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: 8926068
• 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/8926068
• 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; Escherichia coli Infections; Event; Foundations; Functional Gastrointestinal Disorders; Gene Deletion; Genetic Transcription; Genome; Goals; Health; Homeostasis; Hour; Human; In Vitro; Incidence; Infant; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Injury; Intestinal Content; Intestinal Diseases; Intestines; Irritable Bowel Syndrome; Ischemic Bowel Disease; Knowledge; Laboratories; Lead; Link; Maintenance; Mediating; Membrane Protein Traffic; Microfilaments; Microtubules; Military Personnel; Mission; Molecular; Morbidity - disease rate; Morphogenesis; Motor; Mus; Myosin ATPase; Necrotizing Enterocolitis; 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; trafficking; 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）： 紧密连接 (TJ) 经历持续的稳态维持。虽然很明显 TJ 蛋白内化和囊泡回收对于这一过程至关重要，但其潜在机制尚不清楚。使用非生理刺激的研究已将肌动蛋白细胞骨架定义为 TJ 组装和维护的关键贡献者。肌动球蛋白收缩与生理和病理生理性 TJ 屏障丧失有关，例如肿瘤坏死因子 (TNFa) 引发的收缩，驱动 TJ 蛋白 occludin 的下游小凹内吞作用。肠病性大肠杆菌 (EPEC) 感染引起的屏障丧失也涉及类似的过程。虽然两种形式的屏障丧失都是可逆的，但尚不清楚该过程中是否涉及肌动球蛋白松弛或 TJ 蛋白回收。此外，尽管微管在顶端蛋白质回收中发挥了既定的作用，但很少有研究探讨微管对 TJ 稳态和修复的贡献。最近，发现微管的一个特定子集，即微管的平面顶端网络，与 TJ 蛋白扣带蛋白相互作用。这种相互作用受到 AMP 激活蛋白激酶 (AMPK) 介导的 cingulin 磷酸化的调节，并且对于上皮形态发生至关重要。我们的数据表明，微管破坏会阻碍钙转换试验中 TJ 的恢复。此外，EPEC 毒力因子 EspG 会破坏微管，削弱 TJ 屏障的恢复。这些发现表明微管在 TJ 稳态和修复中发挥着意想不到的关键作用。然而，微管促进这些过程的机制尚未被探索。该提案的长期目标是定义 TJ 结构和功能恢复所涉及的分子和细胞过程，并利用这些知识设计促进上皮修复的疗法。本研究的目的是确定肌动蛋白细胞骨架和微管在 TJ 从病理生理损伤中恢复中的作用。中心假设是微丝和微管在炎症和感染等病理损伤破坏后积极参与肠上皮 TJ 的重建。这项研究与退伍军人管理局人群高度相关，因为腹泻在军队中相当普遍，导致很大一部分人在任何特定时间都不适合执行任务，并且与包括肠易激病和炎症性肠病在内的慢性健康后遗症有关。 TJ 维护和恢复所涉及事件的定义将指导制定保护完整 TJ 并帮助 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) 刺激后 TJ 恢复中的作用； 2. 体外确定微管对炎症损伤（TNF）和肠道感染（EPEC）TJ恢复的贡献； 3. 解决微丝和微管在体内病理生理条件（TNF 和 EPEC 感染）中 TJ 恢复中的作用。