Naohiro Yamamoto

Resveratrol inhibits BMP-4-stimulated VEGF synthesis in osteoblasts: Suppression of S6 kinase

Resveratrol is well known as a natural polyphenol abundantly found in red wine. We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates vascular endothelial growth factor (VEGF) synthesis via p70 S6 kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of resveratrol on the BMP-4-stimulated VEGF synthesis in MC3T3-E1 cells. Resveratrol significantly suppressed BMP-4-stimulated release and expression levels of VEGF mRNA. SRT1720, an activator of SIRT1 with potencies greater than resveratrol, also reduced VEGF release and the mRNA levels. Both resveratrol and SRT1720 markedly attenuated the BMP-4-induced phosphorylation of p70 S6 kinase without affecting the BMP-4-induced phosphorylation of Smad1/5/8. These findings strongly suggest that resveratrol attenuates BMP-4-stimulated VEGF synthesis through suppression of the activation of p70 S6 kinase in osteoblasts, and that the inhibitory effect is mediated at least in part by SIRT1 activation.

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation.

Suppression by resveratrol of prostaglandin D2-stimulated osteoprotegerin synthesis in osteoblasts

Resveratrol, a natural polyphenol with health-related properties mainly existing in grape skins and red wine, possesses beneficial effects on human being. We have previously reported that prostaglandin D2 (PGD2) stimulates heat shock protein 27 (HSP27) induction via activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism behind the effect of PGD2 on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the OPG synthesis in MC3T3-E1 cells. PGD2 significantly stimulated both the OPG release and the expression levels of OPG mRNA. Resveratrol and SRT1720, an activator of SIRT1, markedly suppressed the PGD2-induced OPG release and the mRNA levels of OPG. PD98059, a specific MEK inhibitor, SB203580, a specific p38 MAP kinase inhibitor, and SP600125, a specific SAPK/JNK inhibitor suppressed the PGD2-stimulated OPG release. PGD2-induced phosphorylation of p38 MAP kinase and SAPK/JNK was attenuated by resveratrol or SRT1720. However, resveratrol or SRT1720 failed to affect the phosphorylation of myosin phosphatase-targeting subunit-1 (MYPT-1), a downstream substrate of Rho-kinase and p44/p42 MAP kinase. These results strongly suggest that resveratrol suppresses PGD2-stimulated OPG synthesis through inhibiting p38 MAP kinase and SAPK/JNK in osteoblasts, and that the suppressive effect is exerted at the point downstream of Rho-kinase but upstream of p38 MAP kinase or SAPK/JNK.

Rho-kinase limits BMP-4-stimulated osteocalcin synthesis in osteoblasts: Regulation of the p38 MAP kinase pathway

AIM We previously reported that bone morphogenetic protein-4 (BMP-4) stimulates the synthesis of osteocalcin via p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, whereas p44/p42 MAP kinase plays as a negative regulator in the synthesis. In the present study, we investigated whether Rho-kinase is involved in BMP-4-stimulated osteocalcin synthesis in MC3T3-E1 cells. MAIN METHODS The levels of osteocalcin were measured by ELISA. The phosphorylation of each protein kinase was analyzed by Western blotting. The mRNA levels of osteocalcin were determined by real-time RT-PCR. KEY FINDINGS BMP-4 induced the phosphorylation of myosin phosphatase targeting subunit-1 (MYPT-1), a substrate of Rho-kinase. Y27632 or fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly amplified the BMP-4-stimulated osteocalcin synthesis in a dose-dependent manner. The osteocalcin mRNA expression levels induced by BMP-4 were enhanced by Y27632 or fasudil. BMP-4-stimulated osteocalcin release was significantly up-regulated in Rho-knocked down cells with Rho A-siRNA. Y27632 or fasudil failed to affect the BMP-4-induced phosphorylation of SMAD1 or p44/p42 MAP kinase. On the other hand, Y27632 or fasudil markedly strengthened the phosphorylation levels of p38 MAP kinase induced by BMP-4. SIGNIFICANCE These results strongly suggest that Rho-kinase negatively regulates BMP-4-stimulated osteocalcin synthesis via the p38 MAP kinase pathway in osteoblasts.

Resveratrol reduces prostaglandin E1-stimulated osteoprotegerin synthesis in osteoblasts: suppression of stress-activated protein kinase/c-Jun N-terminal kinase.

Resveratrol, a natural polyphenol mainly existing in red grapes and berries, possesses beneficial effects on human being. We have previously reported that prostaglandin E1 (PGE1) stimulates vascular endothelial growth factor synthesis via activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the PGE1-effect on osteoprotegerin (OPG) synthesis and the effect of resveratrol on the synthesis in MC3T3-E1 cells. PGE1 induced the expression levels of OPG mRNA and stimulated the OPG release. Resveratrol significantly reduced the PGE1-induced OPG release and the mRNA expression. SRT1720, an activator of SIRT1, suppressed the release of OPG. The protein levels of SIRT1 were not up-regulated by resveratrol with or without PGE1. Both SB203580 and SP600125, a specific p38 MAP kinase inhibitor and a specific SAPK/JNK inhibitor, respectively, but not PD98059, a specific MEK inhibitor, reduced the PGE1-stimulated OPG release. Resveratrol or SRT1720 failed to affect the phosphorylation of p38 MAP kinase. On the contrary, PGE1-induced phosphorylation of SAPK/JNK was significantly attenuated by both resveratrol and SRT1720. Our results strongly suggest that resveratrol inhibits PGE1-stimulated OPG synthesis via suppressing SAPK/JNK but not p38 MAP kinase in osteoblasts.

Regulation by resveratrol of prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts

Resveratrol is a natural polyphenol found in red grape skins, berries and red wine. Accumulating evidence suggests that resveratrol has various beneficial effects on the human body. In the present study, we investigated the effects of prostaglandin E(2) (PGE(2)) on osteoprotegerin (OPG) synthesis and the effects of resveratrol on OPG synthesis in osteoblast-like MC3T3-E1 cells. PGE(2) significantly stimulated both the release of OPG and the mRNA expression levels of OPG, as shown by OPG assay and real-time RT-PCR, respectively. Resveratrol markedly suppressed the release and the mRNA levels of OPG induced by PGE(2). On the contrary, SRT1720, an activator of sirtuin 1 (SIRT1), hardly affected the PGE(2)-induced release of OPG. PD98059 [a specific inhibitor of the upstream kinase that activates p44/p42 mitogen-activated protein (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 PGE(2)-induced release of OPG. Resveratrol attenuated the PGE(2)-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK. However, SRT1720 failed to affect the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK induced by PGE(2). These results strongly suggest that resveratrol reduces PGE(2)-stimulated OPG synthesis through the inhibition of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK in osteoblasts, and that these suppressive effects are independent of the activation of SIRT1.

Heat shock protein 22 (HSPB8) limits TGF-β-stimulated migration of osteoblasts.

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII.

Rac limits TGF-β-induced VEGF synthesis in osteoblasts

We previously showed that transforming growth factor-β (TGF-β) stimulates vascular endothelial growth factor (VEGF) synthesis via p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rac, which is a member of the Rho family of small GTPases, in the TGF-β-stimulated VEGF synthesis in MC3T3-E1 cells. TGF-β markedly increased the levels of GTP-bound Rac. NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, significantly increased both the release of VEGF and the mRNA expression levels induced by TGF-β. In addition, the release of VEGF stimulated by TGF-β was amplified in Rac-knock down cells. Meanwhile, SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the TGF-β-stimulated VEGF release. However, the phosphorylation of Smad2 or Smad3 induced by TGF-β was hardly affected by NSC23766. On the other hand, NSC23766 enhanced the TGF-β-induced phosphorylation of p38 MAP kinase without affecting the phosphorylation of p44/p42 MAP kinase or SAPK/JNK. Furthermore, the phosphorylation of p38 MAP kinase induced by TGF-β was markedly upregulated in the Rac-knock down cells. These results strongly suggest that Rac negatively regulates the TGF-β-stimulated VEGF synthesis via the inhibition of p38 MAP kinase in osteoblasts.

Resveratrol amplifies BMP‑4‑stimulated osteoprotegerin synthesis via p38 MAP kinase in osteoblasts

Resveratrol is a naturally occurring polyphenol that possesses health‑related properties, and is predominantly found in grapes and berries. Bone morphogenetic protein‑4 (BMP‑4) stimulates osteocalcin synthesis via p38 mitogen‑activated protein (MAP) kinase in osteoblast‑like MC3T3‑E1 cells. The present study aimed to investigate the effects of resveratrol on BMP‑4‑induced osteoprotegerin (OPG) synthesis in MC3T3‑E1 cells. Resveratrol alone had no effect on OPG expression levels, but significantly enhanced BMP‑4‑induced OPG release. In addition, resveratrol markedly amplified the mRNA expression levels of BMP‑4‑induced OPG. SB203580 is an inhibitor of p38 MAP kinase, which was shown to suppress BMP‑4‑stimulated OPG release. BMP‑4‑induced phosphorylation of p38 MAP kinase was also enhanced by resveratrol. Furthermore, SB203580 significantly reduced the resveratrol‑induced amplification of BMP‑4‑stimulated OPG release. These results suggested that resveratrol was able to upregulate BMP‑4‑stimulated OPG synthesis via the amplification of p38 MAP kinase activity in osteoblasts.

Amplification by (-)-epigallocatechin gallate and chlorogenic acid of TNF-α-stimulated interleukin-6 synthesis in osteoblasts.

Polyphenolic compounds in foods and beverages have beneficial effects on human health. (-)-Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), a major flavonoid in green tea and a major phenolic acid in coffee, respectively, have potent properties, including antioxidative effects. Our previous study demonstrated that p70 S6 kinase acts as a negative regulator in tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, the effects of EGCG and CGA on the TNF-α-stimulated interleukin‑6 synthesis were investigated in MC3T3‑E1 cells. EGCG and CGA significantly enhanced TNF-α-stimulated interleukin-6 release. In addition, the interleukin-6 mRNA expression levels induced by TNF‑α were supported by EGCG, as well as CGA. EGCG markedly attenuated the TNF-α-induced phosphorylation of p70 S6 kinase whereas CGA failed to affect the phosphorylation. These results strongly suggest that EGCG and CGA enhance the TNF-α-stimulated interleukin-6 synthesis in osteoblasts, and that the amplifying effect of EGCG, but not CGA, is exerted via inhibiting p70 S6 kinase.