Dexamethasone-Mediated Repression of MUC5AC Gene Expression in Human Lung Epithelial Cells

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-κB 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

IL-1β induction of MUC5AC gene expression is mediated by CREB and NF-κB and repressed by dexamethasone

Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the proinflammatory cytokine interleukin-1 β (IL-1β) via activation of cAMP response element-binding protein (CREB) in the NCI-H292 cancer cell line and nuclear factor-κB (NF-κB) in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NF-κB subunits to the IL-1β-induced upregulation of MUC5AC. Data show that ligand binding of IL-1β to the IL-1β receptor is required to increase MUC5AC mRNA abundance. Chromatin immunoprecipitation analyses show direct binding of CREB to the previously identified cAMP response element site and binding of p65 and p50 subunits to a novel NF-κB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NF-κB site in the distal promoter. P50 binds to both NF-κB sites at 1 h following IL-1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus IL-1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL-1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to a honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases