REGULATION OF CC CHEMOKINES IN HUMAN AIRWAY EPITHELIUM

描述(改编自申请人的摘要)：以前的工作有 证明了呼吸道上皮细胞是三种细胞的重要来源 主要嗜酸性粒细胞活性趋化因子、嗜酸性粒细胞趋化因子、单核细胞趋化蛋白-4和RANTES 糖皮质激素(GC)抑制这些趋化因子的产生，而且两者 这些趋化因子的产生和对GC的敏感性不同。去探索 这些观察，PI提出了3个具体目标：1)识别 IL-4对嗜酸性粒细胞趋化因子和RANTES差异调控的分子基础 在人呼吸道上皮细胞中的表达，特别是转录 以及对这些趋化因子表达的转录后调控。这个 计划的工作是基于观察到在报告基因结构中，IL-4 上调嗜酸性粒细胞趋化因子启动子驱动的结构，这一效应似乎是 依赖于STAT6，同时抑制RANTES启动子的活性。转染腺病毒载体 具有过表达和抑制STAT6的BEAS-2B细胞是计划的。 转录后通路将通过确定 IL-4对嗜酸性粒细胞趋化蛋白mRNA稳定性的影响。2)研究其分子机制。 糖皮质激素抑制上皮细胞嗜酸性粒细胞趋化因子和RANTES的表达。基于 观察到GCs加速嗜酸性粒细胞趋化因子和单核细胞趋化蛋白-4的mRNA的降解， 但不是RANTES，将探索转录后机制，包括 刺激特异性、mRNA失稳的时间进程和持续时间，以及 这些效应的发生需要蛋白质的合成。私家侦探假设 嗜酸粒细胞趋化蛋白3‘非翻译区的富Au元素 参与了这种GC诱导的mRNA失稳。BEAS-2B细胞将是 用瞬时感染的报告基因构建的含有 瞬时可诱导的c-fos/B-球蛋白基因融合表达载体 嗜酸性粒细胞趋化因子或RANTES的3‘非编码区。最后，地方政府监管的机制(S) 转录将根据观察到的GC抑制 嗜酸性粒细胞趋化因子和RANTES启动子驱动的构建。GCs对STAT6的影响 表达和功能将通过缺失和突变来确定 对报告基因的构建进行分析。3)为了表征表达和 CCR-3对上皮细胞的作用，包括因子的测定 CCR3在上皮细胞中的表达；结合，信号特性， 和上皮性CCR3的功能，以及CCR3在呼吸道中的活体表达 正常人和过敏者的上皮细胞。

DESCRIPTION (Adapted from the applicant's abstract): Previous work has demonstrated that airway epithelial cells are an important source of three major eosinophil-active chemokines, eotaxin, MCP-4, and RANTES, that glucocorticoids (GCs) inhibit production of these chemokines, and that both production and sensitivity to GCs differ among these chemokines. To explore these observations, the PI has proposed 3 specific aims: 1) To identify the molecular basis of the differential regulation by IL-4 of eotaxin and RANTES expression in human airway epithelial cells, specifically the transcriptional and post-transcriptional regulation of the expression of these chemokines. The work planned is based on the observation that in reporter gene constructs, IL-4 upregulates eotaxin promoter-driven constructs, an effect which appears to be dependent on STAT6, while it inhibits RANTES promoter activity. Transfection of BEAS-2B cells with overexpressed and repressed STAT6 is planned. Post-transcriptional pathways will be examined by determining the effect of IL-4 on eotaxin mRNA stability. 2) To study the molecular mechanisms by which glucocorticoids inhibit epithelial expression of eotaxin and RANTES. Based on the observation that GCs accelerate the decay of mRNA for eotaxin and MCP-4, but not RANTES, post-transcriptional mechanisms will be explored including the stimulus-specificity, time course and duration of mRNA destabilization, and the need for protein synthesis for these effects to occur. The PI hypothesizes that AU rich-elements in the 3' untranslated region of eotaxin but not RANTES are involved in this GC-induced mRNA destabilization. BEAS-2B cells will be transfected with a transiently infected with reporter constructs containing a transiently inducible c-fos/B-globulin gene fusion plasmid containing the 3'-UTR of eotaxin or RANTES. Finally, the mechanism(s) by which GCs regulate transcription will be determined based on the observation that GCs suppress both eotaxin and RANTES promoter-driven constructs. The effect of GCs on STAT6 expression and function will be determined using deletional and mutational analyses of reporter construct genes.3) To characterize the expression and function of CCR-3 on epithelial cells, including a determination of the factors modulating CCR3 expression in epithelium; the binding, signaling properties, and function of epithelial CCR3; and the expression of CCR3 in vivo in airway epithelial cells in normal and allergic subjects.