title: Cyclic stretch enhances bone morphogenetic protein-2-induced osteoblastic differentiation through the inhibition of Hey1.
abstract: Substantial evidence has indicated that osteoblastic differentiation may be regulated by mechanical loads or bone morphogenetic protein-2 (BMP-2). BMP-2-induced in vivo osteogenesis can be significantly enhanced in the presence of mechanical stimuli, revealing the therapeutic potential of the combined application of BMP-2 and mechanical loads in clinical bone diseases (e.g., bone fractures and osteoporosis); however, the underlying mechanisms remain elusive. In this study, we found that cyclic stretch or BMP-2 alone increased the expression of osteoblastic differentiation markers, including alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2), as shown by RT-qPCR, western blot analysis and ALP activity test. Furthermore, our results revealed that cyclic mechanical stretch with 10% elongation at 0.1 Hz significantly enhanced the BMP-2-induced upregulation of ALP and Runx2 expression in osteoblast-like MC3T3-E1 cells. Cyclic stretch also inhibited the BMP-2-induced upregulation of Hes-related family bHLH transcription factor with YRPW motif 1 (Hey1, measured by RT-qPCR and immunofluorescence staining), a potent negative regulator of osteogenesis. Moreover, the transient transfection of a Hey1 expression plasmid (pcDNA3.1-Hey1) significantly reversed the effects of cyclic stretch on the BMP-2-induced upregulation of differentiation markers in the MC3T3-E1 cells. This revealed the importance of Hey1 in modulating BMP-2-induced osteoblastic differentiation in response to cyclic stretch. Taken together, our results demonstrated that cyclic stretch enhanced the BMP-2‑induced osteoblastic differentiation through the inhibition of Hey1. The present study broadens our fundamental knowledge of osteoblastic mechanotransduction and also sheds new insight into the mechanisms through which the combined application of BMP-2 and mechanical load promotes osteogenesis.
authors.affiliation: State Key Laboratory of Military Stomatology, Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
authors.forename: Zhaobin
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Military Stomatology, Department of Operative Dentistry and Endodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yin
authors.affiliation: Department of Stomatology, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110840, P.R. China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
authors.forename: Da
authors.initials: D
authors.lastname: Jing
authors.affiliation: State Key Laboratory of Military Stomatology, Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
authors.forename: Xue
authors.initials: X
authors.lastname: Feng
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000502:physiology; D010006:Osteoblasts / Q000378:metabolism* / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000502:physiology
chemical_list: D000943:Antigens, Differentiation; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D018797:Cell Cycle Proteins; D050659:Core Binding Factor Alpha 1 Subunit; C120420:Hey1 protein, mouse; C493716:Runx2 protein, mouse; D014157:Transcription Factors; D000469:Alkaline Phosphatase