Regulation and Function of Urocortins and their Receptors

描述（由申请人提供）：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）是中枢神经系统中必不可少的应激介质。因此，对其功能的系统性抑制并不是一种有吸引力的治疗模式。这种肽/受体家族的组成部分在肠道内显著表达，其局部功能仍有待确定。我们从CRFR2杂合小鼠中获得的初步数据表明，CRFR2系统的扰动使小鼠更容易受到突发压力和免疫挑战。我们的研究结果表明，肠道炎症性疾病的新治疗可能需要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症性疾病的类型。我们在结肠炎期间观察到的一个显著特征是Ucn配体及其受体的mRNA和蛋白质水平的相互变化。这些不同的变化可以改变ucn诱导的受体进出质膜的运输。然而，这种不同的转运到回收或降解途径的分子机制仍未被探索。因此，我们的假设是Ucn异构体，通过CRF受体的差异信号传导，在分子，细胞和器官水平上控制肠道炎症。本建议的目的是：具体目标1。明确Ucn/CRFR系统在肠道炎症中的全身性作用。我们将使用药理学和遗传学方法来确定Ucn/CRFR系统在肠道炎症中的全系统作用。我们将确定CRFR2基因敲除和杂合小鼠与野生型小鼠相比，是否对炎症和激动剂治疗表现出不同的敏感性。我们将通过测量体重增加、死亡率、组织学损伤和液体泄漏的变化来确定结肠炎严重程度的改变。具体目标2。利用RNA干扰（RNAi）确定结肠特异性ucn和crfr在肠道炎症中的作用。为了确定Ucn/CRFR系统的结肠特异性作用，我们将建立使用RNAi在结肠中局部敲低Ucns和CRFR2的表达。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ucn和crfr是否足以改善或加剧结肠炎，使用Specific Aim 1中定义的终点。具体目标3。明确ucn1诱导的CRFR1转运的机制和功能。我们将使用共聚焦显微镜和免疫荧光检测Ucn1刺激对CRFR1亚细胞定位的影响。我们将探讨接头/支架蛋白（2-阻滞蛋白和动力蛋白）对crfr脱敏和再敏的能力。我们将通过测量Ucn1刺激的细胞内Ca2+动员来评估Ucn1对CRFR1活性的调节。因此，对尿皮质素及其受体的操作为了解其胃肠道功能提供了一个有效的起点，这可能有助于开发炎症性肠病的新治疗方法。公共卫生相关性：尿皮质素（Ucns）和促肾上腺皮质激素释放因子（CRF）是短蛋白（肽），介导心理、生理和免疫应激源对激素反应、焦虑、情绪、摄食行为和胃肠功能的影响。中枢神经系统表达的CRF和Ucns控制自主神经的活动，从而介导中枢应激源对胃肠运动和分泌的影响。研究表明，患有活动性克罗恩病的人比没有这种疾病的人有更高水平的一种尿皮质素（Ucn1）。我们从CRFR2杂合小鼠中获得的初步数据表明，CRFR2系统的扰动使小鼠更容易受到突发压力和免疫挑战。然而，Ucn肽/受体系统的特定组分在肠道炎症的发生、发展和进展中的确切作用仍有待明确。这项研究的创新之处在于，它调查了Ucn及其受体的局部扰动影响结肠炎症结果的概念。如果成功，这项研究将确定尿皮质素如何帮助维持肠道的正常免疫反应，以及操纵尿皮质素是否有助于阻止肠道炎症。针对这种炎症途径的治疗可以改善炎症性肠病患者的生活质量，并降低终生治疗成本。

DESCRIPTION (provided by applicant): 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 (2-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. PUBLIC HEALTH RELEVANCE: Urocortins (Ucns) and corticotropin-releasing factor (CRF) are short proteins (peptides) that mediate the effects of psychological, physical and immunological stressors on hormonal responses, anxiety, mood, feeding behavior and gastrointestinal functions. CRF and Ucns expressed in the central nervous system control activity of autonomic nerves and thereby mediate the effects of central stressors on gastrointestinal motility and secretion. Studies have shown that people with active Crohn's disease have higher levels of one type of urocortin (Ucn1) than people without the disease. 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. However, the precise roles of specific components of Ucn peptide/receptor system in the initiation, development and progression of intestinal inflammation remain to be defined. This research is innovative in that it investigates the notion that local perturbation of the Ucn and its receptors influences the outcome of colonic inflammation. If successful, this study will establish how urocortins help maintain a normal immune response in the gut and whether manipulation of urocortins can help stop intestinal inflammation. Therapies that target this inflammatory pathway could improve the quality of life for patients with inflammatory bowel diseases and decrease the lifetime cost of treatment.