Airway Inflammatory Pathways Regulating Glucocorticoid Receptor Phosphorylation

项目摘要 气道平滑肌（ASM）质量增加是哮喘气道重塑的主要特征， 哮喘发病率、死亡率和发病机制的重要因素。这种增加的质量是由于 很大程度上是由于ASM细胞的增殖活性增强。不幸的是，这些后一种细胞的增殖是 不受当前哮喘药物的影响，因为该过程似乎对糖皮质激素（GC）不敏感 方面的影响.虽然旨在了解GC不敏感性（GCI）基础的研究集中在以下方面的作用： 免疫细胞，很少有研究者研究GCI在ASM细胞，调节支气管张力的关键细胞。 本研究的长期目标是确定损害ASM增殖敏感性的因素， 哮喘患者对GC的反应，最终目标是建立治疗策略， 绕过GCI目前的建议将研究细胞和分子机制， GFs调节ASM对GCs的敏感性。使用源自患者的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诱导的异常位点特异性 糖皮质激素受体（GR）的磷酸化损害ASM对GC的敏感性。这些数据表明， （以前未被认识到的）GR丝氨酸134（ser 134）残基磷酸化抑制GR信号传导。这些数据 还表明GF诱导的丝氨酸/苏氨酸激酶蛋白激酶B（PKB/Akt）的活化和 丝氨酸/苏氨酸蛋白磷酸酶2A（PP 2A）调节GR-丝氨酸134磷酸化。的理由 建议的研究是，了解GR信号传导和GF诱导的激酶和/或 磷酸酶可能揭示新疗法的发展，以克服GCI的关键信息， 哮喘我们的假设将通过追求四个具体目标进行测试：确定 GF诱导的GR位点特异性异常磷酸化对GR信号传导的影响（目的1）; 激酶和磷酸酶对GF诱导的异常GR位点特异性磷酸化的影响（目的2）;并阐明 类固醇靶基因在调节ASM细胞中GC抗增殖作用中的作用（Aim 3）。为此我们 产生的工具（抗体，突变的构建体），以检查GR-ser 134磷酸化的作用。在所有目标中， 药理学抑制剂、突变的构建体、siRNA和表达载体将用于调节 类固醇辅阻遏物、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的生长将是 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 通过检查上述通路是否被激活，我们在体外观察的临床意义 使用支气管内活组织检查从患有和不患有哮喘的受试者的组织中（目的4）。这一综合 翻译的方法，预计将促进我们的机制，有助于了解 在某些哮喘患者中发生GCI。

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.