Purified Mouse CYP27B1 Can Hydroxylate 2023-Dihydroxyvitamin D3 Producing 1α2023-Trihydroxyvitamin D3 Which Has Altered Biological Activity

摘要

20,23-Dihydroxyvitamin D3 [20,23(OH)2D3] is a biologically active metabolite produced by the action of cytochrome P450scc (CYP11A1) on vitamin D3. It inhibits keratinocyte proliferation, stimulates differentiation, and inhibits nuclear factor-κB activity, working as a vitamin D receptor agonist. We have tested the ability of purified mouse 25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1) to add a 1α-hydroxyl group to this vitamin D analog and determined whether this altered its biological activity. 20,23(OH)2D3 incorporated into phospholipid vesicles was converted to a single product by CYP27B1, confirmed to be 1α,20,23-trihydroxyvitamin D3 [1,20,23(OH)3D3] by mass spectrometry and NMR. The 20,23(OH)2D3 was a relatively poor substrate for CYP27B1 compared with the normal substrate, 25-hydroxyvitamin D3, displaying a 5-fold higher Km and 8-fold lower kcat value. Both 20,23(OH)2D₃ and 1,20,23(OH)₃D₃ decreased neonatal human epidermal keratinocyte proliferation, showing significant effects at a lower concentration (0.1 nM) than that seen for 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] 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)₂D₃ at 24 h. Addition of a 1α-hydroxyl group to 20,23(OH)₂D₃ greatly enhanced its ability to stimulate the expression of the CYP24 gene but not to the extent seen with 1,25(OH)₂D₃. This study shows that purified CYP27B1 can add a 1α-hydroxyl group to 20,23(OH)₂D₃ with the product showing altered biological activity, especially for the stimulation of CYP24 gene expression.