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...

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-B and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GR␣), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GR␤), which does not bind known ligands and attenuates GR␣ action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-B DNA binding site 5 to the hGR promoter that leads to a 1.5-fold increase in GR␣ mRNA and a 2.0-fold increase in GR␤ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-␣ treatment disproportionately increased the steady-state levels of the GR␤ protein isoform over GR␣, making GR␤ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-␣ or IL-1. The increase in GR␤ protein expression correlated with the development of glucocorticoid resistance.

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGR␤) that differs from the wild-type receptor (hGR␣) only at the carboxyl terminus. Previously we have shown that hGR␤ inhibits the transcriptional activity of hGR␣, which is consistent with reports of elevated hGR␤ expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGR␤ in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGR␤ inhibits hGR␣-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGR␤. hGR␤ also inhibits hGR␣-mediated repression of an NF-B-responsive promoter but does not interfere with homologous down-regulation of hGR␣. We show that hGR␤ can associate with the heat shock protein hsp90 although with lower affinity than hGR␣. In addition, hGR␤ binds GRE-containing DNA with a greater capacity than hGR␣ in the absence of glucocorticoids. Glucocorticoid treatment enhances hGR␣, but not hGR␤, binding to DNA. Moreover, we demonstrate that hGR␣ and hGR␤ can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGR␤ resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGR␣-hGR␤ heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGR␤.

No abstract