Mechanisms of Cytoskeletal Regulation of Tight Junction Homeostasis and Repair

细胞骨架对紧密连接稳态和修复的调节机制

• 批准号: 9206081
• 负责人: Gail A Hecht
• 金额: --
• 依托单位: EDWARD HINES JR VA HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-10-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206081
• 关键词: 5' -AMP-activated protein kinase; Actins; Actomyosin; Acute; Address; Affect; Antigens; Apical; Attenuated; Biological Assay; Calcium; Cell physiology; Chronic; Colitis; Collaborations; Cytoskeleton; Data; Data Reporting; Developing Countries; Development; Diarrhea; Diet; Diffusion; Disease; Disease Progression; Disease model; Dynein ATPase; Effectiveness; Endocytosis; Enteral; Epithelial; Epithelial Cells; Event; Foundations; Functional Gastrointestinal Disorders; Gene Deletion; Genetic Transcription; Genome; Goals; Health; Homeostasis; Hour; Human; In Vitro; Incidence; Individual; Infant; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Injectable; Injury; Intestinal Content; Intestinal Diseases; Intestines; Irritable Bowel Syndrome; Ischemic Bowel Disease; Knowledge; Laboratories; Link; Maintenance; Mediating; Membrane Protein Traffic; Microfilaments; Microtubules; Military Personnel; Mission; Molecular; Molecular Target; Morbidity - disease rate; Morphogenesis; Motor; Mus; Myosin ATPase; Necrotizing Enterocolitis; Pathologic; Periodicity; Pharmacology; Phosphorylation; Physiological; Play; Population; Process; Proteins; Publishing; Recovery; Recycling; Regulation; Relaxation; Research; Resolution; Role; Sterility; Stimulus; Structure; TNF gene; Testing; Therapeutic; Tight Junctions; Time; Type III Secretion System Pathway; Veterans; Virulence Factors; base; chelation; clinically relevant; enteropathogenic Escherichia coli; gastrointestinal infection; improved; in vivo; insight; knock-down; microbiota; monolayer; mortality; novel therapeutic intervention; novel therapeutics; occludin; overexpression; polymerization; prevent; protein transport; public health relevance; repaired; restoration; stem; therapy design; treatment strategy

 DESCRIPTION (provided by applicant): Tight junctions (TJs) undergo constant homeostatic maintenance. While it is clear that TJ protein internalization and vesicular recycling are essential for this process, the underlying mechanisms are not defined. Studies using non-physiological stimuli have defined the actin cytoskeleton as a critical contributor to TJ assembly and maintenance. Actomyosin contraction, which has been linked to physiologic and pathophysiologic TJ barrier loss, such as that triggered by tumor necrosis factor (TNFa), drives downstream caveolar endocytosis of the TJ protein occludin. Similar processes have been implicated in barrier loss caused by enteropathogenic E. coli (EPEC) infection. While both forms of barrier loss are reversible, it is not clear if actomyosin relaxation or TJ protein recycling are involved in this process. Further, despite an established role for microtubules in apical protein recycling, few studies have examined the contributions of microtubules to TJ homeostasis and repair. Recently, a specific subset of microtubules, planar apical networks of microtubules, was found to interact with the TJ protein cingulin. This interaction is regulated by AMP-activated protein kinase (AMPK)-mediated phosphorylation of cingulin and is essential to epithelial morphogenesis. Our data show that microtubule disruption impedes TJ recovery in calcium switch assays. In addition, the EPEC virulence factor EspG, which disrupts microtubules, attenuates TJ barrier restoration. These findings indicate that microtubules play an unexpected and critical role in TJ homeostasis and repair. However, the mechanisms by which microtubules contribute to these processes have not been explored. The long-term goal of this proposal is to define the molecular and cellular processes involved in the restoration of TJ structure and function and use this knowledge to design therapies to promote epithelial repair. The objective of the proposed studies is to define the role of the actin cytoskeleton and microtubules in TJ recovery from pathophysiologic insults. The central hypothesis is that microfilaments and microtubules actively participate in reestablishing intestinal epithelial TJs after disruption by pathological insults such as inflammation and infection. This research is highly relevant to the VA population as diarrhea among military troops is quite prevalent rendering a substantial portion unfit for duty at any given time and is associated with chronic health sequelae including irritable bowel disease and inflammatory bowel disease. Definition of the events involved in TJ maintenance and restoration will guide the development of strategies to preserve intact TJs and aid in TJ recovery. The central hypothesis will be tested by 3 aims: 1. Define the role of the actin cytoskeleton in TJ recovery following molecularly targeted, inflammatory (TNF) and infectious (EPEC), stimuli; 2. Determine the contribution of microtubules to TJ restoration from inflammatory insults (TNF) and enteric infection (EPEC) in vitro; and 3. Resolve the role of microfilaments and microtubules in TJ recovery from pathophysiologic conditions (TNF and EPEC infection) in vivo.

 描述（由申请人提供）： 紧密的连接（TJS）经过持续的稳态维护。虽然很明显，TJ蛋白质内在化和囊泡回收对于此过程至关重要，但未定义的基本机制。使用非生理刺激的研究将肌动蛋白细胞骨架定义为TJ组装和维持的关键因素。与生理和病理生理的TJ屏障丧失有关的肌动蛋白收缩，例如由肿瘤坏死因子（TNFA）触发的障碍物损失，驱动了TJ蛋白闭塞的下游洞穴内吞作用。肠病大肠杆菌（EPEC）感染引起的障碍损失已经实施了类似的过程。尽管两种形式的屏障损失都是可逆的，但尚不清楚肌动菌素松弛或TJ蛋白回收是否参与此过程。此外，尽管微管在顶蛋白回收中起着确定的作用，但很少有研究研究微管对TJ稳态和修复的贡献。最近，发现微管的特定子集（计划的微管的顶端网络）与TJ蛋白质c蛋白相互作用。这种相互作用受AMP激活的蛋白激酶（AMPK）介导的葡萄糖磷酸化的调节，对于上皮形态发生至关重要。我们的数据表明，微管破坏阻碍了钙开关测定中的TJ恢复。此外，破坏微管的EPEC病毒因子ESPG减轻了TJ屏障的恢复。这些发现表明，微管在TJ稳态和修复中起着意想不到的至关重要的作用。但是，微管对这些过程的贡献的机制尚未探索。该提案的长期目标是定义恢复TJ结构和功能涉及的分子和细胞过程，并利用这些知识来设计促进的疗法。上皮修复。拟议的研究的目的是定义肌动蛋白细胞骨架和微管在从病理生理损伤中恢复TJ中的作用。中心假设是微丝和微管在被病理损伤（如感染和感染）破坏后积极参与重建肠上皮TJ。这项研究与VA人群高度相关，因为军队中的腹泻非常普遍，在任何给定时间都不适合任何值班，并且与慢性健康后遗症有关，包括肠胃病和炎症性肠病。 TJ维护和恢复中涉及的事件的定义将指导制定保留完整的TJ并有助于TJ恢复的策略。中心假设将通过3个目标进行检验：1。定义肌动蛋白细胞骨架在分子靶向，炎症（TNF）和感染性（EPEC）之后的TJ恢复中的作用。 2。在体外确定微管对TJ恢复（TNF）和肠感染（EPEC）的贡献；和3。在体内从病理生理状况（TNF和EPEC感染）中恢复微丝和微管在体内恢复中的作用。