Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin

Abstract

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Keywords: Haptoglobin; Osteoclast differentiation; Osteoclasts; Transcription factors; Zscan10.