p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.     

Platelet-derived growth factor-BB amplifies PGF2alpha-stimulated VEGF synthesis in osteoblasts: function of phosphatidylinositol 3-kinase

We have reported that prostaglandin F2alpha (PGF2alpha) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In addition, we recently showed that phosphatidylinositol 3 (PI3)-kinase activated by platelet-derived growth factor-BB (PDGF-BB) negatively regulates the interleukin-6 synthesis in these cells. In the present study, we investigated the effect of PDGF-BB on the PGF2alpha-induced VEGF synthesis in MC3T3-E1 cells. PDGF-BB, which alone did not affect the levels of VEGF, significantly enhanced the PGF2alpha-stimulated VEGF synthesis. The amplifying effect of PDGF-BB was dose dependent in the range between 10 and 70 ng/ml. LY294002 or wortmannin, specific inhibitors of PI3-kinase, which by itself failed to affect the PGF2alpha-stimulated VEGF synthesis, significantly suppressed the amplification by PDGF-BB. PD98059, a specific inhibitor of MEK1/2, suppressed the amplification by PDGF-BB of the PGF2alpha-stimulated VEGF synthesis similar to the levels of PGF2alpha with PD98059. PDGF-BB itself induced the phosphorylation of p44/p42 MAP kinase in these cells, and the effects of PDGF-BB and PGF2alpha on the phosphorylation of p44/p42 MAP kinase were additive. Moreover, LY294002 had little effect on the phosphorylation of p44/p42 MAP kinase induced by PGF2alpha with PDGF-BB. These results strongly suggest that PGF2alpha-stimulated VEGF synthesis is amplified by PI3-kinase-mediating PDGF-BB signaling in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Incadronate amplifies prostaglandin F2 alpha-induced vascular endothelial growth factor synthesis in osteoblasts. Enhancement of MAPK activity

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.     

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.     

Sphingosine 1-phosphate amplifies phosphoinositide hydrolysis stimulated by prostaglandin f2 alpha in osteoblasts: involvement of p38MAP kinase

We previously showed that sphingosine 1-phosphate phosphorylates p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine 1-phosphate on phospholipase C-catalyzing phosphoinositide hydrolysis induced by prostaglandin F2alpha (PGF2 alpha) in these cells. Sphingosine 1-phosphate significantly amplified the inositol phosphates formation by PGF2 alpha. Sphingosine 1-phosphate did not enhance the formation induced by NaF, a direct activator of heterotrimeric GTP-binding proteins. PD98059, an inhibitor of the kinase that activates p42/p44 MAP kinase, had little effect on the amplification by sphingosine 1-phosphate. SB203580, an inhibitor of p38 MAP kinase, reduced the effect of sphingosine 1-phosphate on the formation of inositol phosphates by PGF2 alpha. The phosphorylation of p42/p44 MAP kinase by PGF alpha was attenuated by PD98059. SB203580 suppressed the phosphorylation of p38 MAP kinase by PGF2 alpha. Tumor necrosis factor-alpha enhanced the PGF2 alpha-stimulated formation of inositol phosphates. These results strongly suggest that sphingosine 1-phosphate amplifies PGF2 alpha-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in osteoblasts.     

(-)-Epigallocatechin gallate suppresses endothelin-1-induced interleukin-6 synthesis in osteoblasts: inhibition of p44/p42 MAP kinase activation

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.     

(--)-epigallocatechin gallate enhances prostaglandin F2alpha-induced VEGF synthesis via upregulating SAPK/JNK activation in osteoblasts

Catechin, one of the major flavonoids presented in plants such as tea, reportedly suppresses bone resorption. We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. To clarify the mechanism of catechin effect on osteoblasts, we investigated the effect of (--)-epigallocatechin gallate (EGCG), one of the major green tea flavonoids, on the VEGF synthesis by PGF(2alpha) in MC3T3-E1 cells. The PGF(2alpha)-induced VEGF synthesis was significantly enhanced by EGCG. The amplifying effect of EGCG was dose dependent between 10 and 100 microM. EGCG did not affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, and SP600125, a specific inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), reduced the PGF(2alpha)-induced VEGF synthesis. EGCG markedly enhanced the phosphorylation of SAPK/JNK induced by PGF(2alpha) without affecting the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. SP600125 markedly reduced the amplification by EGCG of the SAPK/JNK phosphorylation. In addition, the PGF(2alpha)-induced phosphorylation of c-Jun was amplified by EGCG. These results strongly suggest that EGCG upregulate PGF(2alpha)-stimulated VEGF synthesis resulting from amplifying activation of SAPK/JNK in osteoblasts.     

Involvement of p42/p44 mitogen-activated protein kinase in prostaglandin f(2alpha)-stimulated induction of heat shock protein 27 in osteoblasts

We previously reported that prostaglandin F(2alpha) (PGF(2alpha)) activates both phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells and then induces the activation of protein kinase C (PKC). In this study, we investigated the effect of PGF(2alpha) on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein, in these cells. PGF(2alpha) significantly induced the accumulation of HSP27 dose-dependently within the range of 10 nM to 10 microM. PGF(2alpha) stimulated the increase in the levels of mRNA for HSP27. A total of 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, induced the accumulation of HSP27. The stimulative effect of PGF(2alpha) was reduced in the PKC down-regulated cells. Calphostin C, a specific inhibitor of PKC, suppressed the PGF(2alpha)-induced HSP27 accumulation as well as that induced by TPA. HSP27 induction by PGF(2alpha) was reduced by U-73122, a phospholipase C inhibitor, or propranolol, a phosphatidic acid phosphohydrolase inhibitor. PGF(2alpha) and TPA stimulated p42/p44 mitogen-activated protein (MAP) kinase. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, suppressed the induction of HSP27 stimulated by PGF(2alpha) or TPA. PD98059 and calphostin C reduced the levels of mRNA for HSP27 increased by PGF(2alpha). These results indicate that PGF(2alpha) stimulates the induction of HSP27 via p42/p44 MAP kinase activation, which depends on upstream PKC activation in osteoblasts.     

Protein kinase C activation amplifies prostaglandin F2 alpha-induced prostaglandin E2 synthesis in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin F2 alpha (PGF2 alpha) stimulated arachidonic acid (AA) release in a dose-dependent manner in the range between 1 nM and 10 microM. 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2 alpha in a dose-dependent manner. 4 alpha-phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2 alpha-induced AA release. 1-oleoyl-2-acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2 alpha. H-7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2 alpha-induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2 alpha-induced AA release, while it suppressed the amplification by OAG of PGF2 alpha-induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol trisphosphate stimulated by PGF2 alpha. Under the same condition, PGF2 alpha indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2 alpha-induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2 alpha-induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like cells.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.     

Involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in prostaglandin F2alpha-induced heat shock protein 27 in osteoblasts

We have reported that prostaglandin F2(alpha) (PGF2(alpha)) activates p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, and that p44/p42 MAP kinase plays a role in the PGF2(alpha)-induced heat shock protein 27 (HSP27). In the present study, we investigated the involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase superfamily, in PGF2(alpha)-induced HSP27 in MC3T3-E1 cells. PGF2(alpha) time dependently induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGF2(alpha)-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between 0.1 and 30 microM. SP600125 reduced the PGF2(alpha)-increased level of HSP27 mRNA. SP600125 suppressed the phosphorylation of SAPK/JNK induced by PGF2(alpha), but did not affect the PGF2(alpha)-induced phosphorylation of p44/p42 MAP kinase. On the other hand, PD98059, a specific inhibitor of the upstream kinase of p44/p42 MAP kinase, which reduced the phosphorylation of p44/p42 MAP kinase stimulated by PGF2(alpha), had little effect on the PGF2(alpha)-induced phosphorylation of SAPK/JNK. These results strongly suggest that SAPK/JNK plays a part in PGF2(alpha)-induced HSP27 in addition to p44/p42 MAP kinase in osteoblasts.     

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.     

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.     

Involvement of MAP kinases in TGF-beta-stimulated vascular endothelial growth factor synthesis in osteoblasts

Transforming growth factor-beta (TGF-beta) reportedly induces vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. We have recently shown that TGF-beta activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in these cells. In the present study, we investigated the exact mechanism of TGF-beta behind the synthesis of VEGF in MC3T3-E1 cells. PD98059 and U-0126, specific inhibitors of MEK, suppressed the VEGF synthesis induced by TGF-beta. U-0126 inhibited the TGF-beta-induced p44/p42 MAP kinase phosphorylation. SB203580 and PD169316, inhibitors of p38 MAP kinase, reduced the TGF-beta-stimulated VEGF synthesis. SB202474, a negative control for p38 MAP kinase inhibitor, did not affect the VEGF synthesis. A combination with PD98059 and SB203580 almost completely suppressed the TGF-beta-induced VEGF synthesis. Retinoic acid, which alone failed to affect VEGF synthesis, markedly enhanced the VEGF synthesis stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-increased levels of VEGF mRNA. The amplifications by retinoic acid of TGF-beta-increased VEGF synthesis and levels of VEGF mRNA were reduced by PD98059 or SB203580. The combination of PD98059 and SB203580 almost completely suppressed the enhancement by retinoic acid of VEGF synthesis induced by TGF-beta. Taken together, our results strongly suggest that both p44/p42 MAP kinase and p38 MAP kinase take part in TGF-beta-stimulated VEGF synthesis in osteoblasts, and that retinoic acid upregulates the VEGF synthesis.     

(-)-epigallocatechin gallate inhibits prostaglandin D2-stimulated HSP27 induction via suppression of the p44/p42 MAP kinase pathway in osteoblasts

We previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether (-)-epigallocatechin gallate (EGCG), the major polyphenol found in green tea, affects the induction of HSP27 in these cells and the mechanism. EGCG significantly reduced the HSP27 induction stimulated by PGD2 without affecting the levels of HSP70. The PGD2-induced phosphorylation of p38 MAP kinase or SAPK/JNK was not affected by EGCG. On the contrary, EGCG markedly suppressed the PGD2-induced phosphorylation of p44/p42 MAP kinase and MEK1/2. However, the PGD2-induced phosphorylation of Raf-1 was not inhibited by EGCG. These results strongly suggest that EGCG suppresses the PGD2-stimulated induction of HSP27 at the point between Raf-1 and MEK1/2 in osteoblasts.     

Wnt3a upregulates transforming growth factor-β-stimulated VEGF synthesis in osteoblasts

It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.     

Up-regulation by zinc of FGF-2-induced VEGF release through enhancing p44/p42 MAP kinase activation in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) activates stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 mitogen-activated protein (MAP) kinase resulting in the stimulation of vascular endothelial growth factor (VEGF) release in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether zinc affects the VEGF release by FGF-2 in MC3T3-E1 cells. The FGF-2-induced VEGF release was significantly enhanced by ZnSO(4) but not Na(2)SO(4). The enhancing effect of ZnSO(4) was dose-dependent between 1 and 100 muM. ZnSO(4) markedly enhanced the FGF-2-induced phosphorylation of p44/p42 MAP kinase while having little effect on the SAPK/JNK phosphorylation. PD98059 significantly reduced the amplification by ZnSO(4) of the FGF-2-stimulated VEGF release. Taken together, our findings strongly suggest that zinc enhances FGF-2-stimulated VEGF release resulting from up-regulating activation of p44/p42 MAP kinase in osteoblasts.     

p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts

We previously reported that p70 S6 kinase takes part in bone morphogenetic protein-4 (BMP-4)-stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. Recently, we showed that BMP-4-induced osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in these cells. In the present study, we investigated whether the MAP kinases are involved in the BMP-4-stimulated synthesis of VEGF in MC3T3-E1 cells. PD-98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, failed to affect BMP-4-stimulated VEGF synthesis. SB-203580 and PD-169316, inhibitors of p38 MAP kinase, significantly reduced VEGF synthesis, whereas SB-202474, a negative control for p38 MAP kinase inhibitor, had little effect on VEGF synthesis. The BMP-4-stimulated phosphorylation of p38 MAP kinase was not affected by rapamycin, an inhibitor of p70 S6 kinase. On the contrary, SB-203580 and PD-169316 reduced the BMP-4-stimulated phosphorylation of p70 S6 kinase. In addition, anisomycin, an activator of p38 MAP kinase, phosphorylates p70 S6 kinase, and the phosphorylation was suppressed by SB-203580. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Not BMP-4 but anisomycin weakly induced the phosphorylation of phosphoinositide-dependent kinase-1. However, anisomycin had little effect on phosphorylation of either Akt or the mammalian target of rapamycin. Taken together, our results suggest that p38 MAP kinase functions in BMP-4-stimulated VEGF synthesis as a positive regulator at a point upstream from p70 S6 kinase in osteoblasts.     

SAPK/JNK plays a role in transforming growth factor-beta-induced VEGF synthesis in osteoblasts.

We previously reported that transforming growth factor-beta (TGF-beta) activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase, resulting in the stimulation of vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK), another member of the MAP kinase superfamily, in TGF-beta-induced VEGF synthesis in these cells. TGF-beta markedly induced SAPK/JNK phosphorylation. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced TGF-beta-induced VEGF synthesis. SP600125 suppressed TGF-beta-induced SAPK/JNK phosphorylation. PD98059, an inhibitor of upstream kinase of p44/p42 MAP kinase and SB203580, an inhibitor of p38 MAP kinase, each failed to reduce TGF-beta-induced SAPK/JNK phosphorylation. A combination of SP600125 and PD98059 or SP600125 and SB203580 suppressed TGF-beta-stimulated VEGF synthesis in an additive manner. These results strongly suggest that TGF-beta activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a role in addition to p42/p44 MAP kinase and p38 MAP kinase in TGF-beta-induced VEGF synthesis.