Mechanisms and consequences of cytokine-induced tight junction barrier regulation

描述(由申请人提供)：在炎症性、感染性、缺血性和免疫介导性肠道疾病中，肠道屏障功能受损。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)诱导的屏障丧失的机制和影响，目的是：i)定义肿瘤坏死因子诱导的、肌球蛋白轻链激酶(MLCK)依赖的紧密连接调节；ii)证明MLCK对于肿瘤坏死因子诱导的腹泻是必需的；iii)鉴定和鉴定人MLCK启动子；iv)证明MLCK激活在人类炎症性肠病(IBD)中的作用；v)表明慢性上皮MLCK激活可以刺激黏膜免疫细胞，使小鼠对实验性IBD敏感。我们现在已经开始确定肿瘤坏死因子特异性激活MLCK1的机制，MLCK1是一种长MLCK剪接变异体，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通量而不影响离子的选择性。我们的初步数据显示，在体内表达具有结构性活性的MLCK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激活改变了细胞旁屏障的离子选择性。这是由于粘膜IL-13的产生增加，导致上皮claudin-2的表达产生小的、阳离子选择性的“孔”，允许Na+，而不是大分子，穿过细胞旁途径。这一体内结果强调了穿过紧密连接的两条不同路径的存在及其之间的相互作用；一条允许小的未带电溶质和特定离子通过的高容量“孔”路径，以及一条可被较大的、未带电的大分子渗透但不具有离子选择性的低容量“泄漏”路径。“毛孔”和“渗漏”途径通过粘膜免疫系统相互作用，即产生IL-13，这一事实表明这些途径可能在疾病发病机制中做出了不同的贡献。这些数据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外途径，即“毛孔”和“泄漏”，以不同的方式影响粘膜动态平衡和疾病发病机制。本应用的目的是：1)确定肿瘤坏死因子触发MLCK1的周向转运和酶激活以增加紧密连接的“泄漏”途径的通量的机制；2)确定调节Claudin-2表达和“孔”途径通量的机制；以及3)确定MLCK、occludin和claudin-2的表达对“孔”和“漏”途径功能以及慢性肠道疾病的发生和发展的影响。作为一个整体，这些研究将确定特定的上皮调节过程和免疫信号在调节“毛孔”和“泄漏”途径、细胞旁流量和疾病发病机制中的相对作用。这些数据将提供对肠道屏障功能对人类健康和疾病贡献的机制的新理解，将为开发纠正屏障功能障碍的新治疗策略奠定必要的基础。 与公共卫生相关：肠壁(上皮)必须保持一种屏障，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。建议的研究将促进对屏障调节和调节失调的机制的理解，从而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.