Shingo Kainuma

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.

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.

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.

Heat shock protein 27 (HSPB1) suppresses the PDGF-BB-induced migration of osteoblasts

Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB-induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild-type HSP27-overexpressing cells and the phospho-mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPK, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPK, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27.

(-)-Epigallocatechin gallate but not chlorogenic acid upregulates osteoprotegerin synthesis through regulation of bone morphogenetic protein-4 in osteoblasts

Chlorogenic acid (CGA) is a primary phenolic component of coffee and (-)-epigallocatechin gallate (EGCG) is a primary flavonoid component of green tea, both of which have been documented to possess beneficial health properties. A previous study by the present authors demonstrated that p38 mitogen-activated protein kinase (MAPK) may be associated with osteoprotegerin synthesis stimulated by bone morphogenetic protein-4 (BMP-4) in osteoblast-like MC3T3-E1 cells. In the present study, the effects of CGA and EGCG on BMP-4-stimulated osteoprotegerin synthesis in MC3T3-E1 cells were investigated. It was observed that CGA had no effect on osteoprotegerin release stimulated by BMP-4, whereas EGCG significantly enhanced BMP-4-stimulated osteoprotegerin release (P=0.003). Levels of osteoprotegerin mRNA expression induced by BMP-4 were also significantly increased by EGCG (P=0.03). By contrast, EGCG had no significant effect on phosphorylation of Smad1 or p38 MAPK induced by BMP-4. In addition, EGCG had little effect on BMP-induced phosphorylation of p70 S6 kinase; however rapamycin, as an inhibitor of p70 S6 kinase, significantly suppressed osteoprotegerin release (P=0.007). These data suggest that EGCG but not CGA may upregulate the synthesis of osteoprotegerin induced by BMP-4 in osteoblasts.

Heat Shock Protein 70 Negatively Regulates TGF-β-Stimulated VEGF Synthesis via p38 MAP Kinase in Osteoblasts

Background/Aims: We previously demonstrated that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) through the activation of p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. Heat shock protein70 (HSP70) is a ubiquitously expressed molecular chaperone. In the present study, we investigated the involvement of HSP70 in the TGF-β-stimulated VEGF synthesis and the underlying mechanism in these cells. Methods: Culture MC3T3-E1 cells were stimulated by TGF-β. Released VEGF was measured using an ELISA assay. VEGF mRNA level was quantified by RT-PCR. Phosphorylation of each protein kinase was analyzed by Western blotting. Results: VER-155008 and YM-08, both of HSP70 inhibitors, significantly amplified the TGF-β-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-β was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-β-induced phosphorylation of p38 MAP kinase. The TGF-β-induced phosphorylation of p38 MAP kinase was amplified in HSP70-knockdown cells. SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by these inhibitors of the TGF-β-induced VEGF release. Conclusion: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-β-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.

Mimosine suppresses the PGF2α-induced synthesis of osteoprotegerin but not interleukin-6 in osteoblasts

Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F2α (PGF2α) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF2α induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF2α-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1α. Both mimosine and deferoxamine significantly suppressed the PGF2α-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF2α-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF2α was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF2α‑induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.

Attenuation by incretins of thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

Incretins, the polypeptide hormone glucose- dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) secreted from the small intestine after nutrient ingestion, are generally known to stimulate insulin secretion from pancreatic β-cells. We previously demonstrated that triiodothyronine (T3) stimulates osteocalcin synthesis at least in part through p38 mitogen-activated protein kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of GIP and GLP-1 on T3-stimulated osteocalcin synthesis and the mechanism of action involved in MC3T3-E1 cells. GIP and GLP-1 markedly suppressed the T3-stimulated osteocalcin release. GIP and GLP-1 significantly attenuated the expression levels of osteocalcin mRNA induced by T3. The T3-stimulated transactivation activity of the thyroid hormone-responsive element was reduced by GIP and GLP-1. These results suggest that incretins repressed the T3-stimulated osteocalcin synthesis in osteoblast-like MC3T3-E1 cells, and the suppressive effect of incretins was mediated through transcriptional levels.

Advantages of an on-the-screwhead crosslink connector for atlantoaxial fixation using the Goel/Harms technique

The goal of fixation surgery for atlantoaxial instability is to achieve solid bony fusion. Achieving bony fusion as early as possible is beneficial for patients. Although placement of a transverse cross-link connector (XL) provides greater biomechanical strength, XLs have not been able to be placed when performing the Goel/Harms procedure. Recently, placing a XL on the screw head (on-the-head XL [OH-XL]) has become a viable option during the Goel/Harms procedure. However, there is little evidence demonstrating whether applying an OH-XL achieves early solid bony union. A matched-control study was conducted to investigate whether placing an OH-XLs in C1/2 fixation surgery provides earlier bone union or not. Eighteen patients who underwent atlantoaxial fusion with OH-XLs (X-group), and 17 age and sex-matched patients without OH-XLs (NX-group) were compared. Bony union was assessed using reconstructed sagittal and coronal computed tomography images. Six months after surgery, six patients in the X-group and one patient in the NX-group achieved bony union (p = .0338). One year after surgery, 14 patients in the X-group and 4 patients in the NX-group achieved bone union (p = .0010). Two years after surgery, 17 patients in the X-group and 9 patients in the NX-group achieved bony union (p = .0011). This is the first report of the clinical application of OH-XLs for the Goel/Harms procedure. OH-XLs yield earlier bony fusion; thus, placing OH-XLs is beneficial for atlantoaxial fixation surgery using lateral mass screws of the atlas.

HSP90 limits thrombin‑stimulated IL‑6 synthesis in osteoblast‑like MC3T3‑E1 cells: Regulation of p38 MAPK

Heat shock protein 90 (HSP90), expressed abundantly in a variety of cell types, is a molecular chaperone, and has a central role in protein homeostasis under stress conditions. In our previous study, it was shown that thrombin stimulates interleukin‑6 (IL‑6) synthesis via p44/p42 mitogen‑activated protein kinase (MAPK) and p38 MAPK in osteoblast‑like MC3T3‑E1 cells, and that Rho‑kinase acts as a positive regulator at a point upstream of p38 MAPK, but not p44/p42 MAPK. The present study investigated whether or not HSP90 is involved in the thrombin‑stimulated synthesis of IL‑6 and examined the mechanism by which HSP90 is involved in MC3T3‑E1 cells. Cultured cells were stimulated by treatment with thrombin. IL‑6 concentrations in MC3T3‑E1 cells were determined using an ELISA assay, and levels of phosphorylated p38 MAPK, p44/p42 MAPK and myosin phosphatase targeting subunit, a substrate of Rho‑kinase; were analyzed by western blotting. The 17‑allylamino‑17demethoxy‑geldanamycin (17‑AAG) and 17‑dimethylamino‑ethylamino‑17‑demethoxy‑geldanamycin (17‑DMAG) HSP90 inhibitors significantly enhanced the thrombin‑stimulated release of IL‑6. Geldanamycin, another inhibitor of HSP90, also upregulated the release and mRNA expression of IL‑6. 17‑AAG and geldanamycin markedly potentiated the thrombin‑induced phosphorylation of p38 MAPK without affecting the phosphorylation of p44/p42 MAPK or myosin phosphatase targeting subunit, a substrate of Rho‑kinase. Additionally, the enhancement by 17‑AAG of the thrombin‑stimulated release of IL‑6 was significantly reduced by SB203580, an inhibitor of p38 MAPK. These results suggested that the thrombin‑stimulated synthesis of IL‑6 was limited by HSP90 in osteoblasts, and that the effects of HSP90 were exerted at the point between Rho‑kinase and p38 MAPK.