The Ubiquitin Editing Enzyme A20 Preserves Intestinal Epithelial Cell Homeostasis

描述(申请人提供)：肠上皮功能障碍是炎症性肠病(IBD)的标志。维持肠上皮细胞(IEC)的完整性需要在分化、增殖、存活和细胞死亡之间取得平衡。A20是一种泛素编辑酶，已被认为与人类IBD有关。与人类IBD的潜在作用一致，IECS中A20的特异性缺失使小鼠容易受到化学诱导的结肠炎的影响，而转基因A20的过表达通过支持IEC屏障功能来保护上皮细胞。然而，由于A20已被报道既可抑制依赖于NFκB的生存信号，也可抑制肿瘤坏死因子-α诱导的细胞死亡，目前尚不清楚A20是如何调节细胞内皮细胞的动态平衡的。我们假设A20的功能是由其相关蛋白的作用调节的。我们的初步数据表明，调节细胞命运的一个重要辅助因子是核因子κB家族的A20结合抑制因子(ABIN-1)。与仅有A20上皮缺陷的小鼠形成鲜明对比的是，IECS中A20和ABIN-1的急性基因缺失会导致猖獗的自发性上皮细胞死亡和快速死亡。本研究试图通过以下具体目标来揭示A20和ABIN-1如何维持IEC动态平衡的分子机制：1)确定A20和ABIN-1如何在体内调节IEC动态平衡；2)确定A20和ABIN-1如何合作限制上皮细胞内的细胞死亡。在目标1中，我们提出了一系列体内实验，以检测上皮细胞死亡、屏障功能和粘膜免疫激活对IEC特异性A20和ABIN-1缺失的反应。我们还建议通过遗传学和药理学方法研究肿瘤坏死因子-α和TLR依赖的信号转导的作用。在目标2中，我们将在体外研究A20和ABIN-1调节细胞死亡信号的分子机制。我们计划通过剖析A20和ABIN-1在内源性和外源性凋亡途径中的作用来定位这两种蛋白的作用部位。然后，我们将使用一种候选方法，通过免疫沉淀和蛋白质印迹等经典生化技术来鉴定由A20和ABIN-1调控的特定复合体。了解IEC稳态是如何维持或调节的，将为炎症性肠病的新疗法的开发提供潜在的新线索。这些研究将为在加州大学旧金山分校结肠炎和克罗恩病中心主任Averil Ma博士的指导下进行R01申请奠定基础。职业发展计划包括授课课程、研究研讨会和期刊俱乐部，以及拟议的研究出版物和职业时间表。由在IBD、免疫学、炎症和细胞凋亡生物学领域拥有专业知识的国际知名导师、合作者和顾问组成的职业发展委员会将有助于确保邵逸夫达到拟议的里程碑，成功申请R01拨款，并成为炎症性肠病的独立研究员。

DESCRIPTION (provided by applicant): Intestinal epithelial dysfunction is a hallmark of inflammatory bowel disease (IBD). Maintaining intestinal epithelial cell (IEC) integrity requires a balance of differentiation, proliferation, survival, and cell death. A20 is a ubiquitin-editing enzyme which has been linked to IBD in humans. Consistent with a potential role in human IBD, specific deletion of A20 in IECs renders mice susceptible to chemically induced forms of colitis while transgenic overexpression of A20 protects the epithelium by supporting IEC barrier function. However, as A20 has been reported to both dampen both NFκB-dependent survival signals as well as TNF-α induced apoptotic cell death, it is not clear exactly how A20 regulates IEC homeostasis. We hypothesize that the function of A20 is modulated by the action of its associated proteins. Our preliminary data suggests that one essential cofactor in regulating cell fate is a member of the A20- binding inhibitors of NFκB family (ABIN-1). In stark contrast to mice with epithelial deficiency of A20 alone, acute genetic deletion of both A20 and ABIN-1 in IECs leads to rampant spontaneous epithelial cell death and rapid lethality. This proposal attempts to unravel the molecular mechanisms of how A20 and ABIN-1 maintain IEC homeostasis through the following specific aims: 1) Determine how A20 and ABIN-1 regulate IEC homeostasis in vivo; and 2) determine how A20 and ABIN-1 cooperate to restrict cell death within epithelial cells. In Aim 1, we propose a set of in vivo experiments to examine epithelial cell death, barrier function, and mucosal immune activation in response to IEC-specific deletion of A20 and ABIN-1. We also propose to study the role of TNF-α and TLR-dependent signaling through genetic and pharmacologic approaches. In Aim 2, we will study the molecular mechanism underlying cell death signaling regulation by A20 and ABIN-1 in vitro. We plan to localize the site of action of A20 and ABIN-1 by dissecting the contribution of these two proteins in both the intrinsic and extrinsic apoptotic pathway. We will then use a candidate approach through classical biochemical techniques such as immunoprecipitation and western blot to identify specific complexes regulated by A20 and ABIN-1. Understanding how IEC homeostasis is maintained or modulated will provide potential new leads for the development of novel therapeutics for inflammatory bowel disease. These studies will provide the foundation for an R01 application under the mentorship of Dr. Averil Ma, Director of the Center for Colitis and Crohn's Disease at UCSF. A Career Development Plan including didactic courses, research seminars, and journal clubs has been developed along with a proposed research publication and career timeline. A career development committee of internationally recognized mentors, collaborators, and advisors with expertise in the fields of IBD, immunology, inflammation and apoptosis biology will help ensure that Dr. Shao meets the proposed milestones, successfully applies for an R01 grant, and becomes an independent investigator in inflammatory bowel disease.