Consequences of desmoglein 2 loss for organization and function of intestinal epithelial junctions

桥粒芯糖蛋白 2 损失对肠上皮连接组织和功能的影响

• 批准号: 273723961
• 负责人: Professor Dr. Pavel Strnad
• 金额: --
• 依托单位: Klinik für Gastroenterologie, Stoffwechselerkrankungen und Internistische Intensivmedizin (Med. Klinik III)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/273723961?language=en
• 关键词: Consequences; desmoglein; loss; organization; function

Intestinal epithelia protect the organism against luminal pathogens while enabling selective uptake of nutrients. These dual functions are supported by the apical junctional complex, which is localized at the adluminal plasma membranes of adjacent cells and consists of circumferential tight junction strands (TJs), the adherens junction belt (AJs) and multiple spot-like desmosomal contact sites. While the function of TJs and AJs for intestinal homeostasis has been investigated in detail, much less is known about desmosomes. They are multiprotein complexes with transmembrane cadherins of the desmoglein (Dsg) and desmocollin type at their core that are associated through intracellular plaque proteins to the cytoplasmic keratin intermediate filament cytoskeleton. We recently generated intestine-specific desmoglein 2 gene (DSG2) knockouts to examine desmosomal cadherin-dependent biological functions of intestinal desmosomes. These mice display no obvious clinical phenotype. Yet, the levels of desmosomal, AJ and TJ proteins are significantly altered. Furthermore, the knockout mice are markedly susceptible to chemically-induced colitis as evidenced by increased weight loss, colon shortening, stronger epithelial damage and increased intestinal levels of proinflammatory cytokines. The goal of the project is to examine the consequences of Dsg2 depletion on the organization and function of the apical junctional complex. We propose that compromised function of the apical junctional complex is responsible for the increased susceptibility of DSG2 knockouts to intestinal injury. To pursue our goal and to investigate our hypothesis we will examine our knockout mice and cultured intestinal tumor cells with a stable DSG2 knockdown. First, we will analyze desmosomal composition, ultrastructure and dynamics and desmosome-dependent adhesion. We will perform immunoprecipitation experiments to study how the ablation of Dsg2 affects the interaction of the remaining desmosomal proteins. Secondly, we will investigate the consequences of Dsg2 loss for the function of TJs and AJs. In this context, we will study intestinal barrier integrity and efficiency of resorption and secretion. Thirdly, we will study the effects of Dsg2 depletion on overall intestinal epithelial physiology by examining migration and differentiation of epithelial cells along the crypt-villus axis, by studying mechanical resilience, adhesive properties and susceptibility to apoptosis of intestinal epithelial cells, and by investigating catenin signaling and protection against microbial infections. The project will improve our understanding of the contribution of desmosomal cadherins to desmosomal functions in the context of the apical junctional complex of intestinal epithelia with the long-term goal to elucidate the role of these prominent cell-cell contact sites for the maintenance and control of intestinal epithelial proliferation and differentiation.

肠上皮细胞保护生物体免受管腔病原体的侵害，同时能够选择性地摄取营养物质。这些双重功能由顶端连接复合物支持，顶端连接复合物位于相邻细胞的近腔质膜处，由周向紧密连接链（TJ）、粘附连接带（AJs）和多个点状桥粒接触位点组成。虽然已经详细研究了TJ和AJs在肠道内稳态中的功能，但对桥粒的了解要少得多。它们是多蛋白复合物，在其核心处具有桥粒芯糖蛋白（Dsg）和桥粒胶蛋白类型的跨膜钙粘蛋白，其通过细胞内斑块蛋白与细胞质角蛋白中间丝细胞骨架相关联。最近，我们产生了精氨酸特异性桥粒芯糖蛋白2基因（DSG 2）敲除研究桥粒钙粘蛋白依赖的生物学功能的肠桥粒。这些小鼠没有表现出明显的临床表型。然而，桥粒、AJ和TJ蛋白的水平显著改变。此外，敲除小鼠对化学诱导的结肠炎明显敏感，如通过增加的体重减轻、结肠缩短、更强的上皮损伤和增加的促炎细胞因子的肠水平所证明的。该项目的目标是研究Dsg 2耗竭对顶端连接复合体的组织和功能的影响。我们认为，顶端连接复合体的功能受损是导致DSG 2基因敲除对肠损伤的敏感性增加的原因。为了追求我们的目标并研究我们的假设，我们将检查我们的敲除小鼠和培养的具有稳定DSG 2敲除的肠肿瘤细胞。首先，我们将分析桥粒组成，超微结构和动力学和桥粒依赖的粘附。我们将进行免疫沉淀实验来研究Dsg 2的消融如何影响其余桥粒蛋白的相互作用。其次，我们将研究Dsg 2缺失对TJ和AJs功能的影响。在这种情况下，我们将研究肠屏障的完整性和吸收和分泌的效率。第三，我们将通过检查上皮细胞沿着隐窝-绒毛轴的迁移和分化，通过研究肠上皮细胞的机械弹性、粘附特性和对凋亡的敏感性，以及通过研究连环蛋白信号传导和对微生物感染的保护，来研究Dsg 2缺失对整体肠上皮生理学的影响。该项目将提高我们对桥粒钙粘蛋白在肠上皮顶端连接复合体中对桥粒功能的贡献的理解，其长期目标是阐明这些突出的细胞-细胞接触位点对维持和控制肠上皮增殖和分化的作用。