Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells

Low-intensity pulsed ultrasound (LIPUS) is known to accelerate bone regeneration, but the precise cellular mechanism is still unclear. The purpose of this study was to determine the effect of LIPUS on the differentiation of pluripotent mesenchymal cell line C2C12. The cells were cultured in differentiation medium with or without the addition of LIPUS stimulation. The ultrasound signal consisted of 1.5 MHz at an intensity of 70 mW/cm2 for 20 min for all cultures. To verify the cell lineage after LIPUS stimulation, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (Runx2, Msx2, Dlx5, AJ18), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (C/EBP, PPARgamma) was studied using real-time polymerase chain reaction analysis. The protein expression of Runx2 and activated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) were performed using Western blotting. The mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased markedly by the LIPUS stimulation, whereas the expression of MyoD, C/EBP, and PPARgamma was drastically decreased. In the Western blot analysis, LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. Our study demonstrated that LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK.     

AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway.