FIGURE 1.

Mechanical stretch induces activation of ERK1/2, JNK1/2, and p38α via Ca²⁺ influx in MC3T3-E1 cells. A, activation of MAPKs in response to different magnitudes of mechanical stretch. MC3T3-E1 cells were stimulated with the indicated durations of cyclic stretch for 20 min. The kinase activity of MAPKs was detected by immunoblotting (IB) using P-ERK, P-JNK, and P-p38 antibodies. B and C, MC3T3-E1 cells were pretreated with or without U0126 (10 μm) for 60 min. The cells were then loaded with or without cyclic stretch (1%) for 6 h. Quantitative PCR analysis was then performed as under “Experimental Procedures.” Relative expression levels were normalized on the basis of the expression of GAPDH mRNA. Error bars indicate mean ± S.E. *, p < 0.05, Student's t test. D and E, MC3T3-E1 cells were pretreated with or without SP600125 (10 μm) or SB203580 (10 μm) for 60 min. The cells were then loaded with or without cyclic stretch (1%) for 6 h. The expression levels of Col1a mRNA were measured by quantitative RT-PCR. Relative expression levels were normalized on the basis of the expression of GAPDH mRNA. Error bars indicate mean ± S.E. *, p < 0.05, Student's t test. F and G, mouse calvarium-derived primary osteoblasts were prepared and subjected to quantitative PCR analysis as in B and C. H and I, mouse calvarium-derived primary osteoblasts were prepared and subjected to quantitative PCR analysis as in D and E. J, identification of MAPK isoforms activated by mechanical stretch. MC3T3-E1 cells were transfected with single or combination siRNA as indicated. After 72 h, the cells were subjected to cyclic stretch loading (12%) for 30 min. The lysates were immunoblotted with the indicated antibodies. C indicates control. K, MC3T3-E1 cells were subjected to cyclic stretch for the indicated periods in the presence or absence of 5 mm EGTA. The lysates were immunoblotted with the indicated antibodies. The data in A, J, and K represent one of at least three experiments.