Airway Inflammatory Pathways Regulating Glucocorticoid Receptor Phosphorylation

气道炎症途径调节糖皮质激素受体磷酸化

• 批准号: 8220499
• 负责人: OMAR TLIBA
• 金额: $ 38.25万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8220499
• 关键词: Asthma; Automobile Driving; Biochemistry; Biopsy; Breathing; Cells; Chronic; Data; Dependency; Development; Disease; Failure; Functional disorder; Gene Expression; Gene Targeting; Glucocorticoid Receptor; Glucocorticoids; Goals; Health Care Costs; Human; Immune; Impairment; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Knowledge; Laboratories; Link; Lung; MAP3K5 gene; Mediating; Mission; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular; Mutate; N-terminal; Nature; Nuclear; Outcome; Pathway interactions; Pattern; Pharmacotherapy; Phosphorylation; Phosphotransferases; Play; Predisposition; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Receptor Signaling; Regulation; Relative (related person); Research; Role; Serine; Severities; Severity of illness; Site; Small Interfering RNA; Smooth Muscle Myocytes; Specificity; Steroids; TNF gene; Testing; Tissues; Transactivation; Transcriptional Regulation; Uncertainty; United States National Institutes of Health; airway inflammation; airway obstruction; asthmatic patient; base; burden of illness; clinically relevant; clinically significant; cofactor; cytokine; expression vector; gene induction; glucocorticoid receptor-interacting protein 1; human RIPK1 protein; human TIF2 factor; insight; new therapeutic target; novel; novel therapeutics; promoter; public health relevance; receptor; receptor function; respiratory smooth muscle; serine receptor; stress-activated protein kinase 1; tissue-factor-pathway inhibitor 2; translational approach

DESCRIPTION (provided by applicant): Because airway smooth muscle (ASM) plays a crucial role in driving the excessive airway obstruction and inflammation seen in severe asthma, failure of ASM to properly respond to drug therapy entails serious consequences for asthmatic patients. While much research aimed at understanding the basis of insensitivity to glucocorticoid (GC) therapy in severe asthmatics focused on the role of immune cells, little has been done to clarify the role of ASM cells. The long-term goal of this research is to define the molecular mechanisms mediating inflammation-associated GC insensitivity in clinically relevant non-immune lung cells, with the hope of identifying novel targets to restore steroid responsiveness in severe asthmatics. The current proposal aims to systematically investigate the mechanisms by which inflammatory cytokines control ASM sensitivity to GCs. The proposal relies on exciting preliminary data supporting the central hypothesis that abnormal phosphorylation of the GC receptor (GR) at specific residues provides a mechanism by which inflammatory cytokines impair ASM sensitivity to GCs. Our preliminary data further suggest that the phosphorylation state of the GR at specific residues is regulated by cytokine-mediated activation of Mitogen-Activated Protein Kinases (MAPKs) concomitant with select serine/threonine protein phosphatases (PPs) present in ASM cells. The rationale for the proposed research is that understanding the interplay between GR signaling and inflammatory kinases and/or protein phosphatases may uncover critical information for the development of novel therapeutics to overcome GC insensitivity in severe asthma. This hypothesis will be tested by pursuing three specific aims: to identify the relative contribution of GR phosphorylation-dependent mechanisms in the differential responsiveness of steroid-target genes to GCs in control and GC-insensitive cells and tissues (Aim 1), to clarify the role of MAPKs (Aim 2) and the contribution of PPs (Aim 3) in cytokine-induced abnormal site- specific phosphorylation of the GR. In the first aim, mutated constructs will be used to modulate site-specific phosphorylation of the GR, after which the transcriptional regulation of 10 different steroid-target genes will be analyzed. In the second and third aims, siRNA, mutated constructs, and expression vectors will be used to modulate the expression of MAPKs and PPs, after which site-specific GR phosphorylation, GR-mediated transactivation activities, GR sub-cellular localization, steroid co-factor recruitment to steroid-target gene promoters, and quantitative expression of steroid-target genes will be analyzed using state-of-the-art molecular and biochemistry approaches already established in our laboratories. We will also evaluate the clinical significance of our in vitro observations by examining whether the aforementioned pathways are activated in tissue biopsies from normal subjects and asthmatic patients and correlate the research findings with asthma severity. This integrated translational approach is expected to advance our understanding of the cellular and molecular mechanisms that contribute to the development of steroid insensitivity in severe asthmatic patients. PUBLIC HEALTH RELEVANCE: The public health relevance of the proposed research derives from the potential of uncovering the link between the activities of asthma-related inflammatory molecules and glucocorticoid receptor dysfunction seen in severe asthmatics. Clarifying the nature and the mechanisms underlying this link could provide new therapeutic targets to overcome steroid insensitivity in severe asthmatics. Thus, the proposed research is relevant to the NIH mission that pertains to developing fundamental knowledge that will help to reduce the burdens of illness.

描述（由申请人提供）：由于气道平滑肌（ASM）在导致严重哮喘中出现的过度气道阻塞和炎症方面发挥着至关重要的作用，因此ASM未能对药物治疗做出适当反应会给哮喘患者带来严重后果。虽然许多旨在了解严重哮喘患者对糖皮质激素 (GC) 治疗不敏感的基础的研究都集中在免疫细胞的作用上，但很少有研究来阐明 ASM 细胞的作用。这项研究的长期目标是确定介导临床相关非免疫肺细胞中炎症相关GC不敏感的分子机制，希望找到恢复严重哮喘患者类固醇反应性的新靶点。目前的提案旨在系统地研究炎症细胞因子控制 ASM 对 GC 敏感性的机制。该提议依赖于支持中心假设的令人兴奋的初步数据，即GC受体（GR）在特定残基处的异常磷酸化提供了炎症细胞因子损害ASM对GC敏感性的机制。我们的初步数据进一步表明，GR 在特定残基处的磷酸化状态受到细胞因子介导的丝裂原激活蛋白激酶 (MAPK) 激活以及 ASM 细胞中存在的选定丝氨酸/苏氨酸蛋白磷酸酶 (PP) 的调节。拟议研究的基本原理是，了解 GR 信号传导与炎症激酶和/或蛋白磷酸酶之间的相互作用可能会为开发克服严重哮喘中 GC 不敏感性的新型疗法提供关键信息。该假设将通过追求三个具体目标进行检验：确定 GR 磷酸化依赖性机制在对照和 GC 不敏感细胞和组织中类固醇靶基因对 GC 的差异反应性中的相对贡献（目标 1），以阐明MAPK（目标 2）的作用和 PP（目标 3）在细胞因子诱导的 GR 异常位点特异性磷酸化中的作用。第一个目标是，突变的构建体将用于调节 GR 的位点特异性磷酸化，之后将分析 10 个不同类固醇靶基因的转录调控。在第二个和第三个目标中，siRNA、突变构建体和表达载体将用于调节 MAPK 和 PP 的表达，然后进行位点特异性 GR 磷酸化、GR 介导的反式激活活性、GR 亚细胞定位、类固醇共-将使用我们实验室已经建立的最先进的分子和生物化学方法来分析类固醇靶基因启动子的因子招募和类固醇靶基因的定量表达。我们还将通过检查上述途径在正常受试者和哮喘患者的组织活检中是否被激活来评估体外观察的临床意义，并将研究结果与哮喘严重程度相关联。这种综合转化方法有望促进我们对导致严重哮喘患者类固醇不敏感的细胞和分子机制的理解。 公共健康相关性：拟议研究的公共健康相关性源于揭示哮喘相关炎症分子的活性与严重哮喘患者中糖皮质激素受体功能障碍之间的联系的潜力。阐明这种联系的本质和机制可以提供新的治疗靶点，以克服严重哮喘患者的类固醇不敏感性。因此，拟议的研究与 NIH 的使命相关，该使命涉及发展有助于减轻疾病负担的基础知识。