Abstract
We have previously reported that prostaglandin F2 alpha (PGF2 alpha) 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 PGF2 alpha and the effect of incadronate on the VEGF synthesis in these cells. PGF2 alpha significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pm and 10 microm. Cycloheximide reduced the PGF2 alpha effect. PGF2 alpha increased the levels of mRNA for VEGF. Cloprostenol, a PGF2 alpha-sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF2 alpha-induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF2 alpha-induced VEGF synthesis. The VEGF synthesis induced by PGF2 alpha was significantly attenuated in the PKC down-regulated cells. PGF2 alpha elicited the translocation of PKC beta I from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF2 alpha. Farnesyltransferase inhibitor failed to affect the PGF2 alpha-induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF2 alpha. NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF2 alpha-induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF2 alpha 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 PGF2 alpha 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.
Highlights
We have previously reported that prostaglandin F2␣ (PGF2␣) activates p44/p42 mitogen-activated protein kinase (MAPK) through protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells
To clarify whether FP receptor is required for the PGF2␣-induced vascular endothelial growth factor (VEGF) synthesis or not in MC3T3-E1 cells, we examined the effect of cloprostenol, an FP agonist (30), on the VEGF synthesis
To elucidate whether p44/p42 MAPK is involved in the PGF2␣induced VEGF synthesis in these cells, we examined the effect of PD98059, a specific inhibitor of MEK (35), on the VEGF synthesis induced by PGF2␣
Summary
Materials—PGF2␣, cycloheximide, PGE2, PGD2, indomethacin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Chemical Co. An ECL Western blotting detection system and a QuickPrep total RNA extraction kit were purchased from Amersham Biosciences (Tokyo, Japan). Assay for VEGF—The cells were stimulated by PGF2␣, cloprostenol, PGE2, PGD2, or NaF in 1 ml of ␣-MEM containing 0.3% FCS for the indicated periods. Isolation of RNA and Quantification of mRNA for VEGF—The cultured cells were stimulated by PGF2␣ or PGE2 in 1 ml of ␣-MEN containing 0.3% FCS for 2 h. Analysis of PKCI, p44/p42 MAPK, MEK1/2, or Raf-1—The cultured cells were stimulated by PGF2␣ or TPA in 4 ml of ␣-MEM containing 0.3% FCS for the indicated periods. Assay for VEGF in Mouse Plasma in Vivo—C57B mice, obtained from SLC (Sizuoka, Japan), were divided into four groups, a control group (n ϭ 3), a group treated with incadronate (n ϭ 3), a group treated with cloprostenol (n ϭ 3), and a group treated with both compounds (n ϭ 6).
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