Targeted Ablation of 25-Hydroxyvitamin D-1a-Hydroxylase (CYP27B1) Impairs VDR Subcellular Localization, Nucleocytoplasmic Trafficking and VDR/RXR Interaction in AOH931 Cells
Presentation Number: SAT 354
Date of Presentation: April 1st, 2017
Sylvester Jusu*1, John Presley2 and Richard Kremer3
1McGill University Health Centre, Montreal, QC, Canada, 2McGill University, Montreal, Canada, 3McGill University Health Center, Montreal, QC, Canada
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D, is a pleiotropic fat-soluble hormone that regulates calcium homeostasis via the transcriptional activation of target genes through the nuclear vitamin D receptor (nVDR). This hormonally active form is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1a-hydroxylase (CYP27B1). We used fixed and live cell fluorescence imaging techniques to examine the effects of CYP27B1 knockout on subcellular localization, nucleocytoplasmic trafficking of the vitamin D receptor (VDR) and VDR/RXR interaction in normal mouse MT1107 and CYP27B1 knockout AOH931 cell lines. A GFP tagged human VDR or hRXRα expression plasmid was transfected in both cell lines followed by treatment with either 1,25(OH)2D3 or 25(OH)D3.
We show, through transfection of hVDR and hRXRα GFP-tagged constructs, that both hVDR and hVDR/ hRXRα complex are localized in the nucleus of MT1107 cells treated with either 1α,25(OH)2D3 or 25-hydroxyvitamin D3 [25(OH)D3]. In contrast, in CYP27B1 knockout AOH932 cells impaired nuclear localization of hVDR and hVDR/hRXRα is observed. Furthermore, we demonstrate using Fluorescence Resonance Energy Transfer (FRET) that hVDR/hRXRα interaction is also impaired in AOH931 cells. Lastly, we demonstrate using Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) that the nucleocytoplasmic trafficking of VDR within the nucleus of CYP27B1 knockout AOH931 cells is reduced consistent with its binding to chromatin.
Our results show that CYP27B1 knockout disrupts VDR function by significantly impairing its nuclear localization, nucleocytoplasmic trafficking and hVDR/hRXR interaction consistent with the complex binding to DNA.
Nothing to Disclose: SJ, JP, RK