1alpha,25-dihydroxyvitamin D(3) (1alpha,25(
OH)(2)D(3)), the active metabolite of
vitamin D, mediates many of its effects through the intranuclear
vitamin D receptor (VDR, NR1I1), that belongs to the large superfamily of
nuclear receptors.
Vitamin D receptor can directly regulate gene expression by binding to
vitamin D response elements (VDREs) located in promoter or enhancer regions of various genes. Although numerous synthetic analogs of 1alpha,25(
OH)(2)D(3) have been analysed for VDR binding and transactivation of VDRE-driven gene expression, the
biologic activity of many naturally occurring metabolites has not yet been analyzed in detail. We therefore studied the transactivation properties of 1alpha,24R, 25-trihydroxyvitamin D(3) (1alpha,24R,25(
OH)(3)D(3)), 1alpha, 25-dihydroxy-3-epi-vitamin D(3) (1alpha,25(OH)(2)-3-epi-D(3)), 1alpha,23S,25-trihydroxyvitamin D(3) (1alpha,23S,25(
OH)(3)D(3)), and 1alpha-hydroxy-23-carboxy-24,25,26,27-tetranorvitamin D(3) (1alpha(
OH)-24,25,26,27-tetranor-23-COOH-D(3);
calcitroic acid) using the human G-361
melanoma cell line. Cells were cotransfected with a VDR expression plasmid and
luciferase reporter gene constructs driven by two copies of the VDRE of either the mouse
osteopontin promoter or the 1alpha,25(
OH)(2)D(3) 24-hydroxylase (CYP24) promoter. Treatment with 1alpha,25(
OH)(2)D(3) or the metabolites 1alpha,24R,25(
OH)(3)D(3), 1alpha,25(OH)(2)-3-epi-D(3), and 1alpha,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 even for
calcitroic acid in the presence of overexpressed VDR. The metabolites that were active in the reporter gene assay also induced expression of CYP24
mRNA in the human keratinocyte cell line HaCaT, although with less potency than the parent
hormone. A
ligand-binding assay based on nuclear extracts from COS-1 cells overexpressing human VDR demonstrated that the metabolites, although active in the reporter gene assay, were much less effective in displacing [(3)H]-labeled 1alpha,25(
OH)(2)D(3) from VDR than the parent
hormone. Thus, we report that several natural metabolites of 1alpha,25(
OH)(2)D(3) retain significant
biologic activity mediated through VDR despite their apparent low affinity for VDR.