Oestrogens downregulate tissue factor pathway inhibitor through oestrogen response elements in the 5′-flanking region

Abstract

Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5′-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5′-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5′-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5′-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5′-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells.