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Nuclear factor kappa B (NF-kappa B) is an inducible transcription factor that positively regulates the expression of proimmune and proinflammatory genes, while glucocorticoids are potent suppressors of immune and inflammatory responses. NF-kappa B and the glucocorticoid receptor (GR) physically interact, resulting in repression of NF-kappa B transactivation. In transient cotransfection experiments, we demonstrate a dose-dependent, mutual antagonism between NF-kappa B and GR. Functional dissection of the NF-kappa B p50 and p65 subunits and deletion mutants of GR indicate that the GR antagonism is specific to the p65 subunit of NF-kappa B heterodimer, whereas multiple domains of GR are essential to repress p65-mediated transactivation. Despite its repression of GR transactivation, p65 failed to block the transrepressive GR homologous down-regulation function. We also demonstrate that negative interactions between p65 and GR are not selective for GR, but also occur between NF-kappa B and androgen, progesterone B, and estrogen receptors. However, although each of these members of the steroid hormone receptor family is repressed by NF-kappa B, only GR effectively inhibits p65 transactivation. Further, in cotransfections using a chimeric estrogen-GR, the presence of the GR DNA-binding domain is insufficient to confer mutual antagonism to the p65-estrogen receptor interaction. Selectivity of p65 repression for each steroid receptor is demonstrated by I kappa B rescue from NF-kappa B-mediated inhibition. Together these data suggest that NF-kappa B p65 physically interacts with multiple steroid hormone receptors, and this interaction is sufficient to transrepress each steroid receptor. Further, the NF-kappa B status of a cell has the potential to significantly alter multiple steroid signaling pathways within that cell.

Nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) are transcription factors with opposing actions in the modulation of immune/inflammatory responses. NF-kappaB induces the expression of proinflammatory genes, while GR suppresses immune function in part by suppressing expression of the same genes. Previously, we demonstrated that physiological antagonism between NF-kappaB and GR is due to a mutual transcriptional antagonism that requires the p65 subunit of NF-kappaB and multiple domains of GR (1). To elucidate the mechanism(s) of NF-kappaB p65 and GR transcriptional antagonism, we analyzed the interactions of wild-type p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65 which lacks a transactivation domain) with GR. We show that p65RHD blocks p65-mediated transactivation, yet does not block the repression of GR transactivation by p65, indicating that transcriptional activity by p65 is not required to repress GR function. Both p65 and p65 RHD physically interact with GR, but only intact p65 represses GR-mediated signaling, implicating the p65 transactivation domain in the transcriptional repression of GR. To further characterize p65-GR interactions, we examined the role of the transcriptional co-integrator CREB binding protein (CBP) in their mutual antagonism. GR-mediated repression of p65 transactivation and p65-mediated repression of GR transactivation, as well as the physical interaction between NF-kappaB and GR, are enhanced by CBP. GR bound to the antagonist RU 486, although transcriptionally inactive, retains the ability to repress p65 transactivation. However, CBP does not physically interact with antagonist-bound GR and does not enhance its repressive effect on p65. These data suggest that CBP functions as an integrator of p65/GR physical interaction, rather than as a limiting cofactor for which p65 and GR compete.

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A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed.

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