Transcriptional activation of the wild-type and mutant vitamin D receptors by vitamin D₃ analogs

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   Figure 1

   Introduction of point mutations in the LBD of VDRs and protein expression of mutant receptors. (A) Schematic diagram of the VDR and the location of mutations identified in clinical cases. (B) Expression of recombinant VDRs in TSA201 cells. Nuclear and cytoplasmic extracts from transfected TSA201 cells treated with calcitriol (10.0nM) were analyzed by western blotting using an anti-VDR antibody. Nucleus: nuclear fractions; cytoplasm: cytoplasmic fractions. (C) The effects of 10.0nM calcitriol on the transcriptional activity of the WT and mutant receptors. TSA201 cells were co-transfected with the WT-VDR or mVDR expression plasmids (20 ng), VDRE-tk-Luc (50ng), and pGL4.70 (5ng). The data are presented as the mean ± s.d. of at least three transfections performed in triplicate. RLU, Relative luciferase unit. ***P<0.001 vs WT.
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   Figure 2

   The effect of active synthetic VD₃ analogs on the transcriptional activity of the WT-VDR in TSA201 and HepG2 cells. The effects of different doses of VD₃ analogs on WT-VDR transcriptional activity. (A) TSA201 cells and (B) HepG2 cells transfected as described above were treated with different concentrations of VD₃ analogs. The data are presented as the mean ± s.d. of at least three transfections performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 vs the same dose of calcitriol.
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   Figure 3

   The effects of VD3 analogs on the transcriptional activity of the mVDRs (A–G). TSA201 cells were co-transfected with VDR expression plasmids (pCMX-VDR, 20ng) carrying the indicated mutants, the reporter plasmid VDRE-tk-Luc (50ng), and the internal control plasmid pGL4.70 (5ng) in the absence or presence of 10nM of VD₃ analogs. Transcriptional activation by VD₃ analogs was normalized to that by calcitriol. The data are presented as the mean±s.d. from at least three transfections performed in triplicate. *P<0.05, **P<0.01, ***P<0.001 vs calcitriol.
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   Figure 4

   The effects of different doses of falecalcitriol and maxacalcitriol on the transcriptional activity of VDRs (A–F). TSA201 cells were co-transfected with the expression plasmids (20ng) carrying the wild-type (WT) or indicated mutant VDRs (mVDR), VDRE-tk-Luc (50ng), and pGL4.70 (5ng) in the absence or presence of increasing concentrations of VD3 analogs (0.1–10nM). The data are presented as the mean±s.d. from three transfections performed in triplicate. **P<0.01, ***P<0.001.
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   Figure 5

   Interactions of cofactors with the wild-type VDR in the mammalian two-hybrid assay. TSA201 cells were co-transfected with the VP16-VDR (WT) expression plasmid (50ng), UAS-E1b-TATA-Luc (100ng), and Gal4-cofactor (50ng) in the absence or presence of VD₃ analogs and analyzed for the recruitment of coactivators (A, B and C) and corepressor (D). The data are presented as the mean±s.d. from three transfections performed in triplicate. *P<0.05, **P<0.01.
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   Figure 6

   Transcriptional control of the VDR target genes by calcitriol and falecalcitriol. (A and B) Functional analysis of calcitriol and falecalcitriol in the CYP27B1 promoter activation assay. The human CYP27B1 promoter was cloned and introduced into the 5′ end of the luciferase gene as a reporter construct. TSA201 cells were co-transfected with the VDR expression plasmid (50ng), the reporter construct (100ng), and pGL4.70 (5ng) in the absence or presence of increasing concentrations (0.1–10.0nM) of calcitriol and falecalcitriol. The data are presented as the mean±s.d. from at least three transfections performed in triplicate. *P<0.05 vs calcitriol, ⁺ P<0.05. (C) CYP24A1 mRNA expression in TSA201 cells and ALP, OPN, COL1, and RANKL in MC3T3-E1 cells stimulated by calcitriol and falecalcitriol. Cells were treated with ethanol (vehicle control) or 10.0nM calcitriol or falecalcitriol and analyzed for mRNA expression by real-time quantitative PCR; relative target mRNA expression was calculated after normalization to GAPDH mRNA used as an internal control. The data are presented as the mean±s.d. from three independent experiments performed in triplicate; **P<0.01, ***P<0.001 vs calcitriol.

• © 2016 Society for Endocrinology