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.     

(-)-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 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.     

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.     

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.     

Rho-kinase regulates endothelin-1-stimulated IL-6 synthesis via p38 MAP kinase in osteoblasts

We have previously reported that endothelin-1 (ET-1) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the ET-1-stimulated IL-6 synthesis in MC3T3-E1 cells. ET-1 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific inhibitor of Rho-kinase, significantly suppressed the IL-6 synthesis induced by ET-1 as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, reduced the ET-1-stimulated IL-6 synthesis. Y27632 as well as fasudil attenuated the ET-1-induced phosphorylation of p38 MAP kinase but not p44/p42 MAP kinase. These results strongly suggest that Rho-kinase regulates ET-1-stimulated IL-6 synthesis through p38 MAP kinase activation in osteoblasts.     

Minodronate suppresses prostaglandin F2alpha-induced vascular endothelial growth factor synthesis in osteoblasts.

In our previous study, we showed that prostaglandin F2alpha (PGF2alpha) stimulates vascular endothelial growth factor (VEGF) synthesis via activation of p44/p42 mitogen-activated protein (MAP) kinase via protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells. In addition, we demonstrated that incadronate amplified, and tiludronate suppressed PGF2alpha-induced VEGF synthesis among bisphosphonates, while alendronate or etidronate had no effect. In the present study, we investigated the effects of minodronate, a newly developed bisphosphonate, on PGF (2alpha)-induced VEGF synthesis in MC3T3-E1 cells. Minodronate significantly reduced VEGF synthesis induced by PGF2alpha dose-dependently at levels between 3 and 100 microM. PGF2alpha-stimulated phosphorylation of Raf-1, MEK1/2 and p44/p42 MAP kinase were suppressed by minodronate. 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator VEGF synthesis induced by PKC, was inhibited by minodronate. Minodronate inhibited Raf-1, MEK1/2 and p44/p42 MAP kinase phosphorylation induced by TPA. Mevalonate failed to affect the suppressive effect of minodronate on PGF2alpha-induced VEGF synthesis. Taken together, these results indicate that minodronate suppresses PGF2alpha-stimulated VEGF synthesis at the point between PKC and Raf-1 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.     

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.     

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.     

(-)-Epigallocatechin gallate amplifies interleukin-1-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells

We previously reported that interleukin-1 (IL-1), a potent bone resorptive cytokine, stimulates the synthesis of interleukin-6 (IL-6) via activation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through the inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway. On the other hand, it is recognized that catechin possesses a beneficial property for bone metabolism. Among them, (-)-epigallocatechin gallate (EGCG) is an abundant and major bioactive component. In the present study, we investigated the effect of EGCG on the IL-1 stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. EGCG significantly enhanced the IL-1-stimulated IL-6 synthesis in a dose-dependent manner in the range between 50 and 100 μM. EGCG increased the mRNA levels of IL-6 stimulated by IL-1. IL-1-induced phosphorylation of IκB and NF-κB were suppressed by EGCG. On the other hand, EGCG failed to affect the IL-1-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and AMPK. These results strongly suggest that EGCG enhances IL-1-stimulated IL-6 synthesis through inhibiting the AMPK-IκB/NF-κB pathway at the point between AMPK and IκB/NF-κB in osteoblasts.     

Stress-activated protein kinase/c-Jun N-terminal kinase (JNK) plays a part in endothelin-1-induced vascular endothelial growth factor synthesis in osteoblasts

We previously reported that endothelin-1 (ET-1) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that not p44/p42 MAP kinase but p38 MAP kinase participates in the ET-1-induced vascular endothelial growth factor (VEGF) synthesis. In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) in ET-1-induced VEGF synthesis in these cells. ET-1 significantly induced the phosphorylation of JNK in a dose-dependent manner in the range between 0.1 and 100 nM. SP600125, an inhibitor of JNK, markedly reduced the ET-1-induced VEGF synthesis. A combination of SP600125 and SB203580 additively reduced the ET-1-stimulated VEGF synthesis. SP600125 suppressed the ET-1-induced phosphorylation of JNK, while having no effect on the phosphorylation of p38 MAP kinase elicited by ET-1. SB203580, an inhibitor of p38 MAP kinase, hardly affected the ET-1-induced phosphorylation of JNK. These results strongly suggest that JNK plays a role in ET-1-induced VEGF synthesis in addition to p38 MAP kinase in osteoblasts.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation 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.     

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.     

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-alpha-induced Interleukin-6 Synthesis in Osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl- CHIRO-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.     

Function of Rho-kinase in prostaglandin D2-induced interleukin-6 synthesis in osteoblasts

We have previously reported that prostaglandin D2 (PGD2) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGD2-stimulated IL-6 synthesis in MC3T3-E1 cells. PGD2 time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGD2-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGD2-stimulated IL-6 synthesis. The PGD2-stimulated IL-6 synthesis was reduced by PD98059, a MEK inhibitor, and SB203580, an inhibitor of p38 mitogen-activated protein (MAP) kinase, but not SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). However, Y27632 and fasudil failed to affect the PGD2-induced phosphorylation of p44/p42 MAP kinase. On the other hand, Y27632 as well as fasudil markedly attenuated the PGD2-induced phosphorylation of p38 MAP kinase. In addition, PGD2 additively induced IL-6 synthesis in combination with endothelin-1 which induces IL-6 synthesis through p38 MAP kinase regulated by Rho-kinase. These results strongly suggest that Rho-kinase regulates PGD2-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of mitogen-activated protein kinases

AimWe have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.Main methodsThe levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.Key findingsPDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.SignificanceThese results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.     

Involvement of p38 mitogen-activated protein kinase in basic fibroblast growth factor-induced interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.