Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of
vitamin D, exerts its anti-proliferative activity in
breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of
estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative
vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at -2488 to -2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at -94 to -70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter-reporter constructs revealed that both nVDREs functioned to mediate
calcitriol transrepression. In the electrophoretic mobility shift assay, the
vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of
calcitriol, and the
chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5' hexameric DNA sequence of the distal nVDRE resulted in the loss of
calcitriol-mediated transrepression and the inhibition of
protein-
DNA complex formation, demonstrating the importance of these
nucleotides in VDR
DNA binding and transrepression. A putative
nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced
calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by
calcitriol. The suppression of ER expression and
estrogen-mediated signaling by
calcitriol in BCa cells suggests that
vitamin D may be useful in the treatment of ER+ BCa.