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.