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: 8427314
• 负责人: OMAR TLIBA
• 金额: $ 35.1万
• 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/8427314
• 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.

描述（由申请人提供）：因为气道平滑肌（ASM）在驱动严重哮喘的过度气道阻塞和炎症方面起着至关重要的作用，因此ASM无法适当应对药物治疗对哮喘患者的严重后果。尽管许多研究旨在理解严重哮喘患者中糖皮质激素（GC）治疗的不敏感性的基础，但重点是免疫细胞的作用，但几乎没有采取任何措施来阐明ASM细胞的作用。这项研究的长期目标是定义临床相关的非免疫肺细胞中介导与炎症相关的GC不敏感性的分子机制，以期识别新的靶标在严重哮喘患者中恢复类固醇反应性。当前的建议旨在系统地研究炎症细胞因子控制ASM对GC的敏感性的机制。该提案依靠令人兴奋的初步数据，该数据支持中心假设，即特定残基上GC受体（GR）异常磷酸化提供了一种机制，从而提供了一种机制，炎症细胞因子会损害对GC的敏感性。我们的初步数据进一步表明，特异性残基的GR的磷酸化状态受细胞因子介导的丝裂原激活蛋白激酶（MAPKS）的激活，以及与选择的丝氨酸/苏氨酸蛋白磷酸酶（PPS）相关的，ASM细胞中存在。拟议的研究的基本原理是，了解GR信号传导与炎症激酶和/或蛋白质磷酸酶之间的相互作用可能会发现关键信息，以开发新的治疗剂，以克服严重哮喘中的GC不敏感性。该假设将通过追求三个具体目的来检验：确定GR磷酸化依赖性机制在类固醇靶向基因对对照和GC不敏感细胞和组织中GC的差异反应性中的相对贡献（AIM 1），以阐明MAPK的作用（AIM 2）和PPS（AIM 3）在细胞因子诱导的GR异常位点特异性磷酸化中的作用。在第一个目标中，突变构建体将用于调节GR的位点特异性磷酸化，之后将分析10种不同类固醇靶标基因的转录调节。在第二和第三目的中，将使用siRNA，突变的构建体和表达矢量来调节MAPK和PPS的表达，此后，该位点特异性的GR磷酸化，GR介导的反式激活活性，GR亚细胞定位，类固醇的共同作用将使用在实验室中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标基因启动子的因子募集以及类固醇靶标基因的定量表达。我们还将通过检查是否在正常受试者和哮喘患者的组织活检中激活上述途径并将研究发现与哮喘严重程度相关的组织活检中，以评估我们体外观察的临床意义。这种综合的翻译方法有望促进我们对严重哮喘患者类固醇不敏感性发展的细胞和分子机制的理解。