Steroid and xenobiotic receptor and vitamin D receptor crosstalk mediates CYP24 expression and drug-induced osteomalacia

Abstract

The balance between bioactivation and degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is critical for ensuring appropriate biological effects of vitamin D. Cytochrome P450, family 24-mediated (CYP24-mediated) 24-hydroxylation of 1,25(OH)2D3 is an important step in the catabolism of 1,25(OH)2D3. The enzyme is directly regulated by vitamin D receptor (VDR), and it is expressed mainly in the kidney, where VDR is also abundant. A recent report suggests that activation of steroid and xenobiotic receptor (SXR) also enhances the expression of CYP24, providing a new molecular mechanism of drug-induced osteomalacia. However, here we showed that activation of SXR did not induce CYP24 expression in vitro and in vivo, nor did it transactivate the CYP24 promoter. Instead, SXR inhibited VDR-mediated CYP24 promoter activity, and CYP24 expression was very low in tissues containing high levels of SXR, including the small intestine. Moreover, 1,25(OH)2D3-induced CYP24 expression was enhanced in mice lacking the SXR ortholog pregnane X receptor, and treatment of humans with the SXR agonist rifampicin had no effect on intestinal CYP24 expression, despite demonstration of marked CYP3A4 induction. Combined with our previous findings that CYP3A4, not CYP24, plays the dominant role in hydroxylation of 1,25(OH)2D3 in human liver and intestine, our results indicate that SXR has a dual role in mediating vitamin D catabolism and drug-induced osteomalacia.

Figure 1. Expression of SXR/PXR, VDR, and their target genes in various tissues.

(A) Total RNA was isolated from human liver, intestine, and kidney tissues (n = 3), and the expression of SXR, VDR, CYP3A4, and CYP24 was analyzed by QRT-PCR. (B) Total RNA was isolated from mouse liver, intestine, and kidney tissues (n = 3), and the expression of PXR, VDR, CYP3A11, and CYP24 was analyzed by QRT-PCR.

Figure 2. 1,25(OH)₂ D₃ but not SXR ligands induce CYP24 gene expression in human primary hepatocytes and intestinal cells.

(A and B) Human primary hepatocytes from 2 different donors were treated with 1, 10, or 50 nM of the VDR ligand 1,25(OH)₂D₃ or 10 μM of SXR ligands RIF, CLOT, or RU486 for 24 hours as indicated. Total RNA from each sample was isolated, and the expression of CYP3A4 and CYP24 genes was determined by QRT-PCR assays. (C) Two different immortalized human intestinal cell lines, Caco-2 and LS180, were treated with 1, 10, or 100 nM of the VDR ligand 1,25(OH)₂D₃ or 10 μM of SXR ligands RIF, CLOT, or RU486 for 24 hours as indicated. Total RNA from each sample was isolated, and the expression of CYP3A4 and CYP24 genes was determined by QRT-PCR assays. (D) Human primary hepatocytes from donor 3 were treated with 10 or 50 nM of the VDR ligand 1,25(OH)₂D₃ or 10 μM of SXR ligands RIF, CLOT, or RU486 for 24, 48, or 72 hours as indicated. Total RNA from each sample was isolated, and the expression of CYP24 genes was determined by QRT-PCR assays. (E) LS180 cells were transfected with control vector, VP16, or VP16-SXR expression vector; total RNA from each sample was isolated; and the expression of CYP3A4 and CYP24 genes was determined by QRT-PCR assays.

Figure 3. VDR but not SXR transactivates the CYP24 promoter.

(A) HepG2 cells were transiently transfected with full-length VDR together with a CYP3A4-luc reporter or CYP24-luc reporter and CMX-β-galactosidase transfection control plasmid. After transfection, cells were treated with control medium or medium containing 1 or 10 nM 1,25(OH)₂D₃ for 24 hours. (B) HepG2 cells were transiently transfected with full-length SXR together with a CYP3A4-luc reporter or CYP24-luc reporter and CMX–β-galactosidase transfection control plasmid. After transfection, cells were treated with control medium or medium containing 10 μM CLOT, RIF, or RU486 for 24 hours.

Figure 4. SXR does not bind to the VDRE-1 and VDRE-2 motifs in the CYP24 promoter region.

(A) In vitro–translated VDR, SXR, and RXR, as indicated, were incubated with [³²P]-labeled VDRE-1 or VDRE-2 probe and analyzed by EMSA. Ten- or 50-fold excess of unlabeled VDRE-1 or VDRE-2 probes was used for competition experiments. (B) In vitro–translated SXR and RXR were incubated with a [³²P]-labeled ER6 motif, and 10- or 50-fold excess of unlabeled ER6, VDRE-1, or VDRE-2 probes was used for competition experiments. (C and D) In vitro–translated VDR and RXR were incubated with [³²P]-labeled VDRE-1 (C) or VDRE-2 (D) along with increasing amounts of SXR or RXR protein and analyzed by EMSA.

Figure 5. Induction of duodenal CYP3A4 but not CYP24 expression in healthy volunteers treated with RIF.

The duodenal epithelial biopsy samples were collected from 6 healthy human volunteers before and after 2, 7, or 14 days of oral RIF administration (150 mg every 6 hours). Total RNA was isolated from biopsy samples, and the expression of CYP3A4 and CYP24 was analyzed by QRT-PCR. ND, not detectable. Statistically significant expressions compared with conditions before RIF administration (day 0) are marked with asterisks; *P < 0.05 and **P < 0.01.

Figure 6. Crosstalk between SXR and VDR coordinately regulates CYP24 promoter activity.

HepG2 cells were transiently transfected with SXR or/and VDR expression plasmids along with a CYP24-luc reporter and CMX–β-galactosidase control plasmid, as indicated. (A) After transfection, cells were treated with control medium or medium containing 1, 10, or 100 nM 1,25(OH)₂D₃ and 10 μM RIF as indicated for 24 hours. (B) After transfection, cells were treated with 100 nM 1,25(OH)₂D₃ and 1, 5, and 10 μM RIF for 24 hours. (C) HepG2 cells were transfected with increasing amounts of SXR at 1:1, 2:1, or 4:1 ratio with VDR expression vector. After transfection, cells were treated with 100 nM 1,25(OH)₂D₃.

Figure 7. SXR inhibits VDR effects on SPP and OC promoter activities but not on their common target gene, CYP3A4.

HepG2 cells were transfected with SXR and VDR expression plasmids along with an SPP2-luc reporter (A), OC-luc reporter (B), or CYP3A4-luc (C) reporter and CMX–β-galactosidase control plasmid, as indicated. After transfection, cells were treated with control medium or medium containing 1 or 10 nM 1,25(OH)₂D₃ and 10 μM RIF, as indicated, for 24 hours.

Figure 8. Activation of mouse PXR by PCN does not induce CYP24 expression in mice, and VDR-mediated CYP24 expression is enhanced by PXR knockout.

Ten-week-old male PXR-knockout and C57BL6/J (wild-type) mice (3 per group) were injected intraperitoneally with vehicle control (DMSO), PXR ligand PCN (40 mg/kg), or VDR ligand 1,25(OH)₂D₃ (50 ng/mouse) for 3 consecutive days. Tissues were collected, and gene expression in the specific tissues was determined by QRT-PCR. (A) Expression of the PXR target gene CYP3A4 in PXR-knockout or WT mice was determined by QRT-PCR. Total RNA was isolated from liver and intestine, as indicated. (B) Expression of the VDR target gene CYP24 in PXR-knockout or WT mice was determined by QRT-PCR. Total RNA was isolated from liver, intestine, and kidney, as indicated. *P < 0.05, **P < 0.01, and ^#P < 0.001.