Mechanisms of colonic responses to neurotensin

结肠对神经降压素的反应机制

• 批准号: 6544665
• 负责人: CHARALABOS POTHOULAKIS
• 金额: $ 37.1万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6544665
• 关键词: bacterial toxins; biological signal transduction; colitis; colon; epidermal growth factor; guanine nucleotide binding protein; inflammation; inflammatory bowel diseases; interleukin 8; mitogen activated protein kinase; molecular pathology; neurotensin; nuclear factor kappa beta; protein kinase C; receptor expression; tissue /cell culture; transfection /expression vector

The pathophysiology of inflammatory bowel disease (IBD), and Clostridium difficile toxin-associated colitis, two common GI clinical entities with significant morbidity and mortality, is not completely understood. Recent evidence indicate that binding of neuropeptides to receptors on intestinal epithelial cells plays a critical role in the pathogenesis of colonic inflammation. We showed that neurotensin (NT), a neuropeptide released in the colon in response to several stimuli, triggers NF-kappaB activation and release of proinflammatory cytokines from the colonic mucosa. Moreover, high affinity NT receptors are expressed on colonic epithelial cells and are upregulated in IBD and C. difficile toxin-mediated enterocolitis. However, the molecular and biochemical mechanism(s) by which NT binding triggers these responses are not known. Our hypothesis is that the peptide NT, acting through G -protein coupled type 1 receptors (NTR1) on colonic epithelial cells, stimulates signal transduction pathways involving PKC, MAPK and NF-kappaB leading to IL-8 release. Aim 1 will examine the role of the NF- kappaB/IkappaB system in IL-8 gene expression following neurotensin stimulation in colonic epithelial cells. Experiments in this aim will elucidate the role of MAP kinase pathways in NTR1-induced NF-kappaB activation and IL-8 gene expression and examine the importance of PKC-epidermal growth factor communication in MAP kinase activation following NTR1 stimulation. Aim 2 will determine the functional role of the Rho family of small GTP binding proteins RhoA, Rac, and Cdc42 in NT- induced IL-8 expression and determine whether NT binding to NTR1 activates the Rho family proteins. Experiments in this aim will also identify which heterotrimeric G protein receptor subtype is involved in NTR1-induced NF-kappaB activation and IL-8 gene expression, as well as activation of Rho family proteins. Studies described in aim 3 will identify the structural determinants of NTR1 that mediate NT-induced IL-8 expression. Specifically, we will examine the role of the third intracellular loop and the C-terminus of NTR1 in NT signaling and IL-8 expression in human colonocytes. Studies are also proposed to identify the critical residues within the third intracellular loop and the C-terminus involved in NTR1-mediated IL-8 production. Our results will provide important insights on the role of NT and its high affinity receptor in colonic inflammation and may lead to novel therapeutic approaches for the treatment of intestinal inflammation.

炎症性肠病（IBD）和艰难梭菌毒素相关性结肠炎（两种常见的GI临床实体，具有显著的发病率和死亡率）的病理生理学尚未完全了解。 最近的证据表明，神经肽与肠上皮细胞上的受体的结合在结肠炎症的发病机制中起着关键作用。 我们发现，神经降压素（NT），一种神经肽释放在结肠中响应于几种刺激，触发NF-κ B激活和释放促炎细胞因子从结肠粘膜。 此外，高亲和力NT受体在结肠上皮细胞上表达，并且在IBD和C中上调。艰难梭菌毒素介导的小肠结肠炎。 然而，NT结合触发这些反应的分子和生化机制尚不清楚。 我们的假设是肽NT通过G蛋白偶联的1型受体（NTR 1）作用于结肠上皮细胞，刺激涉及PKC、MAPK和NF-κ B的信号转导途径，导致IL-8释放。 目的1研究NF-κ B/IkappaB系统在神经降压素刺激结肠上皮细胞后IL-8基因表达中的作用。 在这一目标的实验将阐明NTR 1诱导的NF-κ B活化和IL-8基因表达的MAP激酶途径的作用，并检查PKC-表皮生长因子通信在NTR 1刺激后MAP激酶活化的重要性。 目的2将确定Rho家族的小GTP结合蛋白RhoA、Rac和Cdc 42在NT诱导的IL-8表达中的功能作用，并确定NT与NTR 1的结合是否激活Rho家族蛋白。 在此目的的实验还将确定异源三聚体G蛋白受体亚型参与NTR 1诱导的NF-κ B活化和IL-8基因表达，以及Rho家族蛋白的活化。目的3中描述的研究将鉴定NTR 1介导NT诱导的IL-8表达的结构决定簇。具体来说，我们将研究第三个细胞内环和NTR 1的C-末端在NT信号转导和IL-8在人类结肠细胞中的表达的作用。 还提出了研究，以确定在第三个细胞内环和C-末端参与NTR 1介导的IL-8的生产中的关键残基。 我们的研究结果将为NT及其高亲和力受体在结肠炎症中的作用提供重要的见解，并可能为治疗肠道炎症提供新的治疗方法。