Incadronate amplifies prostaglandin F2α-induced vascular endothelial growth factor synthesis in osteoblasts -: Enhancement of MAPK activity

We have previously reported that prostaglandin F-2alpha (PGF(2alpha)) activates p44/p42 mitogen-activated protein kinase (MAPK) through protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of vascular endothelial growth factor (VEGF) synthesis induced by PGF(2alpha) and the effect of incadronate on the VEGF synthesis in these cells. PGF(2alpha) significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pM and 10 muM. Cycloheximide reduced the PGF(2alpha) effect. PGF(2alpha) increased the levels of mRNA for VEGF. Cloprostenol, a PGF(2alpha)-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF(2alpha)-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF(2alpha)-induced VEGF synthesis. The VEGF synthesis induced by PGF(2alpha) was significantly attenuated in the PKC down-regulated cells. PGF(2alpha) elicited the translocation of PKCbetaI from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF(2alpha). Farnesyltransferase inhibitor failed to affect the PGF(2alpha)-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF(2alpha). NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF(2alpha)-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF(2alpha) or 12-O-tetradecanoylphorbol-13-acetate, a PKC activator. Incadronate significantly enhanced the cloprostenol-increased level of VEGF concentration in mouse plasma in vivo. These results strongly suggest that PGF(2alpha) stimulates VEGF synthesis through the PKC-dependent activation of p44/p42 MAPK in osteoblasts and that the incadronate enhances the VEGF synthesis at the point between PKC and Raf-1.