Junctional exocytosis and breakdown of the intestinal barrier in inflammation

炎症中的连接胞吐作用和肠道屏障的破坏

基本信息

批准号：
8243607
负责人：
Andrei Ivanovich Ivanov
金额：
$ 29.64万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2015-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8243607
关键词：
Adherens Junction Affinity Chromatography Aging Alcoholic Liver Diseases Biochemical Biotinylation Celiac Disease Cell membrane Chemicals Colitis Complex Development Digestive System Disorders Disease Endocytosis Epithelial Epithelial Cells Epithelium Event Excision Exocytosis Extracellular Space Free Radicals Functional disorder Gastrointestinal Diseases Genes Genetic Goals Healed Immunoprecipitation In Vitro Inflammation Inflammation Mediators Inflammatory Inflammatory Bowel Diseases Inflammatory Response Inflammatory disease of the intestine Injury Insulin-Dependent Diabetes Mellitus Intestinal Mucosa Intestines Link Maintenance Mediating Mediator of activation protein Membrane Membrane Fusion Missense Mutation Modeling Modification Molecular Morbidity - disease rate Mucositis Mucous Membrane Mus Mutagenesis N-ethylmaleimide-sensitive protein Nitric Oxide Nitric Oxide Donors Organ Pathogenesis Patients Permeability Phosphorylation Play Prevention Protein Isoforms Protein Overexpression Proteins RNA Interference Reactive Oxygen Species Recovery Regulation Research Resistance Risk Role S-nitro-N-acetylpenicillamine SNAP receptor Septic Shock Small Interfering RNA Structure Testing Tight Junctions Tissues Ulcerative Colitis Vesicle cohort cytokine design healing in vivo injured innovation insight intestinal epithelium knock-down microbial monolayer mortality mutant new therapeutic target novel overexpression pathogen prevent protein complex protein expression public health relevance receptor research study restoration seal syntaxin-2 trafficking

项目摘要

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.

描述（申请人提供）：肠上皮通透性增强是肠道炎症性疾病的关键机制。这种肠道渗漏使内脏暴露于管腔微生物产物，因此加剧了粘膜炎症并增加了全身炎症反应的风险。肠道屏障的破坏是由特殊上皮结构、紧密连接 (TJ) 和粘附连接 (AJ) 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，已知会破坏 AJ 和 TJ 结构。了解肠道炎症期间上皮连接解体的机制是本研究的主要目标。一种新兴的观点认为，TJ 和 AJ 经历持续重塑，包括通过内吞作用从质膜上去除老化的连接成分，并通过胞吐作用输送新的 TJ/AJ 蛋白。该提案的一个中心创新假设表明，肠道炎症通过阻止囊泡介导的连接蛋白的胞吐作用来中断上皮 AJ 和 TJ 的稳态重塑。这种 AJ/TJ 胞吐作用的抑制是通过抑制调节囊泡与质膜融合的蛋白质的表达和/或活性来实现的，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 (1SNAP) ）。 SNARE/NSF/1SNAP 介导的连接蛋白运输功能障碍可能最终导致 AJ/TJ 结构缺陷和肠屏障通透性增加。我们将在以下目标中检验这一假设：（1）：研究 SNARE 介导的胞吐作用在体外和体内调节上皮连接结构和功能的作用； (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1SNAP在粘膜损伤和恢复过程中上皮连接的分解和恢复中的作用。这些目标将通过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、亲和层析）、免疫细胞化学和遗传学（SNARE 蛋白的 siRNA 敲低、NSF 和 1SNAP 突变体的过表达）方法的组合来分析囊泡融合机制。意义：拟议的研究将为炎症期间肠粘膜损伤的基本机制提供新的见解。了解这些机制可能会提供新的治疗靶点，以防止消化系统疾病患者肠道屏障的破坏。