Gen Kuroyanagi

Resveratrol suppresses prostaglandin F2α-induced osteoprotegerin synthesis in osteoblasts: Inhibition of the MAP kinase signaling

Resveratrol, a natural polyphenol abundantly found in grape skins and red wine, possesses various beneficial properties for human health. In the present study, we investigated the mechanism underlying the effects of prostaglandin F2α (PGF2α) on osteoprotegerin (OPG) synthesis and of resveratrol on the OPG synthesis in osteoblast-like MC3T3-E1 cells. PGF2α stimulated both the release of the OPG protein and the expression of OPG mRNA. Treatment with PD98059, SB203580 and SP600125, specific inhibitors of MEK1/2, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) all suppressed the OPG release induced by PGF2α. Resveratrol also significantly reduced the PGF2α-stimulated OPG release and the mRNA levels of OPG. Similarly, treatment with SRT1720, an activator of SIRT1, also suppressed the PGF2α-stimulated OPG release. Resveratrol and SRT1720 both attenuated the phosphorylation of p44/p42 MAP kinase, MEK1/2, Raf-1, p38 MAP kinase and SAPK/JNK induced by PGF2α. These findings strongly suggest that resveratrol suppresses PGF2α-stimulated OPG synthesis by inhibiting the MAP kinase pathways in osteoblasts, and that the effect is mediated via SIRT1 activation.

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.

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

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.

Regulation by heat shock protein 22 (HSPB8) of transforming growth factor-α-induced ovary cancer cell migration.

Accumulating evidence suggests that heat shock proteins (HSPs) are implicated in progression of cancer. HSP22 (HSPB8), a small HSP, is recognized to be ubiquitously expressed in various tissues. However, the expression and the role of HSP22 in ovarian cancer remain to be clarified. In the present study, we investigated the involvement of HSP22 in transforming growth factor (TGF)-α-induced migration of ovarian cancer cells. The expression of HSP22 was detected in a serous ovarian cancer cell line, SKOV3.ip1. The migration was reduced by down-regulation of HSP22 expression. The TGF-α-induced migration was reduced by SB203580 (a p38 MAP kinase inhibitor), SP600125 (a SAPK/JNK inhibitor) and Y27632 (a Rho-kinase inhibitor). However, down-regulation of HSP22 had little effect on the TGF-α-induced phosphorylation of p38 MAP kinase, SAPK/JNK and MYPT, a target protein of Rho-kinase. The HSP22 expression was further analyzed in 20 resected specimens of human ovarian serous carcinoma. The expression of HSP22 was detected in all the twenty tissues (8.24-109.22 pg/mg protein), and the cases with highly expression of HSP22 showed a tendency to acquire the progressive ability. Our results strongly suggest that HSP22 acts as a positive regulator in TGF-α-induced migration of ovarian cancer cells, subsequently directing ovarian cancer toward progression.

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.

Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.

We previously reported that the collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets. It has been shown that Rac, which belongs to the Rho family of small GTPases, is involved in the collagen-induced platelet aggregation. In this study, we investigated the role of Rac in the collagen-stimulated release of PDGF-AB and sCD40L in human platelets. Human blood was donated from healthy volunteers and platelet-rich plasma was obtained from the blood samples. The samples were then treated with 1.0 µg/ml collagen for 0, 1, 3, or 5 min and Rac1 activity was determined using the Rac1 Activation Assay kit. We found that collagen stimulated the activation of Rac in human platelets in a time-dependent manner. However, pre-treatment with NSC23766, a selective inhibitor of Rac-guanine nucleotide exchange factor interaction, reduced the collagen-induced platelet aggregation. NSC23766 markedly attenuated not only the collagen-induced p44/p42 MAP kinase phosphorylation, but also the phosphorylation of HSP27 at three serine residues (Ser-15, Ser-78 and Ser-82). In addition, the collagen‑induced release of PDGF-AB and sCD40L was significantly suppressed by NSC23766 in a dose-dependent manner. These results strongly suggest that Rac regulates the collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets, resulting in the stimulation of PDGF-AB secretion and the release of sCD40L.

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.