Regulation of Intestinal Epithelial Restitution

描述（由申请人提供）： 项目摘要/摘要早期粘膜修复是胃肠道重要的一级修复方式，其缺陷性调节是各种关键病理状态的基础，如粘膜出血和急性损伤、胃肠道上皮完整性破坏和屏障功能障碍。由于浅表创伤后急性粘膜损伤和早期快速粘膜上皮再生的确切机制仍不清楚，因此临床上保护GI上皮完整性的有效治疗方法有限，尤其是在外科危重病患者中。在之前的资助期间，我们已经确定了经典瞬时受体电位-1（TRPC 1）介导的Ca 2+信号在调节粘膜损伤后肠上皮恢复中的重要性。然而，确切的上游信号启动TRPC 1通道激活后，粘膜损伤仍然难以捉摸，是这个竞争性更新应用的焦点。最近，基质相互作用分子1（STIM 1）被确定在筛选的分子是必不可少的激活商店经营的钙通道（SOC），而抑制STIM 1的表达减少商店经营的钙通道（SOCE）后，商店耗尽。我们的初步结果表明，a）损伤后质膜上的STIM 1水平显著增加，B）组成型活性STIM 1 EF-手突变体的过表达增加SOCE并增强损伤后的上皮恢复，以及c）STIM 1沉默降低SOCE并抑制过表达TRPC 1的细胞中的上皮恢复。基于这些令人兴奋的观察结果，我们假设STIM 1通过激活TRPC 1通道活性在促进胃肠道粘膜损伤后上皮修复中起重要作用。提出了三个具体目标来检验这一假设。1)确定STIM 1在体外和体内粘膜损伤后肠上皮恢复中的模式和作用。2)表征STIM 1与TRPC 1在肠上皮恢复过程中调节SOCE的功能和物理相互作用。3)明确黏膜损伤后调控STIM 1亚细胞再分布及其表达的细胞信号通路。这些目标的完成将确定最初激活TRPC 1通道后，粘膜损伤的上游信号，也产生一个新的模型，其中STIM 1亚细胞运输和表达的调节过程中恢复。希望我们的研究结果将提供支持性数据，以加强我们的长期目标，即开发胃肠道粘膜损伤相关疾病的治疗方法，并在各种临床条件下保持上皮完整性。 公共卫生相关性： 当前研究的直接目标是确定急性肠粘膜损伤和修复的机制。各种急性胃肠道粘膜损伤和胃炎在我们的VA患者中比在一般公众中观察到的更常见。据报道，退伍军人中胃肠道溃疡和损伤的患病率为24.7%，而对照人群为10.5%。此外，药物，特别是非甾体抗炎药，和应激作为胃肠道粘膜损伤和溃疡的病因正变得越来越重要。这一点对于新的和年轻的持久自由行动/法语国家组织退伍军人尤其重要，他们返回后，与压力有关的疾病的发病率大幅增加。重症患者以及军事战斗情况下应激诱导的粘膜损伤和溃疡的发生率进一步加剧了VA人群中GI粘膜损伤和损伤相关疾病的问题。 然而，由于缺乏对肠粘膜损伤和修复机制的认识，目前临床上预防急性肠粘膜损伤和促进肠粘膜修复的有效方法还很有限。因此，提高对维持肠粘膜完整性和增强修复的过程的理解是该领域治疗举措的第一步，也是当前提案的重点。 基于我们以前的研究和令人兴奋的初步结果，这里提出的研究是确定一种新的蛋白质STIM 1是否发挥重要作用，促进胃肠道粘膜损伤后的恢复Ca 2+信号。这些目标的完成将提供支持性数据，以加强我们的长期目标，即为我们的VA患者开发胃肠道粘膜损伤相关疾病的治疗方法。

DESCRIPTION (provided by applicant): Project Summary/Abstract Early mucosal restitution is an important primary repair modality in the gastrointestinal (GI) tract and its defective regulation underlies various critical pathological states such as mucosal bleeding and acute injury, disruption of GI epithelial integrity, and barrier dysfunction. Since the exact mechanisms underlying acute mucosal injury and early rapid mucosal reepithelialization after superficial wounds are still obscure, effective therapies to preserve GI epithelial integrity in clinic are limited, especially in patients with critical surgical illnesses. During previous funding period, we have established the importance of canonical transient receptor potential-1 (TRPC1)-mediated Ca2+ signaling in regulating intestinal epithelial restitution after mucosal injury. However, the exact upstream signals initiating TRPC1 channel activation after mucosal injury remain elusive and are the focus of this competitive renewal application. Recently, stromal interaction molecule 1 (STIM1) was identified in screens for molecules that are essential for the activation of store-operated Ca2+ channels (SOCs), whereas inhibition of STIM1 expression reduces store-operated Ca2+ entry (SOCE) after store depletion. Our preliminary results indicate that a) levels of levels of STIM1 at the plasma membrane increase significantly after wounding, b) overexpression of the constitutively active STIM1 EF-hand mutant increases SOCE and enhances epithelial restitution after wounding, and c) STIM1 silencing decreases SOCE and represses epithelial restitution in cells overexpressing TRPC1. Based on these exciting observations, we HYPOTHESIZE that STIM1 plays an important role in promoting GI epithelial restitution after mucosal injury by activating TRPC1 channel activity. Three specific aims are proposed to test the hypothesis. 1) To determine the pattern and role of STIM1 in gut epithelial restitution after mucosal injury in vitro as well as in vivo. 2) To characterize functional and physical interactions of STIM1 with TRPC1 in regulating SOCE during intestinal epithelial restitution. 3) To define the cellular signaling pathways regulating STIM1 subcellular redistribution and its expression after mucosal injury. Completion of these aims will identify the up stream signals initially activating TRPC1 channels after mucosal injury and also yield a novel model in which STIM1 subcellular trafficking and expression are regulated during restitution. It is hoped that our findings will provide supportive data to strengthen our long-term goal that is to develop therapeutic approaches for GI mucosal injury-related diseases and for maintaining epithelial integrity under various clinical conditions. PUBLIC HEALTH RELEVANCE: Project Narrative Immediate goal of the current study is to define the mechanisms underlying acute gut mucosal injury and repair. Various acute GI mucosal injury and gastritis are much more common at our VA patients than those observed in the general public. It has been reported that there are 24.7% prevalence of GI ulcers and injury in veteran population, compared with 10.5% in the control population. Furthermore, drugs, specifically NSAIDs, and stress as etiologic factors in GI mucosal injury and ulcerations are becoming increasing important. This is especially relevant to the new and young OEF/OIF veterans who return with a major increase in the prevalence of stress-related disorders. The incidence of stress-induced mucosal injury and ulceration in critically ill intensive patients as well as in military combat situations further aggravate the problem of GI mucosal injury and injury-related disorders in the VA population. However, the effective therapies for preventing acute gut mucosal injury and for promoting mucosal repair in clinic are limited to date, because of lacking knowledge of the mechanisms involved in gut mucosal injury and repair. Thus, improving the understanding of the processes that maintain the integrity of the gut mucosa and enhance repair is the first step towards therapeutic initiatives in this area and is the focus of the current proposal. Based on our previous studies and exciting preliminary results, studies proposed here are to determine whether a novel protein STIM1 plays an important role in promoting GI epithelial restitution after mucosal injury by Ca2+-signaling. Completion of these aims will provide supportive data to strengthen our long-term goal that is to develop therapeutic approaches for GI mucosal injury-related diseases for our VA patients.