Vitamin K2 promotes 1alpha,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3). Vitamin K1 and K2, but not vitamin K3, at 2.5 microM enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K2 (2-methyl-3-all-trans-tetraphenyl-1, 4-naphthoquinone : menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10(-9) M of 1,25(OH)2D3 for 20 or 25 days after confluence. Vitamin K2 promoted the 1,25(OH)2D3-induced mineralization, but slightly inhibited the 1,25(OH)2D3-induced ALP activity. Moreover, vitamin K2 enhanced the 1,25(OH)2D3-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)2D3 treatment. However, vitamin K2 alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1, 25(OH)2D3-treated cells was enhanced by vitamin K2 treatment, but vitamin K2 alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)2D3-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1, 25(OH)2D3 plus vitamin K2-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)2D3-induced mineralization promoted by vitamin K2 was probably due to the enhanced accumulation of osteocalcin induced by vitamin K2 in the cell layer. However, we concluded that the mineralization induced by vitamin K2 alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K2 in the presence of 1,25(OH)2D3 was different from that of vitamin K2 alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.