Mechanisms of colonic responses to neurotensin

炎症性肠病(IBD)和艰难梭菌毒素相关性结肠炎是两种常见的胃肠道疾病临床实体，其发病率和死亡率尚不完全清楚。近年来的研究表明，神经肽与肠上皮细胞上的受体结合在结肠炎的发病机制中起着重要作用。我们发现，神经降压素(NT)是一种在结肠对几种刺激做出反应而释放的神经肽，它触发了NF-kappaB的激活，并从结肠粘膜释放了促炎细胞因子。此外，高亲和力NT受体在结肠上皮细胞上表达，并在IBD和艰难梭菌毒素介导的小肠结肠炎中上调。然而，NT结合触发这些反应的分子和生化机制(S)尚不清楚。我们的假设是，肽NT通过作用于结肠上皮细胞的G蛋白偶联1型受体(NTR1)，刺激涉及PKC、MAPK和NF-kappaB的信号转导通路，从而导致IL-8的释放。目的1研究神经降压素刺激结肠上皮细胞后，NF-kappaB/IkappaB系统在IL-8基因表达中的作用。本实验旨在阐明MAP激酶通路在NTR1诱导的NF-kappaB激活和IL-8基因表达中的作用，并研究PKC-表皮生长因子通讯在NTR1刺激后MAP激酶激活中的重要性。目的2确定Rho家族的小GTP结合蛋白RhoA、Rac和CDC42在NT诱导的IL-8表达中的功能作用，并确定NT与NTR1结合是否激活Rho家族蛋白。这一目标的实验还将确定哪个异源三聚体G蛋白受体亚型参与了NTR1诱导的NF-kappaB激活和IL-8基因表达，以及Rho家族蛋白的激活。目标3中描述的研究将确定介导NT诱导的IL-8表达的NTR1的结构决定因素。具体地说，我们将研究NTR1的第三个细胞内环和C末端在人类结肠细胞NT信号和IL-8表达中的作用。还建议进行研究，以确定参与NTR1介导的IL-8产生的第三胞内环和C末端的关键残基。我们的结果将对NT及其高亲和力受体在结肠炎中的作用提供重要的见解，并可能导致治疗肠炎的新的治疗方法。

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.