Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms

Abstract

The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that gamma-carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-kappaB ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formation.

Osteocyte differentiation and the formation of an interconnected cellular network in vitro

“…In particular, a soft 2D substrate (0.3 kPa) was shown to lead to osteocyte differentiation (Mullen et al, 2013), whereas more rigid 2D substrates (40 kPa) favour osteoblast differentiation (Engler et al, 2004). Osteoblast to osteocyte differentiation has been previously elicited in human primary osteoblast-like cells within 3D collagen gels, with 10 % of cells becoming dendritic and expressing osteocyte-specific gene E11 after 28 d (Atkins et al, 2009). Primary mouse calvarial and MC3T3-E1 cells formed dendrites and expressed osteocyte-specific genes (DMP1, Sost, Phex) after 35 d on type I collagen gels with osteogenic supplements (β-glycerophosphate and ascorbic acid) (Uchihashi et al, 2013).…”

Evidence for the dysregulated expression of TWIST1, TGFβ1 and SMAD3 in differentiating osteoblasts from primary hip osteoarthritis patients

“…Mineral composition analysis was performed as described 22 , but with some modifications. Media were removed and cells washed with 200 mL of dH 2 O for 5 min at room temperature.…”

Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation

“…Five hours later the medium was changed to ·-MEM containing 10% FBS plus antibiotics and cells were treated with TNF· (MC3T3) or RANKL (RAW264.7) to stimulate NF-κB activity, or with TGFß (1 ng/ml) or BMP-2 (0.5 μg/ml) to stimulate SMAD activity and treated with or without TNF·. Some cultures received warfarin (20 μM) in order to suppress Á-carboxylation activity as previously reported (36). Parallel groups received vehicle or MK-7 (0.01-10 μM).…”