Figure 5.

Direct repression of aromatase transcription through promoter II by calcitriol. A, A schematic diagram of the aromatase promoter I.3/II. We identified two putative nVDREs (designated as proximal and distal nVDRE) in the aromatase I.3/II promoter. The putative proximal nVDRE sequence overlaps a CRE that mediates the cAMP stimulation of promoter II. B, Calcitriol suppresses basal and cAMP stimulation of aromatase I.3/II promoter activity in BCa cells. An aromatase I.3/II promoter-luciferase reporter and renilla luciferase plasmids were transiently transfected into MCF-7 cells. Cells were treated with vehicle (Veh) or 100 nm calcitriol (Cal) in the absence (control, Con) or presence 50 μm Fsk or 0.5 mm (Bu)₂cAMP for 24 h. Results are expressed as a percent of relative luciferase activity in vehicle-treated control set at 100%. **, P < 0.01 and ***, P < 0.001 compared with vehicle. ++, P < 0.01 compared with Veh + Fsk or Veh + (Bu)₂cAMP. C, Calcitriol recruits the VDR to the VDREs in the aromatase II promoter. ZR-75-1 cells were exposed to vehicle (Cal−) or 10 nm calcitriol (Cal+) for 2 h, and the cell extracts were subjected to ChIP analyses. Immunoprecipitations were carried out with IgG or a mixture of three αVDR antibodies, followed by PCR using primers designed to amplify fragments of the aromatase promoter II encompassing the proximal nVDRE and the distal nVDRE.