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: 10159949
• 负责人: OMAR TLIBA
• 金额: $ 9.27万
• 依托单位: LONG ISLAND UNIVERSITY C. W. POST CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2022-03-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159949
• 关键词: AKT inhibition; Address; Affect; Age; Antibodies; Asthma; Binding Proteins; Biopsy; Cell model; Cells; Data; Defect; Development; Gender; Gene Expression; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; Growth; Growth Factor; Human; Immune; Immunohistochemistry; Impairment; In Vitro; Inflammatory; Insulin; Insulin-Like Growth-Factor Binding Protein 1; Knowledge; Laboratories; Lesion; MAPK11 gene; Mediating; Mission; Mitogen-Activated Protein Kinases; Molecular; Morbidity - disease rate; Mutate; NCOR1 gene; NRIP1 gene; Nuclear Translocation; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Phosphotyrosine; Play; Process; Protein Serine/Threonine Phosphatase; Protein phosphatase; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor Signaling; Research; Research Personnel; Role; Serine; Signaling Molecule; Site; Small Interfering RNA; Smooth Muscle Myocytes; Specificity; Steroids; Testing; Therapeutic; Tissues; Transactivation; United States National Institutes of Health; Up-Regulation; airway remodeling; burden of illness; clinically significant; cofactor; design; expression vector; functional disability; genetic corepressor; in vivo; inhibitor/antagonist; injured airway; mortality; muscle form; new therapeutic target; novel therapeutics; p38 Mitogen Activated Protein Kinase; prevent; promoter; public health relevance; receptor function; recruit; respiratory smooth muscle; response; tool; translational approach

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.

项目摘要 增加气道平滑肌（ASM）质量，这是哮喘中气道重塑的主要特征， 深刻地有助于哮喘发病率，死亡率和发病机理。这种增加的质量应在 大部分是增强ASM细胞增殖活性。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不敏感（GC） 效果。尽管旨在理解GC不敏感性基础（GCI）的研究重点是 免疫细胞，很少有研究人员研究了ASM细胞中的GCI，即调节支气管运动的关键细胞。 这项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，最终是建立治疗策略 绕过GCI。当前的建议将检查生长的细胞和分子机制 因素（GFS）调节ASM对GC的敏感性。使用来自患者的ASM细胞的独特细胞模型 使用哮喘，我们令人兴奋的初步数据支持了GF引起的异常现场特异性的中心假设 糖皮质激素受体（GR）的磷酸化会损害ASM对GC的敏感性。这些数据现在表明 （以前未接受的）丝氨酸134（Ser134）残基处的GR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）的激活和 丝氨酸/苏氨酸蛋白磷酸酶2a（PP2A）调节GR-SER134磷酸化。理由 拟议的研究是，了解GR信号传导和GF诱导的激酶和/或 磷酸酶可能会发现开发新疗法以克服GCI的关键信息 哮喘。我们的假设将通过追求四个具体目标来检验：确定功能后果 GF诱导的GR信号传导上的异常GR特异性磷酸化（AIM 1）；表征贡献 GF诱导的异常GR特异性磷酸化的激酶和磷酸酶（AIM 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 生成的工具（抗体，突变构建体）检查GR-SER134磷酸化的作用。所有目标， 药理学抑制剂，突变构建体，siRNA和表达载体将用于调节 类固醇共抑制剂，激酶，磷酸酶和类固醇靶向基因的表达，然后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，ASM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 通过检查上述途径是否激活，我们的体外观察的临床意义 在组织中，使用或没有哮喘的受试者的支撑架活检（AIM 4）。这整合了 预计翻译方法将促进我们对有助于促成机制的理解 一些哮喘患者的GCI发育。