Airway Inflammatory Pathways Regulating Glucocorticoid Receptor Phosphorylation

描述(申请人提供)：由于气道平滑肌(ASM)在推动严重哮喘的过度呼吸道阻塞和炎症中起着关键作用，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。虽然许多旨在了解重症哮喘患者对糖皮质激素(GC)治疗不敏感的基础的研究集中在免疫细胞的作用上，但很少有人澄清ASM细胞的作用。这项研究的长期目标是确定在临床相关的非免疫性肺细胞中介导炎症相关GC不敏感性的分子机制，希望找到新的靶点来恢复重症哮喘患者的类固醇反应。目前的建议旨在系统地研究炎性细胞因子控制ASM对GCs敏感性的机制。该提议依赖于令人兴奋的初步数据，支持中心假设，即特定残基上GC受体(GR)的异常磷酸化提供了一种炎性细胞因子削弱ASM对GCs敏感性的机制。我们的初步数据进一步表明，特定残基上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ASM细胞中存在的选择性丝氨酸/苏氨酸蛋白磷酸酶(PPS)的调节。这项研究的基本原理是，了解GR信号与炎性激酶和/或蛋白磷酸酶之间的相互作用可能会揭示出新的治疗方法的开发，以克服严重哮喘患者对GC不敏感的关键信息。这一假说将通过追求三个特定目标来验证：在对照和GC不敏感的细胞和组织中，确定类固醇靶基因对GC的差异反应中GR磷酸化依赖机制的相对贡献(目标1)，阐明MAPKs(目标2)和PPS(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。在第一个目标中，突变的构建体将被用来调节GR的位点特异性磷酸化，然后将分析10个不同的类固醇靶基因的转录调控。在第二和第三个AIMS中，将使用siRNA、突变的构建体和表达载体来调节MAPKs和PPS的表达，之后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特定部位的GR磷酸化、GR介导的反式激活活性、GR亚细胞定位、类固醇辅助因子对类固醇靶基因启动子的募集以及类固醇靶基因的定量表达。我们还将通过检测上述通路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我们的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。这种综合的翻译方法有望促进我们对导致重症哮喘患者激素不敏感的细胞和分子机制的理解。

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.