Natural metabolites of 1 alpha,25-dihydroxyvitamin D-3 retain biologic activity mediated through the vitamin D receptor

1 alpha,25-dihydroxyvitamin D-3 (1 alpha,25(OH)(2)D-3), the active metaboli te of vitamin D, mediates many of its effects through the intranuclear vita min D receptor (VDR, NR1I1), that belongs to the large superfamily of nucle ar receptors. Vitamin D receptor can directly regulate gene expression by b inding to vitamin D response elements (VDREs) located in promoter or enhanc er regions of various genes. Although numerous synthetic analogs of 1 alpha ,25(OH)(2)D-3 have been analysed for VDR binding and transactivation of VDR E-driven gene expression, the biologic activity of many naturally occuring metabolites has not yet been analyzed in detail. We therefore studied the t ransactivation properties of 1 alpha,24R,25-trihydroxyvitamin D-3 (1 alpha, 24R,25(OH)(3)D-3), 1 alpha,25-dihydroxy-3-epi-vitamin D-3 (1 alpha,25(OH)(2 )-3-epi-D-3), 1 alpha,23S,25-trihydroxyvitamin D-3 (1 alpha,23S,25(OH)(3)D- 3), and 1 alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D-3 (1 alpha (OH)-24,25,26,27-tetranor-23-COOH-D-3; calcitroic acid) using the human G-3 61 melanoma cell line. Cells were cotransfected with a VDR expression plasm id and luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse osteopontin promoter or the 1 alpha,25(OH)(2)D-3 24-hy droxylase (CYP24) promoter. Treatment with 1 alpha,25(OH)(2)D-3 or the meta bolites 1 alpha,24R,25(OH)(3)D-3, 1 alpha,25(OH)(2)-3-epi-D-3, and 1 alpha, 23S,25(OH)(3)D-3 resulted in transactivation of both constructs in a time- and dose-dependent manner, and a postitive regulatory effect was observed e ven for calcitroic acid in the presence of overexpressed VDR. The metabolit es that were active in the reporter gene assay also induced expression of C YP24 mRNA in the human keratinocyte cell line HaCaT, although with less pot ency than the parent hormone. A ligand-binding assay based on nuclear extra cts from COS-1 cells overexpressing human VDR demonstrated that the metabol ites, although active in the reporter gene assay, were much less effective in displacing [H-3]-labeled 1 alpha,25(OH)(2)D-3 from VDR than the parent h ormone. Thus, we report that several natural metabolites of 1 alpha,25(OH)( 2)D-3 retain significant biologic activity mediated through VDR despite the ir apparent low affinity for VDR. (C) 2000 Wiley-Liss, Inc.