Characterization of Mechanisms Involved in Transrepression of NF-κB by Activated Glucocorticoid Receptors

Abstract

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment 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 for GR repression of AP-1. Surprisingly, while both AP-1 and NF-B can be inhibited by activated GR, synergistic NF-B/AP-1 activity is largely unaffected. These data suggest that NF-B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.Glucocorticoids have long been used as effective immunosuppressive agents in the treatment of conditions involving T-cell-or cytokine-mediated tissue damage. These steroids have been shown to block inflammation, suppress immune system activation, and act as growth-inhibitory agents both in vivo and in vitro (23). Surprisingly, despite the lengthy history of the use of glucocorticoids as therapeutic agents, the mechanism by which they perform these functions is largely unknown.Studies of the effect of glucocorticoid administration on the immune system have resulted in a number of important observations. Glucocorticoids induce a rapid redistribution of lymphocytes from the circulation to other lymphoid compartments (23). In addition, glucocorticoids potently suppress lymphocyte accessory function, the clonal expansion of T lymphocytes, and the secretion of cytokines (23, 73). Interestingly, cytotoxic Tlymphocyte clones provided with exogenous interleukin 2 (IL-2) are able to proliferate in response to mitogenic stimulation in the presence of glucocorticoids (28). These data suggest that the block of cytokine secretion plays an important role in glucocorticoid-mediated immunosuppression. Indeed, glucocorticoid administration represses the de novo transcription of a number of cytokine gene...

Therapeutic effects of acetylsalicylic acid in giant cell arteritis

“…Steroidmediated repression of NF-B-dependent genes has also been demonstrated in fibroblasts, although I B protein levels remained unchanged. It has been suggested that direct protein-protein interactions between the activated glucocorticoid receptor and the p65 subunit of NF-B can mediate suppression of NF-Bdependent transcriptional activity (43)(44)(45).…”

Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells

“…Another way in which glucocorticoids decrease transcription is by binding directly to positive regulators of transcription, such as the transcription factors AP-1 [37] and nuclear factor κB (NF-κB) [38], thus preventing their interaction with promoter elements and\or translocation to the nucleus. Glucocorticoids can also inhibit transcription of genes driven by NF-κB, by increasing the levels and binding of an inhibitor to NF-κB, inhibitory κ Bα [39].…”

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia

“…This lower transcription appears to be dependent on a reduced activity of NF-B, a transcription factor playing a central role in the activation of TNF gene expression in LPS-activated macrophages [34][35][36]. Inhibition of NF-B activity by GCs is considered to be the consequence of a transrepressive effect of the activated glucocorticoid receptor [34] and/or of the increased transcription of the inhibitor I-B gene [35,37,38]. Although mouse IL-10 promoter contains NF-B-like binding sites [39], our results might suggest that NF-B does not play a central role in the activation of IL-10 gene transcription.…”