Steroid and Xenobiotic Receptor SXR Mediates Vitamin K2-activated Transcription of Extracellular Matrix-related Genes and Collagen Accumulation in Osteoblastic Cells*♦

Tomoe Ichikawa,Kuniko Horie-Inoue,Kazuhiro Ikeda,Bruce Blumberg,Satoshi Inoue

doi:10.1074/jbc.m600896200

Tomoe Ichikawa, Kuniko Horie-Inoue + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m600896200

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Abstract

Vitamin K2 is a critical nutrient required for blood coagulation. It also plays a key role in bone homeostasis and is a clinically effective therapeutic agent for osteoporosis. We previously demonstrated that vitamin K2 is a transcriptional regulator of bone marker genes in osteoblastic cells and that it may potentiate bone formation by activating the steroid and xenobiotic receptor, SXR. To explore the SXR-mediated vitamin K2 signaling network in bone homeostasis, we identified genes up-regulated by both vitamin K2 and the prototypical SXR ligand, rifampicin, in osteoblastic cells using oligonucleotide microarray analysis and quantitative reverse transcription-PCR. Fourteen genes were up-regulated by both ligands. Among these, tsukushi, matrilin-2, and CD14 antigen were shown to be primary SXR target genes. Moreover, collagen accumulation in osteoblastic MG63 cells was enhanced by vitamin K2 treatment. Gain- and loss-of-function analyses showed that the small leucine-rich proteoglycan, tsukushi, contributes to vitamin K2-mediated enhancement of collagen accumulation. Our results suggest a new function for vitamin K2 in bone formation as a transcriptional regulator of extracellular matrix-related genes, that are involved in the collagen assembly.

Highlights

One of the major known functions of vitamin K is the posttranslational modification of vitamin K-dependent proteins containing ␥-carboxyglutamic acid (Gla) residues, most of which are related to coagulation
Our findings indicate that vitamin K2 activates SXR to regulate the transcription of extracellular matrix-related genes that may contribute to collagen assembly
Both vitamin K2 and RIF increased the transcriptional activity of the trimerized SXR response elements derived from the CYP3A4 promoter in MG63 cells transiently transfected with FLAG-SXR or FLAG-VP16CSXR (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—MG63 human osteosarcoma cells, 293T, and COS1 cells were grown in Dulbecco’s modified Eagle’s medium supplement with 10% fetal bovine serum (FBS), 50 units/ml penicillin, and 50 ␮g/ml streptomycin. Preparation of cRNA—Total RNA was extracted from MG63 cells stably expressing FLAG-VP16C-SXR treated with vehicle (0.1% ethanol), MK-4 (10 ␮M), or RIF (10 ␮M) for 48 h. Twenty ␮g of each cRNA sample was fragmented by mild alkaline treatment, at 94 °C for 35 min in fragmentation buffer (200 mM Tris acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) and used to prepare 400 ␮l of master hybridization mix (0.1 mg/ml herring sperm DNA (Promega), 0.5 mg/ml of acetylated bovine serum albumin in hybridization buffer containing 100 mM MES, 1 M [Naϩ], 20 mM EDTA, 0.01% Tween 20). Cells were transfected with siRNA (70 nM) using GeneSilencer reagent (Genlantis, San Diego, CA) for 48 h, and further maintained in the culture medium containing 10% dextran-charcoalstripped FBS with or without ligand stimulation for indicated times. All data are presented in the text and figures as the mean Ϯ S.D

RESULTS

Likely ortholog of chicken tsukushi

DISCUSSION