Mechanisms and consequences of cytokine-induced tight junction barrier regulation

细胞因子诱导的紧密连接屏障调节的机制和后果

基本信息

批准号：
7996729
负责人：
JERROLD R. TURNER
金额：
$ 55.56万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7996729
关键词：
Affect Cations Cells Chronic Data Development Diarrhea Disease Endocytosis Epithelial Epithelium Event Foundations Functional disorder Goals Health Homeostasis Human Immune Immune system In Vitro Inflammatory Inflammatory Bowel Diseases Interleukin-13 Intestinal Content Intestinal Diseases Intestines Ions Knock-out Lead MYLK gene Mediating Modeling Molecular Molecular Chaperones Mus Myosin Light Chain Kinase Pathogenesis Pathway interactions Process Production Productivity Progress Reports Proteins RNA Splicing Regulation Relative (related person)Research Role Signal Transduction TNF gene Tertiary Protein Structure Therapeutic Tight Junctions Tissues Transgenic Model Tumor Necrosis Factor-alpha Tumor Necrosis Factors Variant base cytokine human disease improved in vivo in vivo Model innovation macromolecule novel therapeutic intervention novel therapeutics occludin overexpression programs promoter public health relevance response solute tool trafficking transcription factor

项目摘要

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.

描述（由申请人提供）：肠道屏障功能在炎症，传染性，缺血和免疫介导的肠道疾病中受到损害。在上一个周期中，我们研究了使用体外和体内模型的肿瘤坏死因子（TNF-）诱导的屏障损失的机制和影响： ii）表明MLCK是TNF诱导的腹泻所必需的； iii）识别并表征人类MLCK启动子； iv）记录人类炎症性肠病（IBD）中的MLCK激活； v）表明慢性上皮MLCK激活刺激粘膜免疫细胞，并使小鼠对实验IBD敏感。现在，我们已经开始定义TNF特异性激活MLCK1的机制，MLCK1是一种长长的MLCK剪接变体，以触发紧密连接蛋白闭塞的内吞作用。这会产生紧密的连接“泄漏”，从而允许大型大分子的细胞细胞通量，而不会影响离子选择性。我们的初步数据表明，体内组成型MLCK的表达会增加跨“泄漏”途径的通量。此外，与体外MLCK激活不同，慢性体内MLCK激活改变了细胞细胞屏障的离子选择性。这是由于粘膜IL-13产生的增加，从而诱导上皮claudin-2表达产生小的阳离子选择性的“孔”，从而允许Na+（但不能大的大分子）横穿细胞细胞途径。这种体内结果强调了整个紧密连接的两个不同途径之间的存在以及相互作用。高容量的“孔”途径，允许小的未充电溶质和特定离子通过，并且具有低容量的“泄漏”途径，该途径可渗透到较大的，未充电的大分子，但不是离子选择性的。 “孔”和“泄漏”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理做出不同的贡献。这些和其他数据导致了一个中心假设，即调节的至少两种机制调节独特的细胞细胞途径，即“孔”和“泄漏”，从而差异地影响了粘膜稳态和疾病发病机理。该应用的目的是i）定义TNF触发MLCK1的插座运输和酶促激活的机制，从而增强了跨紧密连接“泄漏”途径的通量； ii）确定调节claudin-2表达和“孔”通路的机制； iii）确定MLCK，Occludin和Claudin-2表达对“孔”和“泄漏”途径功能以及慢性肠道疾病的起始和进展的影响。总体而言，这些研究将定义特定上皮调节过程的相对作用以及免疫信号在调节“孔”和“泄漏”途径旁细胞通量和疾病发病机理中的相对作用。该数据将对肠道屏障功能有助于人类健康和疾病的机制提供新的理解，它将为开发新型治疗策略的基础奠定基础，以纠正障碍功能障碍。公共卫生相关性：肠壁（上皮）必须保持障碍，使肠道含量与身体的其余部分分开。该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一步。拟议的研究将提高对障碍调节和失调的机械理解，从而导致开发新的治疗方法以改善人类健康。