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 NF?B-dependent survival signals as well as TNF-? 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 NF?B 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-? 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.
Human genetic studies have identified TNFAIP3, which encodes for the protein A20, as being an inflammatory bowel disease susceptibility gene. A20 regulates multiple cellular pathways including those involved in inflammation and cell death that are likely to be important in the intestinal epithelium that undergoes rapid turnover and is a target of mucosal inflammation. Since epithelial dysfunction underlies many of the clinical manifestations of Crohn's disease and ulcerative colitis, we propose to study how A20 helps maintain normal epithelial cell function and hope these studies will provide new insights into the development of targeted therapies for inflammatory bowel disease patients.