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 研究表明，气道上皮细胞是三个重要来源， 主要的嗜酸性粒细胞活性趋化因子，嗜酸性粒细胞趋化因子，MCP-4和RANTES， 糖皮质激素（GC）抑制这些趋化因子的产生， 这些趋化因子的产生和对GC的敏感性不同。探讨 根据这些观察，PI提出了3个具体目标：1）确定 IL-4对Eotaxin和RANTES差异调节的分子基础 在人气道上皮细胞中的表达，特别是转录 以及这些趋化因子表达的转录后调节。的 计划的工作是基于观察到在报告基因构建体中，IL-4 上调嗜酸性粒细胞趋化因子启动子驱动的结构，这种作用似乎是 依赖于STAT 6，同时抑制RANTES启动子活性。转染 计划用过表达和抑制的STAT 6的BEAS-2B细胞。 转录后途径将通过确定以下因素的影响来检查： IL-4对Eotaxin mRNA稳定性的影响。2)研究分子机制， 糖皮质激素抑制嗜酸细胞活化趋化因子和RANTES的上皮表达。基于 GC加速嗜酸细胞趋化因子和MCP-4 mRNA衰变的观察， 而不是RANTES，将探讨转录后机制，包括 刺激特异性，mRNA不稳定的时间过程和持续时间，以及 需要蛋白质合成才能产生这些效应。PI假设， 在嗜酸细胞活化趋化因子的3'非翻译区中富含Au的元素，而不是RANTES， 参与了GC诱导的mRNA不稳定。BEAS-2B细胞将在 用瞬时感染的含有 瞬时诱导型c-fos/B-球蛋白基因融合质粒， 嗜酸细胞活化趋化因子或RANTES的3 '-UTR。最后，GC调节的机制 转录将基于GC抑制 嗜酸性粒细胞趋化因子和RANTES启动子驱动的构建体。GCs对STAT 6的影响 表达和功能将使用缺失和突变 分析报告基因构建体。3）表征表达和 CCR-3对上皮细胞的功能，包括确定 调节上皮细胞中的CCR 3表达; CCR 3的结合，信号传导特性， 以及CCR 3在气道中的表达 正常和过敏受试者的上皮细胞。

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.