Vasopressin increases GAGA binding activity to the V1b receptor promoter through transactivation of the MAP kinase pathway.
Previous studies show that binding of nuclear proteins to GAGA repeats (GAGA box) in the vasopressin type 1b receptor (V1bR) promoter is essential for transcriptional initiation of the gene. To determine whether increased vasopressin (VP) during stress activates V1bR expression through the GAGA box, we examined the effects of VP on GAGA binding activity and on the ability of the V1bR promoter to recruit RNA polymerase in the hypothalamic cell line, H32. In chromatin immunoprecipitation assays, VP induced RNA polymerase II recruitment by the wild type V1bR promoter but not by a construct with the major GAGA box deletion. VP (10 min) also increased binding of nuclear proteins to radiolabeled GAGA oligonucleotides in electromobility shift assays. VP-induced GAGA binding activity was potentiated by the protein kinase C inhibitor, calphostin C, and was prevented by the MEK inhibitor, UO126, and the epidermal growth factor receptor (EGFR) inhibitor, AG1478, suggesting that VP activates GAGA binding through transactivation of the EGFR. This was confirmed by western blot experiments showing rapid increases in phospho ERK after incubation with VP, an effect that was potentiated by calphostin C and inhibited by UO12 and AG1478, as well as by the ability of VP to phosphorylate the EGFR. Using receptor selective VP analogs we showed that both V1aR and V1bR subtypes can mediate GAGA binding activation in H32 cells. This study demonstrates that VP stimulates GAGA binding to the V1bR promoter through transactivation of the EGFR and MAP kinase. The data support the hypothesis that VP contributes to pituitary V1bR upregulation during stress through GAGA binding-mediated transcriptional activation.
Volpi
Simona
S
Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, Maryland 20892-1303, USA.
Liu
Ying
Y
Aguilera
Greti
G
D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007031:Hypothalamus / Q000166:cytology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D015533:Transcriptional Activation; D014667:Vasopressins / Q000737:chemistry / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017483:Receptors, Vasopressin; D014667:Vasopressins; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D012319:RNA Polymerase II