Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor.
Transition metal oxyanions, such as molybdate, tungstate and vandadate, have been shown to prevent in vitro hormone-induced activation of the glucocorticoid receptor (GR) by blocking dissociation of the GR/heat shock protein heterocomplex. In this work, we report a novel effect of vanadate: in vivo potentiation of GR-mediated gene expression. In cells stably-transfected with complex (mouse mammary tumor virus (MMTV)) or minimal GR-regulated CAT reporters, treatment with 500muM vanadate caused CAT gene expression to dramatically increase, even at saturating concentrations of dexamethasone; while no such effect was seen in response to RU486 antagonist. Similar treatment with molybdate had no effect on GR activity, suggesting that the response to vanadate was not a general property of transition metal oxyanions. Treatment with vanadate after hormone-induced nuclear translocation of the GR also caused potentiation, demonstrating that vanadate was acting on a post-transformation event, perhaps by affecting the transactivation function of DNA-bound GR. Paradoxically, vanadate caused an apparent but temporary "loss" of GR protein immediately after treatment (as measured by loss of reactivity to BuGR2 antibody and of hormone-binding capacity) that returned to normal at approximately 8h post-treatment, suggesting that potentiation of GR transactivation function (as measured by our CAT assays) was probably occurring during the later stages (8-24h) of this assay. However, gel shift analyses revealed that vanadate could induce binding of the hormone-free GR to glucocorticoid response element (GRE)-containing oligonucleotides immediately after treatment. Thus, the rapid vanadate-induced "loss" of GR was not due to degradation of GR protein. Yet, vanadate in the absence of hormone had no effect on CAT reporter expression, demonstrating that this form of the GR still requires agonist for its enhanced transcriptional activity. As an indication of the potential mechanism of vanadate action, vanadate was found to dramatically stimulate the mitogen-activated protein kinases, ERK-1 and ERK-2. In addition, vanadate potentiation of GR reporter gene expression was completely blocked by the tyrosine kinase inhibitor herbimycin A. Taken as a whole, our results suggest that vanadate can have dramatic and complex effects on GR structure and function, resulting in hormone-free activation of GR DNA-binding function, as well as alterations to the BuGR2 epitope and hormone-binding domains--while at the same time stimulating tyrosine phosphorylation pathways controlling GR-mediated gene transcription.
Li Calzi
Sergio
S
Department of Pharmacology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.
Periyasamy
Sumudra
S
Li
Da Pei
DP
Sánchez
Edwin R
ER
D000818:Animals; D016227:Benzoquinones; D002458:Cell Fractionation; D002460:Cell Line; D003907:Dexamethasone / Q000737:chemistry / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008954:Models, Biological; D011809:Quinones / Q000494:pharmacology; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D014638:Vanadates / Q000494:pharmacology*
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005938:Glucocorticoids; D047029:Lactams, Macrocyclic; D011809:Quinones; D011965:Receptors, Glucocorticoid; D017828:Rifabutin; D014638:Vanadates; C020754:herbimycin; D003907:Dexamethasone; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one