Glucocorticoid Ligands Specify Different Interactions with NF-κB by Allosteric Effects on the Glucocorticoid Receptor DNA Binding Domain

Abstract

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-B function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-B through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-B and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-B with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-B transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-B. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-B, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-Bresponsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligandinduced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.

Anti-inflammatory functions of glucocorticoid-induced genes

“…In HeLa cells dexamethasone promoted recruitment of GR to the CXCL8 promoter and inhibited TNF-induced expression of CXCL8. In contrast the antagonistic ligand RU486 did neither (Garside et al, 2004), suggesting a causal link between GR recruitment and gene suppression. However, in at least one instance recruitment of GR to an inflammatory promoter occurred in the absence of transrepression (Luecke and Yamamoto, 2005;Nissen and Yamamoto, 2000).…”

“…Such recruitment has been demonstrated in very few examples, one of which is CXCL8 (Garside et al, 2004;Luecke and Yamamoto, 2005;Nissen and Yamamoto, 2000). In HeLa cells dexamethasone promoted recruitment of GR to the CXCL8 promoter and inhibited TNF-induced expression of CXCL8.…”

Glucocorticoid receptor overexpression exerts an antisurvival effect on human small cell lung cancer cells

“…In addition, A549 cells transfected with Gc reporter genes showed Gc responses, but in contrast, neither the HEK cells nor the SCLC cell lines show any effect (data not shown, Ray et al, 1996;Garside et al, 2004).…”

“…Reporter assays HEK cells were transfected as described (Ray et al, 1994;Garside et al, 2004) with 10 mg TAT3-luciferase reporter construct and 5 mg pcDNA3-GR or control pcDNA3 using FuGene 6 (Roche). In addition, the cells were transfected with 2 mg pRLCMV to control for transfection efficiency.…”

Glomerulonephritis, Pathogenetic Mechanisms and Therapeutic Options: An Overview

“…Considering these several side effects, steroids should be administered at the lowest dose for the shortest time possible, considering, however, that a suspension with an early recurrence of the disease can often occur. In the future it is hoped to use elective glucocorticoids modulating specific transcriptional effects [23].…”