Low-Intensity Pulsed Ultrasound Induced Osteoblast Differentiation Mediated by the PYK2-ERK2 Signaling in MC3T3-E1 Cells.

Low-intensity pulsed ultrasonic (LIPUS) is a noninvasive force promoting bone regeneration as mechanical stimulation. Integrins mediate numerous intracellular signaling pathways, including mitogen-activated protein kinase (MAPK) and calcium influx through focal adhesion. A non-receptor tyrosine kinase of proline-rich tyrosine kinase 2 (PYK2) associates with integrins at focal adhesions. A previous study reported that intracellular Ca²⁺ levels regulate the activation of PYK2 induced by mechanical strain in osteoblasts. However, the roles of PYK2 on the expression of extracellular matrix (ECM) proteins involved in osteogenesis and osteoblast differentiation induced by LIPUS remain unclear. We aimed to investigate the roles of PYK2 on the expression of transcription factors runt-related transcription factor 2 (Runx2) and osterix, which regulate osteoblast differentiation, ECM proteins, and the downstream intracellular signaling pathway, including PYK2 and focal adhesion kinase (FAK) in MC3T3-E1 cells when treated with LIPUS. LIPUS enhanced the phosphoryla-tion of PYK2 and extracellular signal-regulated kinase2 (ERK2) and induced the expression of Runx2, osterix, type I collagen, osteocalcin, and calcium content in ECM. However, siPYK2 and PYK2 inhibitor PF431396 suppressed these stimulatory effects of LIPUS. These results suggest that LIPUS induces osteoblast differentiation mediated by the PYK2-ERK2 signaling in MC3T3-E1 cells.