I–2 Osteoblast Responses to Low-Intensity Pulsed Ultrasound (LIPUS) are Attenuated by Osteopontin Expression

Objective: Osteopontin (OPN) is a secretory protein considered to be an important marker of osteoblastic cell differentiation. We previously reported that OPN is highly expressed in osteoblasts when their differentiation was inhibited by treatment with lipopolysaccharide (LPS) or unsaturated fatty acids. Here, we examine how OPN expression affects response to LIPUS by osteoblasts. Materials and Methods: The Tet-on inducible expression system was used in MC3T3-E1, a mouse osteoblastic cell line, to produce a MC3T3-E1 Tet-on OPN cell line, which can be induced to overexpress OPN by doxycycline treatment. This cell line was stimulated by LIPUS for 20 minutes. In another experiment, MC3T3-E1 cells were pretreated with recombinant OPN proteins for 6 hours and stimulated by LIPUS. The phosphorylation levels of focal adhesion kinase (FAK) in all cells were evaluated by western blotting. Finally, to examine how endogenous OPN affects cellular responses to LIPUS, differentiated MC3T3-E1 cells were transfected with OPN-specific siRNAs, or pretreated with anti-OPN neutralizing antibodies before LIPUS irradiation. Results: LIPUS-induced FAK phosphorylation was repressed by the induction of OPN expression in MC3T3 -E1 Tet-on OPN cells. FAK phosphorylation was similarly repressed by treatment with recombinant OPN. Conversely, phosphorylation levels of FAK were increased by transfection with OPN siRNA or treatment with OPN-neutralizing antibodies in LIPUS-stimulated MC3T3-E1 cells. Discussion: Osteoblast responses to LIPUS were attenuated by overexpression of OPN or by treatment with recombinant OPN. Therapeutic effects of LIPUS on osteoblasts may be attenuated by OPN expression.