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: 8088800
• 负责人: Aditi Bhargava
• 金额: $ 11.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8088800
• 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; Disease Outcome; Dose; Double-Stranded RNA; Down-Regulation; Dynamin; Employee Strikes; Endocytosis; Exhibits; Extravasation; Family; Feeding behaviors; Figs - dietary; Financial compensation; Gastrointestinal Motility; Gastrointestinal tract structure; Genes; Genetic; Genetic Models; Genus Cola; Glucocorticoids; 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; public health relevance; 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(申请人提供)：促肾上腺皮质激素释放因子(CRF)家族神经肽(CRF和urocortins[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，全身性抑制它们的功能并不是一个有吸引力的治疗模式。这个多肽/受体家族的组成部分在肠道中显著表达，其局部功能仍有待确定。我们对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应激和免疫挑战。我们的结果表明，肠炎性疾病的新疗法可能需要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和蛋白质水平的对等变化。这些不同的变化可以改变UCN诱导的受体从质膜向质膜的运输。然而，这种不同的运输到循环或降解途径的分子机制仍然没有被探索。因此，我们的假设是，UCN亚型通过CRF受体发出不同的信号，在分子、细胞和器官水平上控制肠道炎症。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系统作用。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身作用。我们将确定CRFR2基因敲除和杂合子小鼠与它们的野生型小鼠相比，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变化。具体目的2.利用RNA干扰技术(RNAi)确定结肠特异的UCNs和CRFR在肠道炎症中的作用。为了确定UCN/CRFR系统在结肠中的特异性作用，我们将建立使用RNAi在结肠中局部下调UCNs和CRFR2的表达。我们将确定RNAi在结肠中的空间和时间效应，并使用特定目的1.特定目的3.确定Ucn1诱导的CRFR1运输的机制和功能，评估结肠特异性敲除UCNs和CRFR是否足以改善或加重结肠炎。我们将利用共聚焦显微镜和免疫荧光技术检测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-arrestins和Dynamin)使CRFR脱敏和再增敏的能力。我们将通过测量Ucn1刺激的细胞内钙动员来评估Ucn1对CRFR1活性的调节。因此，对尿皮质素及其受体的操纵为了解其胃肠功能提供了一个有效的起点，这可能有助于开发治疗炎症性肠病的新方法。公共卫生相关性：尿皮质激素(UCNs)和促肾上腺皮质激素释放因子(CRF)是一种短蛋白(肽)，介导心理、身体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。CRF和UCNs在中枢神经系统中表达，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，活动期克罗恩病患者的一种尿皮质素(Ucn1)水平高于没有这种疾病的人。我们对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应激和免疫挑战。然而，UCN多肽/受体系统的特定成分在肠炎的发生、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结果的概念。如果成功，这项研究将确定urocortins如何帮助维持肠道的正常免疫反应，以及urocortins的操作是否有助于阻止肠道炎症。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，降低终生治疗成本。