Steroid Repression of MUC5AC Mucin Gene Expression in Human Lung Epithelial Cells
Open AccessOverproduction of secretory mucins contributes to morbidity/mortality in lung diseases. Inflammatory mediators upregulate mucin genes, but few drugs directly repress mucin gene expression. IL1β has been shown to upregulate the MUC5AC mucin gene via the transcription factors NFκB and CREB. The glucocorticoid dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. Glucocorticoids are commonly used to treat inflammatory diseases but have adverse long-term side-effects due to prolonged GRE-mediated gene regulation. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcription. Thus, they have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases, such as cystic fibrosis (CF). We analyzed VBP15 efficacy as an anti-mucin agent in normal and CF-diseased cells by analyzing the transcription factor activities and promoter binding associated with VBP15-induced MUC5AC transcriptional repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex. Repression continued in the presence of IL1β in both A549 lung cancer cells, and primary differentiated Human Bronchial Epithelial (HBE) cells, demonstrating therapeutic potential. Similar to Dex, VBP15 induced nuclear translocation of GR and repression was abrogated in the presence of the GR antagonist, RU486, indicating GR is necessary for VBP15-induced repression of MUC5AC. Similarly, inhibitors of NFκB and CREB activity reduced baseline expression of MUC5AC indicating a potential target for inhibition of MUC5AC gene transcription. ChIP analysis demonstrated binding of Dex-activated GR and p65 in the Mucin Regulatory Domain (MRD), a composite GRE site of the MUC5AC promoter, but not in cells exposed to VBP15. Interactions of VBP15-activated GR with the p65 subunit of NFκB showed a trend toward increased interaction in the nucleus while increased cytoplasmic interactions of Dex-activated GR with the p65 subunit of NFκB were observed. CF HBE cells that have increased MUC5AC mRNA abundance compared to non-CF HBE cells, showed a significant reduction of MUC5AC mRNA abundance when exposed to VBP15 or Dex. The CF HBE cells lacked the IL1β-induced increase in MUC5AC mRNA abundance observed in non-CF HBE cells consistent with previous studies in A549 and normal HBE cells. There was only a trend toward increased levels of IL1β in the CF HBE apical secretions as compared to the non-CF cells but a significant decrease in IL-1R levels demonstrating altered ability of CF cells to respond to an inflammatory stimulus. In conclusion, VBP15 represents a possible anti-mucin agent in inflammatory lung diseases with reduction of MUC5AC mRNA abundance by a distinct mechanism as compared to Dex.
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