Abstract

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D₃, producing 20S-hydroxyvitamin D₃ [20(OH)D₃] and 20S,23-dihydroxyvitamin D₃ [20,23(OH)₂D₃] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D₃, 17,20-dihydroxyvitamin D₃, and 17,20,23-trihydroxyvitamin D₃. In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D₃ and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D₃ [22(OH)D₃] and 20S,22-dihydroxyvitamin D₃ [20,22(OH)₂D₃]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D₃ to 20,22(OH)₂D₃. The 20,22(OH)₂D₃ could also be produced from 20(OH)D₃ and was metabolized to a trihydroxyvitamin D₃ product. We compared the biological activities of these new derivatives with those of 20(OH)D₃, 20,23(OH)₂D₃, and 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. 1,25(OH)₂D₃, 20(OH)D₃, 22(OH)D₃, 20,23(OH)₂D₃, and 20,22(OH)₂D₃ significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)₂D₃, 20,22(OH)₂D₃, 20(OH)D₃, and 1,25(OH)₂D₃, with 22(OH)D₃ having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)₂D₃. All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D₃ and 20,22(OH)₂D₃ having less effect than 1,25(OH)₂D₃ and 20(OH)D₃. Thus, we have identified 22(OH)D₃ and 20,22(OH)₂D₃ as products of CYP11A1 action on vitamin D₃ and shown that, like 20(OH)D₃ and 20,23(OH)₂D₃, they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D₃.