Synergistic activation of the prolactin promoter by vitamin D receptor andGHF-1: Role of the coactivators, CREB-binding protein and steroid hormone receptor coactivator-1 (SRC-1)

PRL gene expression is dependent on the presence of the pituitary-specific transcription factor GHF-1/Pit-1, which is transcribed in a highly restrict ed manner in cells of the anterior pituitary. In pituitary GH3 cells, vitam in D increases the levels of PRL transcripts and stimulates the PRL promote r. We have analyzed the role of GHF-1 and of the vitamin D receptor (VDR) t o confer vitamin D responsiveness to the PRL promoter. For this purpose we have used nonpituitary HeLa cells, which do not express GHF-1. We found tha t VDR activates the PRL promoter both in a ligand-dependent and -independen t manner through a sequence located between positions -45/-27 in the proxim al 5'-flanking region. This sequence also confers VDR and vitamin D respons iveness to a heterologous promoter. In the context of the PRL gene, VDR req uires the presence of GHF-1 to activate the promoter. Truncation of the las t 12 C-terminal amino acids of VDR, which contain the ligand-dependent acti vation function (AF2), abolishes regulation by vitamin D, suggesting that b inding of coactivators to this region mediates ligand-dependent stimulation of the PRL promoter by the receptor. Indeed, expression of the coactivator s, steroid hormone receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP), significantly enhances the stimulatory effect of vitamin D mediated by the wild-type VDR but not by the AF2 mutant receptor. Furthermore, CBP a lso increases the activation of the PRL promoter by GHF-1 and the ligand-in dependent activation by both wild-type and mutant VDR.