TY - JOUR T1 - Purified Mouse CYP27B1 Can Hydroxylate 20,23-Dihydroxyvitamin D<sub>3</sub>, Producing 1α,20,23-Trihydroxyvitamin D<sub>3</sub>, Which Has Altered Biological Activity JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1553 LP - 1559 DO - 10.1124/dmd.110.034389 VL - 38 IS - 9 AU - Edith K. Y. Tang AU - Wei Li AU - Zorica Janjetovic AU - Minh N. Nguyen AU - Zhao Wang AU - Andrzej Slominski AU - Robert C. Tuckey Y1 - 2010/09/01 UR - http://dmd.aspetjournals.org/content/38/9/1553.abstract N2 - 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. ER -