Mechanism of inflammation-induced steroid resistance

描述（由申请人提供）： 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。候选人目前在宾夕法尼亚大学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。目前和在该奖项的指导阶段（K99）进行的研究的短期目标是描述抑制类固醇疗效降低的分子机制。这项研究将在本奖项的独立阶段（R 00）进行，最终目标是开发新的治疗选择来治疗类固醇抵抗性哮喘。候选人的长期目标是建立自己的独立研究计划，并提交R 01申请。该提议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三种途径损害ASM细胞中的类固醇功能：（i）GR β，一种类固醇受体β同种型，可作为GC作用的抑制剂（将在目标1中解决），（ii）IRF-1，调节许多炎症基因的转录因子（将在目标2中论述），和（iii）丝氨酸/苏氨酸蛋白磷酸酶5（PP 5），其显示在不同细胞系中作为类固醇作用的抑制剂（将在目标3中论述）。本发明的所有三个目的将依赖于多种互补方法，如siRNA技术、报告载体的转染以及组成型活性或显性阴性蛋白的过表达、共免疫沉淀和共定位技术、染色质免疫沉淀和凝胶位移测定。随后，该奖项将大大提高候选人的技术技能，并将扩大他在分子药理学和细胞生物学方面的专业知识。我们相信，我们的建议是相关的公共卫生，因为提高知识的因素，导致类固醇耐药将有助于设计新的治疗药物或替代品，以治疗类固醇耐药哮喘。

DESCRIPTION (provided by applicant): 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 candidate's current research at the University of Pennsylvania focuses in studying steroid resistance in airway smooth muscle (ASM), a tissue that is relevant for lung diseases. The short term goal of the studies performed currently and during the mentored phase of this award (K99) is to delineate the molecular mechanisms that underlay the diminished efficacy of steroids. This study will be pursued during the independent phase of this award (R00) with the ultimate objective to develop new therapeutic options to treat steroid-resistant asthmatics. The candidate's long term goal is to establish his own independent research program and submit an R01 application. The central hypothesis of this proposal is novel and states that pro-asthmatic cytokines impair steroid function in ASM cells through the coordinated activation of three pathways: (i) GR beta, a steroid receptor beta isoform that can act as an inhibitor of GC actions (will be addressed in Aim 1), (ii) IRF-1, a transcription factor that regulates many inflammatory genes (will be addressed in Aim 2), and (iii) Serine/threonine protein phosphatase 5 (PP5), shown to act as an inhibitor of steroid actions in different cell lines (will be addressed in Aim 3). All three Aims of the present proposal will rely on multiple complementary approaches such as siRNA technology, transfection of reporter vectors as well as over-expression of constitutively active or dominant negative proteins, co-immunoprecipitation and co-localization techniques, chromatin immunoprecipitation and gel shift assays. Subsequently, this award will dramatically enhance the candidate's technical skills and will broaden his expertise in molecular pharmacology and cell biology. We believe that our proposal is relevant to public health since improving the knowledge about the factors that lead to steroid resistance will help design new therapeutic agents or alternatives to treat steroid-resistant asthmatics.