Actin dynamics and regulation of epithelial barrier in intestinal inflammation

描述（由申请人提供）：肠上皮屏障的破坏是包括炎症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（TJs）和粘附连接（AJs）的细胞结构调节。TJs和AJs的破坏是炎症肠上皮通透性增加的基础，而在粘膜恢复过程中，接合面重组对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。TJs和AJs与肌动蛋白细胞骨架相关，肌动蛋白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多种细胞功能至关重要，但其参与AJ/TJ调节的研究仍然很少。该提议的一个核心创新假设表明，在粘膜损伤和恢复过程中，f -肌动蛋白的转换在上皮AJs和TJs的重组中起关键作用。我特别提出，交界周肌动蛋白丝的加速转换介导了AJ/TJ的分解和新生AJ样连接的重组，而f -肌动蛋白转换的减少是TJ的建立和屏障重封的先决条件。这一假设将在以下目的中得到验证：(1)研究肌动蛋白丝的转换在上皮AJs和TJs的分解和重组中的作用；(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中的作用；3)分析F-actin交联蛋白在上皮连接的拆卸和重建中的作用。这些目标将通过暴露于炎症介质的体外肠上皮细胞单层以及体内小鼠结肠炎模型和人类患者的组织活检来实现。肌动蛋白丝的转换和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、洗涤剂分离）、免疫细胞化学和遗传（sirna介导的敲除、显性阴性或构成活性突变体的表达、敲除小鼠）方法的组合进行分析。意义：本研究将对炎症期肠黏膜损伤的基本机制提供新的认识。了解这些机制将为消化系统疾病患者提供新的治疗靶点，以防止肠道屏障的破坏和增强肠道屏障的修复。

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.