Cytosolic glucocorticoid receptor interaction with nuclear factor-kappa B proteins in rat liver cells.
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.
Widén
Christina
C
Department of Medical Nutrition, Karolinska Institute, NOVUM, S-141 86 Huddinge, Sweden.
Gustafsson
Jan-Ake
JA
Wikström
Ann-Charlotte
AC
D000315:Adrenalectomy; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002846:Chromatography, Affinity; D003600:Cytosol / Q000378:metabolism; D005260:Female; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D008954:Models, Biological; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D011965:Receptors, Glucocorticoid; D014409:Tumor Necrosis Factor-alpha; D014222:Triamcinolone Acetonide