Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor ␣ heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor ␣ to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.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 ␥-carboxyglutamate (Gla) residues in VK-dependent proteins, such as prothrombin, and factors IX and X (6,10,29). Such VK-induced protein modification also occurs in osteocalcin (7,21) 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 (9,11,28). 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 ne...