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， 发病率和死亡率。 白介素-4（IL-4）和白介素-13（IL- 13） 是T淋巴细胞释放的功能和结构相关的细胞因子 它们是哮喘的中枢介质。 功能相似性 是由于共享的受体亚基，IL-4 Roc。 尽管有许多 由于IL-4和IL- 13之间存在冗余， 在体内和体外，暗示IL- 13在气道中的作用更大， 气道重塑和气道高反应性，这是肺动脉高压的重要特征。 哮喘的发病机制。 此外，我们的初步数据表明， IL-4和IL-13的转录作用在 支气管平滑肌细胞（BSMC）和正常人肺成纤维细胞（NHLF）。 这些差异的机制知之甚少。 在当前 应用程序，我建议研究这些机制（S） 转录差异，并确定差异调节基因 这可能具有功能重要性。 在第一个目标中，我希望 鉴定了许多其他受IL-4和IL-13差异调节的基因。 这将通过研究气道细胞的基因表达谱来实现 在用一系列浓度的IL-4或 IL- 13使用含有14，000个基因的探针的寡核苷酸阵列。 在 第二和第三个目标，我建议确定具体机制， IL-4和IL-13通过以下方式调节不同的转录效应： 系统地检查IL-4和IL-1 3信号通路的组成部分 包括受体和信号传导介质。 这些研究将利用 免疫沉淀、免疫印迹、逆转录酶PCR和 竞争性受体结合测定。 最后，为了检验直接相关性， 这些差异调节的基因在体内，我将检查STAT-6 这些基因在STAT-6敲除小鼠中的依赖性， STAT-6在气道细胞中的重建。 这种方法将使我能够 直接评估已知IL-4和IL- 13的中心组分的重要性 体内的信号通路。 了解IL-4的潜在机制， IL-13在哮喘发病机制中的不同作用以及 识别可能具有功能相关性的关键基因可能最终导致 to better design设计of novel新treatment治疗of asthma哮喘. 连同拟议的 课程，期刊俱乐部和研讨会，并定期与我的会议 导师和咨询委员会，这个培训计划应该让我 发展成为一个成功的独立人士所需的技能和经验 调查员和大学教员。

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.