Regulation and Function of Urocortins and their Receptors

项目总结/摘要 促肾上腺皮质激素释放因子（CRF）家族的神经肽（CRF和尿皮质素[Ucn] 1 - 3）及其 受体（CRFR1，CRFR2）是中枢神经系统中应激的基本介质。因此 它们功能的系统性抑制不是有吸引力的治疗模型。该肽/受体的组分 家族主要在肠道内表达，其局部功能仍有待确定。我们 来自CRFR2杂合子小鼠的初步数据表明，CRFR2系统的扰动使得CRFR2基因表达增加。 小鼠更容易受到突然的压力和免疫挑战。我们的研究结果表明， 肠道炎症性疾病可能需要CRF受体的拮抗剂或激动剂，这取决于 受影响的肠道区域和炎性疾病的类型。我们在结肠炎中观察到的一个显著特征是 Ucn配体及其受体mRNA和蛋白水平的变化是相互的。这些差分 这些变化可以改变Ucn诱导的受体从质膜到质膜的运输。但 这种不同的转运到再循环或降解途径的分子机制仍然未被探索。 因此，我们的假设是，Ucn亚型，通过CRF受体的差异信号传导， 肠道炎症在分子、细胞和器官水平。这项建议的目的是： 具体目标1。明确Ucn/CRFR系统在肠道炎症中的全身作用。我们将 使用药理学和遗传学方法来确定Ucn/CRFR系统在肠道中的全身作用， 炎症我们将确定CRFR2基因敲除小鼠和杂合子小鼠是否表现出对 炎症和激动剂处理的小鼠与其野生型同窝仔相比。我们将决定修改 通过测量体重增加、死亡率、组织学损伤和液体渗漏的变化来评估结肠炎的严重程度。 具体目标2。明确结肠特异性Ucns和CRFRs在肠上皮细胞中的作用， 使用RNA干扰（RNAi）来治疗炎症。为了确定Ucn/CRFR系统的结肠特异性作用， 我们将建立使用RNAi来敲低结肠中Ucns和CRFR2的局部表达。我们将 确定RNAi在结肠中的空间和时间效应，并评估结肠特异性敲低是否 使用特定目标1中定义的终点，Ucns和CRFR足以改善或加重结肠炎。 具体目标3。明确Ucn 1诱导CRFR1转运的机制和功能。我们将 使用共聚焦显微镜检查Ucn1刺激对CRFR1亚细胞定位的影响 和免疫荧光。我们将探讨衔接/支架蛋白（抑制蛋白和 发动蛋白）使CRFR脱敏和再敏化。我们将通过以下方法评估Ucn1对CRFR1活性的调节： 测量Ucn 1刺激的细胞内Ca 2+动员。因此，操纵尿皮质素及其组合物是不可能的。 受体为了解其GI功能提供了有效的起点，这可能有助于 炎症性肠病的新疗法的开发。

PROJECT SUMMARY/ABSTRACT The corticotropin-releasing factor (CRF) family of neuropeptides (CRF and urocortins [Ucn] 1-3) and their receptors (CRFR1, CRFR2) are essential mediators of stress in the central nervous system. Therefore a systemic inhibition of their function is not an attractive therapeutic model. Components of this peptide/receptor family are prominently expressed within the intestine, where their local functions remain to be defined. Our preliminary data from CRFR2 heterozygous mice suggests that perturbation of the CRFR2 system renders the mice more susceptible to sudden stress and immune challenges. Our results suggest that new treatments for intestinal inflammatory diseases may require either antagonists or agonists of CRF receptors, depending upon the affected intestinal region and the type of inflammatory disease. A striking feature we observed during colitis was reciprocal changes in mRNA and protein levels of Ucn ligands and their receptors. These differential changes can alter Ucn-induced trafficking of receptors from and to the plasma membrane. However, the molecular mechanisms of this divergent trafficking to recycling or degradatory pathways remain unexplored. Thus, our hypothesis is that Ucn isoforms, differentially signaling through CRF receptors, govern intestinal inflammation at the molecular, cellular and organ levels. The aims of this proposal are to: Specific Aim 1. To define the systemic role of the Ucn/CRFR system in intestinal inflammation. We will use pharmacological and genetic approaches to define the systemic role of the Ucn/CRFR system in intestinal inflammation. We will determine if CRFR2 knockout and heterozygous mice exhibit differential sensitivity to inflammation and agonist treatment as compared with their wild type littermates. We will determine alterations in colitis severity by measuring changes in body weight gain, mortality, histological damage, and fluid leak. Specific Aim 2. To define the contributions of colon-specific Ucns and CRFRs in intestinal inflammation using RNA interference (RNAi). To determine the colon-specific role of the Ucn/CRFR system, we will establish the use RNAi to knockdown expression of Ucns and CRFR2 locally in the colon. We will determine the spatial and temporal effects of RNAi in the colon, and assess whether colon-specific knockdown of Ucns and CRFRs is sufficient to ameliorate or exacerbate colitis, using end points defined in Specific Aim 1. Specific Aim 3. To define the mechanism and function of Ucn1-induced trafficking of CRFR1. We will examine the influence of Ucn1 stimulation on the subcellular localization of CRFR1 using confocal microscopy and immunofluorescence. We will explore the ability of the adaptor/scaffolding proteins (¿-arrestins and dynamin) to desensitize and resensitize CRFRs. We will assess Ucn1 regulation of CRFR1 activity by measuring Ucn1-stimulated intracellular Ca2+ mobilization. Thus, manipulation of the urocortins and their receptors provides an effective starting point for understanding their GI functions, which could contribute to the development of new treatments for inflammatory bowel disease.