Cytosolic glucocorticoid receptor interaction with nuclear factor-kappaB proteins in rat liver cells

Abstract

The glucocorticoid receptor (GR) acts as an anti-inflammatory factor. To a large extent, this activity is exerted by the interference of pro-inflammatory nuclear factor kappa B (NF-kappa B) activity. In their respective inactive forms, both GR and NF-kappa B reside in the cytoplasm and translocate to the nucleus on relevant stimulation. Previously, p65, a component of the NF-kappa B complex, and GR have been shown to interact physically in vitro, and the interaction is assumed to take place in the nucleus of cells [McKay and Cidlowski (1999) Endocrine Rev. 20, 435-459]. We have studied the interaction between GR and NF-kappa B using in vivo -like conditions. Using immunoaffinity chromatography or immunoprecipitation, combined with Western blotting, we observed that, with endogenous protein levels in cytosolic extracts of rat liver and of H4-II-E-C3 hepatoma cells and in contrast with the current belief, p65, p50 and inhibitory kappa B alpha complex interact with GR, even in the absence of glucocorticoid or an inflammatory signal. The interaction between non-liganded/non-activated GR and p65/p50 has also been verified by both p65 and p50 co-immunoprecipitations. Intracellular localization studies, using Western blotting, revealed that glucocorticoids can decrease tumour necrosis factor alpha (TNFalpha)-induced nuclear entry of p65, whereas glucocorticoid-induced GR translocation was much less affected by TNFalpha. We were also able to demonstrate a nuclear interaction of GR and p65 and p50 using in vivo -like protein concentrations. Furthermore, nuclear GR interaction with heat-shock protein 90 was enhanced distinctly by TNFalpha treatment. In conclusion, our studies suggest a strong interconnectivity between the NF-kappa B and GR-signalling pathways where also, somewhat unexpectedly, a physical interaction in the cytosol constitutes an integral part of GR-NF-kappa B cross-talk.

Glucocorticoid action and novel mechanisms of steroid resistance: role of glucocorticoid receptor-interacting proteins for glucocorticoid responsiveness

“…Contrary to current beliefs, we found that p65, p50 and I-B were co-immunopurifying with cytosolic, nonliganded, non-activated GR (Widén et al 2003). The current understanding of GR and NF-B interaction favours the model that glucocorticoids induce the GR-NF-B interaction and that this interaction would be one of two possible mechanisms for how glucocorticoids counteract inflammation mediated by NF-B.…”

“…Several of the interacting proteins that we have studied have, in turn, been reported to interact with other proteins important for signal transduction. Based on this information we have tested whether the interaction with GR also allows for interaction with previously described secondary interaction partners (Widén et al 2000, Widén et al 2003 and have sometimes found this to be the case (see below).…”

Cross-talk between nuclear receptors and nuclear factor κB

“…Surprisingly, these interactions can occur even in the absence of glucocorticoids or of an inflammatory signal (Wide´n et al, 2003) (Figure 1). Furthermore, the NF-kB-IkB complex had already been found to shuttle between the cytoplasm and the nucleus in the absence of signal (Johnson et al, 1999;Carlotti et al, 2000;Ghosh and Karin, 2002).…”

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

“…Glutathione S-transferase (GST) and GST-RHD fusion proteins were expressed in Escherichia coli strain DH5␣ (Invitrogen) and were purified as described (29). Briefly, a 50-ml culture of the expression vector was stimulated with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside (Sigma) and grown for 3 h. The bacteria were then pelleted and resuspended in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.01% Nonidet P-40) supplemented with protease inhibitors ("Complete"; Roche Applied Science).…”