithelium) must maintain a barrier that keeps the intestinal contents separate from the remainder of the body. This function is frequently compromised in intestinal disease and has been implicated as an early step in disease development. The proposed studies will advance mechanistic understanding of barrier regulation and dysregulation and, therefore, lead to development of novel therapeutic approaches to improve human health.