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Heat shock protein 70 (HSP70) functions as an ATP-dependent molecular chaperone under stress and is involved in protein homeostasis, folding and degradation. HSP70 inhibitors amplify TGF-β-stimulated VEGF synthesis in the mouse osteoblastic MC3T3-E1 cell line. Basic fibroblast growth factor (bFGF) stimulates IL-6 release via p38 MAPK in MC3T3-E1 osteoblast-like cells. In the present study, the effects of HSP70 on the bFGF-stimulated release of IL-6 was evaluated using MC3T3-E1 osteoblast-like cells. IL-6 release and mRNA expression levels were analyzed using ELISA and reverse transcription-quantitative PCR, respectively. Phosphorylation of p38 MAPK and HSP70 was assessed using western blotting. HSP70 inhibitor VER-155008 significantly increased the bFGF-stimulated release of IL-6 in both MC3T3-E1 osteoblastic cells and normal human osteoblasts. Furthermore, VER-155008 significantly enhanced the mRNA expression levels of IL-6 stimulated by bFGF. Western blotting demonstrated a significant increase in the bFGF-stimulated phosphorylation of p38 MAPK in VER-155008-treated MC3T3-E1 cells. A significant increase in the bFGF-stimulated phosphorylation of p38 MAPK was also demonstrated in MC3T3-E1 cells treated with YM-08, another HSP70 inhibitor. VER-155008 or YM-08 did not significantly affect the expression of HSP70 with or without bFGF stimulation. Finally, the specific p38 MAPK inhibitor SB203580 markedly suppressed the enhancing effects of VER-155008 on bFGF-stimulated release of IL-6. Taken together, these results indicated that HSP70 inhibitor amplified bFGF-stimulated release of IL-6 through p38 MAPK activation in the osteoblastic MC3T3-E1 cell line.

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.

Aim In acute medicine, we occasionally treat life‐threatening conditions such as sepsis and trauma, which cause severe thrombocytopenia. Serum thrombopoietin levels have been reported to increase under the condition of thrombocytopenia related to severity. Collagen is a crucial activator of platelets, and Rho family members, such as Rho/Rho‐kinase and Rac, play roles as active molecules involved in the intracellular signaling pathways in platelet activation. The present study aimed to elucidate the effects of thrombopoietin (TPO) on subthreshold low‐dose collagen‐stimulated human platelets in terms of Rho/Rho‐kinase and Rac. Methods Platelet‐rich plasma donated from healthy volunteers was stimulated by the subthreshold low‐dose of collagen after pretreatment with TPO and/or NSC23766, an inhibitor of the Rac‐guanine nucleotide exchange factor interaction, or Y27632, an inhibitor of Rho‐kinase. Platelet aggregation was measured using an aggregometer based on laser‐scattering methods. Proteins involved in intracellular signaling were analyzed using western blotting, and the secretion of platelet‐derived growth factor‐AB from activated platelets was determined using an enzyme‐linked immunosorbent assay. Results Under the existence of TPO, the low dose of collagen remarkably elicited the aggregation and platelet‐derived growth factor‐AB secretion of platelets, which were suppressed by NSC23766 and Y27632. The combination of TPO and collagen considerably induced a transient increase of guanosine triphosphate (GTP)‐binding Rac and GTP‐binding Rho followed by an increase of phosphorylated cofilin, a Rho‐kinase substrate. Conclusion These results strongly suggest that TPO and collagen in low doses cooperatively potentiate human platelet activation through both Rac and Rho/Rho‐kinase mediated pathways. Thrombopoietin and collagen in low doses cooperatively potentiate human platelet activation via both Rac and Rho/Rho‐kinase mediated pathways. The effect of TPO on platelet activation under the stimulation of collagen, even though of which the levels are insufficient to trigger by itself, might indicate a compensatory mechanism to enhance platelet activation in thrombocytopenia.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. However, effective chemopreventive and chemotherapeutic agents for this cancer have not yet been developed. In clinical trials acyclic retinoid (ACR) and vitamin K2 (VK2) decreased the recurrence rate of HCC. In the present study we examined the possible combined effects of ACR or another retinoid 9‐cis retinoic acid (9cRA) plus VK2 in the HuH7 human HCC cell line. We found that the combination of 1.0 µM ACR or 1.0 µM 9cRA plus 10 µM VK2 synergistically inhibited the growth of HuH7 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VK2 also acted synergistically to induce apoptosis in HuH7 cells. Treatment with VK2 alone inhibited phosphorylation of the retinoid X receptor (RXR)α protein, which is regarded as a critical factor for liver carcinogenesis, through inhibition of Ras activation and extracellular signal‐regulated kinase phosphorylation. Moreover, the inhibition of RXRα phosphorylation by VK2 was enhanced when the cells were cotreated with ACR. The combination of retinoids plus VK2 markedly increased both the retinoic acid receptor responsive element and retinoid X receptor responsive element promoter activities in HuH7 cells. Our results suggest that retinoids (especially ACR) and VK2 cooperatively inhibit activation of the Ras/MAPK signaling pathway, subsequently inhibiting the phosphorylation of RXRα and the growth of HCC cells. This combination might therefore be effective for the chemoprevention and chemotherapy of HCC. (Cancer Sci 2007; 98: 431–437)

Incretins, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine cells after food ingestion, are currently recognized to regulate glucose metabolism through insulin secretion. We previously demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces the migration of osteoblast-like MC3T3-E1 cells through mitogen-activated protein (MAP) kinases, including p38 MAP kinase. In the present study, we investigated whether or not incretins affect the osteoblast migration. The PDGF-BB-induced cell migration was significantly reinforced by GLP-1, GIP or cAMP analogues in MC3T3-E1 cells and normal human osteoblasts. The upregulated migration by GLP-1 or cAMP analogues was suppressed by H89, an inhibitor of protein kinase A. The amplification by GLP-1 of migration induced by PDGF-BB was almost completely reduced by SB203580, a p38 MAP kinase inhibitor in MC3T3-E1 cells and normal human osteoblasts. In addition, GIP markedly strengthened the PDGF-BB-induced phosphorylation of p38 MAP kinase. Exendin-4, a GLP-1 analogue, induced Rho A expression and its translocation from cytoplasm to plasma membranes in osteoblasts at the epiphyseal lines of developing mouse femurs in vivo . These results strongly suggest that incretins accelerates the PDGF-BB-induced migration of osteoblasts via protein kinase A, and the up-regulation of p38 MAP kinase is involved in this acceleration. Our findings may highlight the novel potential of incretins to bone physiology and therapeutic strategy against bone repair.

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.

A malfunction in retinoid X receptor (RXR) α due to phosphorylation is associated with the development of hepatocellular carcinoma. However, the precise mechanisms by which phosphorylated RXRα loses its physiological function remain unclear. In the present study we examined whether phosphorylation of RXRα affects its dimeric activity. Fluorescence resonance energy transfer studies and immunoprecipitation assays showed that the physical interaction between RXRα and retinoic acid receptor β was impaired when 293T cells were transfected with phosphomimic mutant RXRα (T82D/S260D), whereas this interaction was activated at a level similar to wild‐type RXRα‐transfected cells when the cells were transfected with an unphosphorylated mutant RXRα (T82A/S260A). Treating the T82A/S260A‐transfected cells with retinoid resulted in a significant increase in the transcriptional activities of the retinoic acid receptor responsive element and RXR responsive element promoters, whereas these transcriptional activities did not increase in the T82D/S260D‐transfected cells. Transfection with T82A/S260A enhanced both the inhibition of cell growth and the induction of apoptosis caused by retinoid, although the T82D/S260D‐transfected cells lost their responsiveness to retinoid. Moreover, transfection with T82A/S260A caused an inhibition of cell growth and a reduction of colony‐forming ability in soft agar in HuH7 human hepatocellular carcinoma cells. These findings suggest that phosphorylation of RXRα abolishes its ability to form homodimers and heterodimers with RXR and retinoic acid receptor β, thus resulting in the loss of cell growth control and the acceleration of cancer development. In conclusion, the inhibition of RXRα phosphorylation and the restoration of its original function as a master regulator of nuclear receptors might therefore be an effective strategy for controlling cancer cell growth. (Cancer Sci 2007; 98: 1868–1874)

Wnt3a is a crucial modulator of bone metabolism through the canonical Wnt/β-catenin signaling pathway in bone-forming osteoblasts. We previously reported that the expression of osteocalcin is stimulated by triiodothyronine (T3) at least in part through the activation of p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on the T3-induced osteocalcin expression in these cells. Wnt3a suppressed the release of osteocalcin induced by T3. The inhibitory effect of Wnt3a was dose-dependent between 0.3 and 30 ng/ml. SB216763, an inhibitor of glycogen synthase kinase-3β, that reduces the phosphorylation of β-catenin, inhibited the T3-induced osteocalcin release. Wnt3a, as well as SB216763, reduced the expression of osteocalcin mRNA induced by T3. The transcriptional activity induced by T3, assessed by a luciferase activity, was also suppressed by both Wnt3a and SB216763. In contrast, Wnt3a did not markedly affect the T3-stimulated phosphorylation of p38 MAP kinase. These results suggested that Wnt3a downregulates the T3-stimulated osteocalcin expression in MC3T3-E1 cells, and the suppressive effect of Wnt3a is independent of p38 MAP kinase.

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 resve ratrol 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 amplifi cation of p38 MAP kinase activity in osteoblasts.