Airway Inflammatory Pathways Regulating Glucocorticoid Receptor Phosphorylation

项目总结 哮喘气道重塑的一个主要特征，即气道平滑肌(ASM)质量增加， 对哮喘的发病率、死亡率和发病机制有着深远的影响。这一增加的质量是由于 很大程度上是由于ASM细胞增殖活性增强所致。不幸的是，后一类细胞的增殖 不受当前哮喘药物的影响，因为这一过程似乎对糖皮质激素(GC)不敏感 效果。虽然旨在了解GC不敏感(GCI)基础的研究主要集中在 在免疫细胞方面，很少有研究人员研究ASM细胞中的GCI，ASM细胞是调节支气管运动张力的关键细胞。 这项研究的长期目标是确定影响ASM增殖剂敏感性的因素 哮喘患者对GC的反应，最终目标是建立治疗策略 绕过GCI。目前的提案将研究生长的细胞和分子机制 因子(GFS)调节ASM对GCs的敏感性。使用来自患者的ASM细胞的独特细胞模型 对于哮喘，我们令人兴奋的初步数据支持了中心假设，即GF诱导的异常部位特异性 糖皮质激素受体(GR)的磷酸化降低ASM对GC的敏感性。这些数据现在表明 丝氨酸134(Ser134)残基的GR磷酸化抑制GR信号转导。这些数据 也提示GF诱导丝氨酸/苏氨酸激酶蛋白激酶B(PKB/Akt)和 丝氨酸/苏氨酸蛋白磷酸酶2A(PP2A)调节GR-Ser134的磷酸化。该计划的基本原理 建议的研究是了解GR信号和GF诱导的激酶和/或 磷酸酶可能为克服GCI的新疗法的开发提供关键信息 哮喘。我们的假设将通过追求四个具体目标来检验：确定 生长因子诱导GR信号的GR位点特异性异常磷酸化(目标1)；以表征其作用 激酶和磷酸酶对生长因子诱导的GR位点特异性异常磷酸化的影响(AIM 2)；并阐明 类固醇靶基因在调节ASM细胞中GC抗增殖效应中的作用(目标3)。为此，我们 生成的工具(抗体、突变的构建体)用于检测GR-ser134磷酸化的作用。在所有目标中， 药物抑制剂、突变的构建体、siRNA和表达载体将被用来调节 类固醇共抑制因子、激酶、磷酸酶和类固醇靶基因的表达，之后GR位点特异性 磷酸化、GR介导的反式激活活性和亚细胞定位以及ASM生长 使用我们实验室已经建立的最先进的方法进行分析。我们还将确定 检测上述通路是否被激活的体外观察的临床意义 使用来自哮喘患者和非哮喘患者的支气管内活检的组织(目标4)。这一集成 翻译方法有望促进我们对有助于 部分哮喘患者GCI的发生。

PROJECT SUMMARY Increased airway smooth muscle (ASM) mass, a major feature of airway remodeling in asthma, profoundly contributes to asthma morbidity, mortality, and pathogenesis. This increased mass is due in large part to enhanced proliferative activity of ASM cells. Unfortunately, proliferation of these latter cells is unaffected by current asthma medications, as the process appears to be insensitive to glucocorticoid (GC) effects. While research aimed at understanding the basis of GC insensitivity (GCI) has focused on the role of immune cells, few investigators have studied GCI in ASM cells, the pivotal cell regulating bronchomotor tone. The long-term goal of this research is to identify factors that impair the sensitivity of the ASM proliferative response to GC in patients with asthma, with the ultimately goal of establishing therapeutic strategies to circumvent GCI. The current proposal will examine the cellular and molecular mechanisms by which growth factors (GFs) modulate ASM sensitivity to GCs. Using unique cellular models of ASM cells derived from patients with asthma, our exciting preliminary data supports the central hypothesis that GF-induced abnormal site-specific phosphorylation of glucocorticoid receptor (GR) impairs ASM sensitivity to GC. These data now suggest that (previously unappreciated) GR phosphorylation at serine 134 (ser134) residue inhibits GR signaling. These data also suggest that GF-induced activation of serine/threonine kinase protein kinase B (PKB/Akt) and serine/threonine protein phosphatase 2A (PP2A) regulates GR-ser134 phosphorylation. The rationale for the proposed research is that understanding the interplay among GR signaling and GF-induced kinases and/or phosphatases may uncover critical information for the development of novel therapeutics to overcome GCI in asthma. Our hypotheses will be tested by pursuing four specific aims: to identify the functional consequences of GF-induced abnormal GR site-specific phosphorylation on GR signaling (Aim 1); to characterize the contribution of kinases and phosphatases to GF-induced abnormal GR site-specific phosphorylation (Aim 2); and to elucidate the role of steroid-target genes in modulating GC anti-proliferative effects in ASM cells (Aim 3). To this end, we generated tools (antibody, mutated constructs) to examine the role of GR-ser134 phosphorylation. In all aims, pharmacological inhibitors, mutated constructs, siRNA, and expression vectors will be used to modulate the expression of steroid co-repressor, kinases, phosphatases, and steroid-target genes, after which GR site-specific phosphorylation, GR-mediated transactivation activities and sub-cellular localization, and ASM growth will be analyzed using state-of-the-art approaches already established in our laboratories. We will also determine the clinical significance of our in vitro observations by examining whether the aforementioned pathways are activated in tissues using endobronchial biopsies from subjects both with and without asthma (Aim 4). This integrated translational approach is expected to advance our understanding of the mechanisms that contribute to the development of GCI in some patients with asthma.