Regulation and Function of Urocortins and their Receptor

总结 炎症性肠病（IBD）是美国最常见的胃肠道疾病之一，费用约为1.7美元 每年治疗10亿次。IBD的病因仍然未知。虽然IBD结果明显受到 通过涉及大脑、肠道、免疫系统和环境的相互作用， 仍然没有得到充分的表征，并已被确定为理解IBD病因的主要差距。我们 该提案通过阐明生物因素，例如， 促肾上腺皮质激素释放因子（CRF）系统，其调节可直接影响肠道和大脑。在 除了IBD的身体症状外，患者还经常经历与压力相关的情绪障碍。通用报告格式 系统通过激活下丘脑-垂体-肾上腺（HPA）轴介导经典的应激反应。 然而，CRF系统的成分产生其功能的细胞机制， IBD期间免疫调节和压力应对作用仍然未知。初步数据显示， 应激受体CRF 2是炎症损伤期间细胞应激反应的关键决定因素。我们的- 这一假说认为，CRF和尿皮质素，通过CRF 1和CRF 2的差异信号传导， 肠道炎症在分子、细胞和器官水平上以性别特异性方式存在。我们提出 以三个具体目标来检验我们的假设在目标1中，我们将确定CRF 2的性别特异性作用 受体在基线时调节GI通透性、器官质量和免疫细胞功能方面的作用 以及结肠炎期间。我们的试验数据显示，雌性小鼠的肠道比雄性小鼠的“泄漏”更多。这种泄漏是 在缺乏CRF 2受体的小鼠中消除。通过测量血管渗漏和免疫细胞的变化， 通过流式细胞术，我们将确定CRF 2受体缺陷是否会改变免疫细胞的募集或迁移 在结肠炎期间，局部在炎症部位、血液和脾脏中。激酶的作用及其 将确定IBD期间表现性别特异性效应的靶点。联合国系统各组成部分的变化 结肠炎期间大脑中的CRF系统将与焦虑和抑郁样行为的变化相关。 在目标2中，我们将确定尿皮质素和CRF激动剂在调节方面的性别特异性作用， 在基线和结肠炎期间的GI渗透性、器官质量和免疫细胞功能。通过使用 目的1中定义的方法，我们将描述4种不同的CRF 2受体激动剂在 以性别特异性方式调节CRF 2的功能。在目标3中，我们将确定 CRF受体（CRF 1：CRF 2）和/或其与支架蛋白和热休克蛋白的结合 导致通过它们的四种配体的差异运输和信号传导。通过制作CRF的标记版本， 受体，并使用免疫沉淀和质谱相结合，我们将确定辅助 与CRF受体相互作用以介导环境特异性信号传导和运输的蛋白质。这项建议会 定义性别特异性信号通路，可用于IBD患者的治疗目的。

SUMMARY Inflammatory bowel disease (IBD) is one of the most prevalent GI diseases in the United States, costing ~ $1.7 billion annually in treatment. The etiology of IBD remains unknown. Although IBD outcome is clearly influenced by interactions involving the brain, gut, immune system, and the environment, the contributing biological factors remain under-characterized and have been identified as a major gap in understanding the etiology of IBD. Our proposal directly addresses this gap by elucidating biological factors, such as the components of the corticotropin-releasing factors (CRF) system, whose modulation can directly influence the gut and the brain. In addition to the physical symptoms of IBD, patients often experience stress-related mood disorders. The CRF system mediates the classic stress response via activation of the hypothalamic-pituitary-adrenal (HPA) axis. However, the cellular mechanisms by which the components of the CRF system bring about their immunomodulatory and stress-coping actions during IBD remain unknown. Our preliminary data shows that the stress receptor CRF2 is a key determinant of cellular stress responses during an inflammatory insult. Our over- arching hypothesis is that CRF and urocortins, differentially signaling through CRF1 and CRF2, govern intestinal inflammation at the molecular, cellular and organ levels in a sex-specific manner. We propose to test our hypothesis in three specific aims. In Aim 1, we will determine the sex-specific role of the CRF2 receptor in modulating aspects of GI permeability, organ mass, and immune cell function at baseline and during colitis. Our pilot data shows that the gut is “leakier” in female than in male mice. This leakiness is abrogated in mice lacking the CRF2 receptor. By measuring vascular leak and immune cell changes by flow cytometry, we will determine whether CRF2 receptor deficiency alters immune cell recruitment or migration during colitis, locally at the site of inflammation, and in the blood and spleen. The role of kinases and their targets in manifesting sex-specific effects during IBD will be ascertained. Changes in the components of the CRF system in the brain during colitis will be correlated with changes in anxiety- and depression-like behaviors. In Aim 2, we will determine the sex-specific role of urocortins and CRF agonists in modulating aspects of GI permeability, organ mass, and immune cell function at baseline and during colitis. By using approaches defined in Aim 1, we will delineate the role of 4 different agonists for the CRF2 receptor in modulating of function of CRF2 in a sex-specific manner. In Aim 3, we will determine if heteromerization of CRF receptors (CRF1:CRF2) and/or their association with scaffold proteins and heat shock proteins results in differential trafficking and signaling by their four ligands. By making tagged versions of CRF receptors and using a combination of immunoprecipitation and mass spectrometry, we will identify ancillary proteins that interact with CRF receptors to mediate context-specific signaling and trafficking. This proposal will define sex-specific signaling pathways that can be targeted for therapeutic purposes for patients with IBD.