Incadronate Amplifies Prostaglandin F2α-induced Vascular Endothelial Growth Factor Synthesis in Osteoblasts

Abstract

We have previously reported that prostaglandin F 2␣ (PGF 2␣ ) 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 2␣ and the effect of incadronate on the VEGF synthesis in these cells. PGF 2␣ significantly stimulated the VEGF synthesis in a dose-dependent manner between 1 pM and 10 M. Cycloheximide reduced the PGF 2␣ effect. PGF 2␣ increased the levels of mRNA for VEGF. Cloprostenol, a PGF 2␣ -sensitive receptor agonist, potently induced the VEGF synthesis. Indomethacin, an inhibitor of cyclooxygenase, significantly reduced the PGF 2␣ -induced VEGF synthesis. Bisindolylmaleimide, an inhibitor of PKC, reduced the PGF 2␣ -induced VEGF synthesis. The VEGF synthesis induced by PGF 2␣ was significantly attenuated in the PKC down-regulated cells. PGF 2␣ elicited the translocation of PKC␤I from cytosol to membrane fraction. PD98059 or U0126, inhibitors of MEK, suppressed the VEGF synthesis induced by PGF 2␣ . Farnesyltransferase inhibitor failed to affect the PGF 2␣ -induced VEGF synthesis. Incadronate enhanced the synthesis of VEGF induced by PGF 2␣ . NaF-induced VEGF synthesis was also amplified by incadronate. PD98059 suppressed the enhancement by incadronate of PGF 2␣ -induced VEGF synthesis. Incadronate markedly enhanced the phosphorylation of Raf-1, MEK1/2, and p44/p42 MAPK induced by PGF 2␣ 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 2␣ 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.Bone metabolism is mainly regulated by two functional cells, osteoblasts and osteoclasts; the former is responsible for bone formation, and the latter for bone resorption (1). Accumulating evidence indicates that bone-resorptive agents such as parathyroid hormone and 1,25-(OH) 2 vitamin D 3 up-regulate RANKL (receptor activator of nuclear factor B) expression through their specific receptors on osteoblasts, suggesting that osteoblasts play pivotal roles in the regulation of bone resorption (2). The bone remodeling results from the coupling bone resorption by activated osteoclasts with subsequent deposition of new matrix by osteoblasts. During the process, capillary endothelial cells provide the microvasculature, and osteoblasts and osteoprogenitor cells, which proliferate locally and differentiate into osteoblasts, migrate into the resorption lacuna.

Inhibition by minodronate of basic fibroblast growth factor-stimulated vascular endothelial growth factor synthesis in osteoblast-like cells

“…In contrast to the inhibitory effect of minodronate presented here, we recently reported that incadronate enhances VEGF synthesis induced by PGF 2α in these cells, though VEGF synthesis was not affected by alendronate or etidronate (20). Therefore, our findings suggest that the suppressive effect of minodronate on bFGF-stimulated VEGF synthesis was a specific effect of this agent, and is not a common effect of bisphosphonates.…”

“…In addition, Flk-1, a VEGF receptor, is reportedly expressed in osteoblasts (27) and involved in bone formation synergistically with BMP-4 (28). In a previous study (20), we reported that incadronate amplifies PGF 2α -induced VEGF synthesis in osteoblast-like MC3T3-E1 cells, while alendronate or ethidronate do not have a similar effect. These findings suggest that bisphosphonates have different molecular mechanisms of action on osteoblasts, most likely due to structural differences.…”

“…In our previous studies, we showed that bFGF positively regulates VEGF synthesis and secretion through p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells (18,19). Furthermore, we demonstrated that incadronate enhances prostaglandin F 2α (PGF 2α )-induced VEGF synthesis through the activation of p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells, while alendronate or etidronate have little such effect (20).…”

Protein kinase C δ regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

“…Although p44/p42 MAPK is highly activated in HCC (Ito et al, 1998;Iyoda et al, 2003), we demonstrated that phosphorylation of HSP27 was not correlated with p44/p42 MAPK activity. It has been reported that PKC is an upstream regulator of the MAPK superfamily cascade (Noguchi et al, 1993;Tanaka et al, 2003;Tokuda et al, 2003). Therefore, we examined whether PKC regulates the HSP27 phosphorylation in HCC cells.…”

“…Protein kinase C (PKC) is reportedly an upstream regulator of MAPK superfamily cascade (Noguchi et al, 1993;Tanaka et al, 2003;Tokuda et al, 2003). PKC is a Ser/Thr protein kinase family with multiple isoforms, its isoforms have been classified into three groups, classical PKC (α, β, γ), novel PKC (δ, ε, η, θ), and atypical PKC (ζ, ι/λ) (Saito et al, 2001).…”