Although it has been reported that vascular endothelial growth factor (VEGF) promotes not only angiogenesis but also osteoclast and osteoblast differentiation, few reports exist regarding VEGF/VEGF receptor (VEGFR) signaling in osteoblasts, which regulate osteoclast differentiation and generate VEGF. This study examined the expression of the bone remodeling factor VEGF-A and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), in murine osteoblastic MC3T3-E1 cells with the application of mechanical stress. The protein concentration of VEGF-A in the mechanical stress group increased markedly compared with the control group, while that of macrophage colony-stimulating factor in the mechanical stress group was lower than in the control group. VEGFR-2 mRNA expression was not detected in osteoblastic MC3T3-E1 cells. Mechanical stress up-regulated VEGF-A, VEGFR-1 and the receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression. In particular, VEGF-A and RANKL mRNA expression increased immediately after mechanical stress. We examined the VEGF/VEGFR system on anti-mouse VEGF neutralizing antibody in osteoblasts with mechanical stress. Neutralizing antibody to VEGF partially inhibited the increase of VEGF-A and RANKL mRNA expression compared with the non-mechanical stress group. VEGFR-1 mRNA expression was completely suppressed to control levels by the neutralizing antibody to VEGF. These findings suggest that mechanical stress up-regulates RANKL expression via the VEGF/VEGFR-1 autocrine pathway in osteoblastic MC3T3-E1 cells, indicating the possibility that, in response to mechanical stress, osteoblasts increase bone resorption by an autocrine up-regulation of VEGF/VEGFR-1 and RANKL expression.

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