Identification of a GABP /  Binding Site Involved in the Induction of Oxytocin Receptor Gene Expression in Human Breast Cells. Potentiation by c-Fos/c-Jun

Abstract

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including the initiation of labor and milk ejection. OTR messenger RNA levels are highly regulated, reaching the greatest concentration in the uterus at the end of gestation, and in the mammary gland during lactation. Factors directly effecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by characterizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region between -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 promoter was identified, by electrophoretic mobility immunoshift assays, to be GABP alpha/beta. Co-transfection of a -85 OTR/luciferase construct with vectors expressing GABP alpha and GABP beta1 had only a modest effect on expression, 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 induction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We showed 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 inhibit serum-induced increases in 125I-labeled OT antagonist binding to Hs578T cells. Our work is the first characterization of the proximal promoter region of the human OTR gene, and it sets the stage for studying regulation of OTR expression in breast cells.

GA-binding protein α/β is a critical regulator of the BRCA1 promoter

“…This has been observed in the oxytocin receptor gene, where GABPa/b alone produced minimal promoter activation, but when cotransfected with fos and jun expression vectors, produced substantial transactivation (Hoare et al, 1999). GABPa/b is ubiquitously expressed and its binding element has been found in a wide variety of promoters.…”

Molecular regulation of the oxytocin receptor in peripheral organs

“…Dexamethasone shows a synergistic effect on serum-treated OTR upregulation in this cell line (Copland et al 1999). Basal promoter activity is conferred by the 85 bp flanking the 5 end of the human OTR gene.…”

“…In this sequence, there is a consensus ets binding sequence. GABP / , which binds to the ets element, cooperates with c-fos/c-jun to activate OTR promoter transcription in Hs578T cells (Hoare et al 1999). Although these transcription factors are important for OTR up-regulation in response to serum stimulation, it has not been determined whether they actually act in vivo under conditions of 100% serum.…”

Expression Profiling of Ductal Carcinoma in Situ by Laser Capture Microdissection and High-Density Oligonucleotide Arrays

“…17 The peptide hormone oxytocin can inhibit growth of breast cancer cells and expression of the corresponding oxytocin receptor has been well characterized in both nonmalignant and malignant mammary epithelium. 18 Intriguingly, transcription of the oxytocin receptor is also regulated by c-fos, 19 a second gene down-regulated in one, but not all three, nonmalignant/malignant specimen pairs.…”