Molecular characterization of vitamin D(3) receptor promoter and 1,25-dihydroxyvitamin D(3) action in breast cancer cells

1,25-dihydroxyvitamin D₃, the active form of vitamin D, mediates gene transcription through the vitamin D receptor, which is expressed in both normal tissues and in tumors, including breast cancers. 1,25-dihydroxyvitamin D₃ inhibits growth and triggers apoptosis in breast cancer cells and vitamin D analogs have been used in clinical trials to treat breast cancer. These studies focused on the promoter region upstream of exon 1c in the human vitamin D receptor gene, which exerts basal and hormonally regulated activity in MCF-7 breast cancer cells. We demonstrate that this vitamin D receptor promoter also mediates basal transcription in T47D breast cancer cells, and that promoter activity is up-regulated following treatment with estrogen, forskolin, 1,25-dihydroxyvitamin D₃, and the phytoestrogens, resveratrol and genistein. We used truncations of the promoter and site-directed mutagenesis to identify three specific Sp-1 sites which independently mediate the effects of estrogen, resveratrol, and 1,25-dihydroxyvitamin D₃ on promoter activity. We also show that the effects of estrogen are dependent on both subtypes of the estrogen receptor whereas the effects of resveratrol are dependent only on estrogen receptor alpha. Because doses of resveratrol that up-regulate the vitamin D receptor do not increase growth of T47D cells, we hypothesized that resveratrol could sensitize cells to the growth inhibitory effects of 1,25-dihydroxyvitamin D₃. We show that resveratrol increased 1,25-dihydroxyvitamin D₃-mediated transactivation of a vitamin D responsive promoter and enhanced T47D cell sensitivity to the growth inhibitory effects of 1,25-dihydroxyvitamin D₃ and the vitamin D analog EB1089. These are the first studies to implicate the Sp-1 transcription factor in regulation of the vitamin D receptor promoter by estrogen, phytoestrogen, and 1,25-dihydroxyvitamin D₃. In addition, we demonstrate that vitamin D receptor up-regulation enhances cell sensitivity to 1,25-dihydroxyvitamin D₃ and its analog EB1089, providing proof of principal that dietary components which transcriptionally up-regulate the vitamin D receptor promoter may enhance the therapeutic efficacy of vitamin D and its analogs.