The number of vitamin D receptor binding sites defines the different vitamin D responsiveness of the CYP24 gene in malignant and normal mammary cells

Abstract

Primary 1α,25-dihydroxyvitamin D3 (1α,25(OH) 2D3)-responding genes are controlled by the vitamin D receptor (VDR) binding to specific sites (VDREs) that are located within the regulatory regions of these genes. According to previous studies, the gene encoding 25-dihydroxyvitamin D3 24-hydroxylase, CYP24, which is the strongest known 1α,25(OH)2D3-responsive gene, has multiple VDREs that locate within the proximal and the distal promoter. However, it has remained unclear, what is the biological role of these regions and how they participate in the regulation of transcription. In this study, we found a different CYP24 expression profile in normal (MCF-10A) and malignant (MCF-7) human mammary cells. Moreover, CYP24 mRNA showed to be three times more stable in MCF-7 cells than in MCF-10A cells. We studied the mechanism of this difference using expression profiling, quantitative chromatin immunoprecipitation and chromosome conformation capture assays. Interestingly, the number of functional VDREs was higher in MCF-7 cells than in MCF-10A cells. Three functional VDREs in MCF-7 cells are connected to linearmRNAaccumulation, whereas only one VDRE seems to lead to stepwise CYP24 mRNA accumulation in MCF-10A cells. The distal VDREs were involved in transcriptional regulation via ligand-dependent, dynamic chromatin looping, which brings cyclically the distal elements together either individually or simultaneously next to the transcription start site. In conclusion, our data suggest that in comparison to normal cells, clearing of 1α,25(OH)2D3 is enhanced in malignant cells due to differences in transcriptional regulation of CYP24 and metabolism of CYP24 mRNA. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.