Junctional exocytosis and breakdown of the intestinal barrier in inflammation

描述（由申请方提供）：肠上皮通透性增强是肠道炎性疾病的关键机制。这种肠道渗漏使内脏暴露于管腔微生物产物，因此加剧粘膜炎症并增加全身炎症反应的风险。肠屏障的破坏是由特化上皮结构、紧密连接（TJ）和粘附连接（AJs）的分解引起的。许多炎症介质，包括细胞因子，一氧化氮和活性氧，已知破坏AJ和TJ结构。了解肠道炎症过程中上皮连接解体的机制是本研究的主要目标。一个新兴的观点是，TJ和AJs经历了连续的重塑，包括通过胞吞作用从质膜上去除老化的连接组分，并通过胞吐作用递送新的TJ/AJ蛋白。这一建议的一个核心创新假设意味着肠道炎症通过阻断囊泡介导的连接蛋白的胞吐作用来中断上皮AJs和TJ的稳态重塑。AJ/TJ胞吐作用的这种抑制通过抑制调节囊泡与质膜融合的蛋白质的表达和/或活性而发生，所述蛋白质即N-乙基马来酰亚胺敏感因子（NSF）、可溶性NSF受体（SNARE）和NSF-附着蛋白（1 SNAP）。SNARE/NSF/1 SNAP介导的连接蛋白运输功能障碍可能导致AJ/TJ结构缺陷和肠屏障通透性增加。我们将从以下几个方面来验证这一假说：（1）在体外和体内研究SNARE介导的胞吐作用在上皮连接结构和功能调节中的作用;（2）确定NSF的氧化修饰在自由基诱导的上皮连接解体中的作用; 3）分析1 SNAP在粘膜损伤和修复过程中上皮连接的解体和恢复中的作用。这些目标将使用暴露于促炎介质的体外肠上皮细胞单层以及肠道炎症的体内小鼠模型来实现。将通过生物化学（生物素化、免疫沉淀、亲和色谱）、免疫细胞化学和遗传（SNARE蛋白的siRNA敲低、NSF和1 SNAP突变体的过表达）方法的组合来分析囊泡融合机制。意义：该研究将为炎症过程中肠粘膜损伤的基本机制提供新的见解。了解这些机制将可能提供新的治疗靶点，以防止消化系统疾病患者的肠道屏障破坏。

DESCRIPTION (provided by applicant): Enhanced permeability of intestinal epithelium is a key mechanism of inflammatory diseases of the gut. Such intestinal leakiness exposes internal organs to luminal microbial products, therefore exaggerating mucosal inflammation and increasing the risk of systemic inflammatory responses. Breakdown of the intestinal barrier is caused by disassembly of specialized epithelial structures, tight junctions (TJs) and adherens junctions (AJs). Many inflammatory mediators, including cytokines, nitric oxide and reactive oxygen species, are known to disrupt AJ and TJ structure. Understanding mechanisms of epithelial junctional disassembly during intestinal inflammation represents the major goal of the proposed study. An emerging view is that TJs and AJs undergo a continuous remodeling consisting of the removal of aging junctional components from the plasma membrane by endocytosis and delivery of new TJ/AJ proteins via exocytosis. A central innovative hypothesis of this proposal implies that intestinal inflammation interrupts a steady-state remodeling of epithelial AJs and TJs by blocking vesicle-mediated exocytosis of junctional proteins. This suppression of AJ/TJ exocytosis occurs via inhibition of expression and/or activity of proteins regulating vesicle fusion with the plasma membrane, namely the N-ethylmaleimide sensitive factor (NSF), soluble NSF receptors (SNAREs) and NSF- attachment protein (1SNAP). Dysfunction of SNARE/NSF/1SNAP-mediated trafficking of junctional proteins is likely to eventuate in the defective AJ/TJ structure and increased intestinal barrier permeability. We will test this hypothesis in the following Aims: (1): to investigate the involvement of SNARE-mediated exocytosis in regulation of epithelial junctional structure and functions in vitro and in vivo; (2) to determine the role of oxidative modification of NSF in free-radical induced disassembly of epithelial junctions; 3) to analyze the role of 1SNAP in disassembly and recovery of epithelial junctions during mucosal damage and restitution. These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to proinflammatory mediators as well as in vivo murine models of intestinal inflammation. Vesicle fusion machinery will be analyzed by a combination of biochemical (biotinylation, immunoprecipitation, affinity chromatography), immunocytochemical and genetic (siRNA knock-down of SNARE proteins, overexpression of NSF and 1SNAP mutants) approaches. Significance: the proposed study will provide new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will potentially provide new therapeutic targets to prevent breakdown of the intestinal barrier in patients with digestive diseases.