REGULATION OF CC CHEMOKINES IN HUMAN AIRWAY EPITHELIUM

人类气道上皮中 CC 趋化因子的调节

• 批准号: 6632166
• 负责人: CRISTIANA STELLATO
• 金额: $ 28.61万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6632166
• 关键词: chemokine; cytokine receptors; eosinophil; gene induction /repression; glucocorticoids; hormone regulation /control mechanism; human tissue; immunoregulation; interleukin 4; respiratory epithelium

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.

描述（改编自申请人的摘要）：以前的工作已经 证明气道上皮细胞是三种重要的来源 主要的嗜酸性粒细胞活性趋化因子、eotaxin、MCP-4 和 RANTES， 糖皮质激素（GC）抑制这些趋化因子的产生，并且两者 这些趋化因子的产生和对 GC 的敏感性不同。探索 根据这些观察结果，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。这 计划的工作基于以下观察：在报告基因构建体中，IL-4 上调eotaxin启动子驱动的构建体，这种效应似乎是 依赖于 STAT6，同时抑制 RANTES 启动子活性。转染 计划使用过度表达和抑制 STAT6 的 BEAS-2B 细胞。 将通过确定以下因素的影响来检查转录后途径： IL-4 对嗜酸细胞趋化因子 mRNA 稳定性的影响。 2）研究其分子机制 糖皮质激素抑制eotaxin和RANTES的上皮表达。基于 观察到 GC 加速嗜酸细胞趋化因子和 MCP-4 mRNA 的衰减， 但 RANTES 除外，我们将探讨转录后机制，包括 刺激特异性、mRNA 不稳定的时间过程和持续时间，以及 这些效应的发生需要蛋白质合成。 PI 假设 嗜酸细胞活化趋化因子 3' 非翻译区（但 RANTES 除外）中的所有丰富元素是 参与GC诱导的mRNA不稳定。 BEAS-2B 细胞将 用瞬时感染的报告构建体转染，其中包含 瞬时诱导型 c-fos/B-球蛋白基因融合质粒，含有 eotaxin 或 RANTES 的 3'-UTR。最后，GC 的调节机制 转录将根据 GC 抑制的观察结果来确定 eotaxin 和 RANTES 启动子驱动的构建体。 GC对STAT6的影响 将使用缺失和突变来确定表达和功能 报告构建基因的分析。3) 表征表达和 CCR-3 对上皮细胞的功能，包括因素的确定 调节上皮细胞中 CCR3 的表达；结合、信号传导特性、 和上皮细胞CCR3的功能；以及CCR3在体内气道中的表达 正常和过敏受试者的上皮细胞。