Abstract
Glucocorticoids inhibit NF-kappaB signaling by interfering with the NF-kappaB transcription factor RelA. Previous studies have identified the DNA-binding domain (DBD) in the glucocorticoid receptor (GR) as the major region responsible for this repressive activity. Using GR mutants with chimeric DBDs the repressive function was found to be located in the C-terminal zinc finger. As predicted from these results the mineralocorticoid receptor that contains a C-terminal zinc finger identical to that of the GR was also able to repress RelA-dependent transcription. Mutation of a conserved arginine or a lysine in the second zinc finger of the GR DBD (Arg-488 or Lys-490 in the rat GR) abolished the ability of GR to inhibit RelA activity. In contrast, C-terminal zinc finger GR mutants with mutations in the dimerization box or mutations necessary for full transcriptional GR activity were still able to repress RelA-dependent transcription. In addition, we found that the steroid analog ZK98299 known to induce GR transrepression of AP-1 had no inhibitory effect on RelA activity. In summary, these results demonstrate that the inhibition of NF-kappaB by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR. Furthermore, the results from the use of mineralocorticoid receptor and anti-glucocorticoids suggest that the mechanisms for GR-mediated repression of NF-kappaB and AP-1 are different.
Highlights
Glucocorticoids inhibit NF-B signaling by interfering with the NF-B transcription factor RelA
To further determine if a particular region in the glucocorticoid receptor (GR) DNA-binding domain (DBD) is responsible for the functional interference with NF-B activity, GR mutants were created in which individual parts of the DBD were replaced by the corresponding regions of the TR DBD
To identify which subdomain in the GR DBD is responsible for the repression of RelA activity, we have used GR mutants in which various parts of the GR DBD have been replaced with the corresponding regions of the non-repressive TR DBD
Summary
We found that the steroid analog ZK98299 known to induce GR transrepression of AP-1 had no inhibitory effect on RelA activity These results demonstrate that the inhibition of NF-B by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR. One function of the DBD is to discriminate between different response elements, determining target genes to be activated [5, 6] This function is achieved by a few amino acids localized in the C-terminal part of the N-terminal zinc finger, the so-called the P box. DNA binding of the ligand-activated GR results in an increased rate of formation of transcriptionally competent pre-initiation complexes This is thought to be achieved by protein-protein interactions between the receptors and different components of the transcriptional machinery [8, 9]. We analyzed the ability of glucocorticoid antagonists to cause repression of NF-B activity
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