Regulation and Function of Urocortins and their Receptors

尿皮质素及其受体的调节和功能

• 批准号: 8055056
• 负责人: Aditi Bhargava
• 金额: $ 46.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055056
• 关键词: Abbreviations; Adrenal Glands; Affect; Agonist; Anxiety; Arrestins; Brain region; Cell Line; Cell membrane; Cells; Chronic; Clostridium difficile; Colitis; Colon; Confocal Microscopy; Corticotropin-Releasing Hormone; Corticotropin-Releasing Hormone Receptors; Crohn' s disease; Data; Development; Disease; Dose; Double-Stranded RNA; Down-Regulation; Dynamin; Employee Strikes; Endocytosis; Exhibits; Extravasation; Family; Feeding behaviors; Financial compensation; Gastrointestinal Motility; Gastrointestinal tract structure; Genes; Genetic; Genetic Models; Glucocorticoids; Health; Hormonal; Hypothalamic structure; Ileitis; Immune; Immune response; Immune system; Immunofluorescence Immunologic; Immunohistochemistry; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Interleukin-10; Intestines; Knock-out; Knockout Mice; Ligands; Link; Liquid substance; Measures; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Modification; Molecular; Moods; Mus; Neuraxis; Neurons; Neuropeptides; Neurosecretory Systems; Organ; Outcome; Pathway interactions; Patients; Peptide Receptor; Peptides; Piroxicam; Pituitary Hormones; Plasma; Play; Process; Production; Protein Isoforms; Proteins; Quality of life; RNA Interference; Recycling; Regulation; Research; Role; Scaffolding Protein; Severities; Signal Transduction; Stress; Sulfonic Acids; System; Testing; Therapeutic; Tissues; Toxin; Trinitrobenzenes; Ubiquitin; Weight Gain; arrestin 2; autonomic nerve; biological adaptation to stress; cytokine; defined contribution; desensitization; gastrointestinal; gastrointestinal function; hypothalamic-pituitary-adrenal axis; ileum; immunoreactivity; improved; innovation; life time cost; mortality; psychologic; receptor; receptor function; receptor recycling; response; single molecule; stressor; trafficking; urocortin

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.

描述（由申请人提供）：促肾上腺皮质激素释放因子 (CRF) 神经肽家族（CRF 和尿皮质素 [Ucn] 1-3）及其受体（CRFR1、CRFR2）是中枢神经系统应激的重要介质。因此，对其功能的系统性抑制并不是有吸引力的治疗模型。该肽/受体家族的成分在肠道内显着表达，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 系统的扰动使小鼠更容易受到突然的压力和免疫挑战。我们的结果表明，肠道炎症性疾病的新疗法可能需要 CRF 受体的拮抗剂或激动剂，具体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着特征是 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 诱导的受体往返于质膜的运输。然而，这种不同运输到回收或降解途径的分子机制仍有待探索。因此，我们的假设是 Ucn 亚型通过 CRF 受体发出差异性信号，在分子、细胞和器官水平上控制肠道炎症。该提案的目的是： 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 系统在肠道炎症中的系统作用。我们将确定 CRFR2 敲除小鼠和杂合小鼠与野生型同窝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、组织学损伤和液体渗漏的变化来确定结肠炎严重程度的变化。具体目标 2. 使用 RNA 干扰 (RNAi) 确定结肠特异性 Ucns 和 CRFRs 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 来敲低结肠局部 Ucns 和 CRFR2 表达的方法。我们将确定 RNAi 在结肠中的空间和时间效应，并使用特定目标 1 中定义的终点评估 Ucns 和 CRFRs 的结肠特异性敲低是否足以改善或加剧结肠炎。特定目标 3。定义机制以及 Ucn1 诱导的 CRFR1 运输的功能。我们将使用共聚焦显微镜和免疫荧光检查 Ucn1 刺激对 CRFR1 亚细胞定位的影响。我们将探索接头/支架蛋白（2-抑制蛋白和动力蛋白）使 CRFR 脱敏和重新敏化的能力。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，操纵尿皮质素及其受体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疗法。公共健康相关性：尿皮质素 (Ucns) 和促肾上腺皮质激素释放因子 (CRF) 是短蛋白（肽），可调节心理、身体和免疫应激源对荷尔蒙反应、焦虑、情绪、进食行为和胃肠功能的影响。在中枢神经系统中表达的 CRF 和 Ucns 控制自主神经的活动，从而介导中枢应激源对胃肠道运动和分泌的影响。研究表明，活动性克罗恩病患者的一种尿皮质素 (Ucn1) 水平高于未患该病的患者。我们来自 CRFR2 杂合子小鼠的初步数据表明，CRFR2 系统的扰动使小鼠更容易受到突然的压力和免疫挑战。然而，UCN肽/受体系统的特定成分在肠道炎症的发生、发展和进展中的确切作用仍有待确定。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正常的免疫反应，以及操纵尿皮质素是否可以帮助阻止肠道炎症。针对这一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。