Vitamin K2 Regulation of Bone Homeostasis Is Mediated by the Steroid and Xenobiotic Receptor SXR

Michelle M Tabb,Aixu Sun,Changcheng Zhou,Felix Grün,Jody Errandi,Kimberly Romero,Hang Pham,Satoshi Inoue,Shyamali Mallick,Min Lin,Barry M Forman,Bruce Blumberg

doi:10.1074/jbc.m303136200

Abstract

Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for gamma-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis.

Highlights

Vitamin K2 is a critical nutrient required for blood clotting that plays an important role in bone formation
To ascertain whether SXR might be mediating the effects of vitamin K2, we first determined whether SXR was expressed in a panel of osteosarcoma cell lines using RT-PCR
Similar to its role in promoting blood clotting, vitamin K2 is an essential cofactor for ␥-carboxylase, an enzyme that catalyzes the conversion of specific glutamic acid residues to glutamic acid to ␥-carboxyglutamyl (Gla) residues

Summary

EXPERIMENTAL PROCEDURES

SXR Detection by RT-PCR—HOS, MG-63, Saos-2, LS180, and HeLa cells were cultured in phenol red-free DMEM supplemented with 10% resin charcoal-stripped FBS. Quantitative Real Time RT-PCR Analysis of Bone Biomarker Genes in Osteosarcoma Cell Lines—Human osteosarcoma cell lines HOS, MG63, and Saos-2 were obtained from American Type Culture Collection (Manassas, VA) and cultured in phenol red-free DMEM supplemented with 10% resin charcoal-stripped FBS. The human cell lines HOS, MG-63, Saos-2, LS180, and HeLa were cultured in phenol red-free DMEM supplemented with 10% resin charcoal-stripped FBS. Total RNA was isolated, reverse transcribed, and analyzed using the following mouse primer sets: MGP (F, 5Ј-TCTCACGAAAGCATGGAGTC-3Ј; R, 5Ј-ATCTCGTAGGCAGGCTTGTT-3Ј), OPG (F, 5ЈCTGCTGAAGCTGTGGAAACA-3Ј; R, 5Ј-AAGCTGCTCTGTGGTGAGGT-3Ј), and glyceraldehyde-3-phosphate dehydrogenase (F, 5Ј-AACTTTGGCATTGTGGAAGG-3Ј; R, 5Ј-GGATGCAGGGATGATGTTCT-3Ј). All data are presented in the text and figures as the mean Ϯ S.E

RESULTS

DISCUSSION

Bruce Blumberg