Chen Yajun, Nickola Tracey J, DiFronzo Nancy L, Colberg-Poley Anamaris M, Rose Mary C
Center for Genetic Medicine Research, Children's Research Institute, Washington, DC 20010, USA.
Am J Respir Cell Mol Biol. 2006 Mar;34(3):338-47. doi: 10.1165/rcmb.2005-0176OC. Epub 2005 Oct 20.
Glucocorticoids regulate gene expression via binding of the ligand-activated glucocorticoid receptor (GR) to glucocorticoid-responsive elements (GRE) in target gene promoters. The MUC5AC gene, which encodes the protein backbone of an abundant secreted airway mucin, has several putative GRE cis-elements in its 5' sequence. Mechanism(s) whereby glucocorticoids regulate mucin genes have not previously been described. In this study, the glucocorticoid dexamethasone (Dex) decreased MUC5AC mRNA abundance in A549 and NCI-H292 cell lines and primary differentiated normal bronchial epithelial cells by 50-80%, suggesting a common mechanism of MUC5AC gene repression in human lung epithelial cells. Kinetic analyses showed that MUC5AC mRNA was not significantly decreased until 6 h after Dex exposure, and that nuclear translocation of GR was biphasic, suggesting that Dex-mediated cis-repression of MUC5AC gene expression was a delayed response of GR translocation. Transfection analyses demonstrated that Dex transcriptionally repressed the MUC5AC promoter. Electrophoretic mobility shift assays with wild-type and mutant oligonucleotide probes showed that GR bound to two GRE cis-sites (nucleotides -930 to -912 and -369 to -351) in the MUC5AC promoter. Analyses of mutated MUC5AC promoter constructs demonstrated that NF-kappaB cis-sites were not involved in Dex-mediated repression of MUC5AC. Dex did not alter mRNA stability of MUC5AC transcripts. Taken together, the data indicate that Dex transcriptionally mediates repression of MUC5AC gene expression in human lung epithelial cells at quiescent states after binding of GR to one or more GRE cis-elements in the MUC5AC promoter.
糖皮质激素通过配体激活的糖皮质激素受体（GR）与靶基因启动子中的糖皮质激素反应元件（GRE）结合来调节基因表达。编码丰富分泌性气道粘蛋白蛋白质骨架的MUC5AC基因，在其5'序列中有几个假定的GRE顺式元件。此前尚未描述糖皮质激素调节粘蛋白基因的机制。在本研究中，糖皮质激素地塞米松（Dex）使A549和NCI-H292细胞系以及原代分化的正常支气管上皮细胞中的MUC5AC mRNA丰度降低了50-80%，提示人肺上皮细胞中存在MUC5AC基因抑制的共同机制。动力学分析表明，直到Dex暴露6小时后MUC5AC mRNA才显著降低，并且GR的核转位是双相的，提示Dex介导的MUC5AC基因表达顺式抑制是GR转位的延迟反应。转染分析表明Dex转录抑制MUC5AC启动子。用野生型和突变型寡核苷酸探针进行的电泳迁移率变动分析表明，GR与MUC5AC启动子中的两个GRE顺式位点（核苷酸-930至-912和-369至-351）结合。对突变的MUC5AC启动子构建体的分析表明，NF-κB顺式位点不参与Dex介导的MUC5AC抑制。Dex未改变MUC5AC转录本的mRNA稳定性。综上所述，数据表明，在GR与MUC5AC启动子中的一个或多个GRE顺式元件结合后，Dex在静止状态下转录介导人肺上皮细胞中MUC5AC基因表达的抑制。
