Comprehensive Analysis of Transcriptional Regulation by Steroid and Xenobiotic Receptor in Osteoblastic Cells Using RNA-Sequencing

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 338-365-Metabolic & Stress Receptors in Energy Homeostasis
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-355
Kotaro Azuma*1, Tomohiko Urano2, Yasuyoshi Ouchi2 and Satoshi Inoue3
1University of Tokyo, Tokyo, Japan, 2Graduate School of Medicine, the University of Tokyo, Tokyo, Japan, 3Univ of Tokyo Hosp, Tokyo, Japan

Vitamin K2 (menatetrenone: MK4) is used as a drug for osteoporosis in Asian countries and shown to prevent bone fracture. As a novel mechanism of vitamin K action on bone tissues, we demonstrated that MK4 serves as a ligand for steroid and xenobiotic receptor (SXR) (1), and identified SXR target genes, such as tsukushi, using osteoblastic cells (2). We further revealed that mice lacking pregnane X receptor (PXR), the murine ortholog of human SXR, displayed osteopenic phenotype (3), indicating involvement of SXR/PXR signaling in bone metabolism in vivo.


Recently, RNA products that are not translated into proteins have been noticed to play biological roles. The aim of the present study is to investigate SXR-mediated transcriptional regulation in osteoblastic cells by using RNA-sequencing in search for target transcripts including novel noncoding RNAs.


We established human MG63 osteoblastic cells overexpressing SXR constitutively. These cells were stimulated by 10 μM rifampicin as an SXR ligand, 10 μM MK4, or ethanol (vehicle) for 24 h, then RNA was isolated from these cells. RNA-sequencing was carried out with the Illumina Genome Analyzer IIx platform (GAIIx) using 41-bp single read. These data were mapped on genome database using Top Hat program, then assembled by Cufflikns program. We employed –g option to include novel transcripts into analysis. Next, we focused on transcripts induced by both rifampicin and MK4 in intergenic region. Among intergenic transcripts, 640 transcripts were significantly induced by MK4 treatment. Then, we selected 108 transcripts which had more than 20 FPKM (fragments per kilobase of exon per million mapped fragments). Eight transcripts of them were significantly induced also by treatment of rifampicin. Among them, we finally focused 3 transcripts as candidates of noncoding RNA induced by SXR ligands. One locating in chromosome 8 is a known non-coding RNA which is described in NONCODE database. Other two RNAs locating in chromosome 11 and 18 were novel ones which are not described in database. Biological significance of these transcripts is investigated by knocking them down.


We identified noncoding RNAs regulated by SXR in osteoblastic cells using RNA-sequencing. Vitamin K may affect bone metabolism by inducing functional non-coding RNA in osteoblasts.

(1) Tabb MM et al. J Biol Chem 2003; 278:43919. (2) Ichikawa T et al. J Biol Chem 2006; 281:16927. (3) Azuma K et al. J Endocrinol 2010; 207:257.

Nothing to Disclose: KA, TU, YO, SI

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