DEPTOR regulates osteogenic differentiation via inhibiting MEG3-mediated activation of BMP4 signaling and is involved in osteoporosis

Si Chen,Lingfei Jia,Yunfei Zheng,Shan Zhang,Yongsheng Zhou

doi:10.1186/s13287-018-0935-9

Abstract

BackgroundThe mammalian target of rapamycin (mTOR) pathway plays a significant role in osteogenic differentiation and bone maintenance. As the only known endogenous inhibitor of mTOR function, DEP domain containing mTOR interacting protein (DEPTOR) is potentially involved in stem cell differentiation, although the pathophysiological significance and its molecular mechanisms remain unclear. The present study aimed to elucidate the effects of DEPTOR on the progress of osteoporosis and investigate the underlying molecular mechanisms of osteogenic regulation.MethodsAn ovariectomy mouse model with decreased bone formation and osteogenic induction with bone marrow mesenchymal stem cells (BMSCs) were used to investigate the relationship between DEPTOR and osteogenic events. A loss-of-function investigation was then performed to explore the role of DEPTOR in the osteogenic differentiation of BMSCs both in vitro and in vivo. Finally, long noncoding RNA (lncRNA) and mRNA sequences were investigated to reveal the underlying mechanisms of DEPTOR in osteogenic regulation. RNA interference, western blotting, and chromatin immunoprecipitation assays were performed for further mechanistic determination.ResultsThe results indicated that DEPTOR contributes to the progress of osteoporosis, and higher expression of Deptor was observed in osteoporotic bones. The expression of DEPTOR was reduced during the osteogenic differentiation of BMSCs, and knockdown of DEPTOR promoted BMSC osteogenesis in vitro and in vivo. lncRNA and mRNA sequences indicated that knockdown of DEPTOR upregulated the expression of maternally expressed 3 (nonprotein coding) (MEG3), which subsequently activated bone morphogenetic protein 4 (BMP4) signaling. Furthermore, DEPTOR could bind to a specific region (− 1000 bp ~ 0) of the MEG3 promoter to regulate its transcription, and inhibition of MEG3 reduced BMP4 activation triggered by DEPTOR knockdown.ConclusionsTaken together, our study revealed a novel function of DEPTOR in osteogenic differentiation by inhibiting MEG3-mediated activation of BMP4 signaling, which suggested that DEPTOR could be a therapeutic target for bone loss diseases and skeletal tissue regeneration.

Highlights

The mammalian target of rapamycin pathway plays a significant role in osteogenic differentiation and bone maintenance
Cells between passages 3 and 5 were utilized for the experiments and all of the in-vitro experiments were repeated in triplicate. Human bone marrow mesenchymal stem cell (hBMSC) were cultured in proliferation medium (PM) containing fresh α-MEM, 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) antibiotics at 37 °C in an incubator with an atmosphere consisting of 95% air and 5% CO2, with 100% relative humidity
High DEP domain containing mammalian target of rapamycin (mTOR) interacting protein (DEPTOR) levels are associated with reduced bone formation in vivo To explore the role of DEPTOR in biological events, we established its global expression profile in Bone marrow mesenchymal stem cell (BMSC) with DEPTOR knockdown, where NC was utilized as the scramble control

Summary

Introduction

The mammalian target of rapamycin (mTOR) pathway plays a significant role in osteogenic differentiation and bone maintenance. As the only known endogenous inhibitor of mTOR function, DEP domain containing mTOR interacting protein (DEPTOR) is potentially involved in stem cell differentiation, the pathophysiological significance and its molecular mechanisms remain unclear. Osteoporosis is one of the most severe skeletal diseases in the elderly population and postmenopausal women [1] It is characterized by decreased bone mineral density and destruction of the microarchitectural bone structure, resulting in higher susceptibility to bone fracture [2, 3]. Emerging studies have demonstrated that mTOR signaling plays a critical role in the osteogenic differentiation of MSCs [10,11,12]. Recent genetic research in mice indicated that impairment of mTORC1 function resulted in increased osteogenesis, while impairment of mTORC2 function led to reduced osteogenesis [12]

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