Production of 22-Hydroxy Metabolites of Vitamin D3 by Cytochrome P450scc (CYP11A1) and Analysis of Their Biological Activities on Skin Cells

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D-3, producing 20S-hydroxyvitamin D-3 [20(OH)D-3] and 20S,23-dihydroxyvitamin D-3 [20,23(OH)(2)D-3] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D-3, 17,20-dihydroxyvitamin D-3, and 17,20,23-trihydroxyvitamin D-3. In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D-3 and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D-3 [22(OH)D-3] and 20S, 22-dihydroxyvitamin D-3 [20,22(OH)(2)D-3]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D-3 to 20,22(OH)(2)D-3. The 20,22(OH)(2)D-3 could also be produced from 20(OH) D3 and was metabolized to a trihydroxyvitamin D-3 product. We compared the biological activities of these new derivatives with those of 20(OH)D-3, 20,23(OH)(2)D-3, and 1 alpha,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3]. 1,25(OH)(2)D-3, 20(OH)D-3, 22(OH)D-3, 20,23(OH)(2)D-3, and 20,22(OH)(2)D-3 significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)(2)D-3, 20,22(OH)(2)D-3, 20(OH)D-3, and 1,25(OH)(2)D-3, with 22(OH)D-3 having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)(2)D-3. All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D-3 and 20,22(OH)(2)D-3 having less effect than 1,25(OH)(2)D-3 and 20(OH)D-3. Thus, we have identified 22(OH)D-3 and 20,22(OH)(2)D-3 as products of CYP11A1 action on vitamin D-3 and shown that, like 20(OH)D-3 and 20,23(OH)(2)D-3, 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 D-3.