Metabolism of 1alpha-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1alpha,20-dihydroxyvitamin D3

Abstract

Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1alpha-hydroxyvitamin D3 and whether products were biologically active. The major product of 1alpha-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1alpha,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1alpha,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The Km for 1alpha-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The kcat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1alpha,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1alpha,25-dihydroxyvitamin D3. 1alpha,20-Dihydroxyvitamin D3 (10 microM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1alpha,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1alpha,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically.

Fig. 1

Chromatogram showing products of 1(OH)D3 metabolism by P450scc. (A) 1(OH)D3 (50 μM) dissolved in cyclodextrin to a final concentration of 0.45%, was incubated with 1.0 μM P450scc for 1 h in a reconstituted system containing adrenodoxin and adrenodoxin reductase. Samples were extracted and analyzed by reverse-phase HPLC. (B) Control incubation (zero time) showing the 1(OH)D3 substrate. The identification of the number of hydroxyl groups in the products is described in the text. RT, retention time in minutes.

Fig. 2

Time course for metabolism of 1(OH)D3 by P450scc in cyclodextrin and in phospholipid vesicles. (A) 1(OH)D3 (50 μM) dissolved in cyclodextrin to a final concentration of 0.45%, was incubated with 2.0 μM P450scc. (B) Vesicles containing 0.1 mol 1(OH)D3/mol phospholipid were incubated with 2.0 μM P450scc. Samples were analyzed by HPLC as described for Fig. 1. RT, retention time in minutes.

Fig. 3

NMR identification of the major product as 1,20(OH)₂ D3. (A) 1D proton NMR; (B) 2D proton–proton TOCSY NMR spectrum; and (C) 2D proton–carbon HSQC spectrum. In 1D proton NMR, 21-Me shifted downfield and became a singlet as a result of hydroxylation at position 20. S denotes solvent peaks from Methanol-D at 3.31 and 4.81 ppm. The solvent peak at 4.81 ppm is coincidently overlapped with one proton from 19-CH2. Solvent suppression of this solvent peak also reduced the intensity from this proton. ** denotes impurities.

Fig. 4

The structure of 1α,20-dihydroxyvitamin D3 (1,20(OH)₂D3).

Fig. 5

Time course for metabolism of 1,20(OH)₂D3 in cyclodextrin. 1,20(OH)₂D3 (50 μM) dissolved in cyclodextrin to a final concentration of 0.45%, was incubated with 2.0 μM P450scc. RT, retention time in minutes.

Fig. 6

Chromatogram showing metabolism of trihydroxyvitamin D3 (RT = 26 min). The (OH)₃D3 product of P450scc action on 1(OH)D3 with a retention time of 26 min in Fig. 1 was purified, incorporated into phospholipid vesicles at a molar ratio to phospholipid of 0.06, incubated with P450scc (1 μM) for 2 h and products analyzed by HPLC with gradient elution. RT, retention time in minutes.

Fig. 7

Suppression of [³H]thymidine incorporation into DNA by 1,20(OH)₂D3. HaCaT keratinocytes were treated with 1,20(OH)₂D3 for (A) 24 h or (B) 48 h. Data are presented as means ± SEM (n = 16). *P < 0.05 versus control.

Fig. 8

Relative cell number (protein concentration) following 1,20(OH)₂D3 treatment. HaCaT keratinocytes were treated with 1,20(OH)₂D3 for 48 h. Cellular protein concentration was measured by the sulforhodamine B assay. Data are presented as means ± SEM (n = 6). *P < 0.05 versus control.

Fig. 9

1,20(OH)₂D3 increases CYP24 mRNA levels in HaCaT keratinocytes. HaCaT keratinocytes were treated with 0.1 μM or 1.0 μM 1,20(OH)₂D3 for 6 h (A) or 24 h (B). Data are presented as means ± SEM (n = 3). *P < 0.05, **P < 0.001.

Fig. 10

Pathways for metabolism of 1α-hydroxyvitamin D3 by P450scc. The major pathway leading to the production of tetrahydroxyvitamin D3 is shown in bold. Arrows with crossed lines indicate that these hydroxy-metabolites are not further hydroxylated by P450scc. RT, retention time in minutes (see Fig. 1).