Accumulating evidence indicates that menaquinone-4 (MK-4), a vitamin K 2 with four isoprene units, inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colonyforming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture, to learn whether the enhancement of the differentiation of CFU-Fs from progenitor cells might relate to inhibition of osteoclast formation. Human bone marrow cells were grown in -minimal essential medium with horse serum in the presence of MK-4 until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP + ) were considered to have osteogenic potential. MK-4 stimulated the number of CFU-F/ALP + colonies in the presence or absence of dexamethasone. The stimulation was also seen in vitamin K 1 treatment. These cells had the ability to mineralize in the presence ofglycerophosphate.In contrast, both MK-4 and vitamin K 1 inhibited 1,25 dihydroxyvitamin D 3 -induced osteoclast formation and increased stromal cell formation in human bone marrow culture. These stromal cells expressed ALP and Cbfa1. Moreover, both types of vitamin K treatment decreased the expression of receptor activator of nuclear factor B ligand/osteoclast differentiation factor (RANKL/ODF) and enhanced the expression of osteoprotegerin/osteoclast inhibitory factor (OPG/OCIF) in the stromal cells. The effective concentrations were 1·0 µM and 10 µM for the expression of RANKL/ODF and OPG/OCIF respectively.Vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.
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.
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.
It has been reported that vitamin K2 (menaquinone-4) promoted 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-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)2D3 and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)2D3 or 25 microM 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 microM), whereas it was almost not inhibited by 1,25(OH)2D3 (0.1-10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 microM epoxide in the presence or absence of warfarin and 1,25(OH)2D3. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5-25 microM), whereas it was not affected by 1,25(OH)2D3 (0.1-1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)2D3 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)2D3. 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)2D3 caused mineralization in cooperation with vitamin K2 was clarified.
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