Actin dynamics and regulation of epithelial barrier in intestinal inflammation

描述（由申请人提供）：肠上皮屏障的破坏代表了包括炎性肠病在内的各种胃肠道疾病的早期和重要表现。上皮屏障的完整性由称为紧密连接（TJ）和粘附连接（AJs）的细胞结构调节。TJ和AJs的破坏是炎症肠上皮通透性增加的基础，而连接重组对于粘膜恢复期间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AJs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，经历恒定的周转（解聚和聚合）。丝状肌动蛋白的这种周转对于多种细胞功能是至关重要的，然而其在AJ/TJ调节中的参与仍然很少被研究。这一建议的一个核心创新假设意味着，F-肌动蛋白营业额在粘膜损伤和恢复过程中上皮AJs和TJs的重组中起着关键作用。特别是，我建议，加快周转的perijunctional肌动蛋白丝介导的AJ/TJ拆卸和新生AJ-like路口的改造，而减少F-肌动蛋白营业额的先决条件，建立TJ和屏障resealing。本研究的主要目的是：（1）探讨肌动蛋白丝更新在肠上皮AJs和TJs解体和重组中的作用;（2）探讨肌动蛋白解聚因子（ADF）/cofilin蛋白在肠上皮损伤和修复过程中连接重构的作用; 3）分析F-肌动蛋白交联蛋白在上皮连接解体和重建中的作用。这些目标将使用暴露于炎症介质的体外肠上皮细胞单层以及结肠炎的体内小鼠模型和人类患者的组织活检来实现。将通过生物化学（肌动蛋白单体螯合、免疫印迹、去污剂分级分离）、免疫细胞化学和遗传（siRNA介导的敲除、显性阴性或组成型活性突变体表达、敲除小鼠）方法的组合分析肌动蛋白丝的周转以及肌动蛋白解聚和交联蛋白的功能。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和增强修复。 公共卫生相关性：拟议的研究旨在了解肠上皮屏障破坏和恢复的机制。屏障破坏是不同胃肠疾病的常见表现，包括溃疡性结肠炎、克罗恩病、乳糜泻和感染性结肠炎。此外，肠道屏障功能障碍有助于其他疾病的发展，如脓毒性休克、酒精性肝病和I型糖尿病。该项目将通过探索一种新的上皮相关机制来了解胃肠道疾病的发病机制，该机制涉及粘膜炎症的启动和/或加剧。此外，它可以提供新的目标，为药理学预防肠屏障破坏和加速愈合受损的肠粘膜。这可能导致大量炎症性疾病患者的发病率和死亡率降低。

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. PUBLIC HEALTH RELEVANCE: The proposed research is aimed to understand mechanisms underlying disruption and restoration of the intestinal epithelial barrier. The barrier breakdown is a common manifestation of different gastroenterological disorders including ulcerative colitis, Crohn's disease, celiac diseases and infectious colitis. Furthermore, dysfunctions of the gut barrier contribute to the development of other diseases such as septic shock, alcoholic liver disease and type I diabetes. This project will provide new insights into understanding the pathogenesis of gastrointestinal disorders by exploring a novel epithelium-related mechanism involving in initiation and/or exaggeration of mucosal inflammation. Furthermore, it may provide novel targets for pharmacological prevention of the intestinal barrier breakdown and for accelerated healing of the injured gut mucosa. This may result in decreased morbidity and mortality of a large cohort of patients with inflammatory disorders.