Inhibitory mechanism on NF-kB transactivation by dexamethasone in pulmonary epithelial cells

Abstract

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-. The goal of this study is to investigate the mechanism by which GR repress NF- activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig-NF- luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF--induced NF- activation and the overexpression of GR showed dose-dependent reduction of TNF--induced NF- activity in both cell lines. However, DNA binding of NF- induced by TNF- in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF- complex induced by Western blot showed that degradation induced by TNF- was not affected by dexamethasone and was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF- in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.