DEFINING THE IN VIVO ROLE OF DAP1 IN AUTOPHAGY AND COLITIS

定义 DAP1 在自噬和结肠炎中的体内作用

• 批准号: 8675731
• 负责人: Broc Taylor McCune
• 金额: $ 2.9万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8675731
• 关键词: Animal Model; Animals; Autophagocytosis; Bacteria; Cells; Colitis; Colorectal Cancer; Complex; Disease; Epithelium; Etiology; Genes; Genetic; Goals; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Interleukin-10; Knock-in Mouse; Lead; Link; Modeling; Mus; Pathology; Pathway interactions; Predisposition; Prevention; Process; Quality of life; Recycling; Regulation; Resistance; Risk Factors; Role; Southern Blotting; Starvation; Testing; Tissues; Transformed Cell Line; Ulcerative Colitis; Virus; Virus Diseases; cell transformation; cytokine; defined contribution; gastrointestinal; gene discovery; human FRAP1 protein; in vivo; mTOR Inhibitor; macrophage; mouse development; mouse model; protein expression; public health relevance; tool; tumor; tumor initiation; tumorigenesis

DESCRIPTION (provided by applicant): The etiology of colorectal cancer (CRC) is complex and multifactorial, however Inflammatory Bowel Disease (IBD) is demonstrated to be a strong risk factor for CRC. Autophagy, the process of degrading and recycling cellular parts, is strongly associated with IBD. Autophagy is likely to be of importance in both IBD and CRC. Unfortunately, the role of autophagy in IBD and CRC remains understudied due to insufficient availability of genetic tools. Dap1 may provide a link between CRC, IBD, and autophagy. An intronic SNP within DAP1 is associated with UC, and Dap1 is highly over-expressed in many tumors, including CRC. Dap1 negatively regulates autophagy in starvation conditions in transformed cell lines. Furthermore, Dap1 is phosphorylated by mTOR, an important molecule in colitis and tumorigenesis. Therefore, we hypothesize that by regulating autophagy, Dap1 expression contributes to ulcerative colitis susceptibility, then in conjunction with IBD, accelerates tumorigenesis resulting in colorectal cancer. Our long-term goals are to delineate the role of Dap1 in IBD, then to test its role in IBD-initiated CRC. We believe this approach will allow us to better understand the spatio-temporal role of autophagy and mTOR in these diseases, and provide a targeted approach to therapies modulating these pathways in IBD and CRC. The focus of this proposal will be to test the following aims: Aim 1. Confirm that Dap1 negatively regulates autophagy, and is regulated by mTOR in primary cells. We will approach this by culturing Dap1 deficient primary cells and analyze autophagy after starvation and/or treatment with mTOR inhibitors. Aim 2. Define Dap1 contributions to colonic inflammation in primary cells. We will culture primary colonic epithelium and macrophages that lack Dap1 expression, analyze inflammatory cytokines after stimulating with bacteria and virus. Aim 3. Define contribution of Dap1 to colitis in vivo. We will test if Dap1 deficiency protects mice from gastrointestinal pathologies occurring in mice hypomorphic for Atg16L1 after virus infection, determine if Dap1-/- mice are resistant to colitis induced by IL10 deficiencies, and analyze inflammatory responses to colonic wounding in Dap1-/- mice.

描述（由申请人提供）：结直肠癌（CRC）的病因复杂且多因素，但炎症性肠病（IBD）被证明是CRC的一个强风险因素。自噬是降解和回收细胞部分的过程，与IBD密切相关。自噬可能在IBD和CRC中都很重要。不幸的是，由于遗传工具的可用性不足，自噬在IBD和CRC中的作用仍然研究不足。Dap 1可能提供CRC、IBD和自噬之间的联系。DAP 1内的内含子SNP与UC相关，并且Dap 1在许多肿瘤中高度过表达，包括CRC。在转化细胞系中，Dap 1在饥饿条件下负调节自噬。此外，Dap 1被mTOR磷酸化，mTOR是结肠炎和肿瘤发生中的重要分子。因此，我们假设通过调节自噬，Dap 1表达有助于溃疡性结肠炎的易感性，然后与IBD结合，加速肿瘤发生，导致结直肠癌。我们的长期目标是描述Dap 1在IBD中的作用，然后测试其在IBD引发的CRC中的作用。我们相信这种方法将使我们能够更好地了解自噬和mTOR在这些疾病中的时空作用，并为IBD和CRC中调节这些途径的疗法提供靶向方法。本提案的重点将是检验以下目标：目标1。证实Dap 1负调控自噬，并在原代细胞中受mTOR调控。我们将通过培养Dap 1缺陷的原代细胞来解决这个问题，并分析饥饿和/或用mTOR抑制剂治疗后的自噬。目标2.定义Dap 1对原代细胞中结肠炎症的贡献。我们将培养原代结肠上皮和缺乏Dap 1表达的巨噬细胞，分析细菌和病毒刺激后的炎性细胞因子。目标3.定义Dap 1对体内结肠炎的贡献。我们将测试Dap 1缺陷是否保护小鼠免受病毒感染后Atg 16 L1亚型小鼠中发生的胃肠道病变，确定Dap 1-/-小鼠是否对IL 10缺陷诱导的结肠炎具有抗性，并分析Dap 1-/-小鼠对结肠损伤的炎症反应。