CAR repression of vitamin D3 activation via SMRT. A, Huh7 cells were transfected with or without pcDNA3.1/mVDR and pCR3/hCAR. After 24 h, cells were treated with DMSO, VD3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 24 h before being harvested for qRT-PCR. Relative CYP24A1 mRNA levels were expressed by taking the level of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. B, pGL3/CYP24A1-3 kb was transfected with or without pcDNA3.1/mVDR and pCR3/hCAR into Huh7 cells. After 24 h, cells were treated with DMSO, VD3 (10 nM), CITCO (25, 75, and 250 nM), and PK11195 (1, 3, and 10 μM) for an additional 24 h before being harvested for luciferase assays. Relative luciferase activities were expressed relative to the activity of the DMSO-treated cells transfected with empty plasmid as 1. Bars, mean ± S.D. C, Huh7 cells were transfected with pcDNA3.1/mVDR and pcDNA3.1/mVDR plus pCR3/hCAR. After 24 h, cells were treated with DMSO, VD3 (10 nM), CITCO (250 nM), and/or PK11195 (10 μM) for an additional 2 h before being harvested for ChIP assays. Cross-linked chromatin-protein complexes were immunoprecipitated with anti-VDR (αVDR), anti-CAR (αCAR), anti-SMRT (αSMRT), or normal mouse IgG (αIgG). By scanning three independent assays, the relative intensities of the bands were calculated based the inputs: in the VDR binding, 3.4 ± 0.8, 3.3 ± 1.0, 3.4 ± 1.3, and 3.4 ± 1.2% for VDR plus VD3, VDR plus CAR and VD3, VDR plus CAR, VD3, and CITCO, and VDR plus CAR, VD3, and PK11195, respectively; in the CAR binding, 0.1 ± 0.3, 1.0 ± 0.3, 2.9 ± 0.5, and 3.7 ± 1.2% for VDR plus VD3, VDR plus CAR and VD3, VDR plus CAR, VD3, and CITCO, and VDR plus CAR, VD3, and PK11195, respectively; in the SMRT binding, 0.0 ± 0.0, 0.8 ± 0.1, 2.0 ± 0.5, and 2.1 ± 0.3% for VDR plus VD3, VDR plus CAR and VD3, VDR plus CAR, VD3, and CITCO, and VDR plus CAR, VD3, and PK11195, respectively.