Gene Regulation by IL-4 and IL-13 in Airway Cells

描述(由申请人提供)： 哮喘是一种复杂的疾病，发病率不断增加，引起显著的 发病率和死亡率。白介素4和白介素13 T淋巴细胞释放的功能和结构相关的细胞因子 它们被认为是哮喘的中枢调节因子。功能相似性 均归因于共有的受体亚单位IL-4Roc。尽管有很多人 冗余，IL-4和IL-13之间存在显著的功能差异 在体内和体外，IL-13在呼吸道中的作用更大 重塑和气道高反应性，这是高血压的重要特征 哮喘的发病机制。此外，我们的初步数据表明 IL-4和IL-13转录效应的重要差异 支气管平滑肌细胞(BSMC)和正常人肺成纤维细胞(NHLF)。 造成这些差异的机制(S)还知之甚少。在当前 应用，我建议研究这些背后的机制(S) 转录差异和鉴定差异调控基因 这可能具有功能意义。在第一个目标中，我希望 确定许多受IL-4和IL-13差异调控的其他基因。 这将通过研究呼吸道细胞的基因表达谱来实现 在用一定浓度的IL-4或 IL-13使用包含14,000个基因探针的寡核苷酸阵列。在……里面 第二和第三个目标，我建议通过以下方式确定具体机制(S) IL-4和IL-13通过哪些途径调节差异转录效应 系统检测IL-4和IL-1 3信号通路的组成 包括受体和信号介体。这些研究将利用 免疫沉淀、免疫印迹、逆转录酶聚合酶链式反应 竞争性受体结合分析。最后，要检查直接关联 在体内这些差异调控的基因中，我将检查STAT-6 组织特异性STAT-6基因敲除小鼠对这些基因的依赖性 STAT-6在气道细胞中的重组。这种方法将使我能够 直接评估已知IL-4和IL-13的中心成分的重要性 体内的信号通路。理解IL-4和IL-4的作用机制(S) 白介素I 3‘S在哮喘发病机制中的不同贡献 识别可能与功能相关的关键基因最终可能导致 以更好地设计治疗哮喘的新疗法。连同建议的 课程作业、杂志俱乐部和研讨会，并定期与我的 导师和顾问委员会，这个培训计划应该能让我 培养成为成功的独立人士所需的技能和经验 调查员和大学教职员工。

DESCRIPTION (provided by applicant): Asthma is a complex disease with increasing incidence causing significant morbidity and mortality. Interleukin-4 (IL-4) and interleukin- 13 (IL- 13) are functionally and structurally related cytokines released by T lymphocytes that are implicated as central mediators of asthma. Functional similarities are attributed to the shared receptor subunit, IL-4Roc. Despite many redundancies, substantial functional differences exist between IL-4 and IL- 13 in vivo and in vitro, implicating a greater role for IL- 13 in airway remodeling and airway hyperreactivity, which are important features in the pathogenesis of asthma. Furthermore, our preliminary data demonstrate important differences in transcriptional effects of IL-4 and IL-13 in bronchial smooth muscle cells (BSMC) and normal human lung fibroblasts (NHLF). The mechanism(s) for these differences are poorly understood. In the current application, I propose to study the mechanism(s) underlying these transcriptional differences and to identify the differentially regulated genes which may be of functional significance. In the first aim, I expect to identify numerous additional genes differentially regulated by IL-4andIL-13. This will be accomplished by gene expression profiling of airway cells studied at multiple times after stimulation with a range of concentrations of IL-4 or IL- 13 using oligonucleotide arrays containing probes for 14,000 genes. In the second and third aims, I propose to identify the specific mechanism(s) by which IL-4 and IL-13 regulate differential transcriptional effects by systematically examining components of IL4 and IL-1 3 signaling pathways including receptors and signaling mediators. These studies will utilize immunoprecipitation, immunoblotting, reverse transcriptase PCR, and competitive receptor binding assays. Finally, to examine the direct relevance of these differentially regulated genes in vivo, I will examine STAT-6 dependence of these genes in STAT-6 knockout mice with tissue-specific reconstitution of STAT-6 in air-way cells. This approach will allow me to directly assess the importance of a central component of known IL-4 and IL- 13 signaling pathways in vivo. Understanding mechanism(s) underlying IL-4 and IL- I 3's differential contributions to asthma pathogenesis as well as identifying key genes which may be of functional relevance may ultimately lead to better design of novel treatments of asthma. Together with proposed coursework, journal clubs and seminars, and with regular meetings with my mentor and advisory committee, this training program should allow me to develop the skills and experience necessary to become a successful independent investigator and University faculty member.