CHARACTERIZATION OF MECHANISMS INVOLVED IN TRANSREPRESSION OF NF-KAPPA-B BY ACTIVATED GLUCOCORTICOID RECEPTORS

Glucocorticoids are potent immunosuppressants which work in part by in hibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhib ited by the synthetic glucocorticoid dexamethasone (DEX). In transfect ion experiments, DEX treatment in the presence of cotransfected glucoc orticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expressio n and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF- kappa B subunits p65 and p50. In addition, we demonstrate that the abi lity of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX a nd GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-k appa B at the levels of both DNA binding acid transcriptional activati on. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data revea l a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild type GR and DEX treat ment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required f or GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activit y is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expressi on and that cross-coupling of NF-kappa B and GR plays an important rol e in glucocorticoid-mediated repression of cytokine transcription.