Mechanisms of Steroid Resistance in Airway Smooth Muscle Cells

虽然类固醇在控制哮喘方面非常有效，但一些患者即使对大剂量也没有反应。类固醇耐药是一个名副其实的健康挑战，因为缺乏治疗替代方案，而且由于类固醇耐药患者占哮喘相关医疗费用的50%以上，这是一种经济负担。 获奖者的研究重点是研究呼吸道平滑肌(ASM)的类固醇抵抗，ASM是一种与肺部疾病相关的组织。这项研究的短期目标是在K99奖为期一年的指导阶段实现的，确定了一些负责调节IRF-1诱导的ASM细胞类固醇功能抑制的分子机制。有趣的是，在细胞因子处理的细胞中，降低IRF-1水平仅能恢复部分类固醇反应，这表明除IRF-1外，其他途径可能参与细胞因子诱导的类固醇耐药，这也是以下研究的目的 杆状阶段。因此，ROO奖的主要目的是调查其他炎症分子在细胞因子诱导的类固醇耐药中的作用，最终目的是寻找新的潜在治疗方案来治疗类固醇耐药哮喘患者。Roo提案的中心假设是新颖的，它指出，促哮喘细胞因子通过两条IRF-1非依赖通路的协调激活来损害ASM细胞的类固醇功能：(I)糖皮质激素受体β亚型(GRP)，一种类固醇受体β亚型，可以作为GC作用的抑制因子(将在Aim 1中讨论)，以及(Ii)丝氨酸/苏氨酸蛋白磷酸酶5(PP5)，在不同的细胞系中被证明是类固醇作用的抑制因子(将在Aim中解决 2)。本提议的后两个目标将依赖于多种互补的方法，如siRNA技术、报告载体的转染以及过度表达 组成活性或显性负性蛋白、免疫共沉淀和共定位 技术、染色质免疫沉淀和凝胶移位分析。随后，这一奖项将极大地加强申请人的独立性，拓宽他在分子药理学和细胞生物学方面的专业知识，重要的是将产生他计划的R01申请所需的高质量数据。

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.