Yoshiteru Hanai

()-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 F2α‐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 F2α (PGF2α) 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 PGF2α in MC3T3‐E1 cells. The PGF2α‐induced VEGF synthesis was significantly enhanced by EGCG. The amplifying effect of EGCG was dose dependent between 10 and 100 µM. EGCG did not affect the PGF2α‐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 PGF2α‐induced VEGF synthesis. EGCG markedly enhanced the phosphorylation of SAPK/JNK induced by PGF2α without affecting the PGF2α‐induced phosphorylation of p38 MAP kinase. SP600125 markedly reduced the amplification by EGCG of the SAPK/JNK phosphorylation. In addition, the PGF2α‐induced phosphorylation of c‐Jun was amplified by EGCG. These results strongly suggest that EGCG upregulate PGF2α‐stimulated VEGF synthesis resulting from amplifying activation of SAPK/JNK in osteoblasts. J. Cell. Biochem. 100: 1146–1153, 2007.

Involvement of p44/p42 MAP kinase in insulin-like growth factor-I-induced alkaline phosphatase activity in osteoblast-like-MC3T3-E1 cells

It has been shown that insulin-like growth factor-I (IGF-I) stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated the involvement of the mitogen-activated protein (MAP) kinase superfamily in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. IGF-I-stimulated alkaline phosphatase activity dose dependently in the range between 1 nM and 0.1 microM. IGF-I induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase but not stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). PD98059 and U0126, specific inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. On the contrary, SB203580 and PD169316, specific inhibitors of p38 MAP kinase, failed to affect the activity induced by IGF-I. Specific inhibitors for phosphatidylinositol 3-kinase (PI3K)/Akt pathway (LY294002 and wortmannin) also had no significant effect on IGF-I-induced p44/p42 MAP kinase phosphorylation. The phosphorylation of p44/p42 MAP kinase induced by IGF-I was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase among the MAP kinase superfamily plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells.

(−)-Epigallocatechin gallate reduces transforming growth factor β-stimulated HSP27 induction through the suppression of stress-activated protein kinase/c-Jun N-terminal kinase in osteoblasts

We previously reported that transforming growth factor-beta (TGF-beta) stimulates heat shock protein 27 (HSP27) induction through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) 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 TGF-beta-stimulated induction of HSP27 in these cells, and its underlying mechanism. EGCG significantly suppressed the HSP27 induction stimulated by TGF-beta in a dose-dependent manner between 10 and 30 microM without affecting the HSP70 levels. TGF-beta with or without EGCG did not affect the advanced oxidation protein products. The TGF-beta-induced phosphorylation of p38 MAP kinase and ERK1/2 was not affected by EGCG. SP600125, a specific inhibitor of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), markedly reduced the HSP27 expression induced by TGF-beta. EGCG significantly suppressed the TGF-beta-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of Smad2. EGCG attenuated the phosphorylation of both MKK4 and TAK1 induced by TGF-beta. These results strongly suggest that EGCG suppresses the TGF-beta-stimulated induction of HSP27 via the attenuation of the SAPK/JNK pathway in osteoblasts, and that this effect is exerted at a point upstream from TAK1.

Phosphatidylinositol 3-kinase/Akt plays a role in sphingosine 1-phosphate-stimulated HSP27 induction in osteoblasts

We previously reported that p38 mitogen‐activated protein (MAP) kinase plays a part in sphingosine 1‐phosphate‐stimulated heat shock protein 27 (HSP27) induction in osteoblast‐like MC3T3‐E1 cells. In the present study, we investigated whether phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) is involved in the induction of HSP27 in these cells. Sphingosine 1‐phosphate time dependently induced the phosphorylation of Akt. Akt inhibitor, 1L‐6‐hydroxymethyl‐chiro‐inositol 2‐(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate, reduced the HSP27 induction stimulated by sphingosine 1‐phosphate. The sphingosine 1‐phosphate‐induced phosphorylation of GSK‐3β was suppressed by Akt inhibitor. The sphingosine 1‐phosphate‐induced HSP27 levels were attenuated by LY294002 or wortmannin, PI3K inhibitors. Furthermore, LY294002 or Akt inhibitor did not affect the sphingosine 1‐phosphate‐induced phosphorylation of p38 MAP kinase and SB203580, a p38 MAP kinase inhibitor, had little effect on the phosphorylation of Akt. These results suggest that PI3K/Akt plays a part in the sphingosine 1‐phosphate‐stimulated induction of HSP27, maybe independently of p38 MAP kinase, in osteoblasts. J. Cell. Biochem. 98: 1249–1256, 2006.

Potentiation by platelet-derived growth factor-BB of FGF-2-stimulated VEGF release in osteoblasts

We previously reported that basic fibroblast growth factor (FGF-2) stimulates the release of vascular endothelial growth factor (VEGF) via p44/p42 mitogenactivated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblastlike MC3T3-E1 cells. In the present study, we investigated the effect of platelet-derived growth factor-BB (PDGF-BB) on FGF-2-induced VEGF release in MC3T3-E1 cells. PDGF-BB significantly enhanced the FGF-2-stimulated VEGF release. The amplifying effect of PDGF-BB was dose dependent in the range between 0.1 and 30 ng/ml. AG1295, a selective inhibitor of PDGF receptor kinase, which reduced the autophosphorylation of PDGF receptor-®, suppressed the enhancement by PDGF-BB without affecting the FGF-2 effect. PDGF-BB failed to strengthen the FGF-2-induced phosphorylation of p44/p42 MAP kinase or SAPK/JNK. The amplification by PDGF-BB of FGF-2-stimulated VEGF release was reduced by PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK. These results strongly suggest that PDGF-BB potentiates FGF-2-stimulated VEGF release at a point downstream from p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Negative Regulation by p70 S6 Kinase of FGF‐2–Stimulated VEGF Release Through Stress‐Activated Protein Kinase/c‐Jun N‐Terminal Kinase in Osteoblasts

To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF‐2–stimulated VEGF release in osteoblast‐like MC3T3‐E1 cells. In this study, we show that p70 S6 kinase activated by FGF‐2 negatively regulates VEGF release through SAPK/JNK in osteoblasts.

Phosphatidylinositol 3‐kinase/Akt auto‐regulates PDGF‐BB‐stimulated interleukin‐6 synthesis in osteoblasts

It has been reported that platelet‐derived growth factor (PDGF)‐BB stimulates the synthesis of interleukin (IL)‐6 in osteoblasts. In the present study, we investigated whether the phosphatidylinositol 3‐kinase (PI3K)/Akt is involved in the PDGF‐BB‐induced IL‐6 synthesis in osteoblast‐like MC3T3‐E1 cells. PDGF‐BB markedly induced the phosphorylation of Akt and GSK‐3β. Akt inhibitor, 1L‐6‐hydroxymethyl‐chiro‐inositol 2‐(R)‐2‐O‐methyl‐3‐O‐octadecylcarbonate, significantly amplified the synthesis of IL‐6 by PDGF‐BB. The PDGF‐BB‐induced GSK‐3β phosphorylation was suppressed by the Akt inhibitor. The IL‐6 synthesis stimulated by PDGF‐BB was markedly enhanced by LY294002 and wortmannin, inhibitors of PI3K. Wortmannin and LY294002 suppressed the PDGF‐BB‐induced phosphorylation of Akt and GSK‐3β. Taken together, these results strongly suggest that PI3K/Akt negatively regulates the PDGF‐BB‐stimulated IL‐6 synthesis in osteoblasts. J. Cell. Biochem. 99: 1564–1571, 2006.

Activation of phosphatidylinositol 3-kinase/akt limits FGF-2-induced VEGF release 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 release of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the role of Akt/protein kinase B in the FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of Akt and GSK-3beta, a downstream element of Akt. The Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, significantly amplified the FGF-2-induced VEGF release, in a dose-dependent manner between 1 and 70microM, while it suppressed the FGF-2-induced phosphorylation of GSK-3beta. The phosphorylation of Akt induced by FGF-2 was markedly attenuated by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) in osteoblast-like MC3T3-E1 cells. Both wortmannin and LY294002 enhanced the FGF-2-induced VEGF release. In addition, Akt inhibitor had no significant effect on the FGF-2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK. Furthermore, the FGF-2-induced Akt phosphorylation was not affected by PD98059, a MEK inhibitor, or SP600125, a SAPK/JNK inhibitor. Taken together, our findings strongly suggest that PI3-kinase/Akt plays an inhibitory role in FGF-2-induced VEGF release in osteoblasts.

Modulation by the steroid/thyroid hormone superfamily of TGF‐β‐stimulated VEGF release from vascular smooth muscle cells

We previously reported that transforming growth factor‐β (TGF‐β) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen‐activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF‐β‐stimulated VEGF release from A10 cells. Retinoic acid or 1,25‐dihydroxyvitamin D3 enhanced TGF‐β‐induced VEGF release in a concentration‐dependent manner, whereas dexamethasone or corticosterone suppressed TGF‐β‐induced VEGF release. 1,25‐Dihydroxyvitamin D3 and TGF‐β stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF‐β or 1,25‐dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25‐dihydroxyvitamin D3, and glucocorticoids affect TGF‐β‐stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25‐dihydroxyvitamin D3 occurs, in part, via modification of TGF‐β‐induced activation of p38 MAP kinase. J. Cell. Biochem.