LIPUS Induces NFATc1 Nuclear Translocation via P2X7 in Osteoblasts

Objectives: Low-intensity pulsed ultrasound (LIPUS) is characterized by ultrasound power of less than 100 mW/cm², no return of sonic energy to heat, and no cavitation effect, and noninvasively promotes bone formation. It has been reported that continuous LIPUS stimulation induces osteoblast differentiation via the P2X7 receptor (Manaka et al., FEBS letters, 2015). The activation of P2X7 promotes the osteogenic potential of osteoblasts via intracellular Ca2+ elevation and activation of the transcription factor nuclear factor of activated T cell c1 (NFATc1) (Grol et al., J Cell Science, 2013). To elucidate the detailed molecular mechanism of the effect of LIPUS stimulation on osteoblast differentiation, we focused on NFATc1 activation. We examined the effect of LIPUS stimulation on nuclear translocation of NFATc1 in this study.
Methods: MC3T3-E1 cells were plated on a 6-well plate， incubated in the presence or absence of competitive P2X7 selective antagonist (A804598)， and then stimulated with LIPUS (1.5 MHz；pulsed-wave mode intensity of 30 mW/cm²) for 20 min using OSTEOTRON V. Samples were fixed after LIPUS stimulation, and the nuclear translocation of NFATc1 was examined by fluorescent immunostaining.
Results: LIPUS also enhanced the nuclear translocation of NFATc1. A804598 blocked the stimulatory effects of LIPUS on NFATc1 nuclear translocation.
Conclusions: These results suggest that LIPUS induces NFATc1-mediated osteoblast differentiation.