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

Lee, Kye Young; Kim, Yoon Seop; Ko, Mi Hye; Park, Jae Seok; Jee, Young Koo; Kim, Keun Youl; Kwak, Sahng June

Abstract

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine gene activated by NF-ÎºB. The goal of this study is to investigate the mechanism by which GR repress NF-ÎºB activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelial cell lines. Using IgGÎº-NF-ÎºB luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-Î±-induced NF-ÎºB activation and the overexpression of GR showed dose-dependent reduction of TNF-Î±-induced NF-ÎºB activity in both cell lines. However, DNA binding of NF-ÎºB induced by TNF-Î± in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-ÎºB complex induced by TNF-Î± Western blot showed that IÎºBÎ± degradation induced by TNF-Î± was not affected by dexamethasone and IÎºBÎº was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Ga14-p65TA1 or TA2 fusion protein expression system together with 5xGa14-luciferase vector. Co-transfection of GR with Ga14-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 or SRC-1 expression vectors. These results suggest that Gr-mediated transrepression of NF-ÎºB in lung epithelial cells is through competing for binding to limiting amount of transcriptional coactivators, CBP or SRC-1.