The K vitamins are a group of fat-soluble vitamins that occur in two natural forms: phyloquinones (K1) and menaquinones (K2). Vitamin K (VK) has classically been associated with blood coagulation (31
). In its canonical role, VK serves as a coenzyme for VK-dependent carboxylase. This enzyme converts glutamate residues into γ-carboxyglutamate (Gla) residues in VK-dependent proteins, such as prothrombin, and factors IX and X (6
). Such VK-induced protein modification also occurs in osteocalcin (7
) and matrix Gla protein (MGP) (22
). Thus, VK may exert beneficial effects on bone formation and remodeling. In fact, animal studies suggest that VK deficiency results in a reduction in bone mass together with hypocarboxylation of osteocalcin (25
Clinically, the most common form of K2, menaquinone 4 (MK4), has been shown to prevent bone fractures (3
). This osteoprotective effect is more pronounced in K2 than in K1, and hence MK4 has been used to treat osteoporotic patients in Japan (9
). However, the bone phenotypic abnormalities of mice deficient in osteocalcin and MGP do not fully support the classical view that the osteoprotective action of VK is the result of the modification of skeletal proteins. These mice, which are genetically deficient for osteocalcin or MGP, exhibited bone mass increases instead of losses (4
). This suggests that the osteoprotective action of VK is mediated by another pathway.
MK4 recently has been shown to act as a ligand for the steroid and xenobiotic receptor (SXR) in human osteoblastic cells (33
). It transcriptionally regulates gene expression and represents a new pathway of VK action. The SXR and its mouse homolog, the pregnane X receptor (PXR), respond to xenobiotics and pregnenones. PXR and SXR are members of the nuclear receptor (NR) gene superfamily and bind to specific DNA elements (PXR-responsive elements [PXRE]) as heterodimers with one of the retinoid X receptor (RXR) subtypes (α, β, and γ) (1
). Like the other NR members, ligand binding to PXR/SXR induces dissociation of corepressors and recruitment of coactivators for ligand-induced transactivation in the target gene promoters (17
). Thus, these findings suggest that it is feasible that the osteoprotective VK action mediates its transcriptional control of the VK target genes via PXR/SXR. In fact, several PXR/SXR genes recently have been shown to transcriptionally respond to VK (8
To test this idea, we screened VK target genes in an osteoblastic cell line (MC3T3-E1) treated with MK4 with two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A prime osteoblastogenic factor, Msx2, was identified, and a PXRE was located in its gene promoter. MK4 interacted with the PXRE via PXR/RXRα binding in vivo and in vitro. Osteoblast genesis in MC3T3-E1 cells was induced by MK4, but knockdown of Msx2 by RNA interference (RNAi) abrogated the MK4 effect. The present study suggests that Msx2 is a target gene for VK-activated PXR/SXR. It implies that the osteoprotective VK action takes place, at least in part, on a genomic level by stimulating osteoblast differentiation through Msx2 gene induction.