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，但有些患者甚至对高剂量也没有反应。由于缺乏治疗替代品和经济负担，类固醇耐药是一个名副其实的健康挑战，因为类固醇耐药患者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）中的类固醇抵抗，这是一种与肺部疾病相关的组织。这些研究的短期目标在K99奖的一年指导阶段实现，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水平仅部分恢复了马槟榔处理的细胞中的类固醇反应性，这表明除IRF-1以外的途径可能参与马槟榔诱导的类固醇抗性，并且是以下研究的目的： ROD阶段。因此，ROO奖的主要目标是研究其他炎症分子在尼古丁诱导的类固醇抵抗中的作用，最终目标是产生治疗类固醇抵抗性哮喘的新的潜在治疗选择。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非依赖性途径来损害ASM细胞中的类固醇功能：（i）糖皮质激素受体β同种型（GRP），一种类固醇受体β同种型，其可充当GC作用的抑制剂，（ii）丝氨酸/苏氨酸蛋白磷酸酶5（PP 5），其显示在不同细胞系中充当类固醇作用的抑制剂（将在Aim 1中论述 2）。本发明的后两个目的将依赖于多种互补方法，例如siRNA技术、报告载体的转染以及过表达 组成型活性或显性阴性蛋白质，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，该奖项将大大加强申请人的独立性，将扩大他在分子药理学和细胞生物学方面的专业知识，重要的是将为他的R 01申请提供高质量的数据。

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.