Mechanisms and consequences of cytokine-induced tight junction barrier regulation

描述（由申请人提供）：肠道屏障功能在炎症、感染性、缺血性和免疫介导的肠道疾病中受损。在上一个周期中，我们使用体外和体内模型研究了肿瘤坏死因子（TNF-）诱导屏障丧失的机制和影响：i)定义TNF诱导的肌球蛋白轻链激酶（MLCK-）依赖的紧密连接调节；ii)表明tnf诱导的腹泻需要MLCK；iii)鉴定和表征人类MLCK启动子；iv)记录MLCK在人类炎症性肠病（IBD）中的激活；和v)表明，慢性上皮细胞MLCK激活刺激粘膜免疫细胞，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MLCK1的机制，MLCK1是一种长MLCK剪接变体，可以触发紧密连接蛋白occludin的内吞作用。这就产生了紧密的连接“泄漏”，允许大分子的细胞旁流动，而不影响离子的选择性。我们的初步数据表明，体内组成活性MLCK的表达增加了通过“泄漏”途径的通量。此外，与体外MLCK激活不同，体内慢性MLCK激活会改变细胞旁屏障的离子选择性。这是由于粘膜IL-13分泌增加，诱导上皮细胞clclin -2表达，形成小的、阳离子选择性的“孔”，允许Na+（而不是大分子）通过细胞旁通路。这在体内的结果强调存在，以及相互作用，两个不同的途径在紧密连接；一种是允许小的不带电溶质和特定离子通过的高容量“孔”途径，另一种是允许较大的不带电大分子渗透的低容量“漏”途径，但不具有离子选择性。“孔”和“漏”途径通过粘膜免疫系统（即IL-13的产生）相互作用，这一事实表明，这些途径可能对疾病的发病机制有不同的贡献。这些和其他数据导致了一个中心假设，即至少有两种调节机制调节独特的细胞旁通路，即。“孔”和“漏”对粘膜稳态和疾病发病机制的不同影响。本申请的目的是：i)定义TNF触发MLCK1环结运输和酶激活的机制，以增强紧密结“泄漏”途径的通量；Ii)确定调节claudin-2表达和“孔”通路通量的机制；iii)确定MLCK、occludin和claudin-2表达对“孔”和“漏”通路功能以及慢性肠道疾病的发生和进展的影响。总的来说，这些研究将确定特异性上皮调节过程和免疫信号在调节“孔”和“漏”途径、细胞旁通量和疾病发病机制中的相对作用。这些数据将为肠道屏障功能对人类健康和疾病的影响机制提供新的认识，为开发纠正屏障功能障碍的新治疗策略奠定必要的基础。

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.