Polyphenolic compounds in beverages may have benefits in the prevention of osteoporosis. It has been demonstrated previously that insulin-like growth factor-I (IGF-I) could stimulate the migration of osteoblasts. In the present study, it was investigated whether chlorogenic acid, a major polyphenol in coffee, and (-)-epigallocatechin gallate (EGCG), a major polyphenol in green tea, could affect this IGF-I-stimulated migration of osteoblast-like MC3T3-E1 cells. The IGF-I-stimulated osteoblast migration, evaluated by Transwell cell migration and wound-healing assays, was inhibited by EGCG but not chlorogenic acid. IGF-I induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p70 S6 kinase and Akt. The IGF-I-induced migration was suppressed by PD98059, a MAP kinase kinase 1/2 inhibitor, and deguelin, an Akt inhibitor, but not rapamycin, an inhibitor of the upstream kinase of p70 S6 kinase (mammalian target of rapamycin). EGCG attenuated the IGF-I-induced phosphorylation of p44/p42 MAP kinase but not Akt. Taken together, the present results suggest that EGCG inhibits IGF-I-induced osteoblast migration via p44/p42 MAP kinase.

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.

It has been previously reported that endothelin‑1 (ET‑1) stimulates the induction of heat shock protein (HSP) 27 through the activation of p38 mitogen‑activated protein (MAP) kinase and stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. The present study investigated whether HSP90, a high‑molecular‑weight HSP, was implicated in the ET‑1‑stimulated HSP27 induction in MC3T3‑E1 cells. The effects of HSP90 inhibitors on the induction of HSP27 were examined. The HSP90 inhibitors geldanamycin and 17‑demethoxygeldanamycin (17‑DMAG) significantly amplified HSP27 induction stimulated by ET‑1 in a dose‑dependent manner. In addition, onalespib (another HSP90 inhibitor) significantly strengthened the ET‑1‑induced HSP27 protein levels. The ET‑1‑stimulated phosphorylation of p38 MAP kinase was minimally affected by geldanamycin, 17‑DMAG or onalespib. Onalespib and 17‑DMAG significantly enhanced the ET‑1‑induced phosphorylation of SAPK/JNK. In addition, SP600125, a SAPK/JNK inhibitor, notably reduced the amplification by onalespib of ET‑1‑induced HSP27. These results suggest that HSP90 limits ET‑1‑stimulated HSP27 induction at a point upstream of SAPK/JNK in osteoblasts. These results suggest that HSP90 may be a novel clinical target for metabolic bone diseases, including osteoporosis.

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.

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.