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（由申请方提供）：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（CRFR 1、CRFR 2）是中枢神经系统中应激的重要介质。因此，它们的功能的系统性抑制不是有吸引力的治疗模型。该肽/受体家族的组分在肠内显著表达，其中它们的局部功能仍有待确定。我们从CRFR 2杂合子小鼠获得的初步数据表明，CRFR 2系统的扰动使小鼠对突发应激和免疫挑战更敏感。我们的研究结果表明，新的治疗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和蛋白水平的相互变化。这些差异性变化可以改变Ucn诱导的受体从质膜到质膜的运输。然而，这种不同的贩运到回收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，差异通过CRF受体信号，在分子，细胞和器官水平上管理肠道炎症。本提案的目的是：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的方法来确定Ucn/CRFR系统在肠道炎症中的系统作用。我们将确定CRFR 2敲除和杂合子小鼠与其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。利用RNA干扰技术研究结肠特异性Ucns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来敲低结肠中Ucn和CRFR 2的局部表达。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CRFR的结肠特异性敲低是否足以改善或加重结肠炎。具体目标3。明确Ucn 1诱导CRFR 1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查Ucn 1刺激对CRFR 1亚细胞定位的影响。我们将探讨衔接/支架蛋白（2-arrestins和发动蛋白）的脱敏和再敏化CRFR的能力。我们将通过测量Ucn 1刺激的细胞内Ca 2+动员来评估Ucn 1对CRFR 1活性的调节。因此，操纵尿皮质素及其受体为了解其GI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）是调节心理、生理和免疫应激对激素反应、焦虑、情绪、进食行为和胃肠道功能的影响的短蛋白（肽）。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（Ucn 1）。我们从CRFR 2杂合子小鼠获得的初步数据表明，CRFR 2系统的扰动使小鼠对突发应激和免疫挑战更敏感。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确定。这项研究是创新的，因为它调查的概念，局部扰动的UCN及其受体影响结肠炎症的结果。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，以及尿皮质素的操作是否有助于阻止肠道炎症。针对这种炎症通路的治疗可以改善炎症性肠病患者的生活质量，并降低终身治疗成本。