Mechanisms of Cytoskeletal Regulation of Tight Junction Homeostasis and Repair

 描述(由申请人提供)： 紧密连接(TJ)需要持续的动态平衡维持。很明显，TJ蛋白内化和囊泡循环是这一过程的关键，但其潜在的机制尚不清楚。使用非生理刺激的研究已经将肌动蛋白细胞骨架定义为TJ组装和维持的关键贡献者。肌动球蛋白收缩与TJ的生理和病理生理屏障丧失有关，如由肿瘤坏死因子(TNFa)触发的TJ屏障丧失，驱动TJ蛋白阻滞素的下游空泡内吞。类似的过程也与肠道致病性大肠杆菌(EPEC)感染引起的屏障丧失有关。虽然这两种形式的屏障丧失都是可逆的，但尚不清楚肌动球蛋白松弛或TJ蛋白循环是否参与了这一过程。此外，尽管微管在心尖蛋白循环中的作用已被证实，但很少有研究检查微管对TJ动态平衡和修复的贡献。最近，人们发现微管的一个特殊亚群，即微管的平面顶端网络，与TJ蛋白cinglin相互作用。这种相互作用由AMP激活的蛋白激酶(AMPK)介导的cinglin的磷酸化来调节，对上皮的形态发生是必不可少的。我们的数据显示，在钙开关测定中，微管破裂阻碍了TJ的恢复。此外，EPEC毒力因子ESPG破坏微管，削弱TJ屏障的恢复。这些发现表明，微管在TJ内稳态和修复中发挥着意想不到的关键作用。然而，微管在这些过程中的作用机制还没有被探索。这项建议的长期目标是确定TJ结构和功能恢复所涉及的分子和细胞过程，并利用这一知识设计促进上皮修复的治疗方法。本研究的目的是明确肌动蛋白细胞骨架和微管在TJ从病理生理损伤中恢复的作用。中心假设是微丝和微管在受到炎症和感染等病理损伤后，积极参与重建肠上皮细胞。这项研究与退伍军人群体高度相关，因为部队中的腹泻相当普遍，导致在任何给定时间都有相当一部分人不适合执行任务，并与慢性健康后遗症有关，包括肠易激疾病和炎症性肠病。确定TJ维护和修复所涉及的事件将指导制定保存完好TJ和帮助TJ恢复的战略。中心假设将通过三个目标得到验证：1.确定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.确定微管在体外炎性损伤(TNF)和肠道感染(EPEC)恢复TJ中的作用；以及3.确定微丝和微管在体内TJ从病理生理条件(肿瘤坏死因子和EPEC感染)恢复中的作用。

 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.