Oestrogen receptors pathways to oestrogen responsive elements: The transactivation function-1 acts as the keystone of oestrogen receptor (ER)β-mediated transcriptional repression of ERα

Abstract

Oestrogen receptors (ER)α and β modify the expression of genes involved in cell growth, proliferation and differentiation through binding to oestrogen response elements (EREs) located in a number of gene promoters. Transient transfection of different luciferase reporter vectors 3xEREs-Vit, 2xEREs-tk and ERE-C3 showed that the transactivation capacity of both ER subtypes was influenced by 1) the nature of the inducer (oestradiol (E 2), phyto- and anti-oestrogen (AE)), 2) the structure of the promoter (nucleotidic sequence, number of ERE, length of the promoter sequence) and 3) the cell line (containing endogenous ER (MCF-7) or in which ER was stably expressed (MDA-MB-231-HE-5 (ERα+) or MDA-MB-231-HERB (ERβ+)). ER subtype did not display the same efficacy on the different constructions in the presence of E 2 and of AE according to the cell (e.g. in MCF-7 cells: tk≫Vit≫C3∼0 while in MDA-MB-231 cells: Vit≫tk∼C3). E 2 response was higher in MCF-7 cells, probably due to higher ER expression level (maximal at 10 −10 M instead of 10 −8 M for E 2 in HE-5 cells). Finally, the same ligand could exert opposite activities on the same promoter according to the ER isoform expressed: in the MDA-MB-231 cells, AE acted as inducers of the C3 promoter via ERβ whereas ERα/AE complexes down-regulated this promoter. Approximately 70% of breast tumours express ER and most tumour cells coexpress both ER isotypes. Thus, different types of ER dimers can be formed in such tumours (ERβ or ERα homodimers or ERα/ERβ heterodimers). We therefore studied the influence of the coexistence of the two ERs on the ligand-induced transcriptional process following transient transfection of ERα in ERβ+ cells, and inversely ERβ in ERα+ cells. ERβ-transfection inhibited the E 2- and genistein-induced ERα-dependent transcription on all promoters in all cell lines except C3 in MCF-7; this inhibitory effect was lost following transfection of ERβ deleted of its AF-1 (ERβ-AF-2). These results suggest that the dominant negative properties of ERβ are mainly due to its AF-1 function. Interestingly, transfection of an ERβ-AF-2 construct into MCF-7 cells potentiated the transcription inhibitory capacity of 4-OH-tamoxifen (OHT) on the Vit and tk promoters. Thus, (1) OHT exerts an agonistic activity through the AF-1 function of ER and (2) expression of ERβ in breast cancer cells seems to favour the AE treatment. Contrary to ERβ, ERα-transfection had little effect on ERβ transactivation capacity in HERB cells. Finally, the ratio ERα/ERβ constitutes one decisive parameters to orientate the transcriptional mechanism of a target gene in the presence of agonist as well as of antagonist ligands.