Vitamin K 2 Enhances Osteocalcin Accumulation in the Extracellular Matrix of Human Osteoblasts In Vitro

The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated by using a human intact osteocalcin‐specific assay system. Human osteoblasts produced osteocalcin by treatment with 10 −9 M 1,25‐dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) for 20 days in culture. With the addition of vitamin K 2 (1.5–5.0 μM), osteocalcin accumulation in the extracellular matrix of the osteoblasts was increased, but the osteocalcin content in the conditioned medium decreased, in comparison with that treated with 10 −9 M 1,25(OH) 2 D 3 alone. The enhancement of osteocalcin accumulation induced by vitamin K 2 was dependent on the duration of the treatment. The vitamin K 2 plus 1,25(OH) 2 D 3 ‐induced osteocalcin accumulation was blocked by the addition of warfarin 2 days before the vitamin treatment. At that time, warfarin significantly reduced the mineralization by osteoblasts in vitro. Osteocalcin accumulated in the extracellular matrix was almost completely precipitated by a low concentration of hydroxyapatite, 10 mg/ml. Moreover, the γ‐carboxyglutamic acid (Gla)‐containing osteocalcin level was increased by the vitamin K 2 plus 1,25(OH) 2 D 3 treatment. These results proved that vitamin K 2 increased Gla‐containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. Vitamin K 2 also enhanced the 1,25(OH) 2 D 3 ‐induced osteocalcin mRNA level, but vitamin K 2 alone did not show osteocalcin mRNA expression. We thus demonstrated that vitamin K 2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH) 2 D 3 in human osteoblasts in culture.