| It is well established that bone mass and architecture depend on the mechanical stimuli applied to the skeleton. Bone loss occurs in over-loading condition, while bone mass increases after appropriate skeletal loading. These changes in mechanical stimulus modulate bone turnover mainly through the osteoblast lineage. However, the signaling pathways involved in osteoblastic mechanotransduction are not fully understood. Among the early events linked to the response to mechanical strain, the expression of the immediate early gene, c-fos, has been specifically connected to bone biology. The c-Fos protein is an important component of the transcription factor activating protein-1 (AP-1), which can alter relative gene expression. In an effort to better understand what processes are involved in mechanotransduction, we have examined whether and how soon c-fos induction occurs in human MG-63 osteoblast shortly after the application of the mechanical stimuli lonely and coupled with the inhibitor of the relative signaling pathways.MG-63 were cultured in DMEM with 10% FBS for 48 hours and starved in DMEM with no FBS for 12 hours, then subjected to mechanicalstrain by self-made four-point bending system at a 0.5Hz frequency for 5min, 15min, 30min, lh, 3h, 6h, respectively. In each time-phase, the cells were loaded with tension and compression stress at lOOOustrain and 4000ustrain respectively, c-fos mRNA were analyzed by Real-time PCR and c-Fos protein by Western Blotting. Changes in the experiments were expressed as ratio (±standard error of the mean) to the controls. Statistical significance was determined for each comparison using the one-way ANOVA and multiple comparisons using the SNK, and p<0.05 was statistically considered significant. The ratios quoted were average changes from three independent experiments.Treatment of cultures with agonists: Before loading, the MG-63 cells were treated with Genistein, the inhibitor of Tyrosine kinase, and Cytochalasin D, which represses the formation of the stress fiber, for 30min and lhr respectively.According to this research, we found: application of mechanical strain to MG-63 cells produced a rapid increase in expression of c-fos mRNA and c-Fos protein within 15min and peaked at 30min-lh, then significantly reduced after lhr and returned to baseline by 3-6h.; the effect of 1000|j.strain mechanical stimulus on the expression of osteoblast c-fos mRNA was stronger than that of 4000^strain; difference mechanical stimuli could induce difference changes of c-fos mRNA and c-Fos protein; both Genistein and Cytochalasin D had obvious repression on the expression of c-fos gene, the repression of Genistein was more important in the expression of c-fos induced by the tension stimulus, while the induced expression of c-fos by the compression stimulus was mainly inhibited byCytochalasin D.So based on the results, we have these conclusions: acted as an immediate-early gene, c-fos could make a rapid and temporal response to mechanical strain; the mechanical stimulus at physiologic magnitude could induce the largest expression of c-fos; the mechanical tension and compression stress could induce c-fos expression through multiple signaling pathways, while the expression of c-fos by tension was mostly induced through the tyrosine kinase pathways and the compression stimulus induced c-fos expression mainly through the reorganization of the cytoskeleton.
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