1,25-dihydroxyvitamin D-3 promotes vitamin K-2 metabolism in human osteoblasts

It has been reported that vitamin K-2 (menaquinone-4) promoted 1,25-dihydroxyvitamin D-3 (1,25 (OH)(2)D-3)-induced mineralization and enhanced gamma -carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of gamma -glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)(2)D-3 and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)(2)D-3 or 25 muM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5-25 muM), whereas it was almost not inhibited by 1,25(OH)(2)D-3 (0.1-10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 muM epoxide in the presence or absence of warfarin and 1,25(OH)(2)D-3. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5-25 muM), whereas it was not affected by 1,25(OH)(2)D-3 (0.1-1 muM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)(2)D-3 on the activity of epoxide reductase. However, the activity of epoxidase, that is the gamma -glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)(2)D-3. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)(2)D-3 caused mineralization in cooperation with vitamin K-2 was clarified.