Regulation of Intestinal Tight Junction Barrier and Inflammation by Autophagy

项目摘要 我们实验室的长期目标是阐明肠道内稳态的分子基础及其在肠道内的作用。 肠道炎症的失调，并开发新的方法来预防和治疗 炎症性肠病（IBD）。小肠顶端的细胞间紧密连接（TJ） 上皮作为细胞旁屏障并防止有害管腔抗原渗透。肠丢失 TJ屏障功能是肠道疾病和IBD的关键致病因素。自噬（macroautophagy） 一种细胞内降解系统，其将不必要或功能失调的细胞货物 在双膜囊泡（自噬体）内到达溶酶体。新出现的证据表明， 自噬在IBD的易感性、病因学和进展中起重要作用。虽然临床数据 动物研究表明，IBD患者肠道TJ屏障缺陷与肠道炎症之间存在直接联系， IBD患者和动物模型，自噬在肠上皮TJ屏障调节中的作用 仍然未知。我们的初步研究表明，自噬在增强细胞增殖中起着关键作用。 肠TJ屏障。具体地说，自噬通过降解孔来降低细胞旁TJ渗透性。 形成紧密连接蛋白claudin-2并增加屏障保护性跨膜TJ的蛋白水平 蛋白闭合蛋白自噬的诱导导致来自人的claudin-2的溶酶体靶向的选择性增加。 膜，并导致膜保留occludin的增加。因此，我们的中心假设是， 自噬选择性地调节TJ膜蛋白组成，以诱导肠上皮细胞的增强。 TJ屏障。我们将讨论我们的假设与具体目标（1）描绘细胞内囊泡 自噬增强肠上皮TJ屏障的转运机制;（2）阐明 细胞内信号传导在肠TJ屏障自噬调节中的机制作用;和（3） 阐明自噬介导的增强小鼠肠道TJ屏障功能的保护作用 IBD模型我们将使用创新的技术工具，如体内siRNA技术，体内全长小鼠， 结肠灌注，在体肠TJ蛋白运输，和高含量定量成像，以研究我们的 具体目标。这一提议将为自噬在细胞凋亡中的关键作用提供新的见解。 肠屏障的稳态和桥梁差距的科学知识，将是重要的治疗 努力对抗IBD。

Project Summary The long-term goal of our laboratory is to elucidate the molecular basis for intestinal homeostasis and its dysregulation in intestinal inflammation, and to develop novel approaches for prevention and therapy of inflammatory bowel diseases (IBD). The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and IBD. Autophagy (macroautophagy) is an intracellular degradation system that delivers unnecessary or dysfunctional cellular cargo sequestered inside double-membrane vesicles (autophagosomes) to the lysosome. Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. We will address our hypothesis with the specific aims of (1) To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier; (2) To elucidate the mechanistic role of intracellular signaling in autophagy regulation of intestinal TJ barrier; and (3) To delineate the protective role of autophagy-mediated enhancement of intestinal TJ barrier function in murine models of IBD. We will use innovative technical tools such as in vivo siRNA technology, in vivo full length mice colon perfusion, in vivo intestinal TJ protein trafficking, and high content quantitative imaging to study our specific aims. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.