Purified mouse CYP27B1 can hydroxylate 20,23-dihydroxyvitamin D3, producing 1alpha,20,23-trihydroxyvitamin D3, which has altered biological activity

Abstract

20,23-Dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] is a biologically active metabolite produced by the action of cytochrome P450scc (CYP11A1) on vitamin D(3). It inhibits keratinocyte proliferation, stimulates differentiation, and inhibits nuclear factor-kappaB activity, working as a vitamin D receptor agonist. We have tested the ability of purified mouse 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) to add a 1alpha-hydroxyl group to this vitamin D analog and determined whether this altered its biological activity. 20,23(OH)(2)D(3) incorporated into phospholipid vesicles was converted to a single product by CYP27B1, confirmed to be 1alpha,20,23-trihydroxyvitamin D(3) [1,20,23(OH)(3)D(3)] by mass spectrometry and NMR. The 20,23(OH)(2)D(3) was a relatively poor substrate for CYP27B1 compared with the normal substrate, 25-hydroxyvitamin D(3), displaying a 5-fold higher K(m) and 8-fold lower k(cat) value. Both 20,23(OH)(2)D(3) and 1,20,23(OH)(3)D(3) decreased neonatal human epidermal keratinocyte proliferation, showing significant effects at a lower concentration (0.1 nM) than that seen for 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] at 24 h of treatment. Both compounds also decreased cell biomass relative to that of control cells, measured by staining with sulforhodamine B. They caused little stimulation of the expression of the vitamin D receptor at the mRNA level compared with the 30-fold induction observed with the same concentration (100 nM) of 1,25(OH)(2)D(3) at 24 h. Addition of a 1alpha-hydroxyl group to 20,23(OH)(2)D(3) greatly enhanced its ability to stimulate the expression of the CYP24 gene but not to the extent seen with 1,25(OH)(2)D(3). This study shows that purified CYP27B1 can add a 1alpha-hydroxyl group to 20,23(OH)(2)D(3) with the product showing altered biological activity, especially for the stimulation of CYP24 gene expression.

Fig. 1.

Chromatogram showing the metabolism of 20,23(OH)₂D₃ by mouse CYP27B1. A, 20,23(OH)₂D₃ was incorporated into phospholipid vesicles at a ratio of 0.025 mol/mol phospholipid and was incubated with 0.6 μM CYP27B1 for 1 h in a reconstituted system containing adrenodoxin and adrenodoxin reductase. Samples were extracted using dichloromethane and analyzed by reverse-phase HPLC. B, control incubation without adrenodoxin showing the substrate but the absence of product.

Fig. 2.

Mass spectra of 1,20,23(OH)₃D₃. A, ESI source showing the molecular ion. B, APCI source showing the progressive loss of water molecules. Both measurements were done in the positive ion mode.

Fig. 3.

One-dimensional proton (A), 2D ¹H-¹³C HSQC (B), 2D ¹H-¹H TOCSY (C), and 2D ¹H-¹H correlation spectroscopy (COSY) (D) NMR spectra of the metabolite. EtOH, ethanol.

Fig. 4.

1,20,23(OH)₃D₃ inhibits HEKn proliferation. HEKn cells were seeded in 96-well plates and treated with a range of concentrations of 1,25(OH)₂D₃, 20,23(OH)₂D₃, or 1,20,23(OH)₃D₃ or with ethanol vehicle for 24 or 48 h. Uptake of MTT reagent was measured from the formazan product measured at 570 nm. n = 6. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control.

Fig. 5.

Effect of 1,20,23(OH)₃D₃ on HEKn biomass. HEKn cells were seeded in 96-well plates and were treated for 24 h with a range of concentrations of 1,25(OH)₂D₃, 20,23(OH)₂D₃, or 1,20,23(OH)₃D₃, or with ethanol vehicle. Cells were stained with sulforhodamine B and lysed, and the absorbance was measured at 565 nm. n = 6. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control.

Fig. 6.

Effect of 1,20,23(OH)₃D₃ on VDR expression. RNA from HEKn cells treated with 1,25(OH)₂D₃, 20,23(OH)₂D₃, or 1,20,23(OH)₃D₃ for 6 h (A) or 24 h (B) was extracted and reverse-transcribed. Real-time PCR was performed using TaqMan PCR Master Mix and primers specific for the VDR gene. The amounts of VDR mRNA were normalized by the comparative C_t method, using cyclophilin B as a housekeeping gene. n = 3. **, P < 0.01; ***, P < 0.001.

Fig. 7.

Effect of 1,20,23(OH)₃D₃ on CYP24 expression. HEKn cells were treated for 6 h (A) or 24 h (B) as for Fig. 6, and the RNA was extracted and reverse-transcribed. Real-time PCR was performed using TaqMan PCR Master Mix and primers specific for the CYP24 gene. The amounts of CYP24 mRNA were normalized by the comparative C_t method, using cyclophilin B as a housekeeping gene. n = 3. ***, P < 0.001.