Intermittent compressive force induces human mandibular-derived osteoblast differentiation via WNT/β-catenin...

Mechanical force induces an efflux of ATP that regulates osteoblast differentiation. However, the effect of mechanical force-induced ATP efflux on WNT/β-catenin signaling remains unclarified. The aim of this study was to investigate the effect of intermittent compressive force (ICF) and ICF-induced extracellular ATP on osteoblast differentiation via WNT/β-catenin signaling in human mandibular-derived osteoblast precursors (hMOBPs). The hMOBPs were subjected to ICF (1.5 g/cm² , 0.3 Hz) for 20 h. To investigate the role of ATP, Apyrase (0.5 units/mL), an enzyme that hydrolyzes ATP, was added 30 min before ICF was applied. The extracellular ATP levels were measured immediately after ICF was removed. The mRNA expression of osteogenic related genes, including WNT was evaluated via quantitative real time polymerase chain reaction. In vitro mineralization was determined by Alizarin Red S staining. The localization of β-catenin was detected using immunofluorescence staining and lentiviral-TOP-dGFP reporter assay. The results demonstrated that ICF increased ATP efflux and in vitro mineralization by hMOBPs. In addition, OSX, ALP, and WNT3A mRNA expression and β-catenin nuclear translocation increased when ICF was applied. The upregulation of these genes was reduced by Apyrase, suggesting the role of ICF-induced ATP on osteoblast differentiation. Notably, ICF altered the mRNA expression of purinergic 2X receptors (P2XRs). A P2X1R antagonist (NF449) downregulated ICF-induced WNT3A, OSX, and ALP mRNA expression. Moreover, when 25 μM α, β-meATP, a P2X1R agonist, was added, WNT3A, and OSX expression increased. In conclusion, our results demonstrate that ICF-induced ATP enhanced hMOBP differentiation. This enhancement was associated with WNT/β-catenin signaling and P2X1R activation.