Identification of a GABP alpha/beta binding site involved in the inductionof oxytocin receptor gene expression in human breast cells. Potentiation by c-Fos/c-Jun

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including th e initiation of labor and milk ejection. OTR messenger RNA levels are highl y regulated, reaching the greatest concentration in the uterus at the end o f gestation, and in the mammary gland during lactation. Factors directly ef fecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by charac terizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region betwee n -85 and -65 that was required for both basal and serum-induced expression in a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OTRs. This DNA region contains an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif. The specific Ets factor binding to the OTR promo ter was identified, by electrophoretic mobility immunoshift assays, to be G ABP alpha/beta. Cotransfection of a -85 OTR/luciferase construct with vecto rs expressing GABP alpha and GABP beta 1 had only a modest effect on expres sion, but cotransfection with GABP alpha/beta- with c-Fos/c-Jun-expressing plasmids resulted in an increase of almost 10-fold in luciferase activity. Mutation of either the GABP- or CRE-like binding sites obliterated the indu ction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We s howed the requirement for GABP alpha/beta and c-Fos/c-Jun in endogenous OTR gene expression using oligonucleotide GABP and AP-1 binding decoys to inhi bit serum-induced increases in I-125-labeled OT antagonist binding to Hs578 T cells. Our work is the first characterization of the proximal promoter re gion of the human OTR gene, and it sets the stage for studying regulation o f OTR expression in breast cells.