FIGURE 4.

Mechanical stretch enhanced Fn14 expression via the ASK1-JNK pathway. A and B, MC3T3-E1 cells were transfected with siRNA as a negative control or with siRNA for ASK1 (ASK1 #1). After 72 h, the cells were subjected to cyclic stretch stimulation (12%) for 6 h. The expression levels of Fn14 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. C and D, MC3T3-E1 cells were pretreated with dimethyl sulfoxide, SP600125 (10 μm), or SB203580 (10 μm) for 60 min and then stimulated with cyclic stretch (12%) for 6 h. The expression levels of Fn14 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. E and F, mouse calvarium-derived primary osteoblasts were prepared and subjected to quantitative PCR analysis as in C and D. G, MC3T3-E1 cells were pretreated with dimethyl sulfoxide (DMSO), SP600125 (10 μm), or SB203580 (10 μm) for 60 min and then stimulated with cyclic stretch (12%) for the indicated periods. The cells were then lysed and subjected to immunoblotting (IB) using the indicated antibodies. H, MC3T3-E1 cells were transfected with single siRNAs or a combination of siRNAs as indicated. After 72 h, the cells were subjected to cyclic stretch loading (12%) for 6 h. The cells were then lysed and analyzed by immunoblotting using the indicated antibodies. C indicates control. G and H, the data represent one of at least three experiments.