Mechanisms of Steroid Resistance in Airway Smooth Muscle Cells

气道平滑肌细胞类固醇抵抗机制

• 批准号: 7684321
• 负责人: OMAR TLIBA
• 金额: $ 24.42万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684321
• 关键词: A549; Accounting; Address; Affect; Agreement; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antiinflammatory Effect; Asthma; Award; Binding; Binding Sites; Biological Assay; Biotechnology; Brefeldin A; Cell Adhesion Molecules; Cell Line; Cell Nucleus; Cells; Cellular biology; Co-Immunoprecipitations; Color; Complex; DNA Binding; Data; Data Quality; Detection; Development; Dimerization; Dominant-Negative Mutation; Dose; EMSA; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Figs - dietary; Flow Cytometry; Fractalkine; Functional disorder; Galactosidase; Gene Expression; General Transcription Factors; Genes; Glucocorticoids; Goals; Growth Factor; Health; Health Care Costs; Homo; Hour; Human; IRF1 gene; Immune; Immunoblotting; Immunoprecipitation; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Intercellular adhesion molecule 1; Interferons; Intracellular Transport; Ionomycin; Knowledge; Length; Ligands; Lung diseases; Malignant Epithelial Cell; Mediating; Mentors; Molecular; Nuclear; Nuclear Export; Nuclear Extract; PP5 protein-serine-threonine phosphatase; Pathway interactions; Patients; Pharmacology; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Plasmids; Promega; Property; Protein Isoforms; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Public Health; RANTES; Rattus; Recruitment Activity; Regulation; Relative (related person); Reporter; Reporting; Research; Research Personnel; Resistance; Role; SF1; Serine; Signal Transduction; Site; Small Interfering RNA; Smooth Muscle Myocytes; Steroid Receptors; Steroid Resistance; Steroid therapy; Steroids; Techniques; Technology; Testing; Therapeutic; Threonine; Time; Tissues; Transactivation; Transcription Factor AP-1; Transfection; Up-Regulation; Western Blotting; airway inflammation; cell growth; cell transformation; cell type; chemokine; chromatin immunoprecipitation; cofactor; cytokine; design; experience; expression vector; gene repression; glucocorticoid receptor beta; immunocytochemistry; improved; inhibitor/antagonist; knock-down; lung Carcinoma; mRNA Expression; monomer; novel; novel therapeutics; nuclear factors of activated T-cells; nucleocytoplasmic transport; overexpression; promoter; receptor; research study; respiratory smooth muscle; retinal rods; trafficking; transcription factor; vector

Although steroids are highly effective in the control of asthma, some patients fail to respond even to high doses. Steroid resistance is a veritable health challenge due to the absence of therapeutic alternatives and a financial burden as steroid-resistant patients account for more than 50% of asthma-related healthcare costs. The awardee's research focuses on studying steroid resistance in airway smooth muscle (ASM), a tissue that is relevant for lung diseases. The short-term goal of the studies was achieved during the one-year mentored phase of the K99 award with the identification of some of the molecular mechanisms responsible for mediating IRF-1-induced inhibition of steroid function in ASM cells. Interestingly, decreasing IRF-1 levels restores only partially steroid responsiveness in cytokine-treated cells suggesting that pathways, other than IRF-1, could be involved in cytokine-induced steroid resistance and are the aims of, the studies under the ROD phase. Thus, the main goal of ROO award is to investigate the contribution of other inflammatory molecules in cytokine-induced steroid resistance with the ultimate objective to generate novel potential therapeutic options to treat steroid-resistant asthmatics. The central hypothesis of the ROO proposal is novel and states that pro-asthmatic cytokines impair steroid function in ASM cells through the coordinated activation of two IRF-1-independent pathways: (i) glucocorticoid receptor beta isoform (GRP), a steroid receptor beta isoform that can act as an inhibitor of GC actions (will be addressed in Aim 1), and (ii) Serine/threonine protein phosphatase 5 (PP5), shown to act as an inhibitor of steroid actions in different cell lines (will be addressed in Aim 2). These latter two aims of the present proposal will rely on multiple complementary approaches such as siRNA technology, transfection of reporter vectors as well as overexpression of constitutively active or dominant negative proteins, co-immunoprecipitation and co-locaiization techniques, chromatin immunoprecipitation and gel shift assays. Subsequently, this award will dramatically strenghten the applicant's independence, will broaden his expertise in molecular pharmacology and cell biology, and importantly will yield high quality data necessary for his projected R01 application.

尽管类固醇在控制哮喘方面非常有效，但有些患者即使对高剂量也没有反应。由于缺乏治疗替代方案，类固醇耐药性是一个名副其实的健康挑战，而且类固醇耐药性患者的经济负担占哮喘相关医疗费用的 50% 以上。 获奖者的研究重点是研究气道平滑肌（ASM）的类固醇抵抗力，这是一种与肺部疾病相关的组织。这些研究的短期目标是在 K99 奖为期一年的指导阶段实现的，确定了一些负责介导 IRF-1 诱导的 ASM 细胞类固醇功能抑制的分子机制。有趣的是，降低 IRF-1 水平只能恢复细胞因子处理细胞中的部分类固醇反应性，这表明除 IRF-1 之外的途径可能参与细胞因子诱导的类固醇抵抗，并且是以下研究的目的 杆阶段。因此，ROO 奖的主要目标是研究其他炎症分子在细胞因子诱导的类固醇抵抗中的作用，最终目标是产生新的潜在治疗方案来治疗类固醇抵抗性哮喘。 ROO提案的中心假设是新颖的，并指出促哮喘细胞因子通过协调激活两个IRF-1独立途径来损害ASM细胞中的类固醇功能：（i）糖皮质激素受体β亚型（GRP），一种类固醇受体β亚型，可以充当GC作用的抑制剂（将在目标1中解决），以及（ii）丝氨酸/苏氨酸 蛋白磷酸酶 5 (PP5)，显示在不同细胞系中可作为类固醇作用的抑制剂（将在 Aim 中讨论） 2）。本提案的后两个目标将依赖于多种互补方法，例如 siRNA 技术、报告载体转染以及过表达 组成型活性或显性失活蛋白、免疫共沉淀和共定位 技术、染色质免疫沉淀和凝胶位移测定。随后，该奖项将极大地加强申请人的独立性，将拓宽他在分子药理学和细胞生物学方面的专业知识，更重要的是，将为其预计的 R01 应用提供所需的高质量数据。