Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

Abstract

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 α 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 α 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.

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Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

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We thank P. Chambon for kindly providing ERαKO mice; S. Takezawa, F. Ohtake, M. S. Kim, S. Fujiyama, Y. Mezaki, R. Fujiki, M. Kouzu-Fujita, T. Matsumoto, and Y. Imai for technical assistance; T. Yoshizawa and H. Kawashima (Niigata University) for the kind gift of Max2 expression vector; Eisai Co., Ltd., for the gift of MK4; T. Matsumoto, D. Inoue, and R. Okazaki for helpful discussions; and H. Higuchi and K. Hiraga for manuscript preparation.

This work was supported in part by a Grant-in-Aid for Basic Research Activities for Innovative Bioscience (BRAIN).