Actin dynamics and regulation of epithelial barrier in intestinal inflammation

肠道炎症中肌动蛋白动力学和上皮屏障的调节

基本信息

批准号：
8587379
负责人：
Andrei Ivanovich Ivanov
金额：
$ 0.11万
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：
2010-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8587379
关键词：
Actinin Actins Address Adherens Junction Alcoholic Liver Diseases Apical Attenuated Biochemical Biopsy Celiac Disease Cell physiology Cells Cellular Structures Colitis Colon Complex Crohn&apos s disease Cytoskeleton Data Detergents Development Digestive System Disorders Disease Disease remission Dominant-Negative Mutation Epithelial Epithelial Cells Epithelium F-Actin Fractionation Functional disorder Gastrointestinal Diseases Genes Genetic Goals Healed Human Immunoblotting Immunofluorescence Immunologic In Vitro Inflammation Inflammation Mediators Inflammatory Inflammatory Bowel Diseases Inflammatory disease of the intestine Injury Insulin-Dependent Diabetes Mellitus Intestinal Mucosa Intestines Knockout Mice LIM Domain Kinase 1 Label Mediating Microfilaments Modeling Molecular Morbidity - disease rate Mucositis Mucous Membrane Mus Pathogenesis Patients Permeability Phosphoric Monoester Hydrolases Phosphorylation Physiological Play Polymers Prevention Proteins Recovery Regulation Relapse Relative (related person)Research Role Septic Shock Small Interfering RNA Staging Structure System Techniques Testing Tight Junctions Tissues Ulcerative Colitis actin depolymerizing factor cofilin cohort crosslink depolymerization healing in vivo inhibitor/antagonist injured innovation insight intestinal epithelium knock-down link protein monolayer monomer mortality mouse model mutant new therapeutic target novel overexpression pathogen polymerization prevent public health relevance restoration

项目摘要

DESCRIPTION (provided by applicant): Breakdown of the intestinal epithelial barrier represents an early and important manifestation of various gastrointestinal disorders including inflammatory bowel disease. The integrity of the epithelial barrier is regulated by cellular structures known as tight junctions (TJs) and adherens junctions (AJs). Disruption of TJs and AJs underlies increased permeability of inflamed intestinal epithelium, whereas junctional reassembly is crucial for recovery of the epithelial barrier during mucosal restitution. Understanding mechanisms of AJ and TJ remodeling during intestinal epithelial damage and restitution represents the major goal of the proposed study. TJs and AJs associate with the actin cytoskeleton which is a crucial regulator of junctional reorganizations and integrity. Actin filaments are highly dynamic polymers undergoing a constant turnover (depolymerization and polymerization). Such a turnover of filamentous (F) actin is critical for a variety of cellular functions, however its involvement in AJ/TJ regulation remains poorly investigated. A central innovative hypothesis of this proposal implies that F-actin turnover plays a key role in reorganizations of epithelial AJs and TJs during mucosal damage and restitution. Particularly, I propose that accelerated turnover of perijunctional actin filaments mediates both AJ/TJ disassembly and reformation of nascent AJ-like junctions, whereas decreased F-actin turnover is prerequisite for the establishment of TJs and barrier resealing. This hypothesis will be tested in the following Aims: (1) To examine the role of actin filament turnover in disassembly and reformation of epithelial AJs and TJs; (2) to investigate the involvement of actin-depolymerizing factor (ADF)/cofilin proteins in junctional remodeling during intestinal epithelial damage and restitution; 3) to analyze the role of F-actin cross-linking proteins in disassembly and reestablishment of epithelial junctions. These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to inflammatory mediators as well as in vivo mouse model of colitis and tissue biopsies of human patients. Turnover of actin filaments and functions of actin- depolymerizing and cross-linking proteins will be analyzed by a combination of biochemical (actin- monomer sequestration, immunoblotting, detergent fractionation), immunocytochemical and genetic (siRNA-mediated knock-downs, expression of dominant-negative or constitutively active mutants, knockout mice) approaches. Significance: the proposed study will provide new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will provide new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases.

描述（由申请人提供）：肠上皮屏障的破坏代表了包括炎症性肠病在内的各种胃肠道疾病的早期且重要的表现。上皮屏障的完整性受到称为紧密连接 (TJ) 和粘附连接 (AJ) 的细胞结构的调节。 TJ 和 AJ 的破坏是发炎肠上皮通透性增加的基础，而连接重新组装对于粘膜恢复过程中上皮屏障的恢复至关重要。了解肠上皮损伤和恢复过程中 AJ 和 TJ 重塑的机制是本研究的主要目标。 TJ 和 AJ 与肌动蛋白细胞骨架相关，肌动蛋白细胞骨架是连接重组和完整性的关键调节因子。肌动蛋白丝是高度动态的聚合物，经历不断的周转（解聚和聚合）。丝状 (F) 肌动蛋白的这种转换对于多种细胞功能至关重要，但其在 AJ/TJ 调节中的作用仍鲜有研究。该提案的一个中心创新假设表明，F-肌动蛋白周转在粘膜损伤和恢复过程中上皮 AJ 和 TJ 的重组中发挥着关键作用。特别是，我认为连接周围肌动蛋白丝的加速周转介导 AJ/TJ 解体和新生 AJ 样连接的重组，而 F-肌动蛋白周转的减少是 TJ 建立和屏障重新密封的先决条件。该假设将在以下目的中得到检验：（1）研究肌动蛋白丝周转在上皮AJ和TJ的分解和重组中的作用；（2）研究肌动蛋白解聚因子（ADF）/丝切蛋白在肠上皮损伤和恢复过程中连接重塑中的作用； 3）分析F-肌动蛋白交联蛋白在上皮连接的分解和重建中的作用。这些目标将通过使用暴露于炎症介质的体外肠上皮细胞单层以及体内结肠炎小鼠模型和人类患者的组织活检来实现。肌动蛋白丝的周转以及肌动蛋白解聚和交联蛋白的功能将通过生物化学（肌动蛋白单体隔离、免疫印迹、去污剂分级分离）、免疫细胞化学和遗传学（siRNA介导的敲低、显性蛋白的表达）的组合进行分析。阴性或组成型活性突变体、基因敲除小鼠）方法。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。了解这些机制将为预防消化系统疾病患者肠道屏障的破坏和增强修复提供新的治疗靶点。