RESUMEN
Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.
Asunto(s)
Alprostadil , Interleucina-6 , Interleucina-6/metabolismo , Alprostadil/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Osteoblastos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Heat shock protein (HSP) 90 functions as a molecular chaperone and is constitutively expressed and induced in response to stress in many cell types. We have previously demonstrated that transforming growth factor-ß (TGF-ß), the most abundant cytokine in bone cells, induces the expression of HSP27 through Smad2, p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in mouse osteoblastic MC3T3-E1 cells. This study investigated the effects of HSP90 on the TGF-ß-induced HSP27 expression and the underlying mechanism in mouse osteoblastic MC3T3-E1 cells. METHODS: Clonal osteoblastic MC3T3-E1 cells were treated with the HSP90 inhibitors and then stimulated with TGF-ß. HSP27 expression and the phosphorylation of Smad2, p44/p42 MAPK, p38 MAPK, and SAPK/JNK were evaluated by western blot analysis. RESULT: HSP90 inhibitors 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib significantly enhanced the TGF-ß-induced HSP27 expression. TGF-ß inhibitor SB431542 reduced the enhancement by 17-DMAG or onalespib of the TGF-ß-induced HSP27 expression levels. HSP90 inhibitors, geldanamycin, onalespib, and 17-DMAG did not affect the TGF-ß-stimulated phosphorylation of Smad2. Geldanamycin did not affect the TGF-ß-stimulated phosphorylation of p44/p42 MAPK or p38 MAPK but significantly enhanced the TGF-ß-stimulated phosphorylation of SAPK/JNK. Onalespib also increased the TGF-ß-stimulated phosphorylation of SAPK/JNK. Furthermore, SP600125, a specific inhibitor for SAPK/JNK, significantly suppressed onalespib or geldanamycin's enhancing effect of the TGF-ß-induced HSP27 expression levels. CONCLUSION: Our results strongly suggest that HSP90 inhibitors upregulated the TGF-ß-induced HSP27 expression and that these effects of HSP90 inhibitors were mediated through SAPK/JNK pathway in osteoblasts.
Asunto(s)
Proteínas de Choque Térmico HSP27 , Factor de Crecimiento Transformador beta , Animales , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/farmacología , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacología , Humanos , Ratones , Osteoblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
Incretins including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), which are secreted from the small intestine after oral food ingestion, are currently well-known to stimulate insulin secretion from pancreatic ß-cells and used for the treatment of type 2 diabetes mellitus. We have previously reported that prostaglandin F2α (PGF2α) stimulates the synthesis of interleukin-6 (IL-6) and osteoprotegerin in osteoblast-like MC3T3-E1 cells, and that IL-6 and osteoprotegerin release are mediated through the p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathways. In the present study, we investigated the effects of incretins including GLP-1 and GIP, on the PGF2α-induced synthesis of IL-6 and osteoprotegerin and examined the detailed mechanism in osteoblast-like MC3T3-E1 cells. We found that GIP and GLP-1 significantly stimulated the PGF2α-induced synthesis of IL-6 in osteoblast-like MC3T3-E1 cells. In addition, GIP and GLP-1 significantly enhanced the PGF2α-induced mRNA expression levels of IL-6. On the other hand, GIP and GLP-1 markedly stimulated the PGF2α-induced synthesis of osteoprotegerin. However, the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or JNK induced by PGF2α was not affected by GIP or GLP-1. Therefore, these results strongly suggest that incretins enhance the PGF2α-induced synthesis of IL-6 and osteoprotegerin in osteoblast-like MC3T3-E1 cells. However, these syntheses are not mediated through p44/p42 MAP kinase, p38 MAP kinase, or JNK pathways.
Asunto(s)
Dinoprost/farmacología , Polipéptido Inhibidor Gástrico/farmacología , Péptido 1 Similar al Glucagón/farmacología , Incretinas/metabolismo , Interleucina-6/biosíntesis , Osteoblastos/metabolismo , Osteoprotegerina/biosíntesis , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: We have demonstrated that epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells is mediated through p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK), and Akt.The molecular chaperone heat shock protein 90 (HSP90) is abundantly expressed in osteoblasts. However, the role of HSP90 in osteoblast migration remains obscure. OBJECTIVE: In this study, we investigated the effect of HSP90 inhibitors on the EGF-induced migration of MC3T3-E1 cells and the mechanism. METHODS: Clonal osteoblast-like MC3T3-E1 cells were treated with the HSP90 inhibitors geldanamycin or onalespib and then stimulated with EGF. Cell migration was evaluated using the transwell cell migration assay and wound-healing assay. The viability of MC3T3-E1 cells was analyzed using the Cell Counting Kit-8. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, Akt, and protein kinase-like endoplasmic reticulum kinase (PERK) was evaluated by western blot analysis. RESULTS: EGF-induced migration was significantly suppressed by geldanamycin and onalespib, evaluated by both transwell cell migration assay and wound-healing assay. Geldanamycin and onalespib did not significantly alter cell viability. Geldanamycin and onalespib markedly reduced the EGF-induced phosphorylation of p44/p42 MAP kinase, but not p38 MAP kinase or Akt. By contrast, geldanamycin and onalespib increased the EGF-induced phosphorylation of SAPK/JNK. PERK phosphorylation was not significantly affected by geldanamycin or onalespib. CONCLUSION: Our results strongly suggest that HSP90 inhibitors reduce the EGF-induced osteoblast migration through the p44/p42 MAP kinase.
Asunto(s)
Factor de Crecimiento Epidérmico , Proteína Quinasa 1 Activada por Mitógenos , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/farmacología , Osteoblastos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacologíaRESUMEN
BACKGROUND: Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. METHODS: MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. RESULTS: The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. CONCLUSIONS: Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.
Asunto(s)
Dinoprost/farmacología , Proteínas de Choque Térmico/fisiología , Interleucina-6/metabolismo , Chaperonas Moleculares/fisiología , Osteoblastos/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/farmacología , Fosforilación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.
Asunto(s)
Adenosina Trifosfato/farmacología , Benzamidas/farmacología , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Interleucina-6/biosíntesis , Isoindoles/farmacología , Lactamas Macrocíclicas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Osteoblastos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Proteínas HSP90 de Choque Térmico/metabolismo , RatonesRESUMEN
Resveratrol is a natural polyphenol with beneficial antioxidant properties. It suppresses the migration of osteoblast-like MC3T3-E1 cells induced by epidermal growth factor, via SIRT1-mediated inhibition of SAPK/JNK and Akt. Moreover, insulin-like growth factor-I (IGF-I) stimulates the migration involving the pathways of p44/p42 mitogen-activated protein (MAP) kinase and Akt. Therefore, we investigated the effects of resveratrol on IGF-I-induced cell migration. Resveratrol and SRT1720, an activator of SIRT1, suppressed IGF-I-induced migration. Inauhzin, a SIRT1 inhibitor, significantly rescued the inhibition of IGF-I-induced cell migration by resveratrol. Resveratrol inhibited IGF-I-induced phosphorylation of p44/p42 MAP kinase but not Akt. SRT1720 inhibited IGF-I-induced phosphorylation of p44/p42 MAP kinase. Furthermore, PD98059, p44/p42 MAP kinase inhibitor, alone suppressed IGF-I-induced osteoblast migration, but did not affect the suppressive effect of resveratrol when administered concomitantly. These findings strongly suggest that resveratrol suppresses IGF-I-induced osteoblast migration via SIRT1 activation at least partially by attenuating the p44/p42 MAP kinase pathway.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Resveratrol/farmacología , Células 3T3 , Animales , Activación Enzimática/efectos de los fármacos , Ratones , Osteoblastos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuina 1/metabolismoRESUMEN
Osteoprotegerin (OPG) synthesized by osteoblasts is currently considered a crucial regulator to suppress the formation and function of osteoclasts. We previously showed that the synthesis of OPG is stimulated by prostaglandin F2α (PGF2α) in the involvement of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. We also found that Rho-kinase is involved in the signaling of PGF2α upstream of p38 MAPK in these cells. Tramadol is widely used to treat chronic pain, such as low back pain associated with osteoporosis. We investigated whether or not tramadol affects the OPG release induced by PGF2α in osteoblast-like MC3T3-E1 cells. The levels of OPG in the conditioned medium were measured by an enzyme-linked immunosorbent assay. The mRNA expression of OPG was determined with real-time reverse transcription polymerase chain reaction. The phosphorylation of target protein was determined with a Western blot analysis. PGF2α induced the release and the mRNA expression of OPG, which tramadol significantly enhanced. Morphine, a selective µ-opioid receptor (MOR) agonist, also enhanced the PGF2α-induced OPG release. In addition, naloxone, a MOR antagonist, suppressed the enhancement by tramadol or morphine of the PGF2α-induced OPG synthesis. Tramadol upregulated the phosphorylation of SAPK/JNK and p38 MAPK stimulated by PGF2α but not that of p44/p42 MAPK or myosin phosphatase targeting protein (MYPT), a substrate of Rho-kinase. The inhibitors of both p38 MAPK and SAPK/JNK, SB203580 and SP600125, respectively, reduced the tramadol amplification of OPG release stimulated by PGF2α. The present results strongly suggest that tramadol enhances the synthesis of OPG stimulated by PGF2α through MOR in osteoblasts, and that the amplifying effect is exerted at upstream of p38 MAPK and SAPK/JNK but downstream of Rho-kinase.
RESUMEN
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.
Asunto(s)
Becaplermina/metabolismo , Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Incretinas/farmacología , Osteoblastos/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Becaplermina/genética , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Ratones , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Fosforilación , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Wnt3a is a crucial modulator of bone metabolism through the canonical Wnt/ß-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-3ß, that reduces the phosphorylation of ß-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.
RESUMEN
Migration of osteoblasts to the sites resorbed by osteoclasts is an essential step in bone remodeling. However, the exact mechanism of osteoblast migration is still not known. We have shown that platelet-derived growth factor (PDGF)-BB induces the migration of osteoblast-like MC3T3-E1 cells through the activation of p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and p44/p42 MAP kinase. Evidence is accumulating that heat shock protein 90 (HSP90) acts as a central regulator of proteostasis under stress conditions and physiological cell functions. In the present study, using transwell cell migration assay and wound-healing assay, we investigated the involvement of HSP90 in the PDGF-BB-stimulated migration of MC3T3-E1 cells, and the underlying signaling mechanism estimated by Western blot analyses. Onalespib, an HSP90 inhibitor, significantly reduced the PDGF-BB-stimulated migration evaluated by the two types of migration assays. The cell migration was also suppressed by geldanamycin, another type of HSP90 inhibitor. Onalespib markedly attenuated the PDGF-BB-elicited phosphorylation of p44/p42 MAP kinase without affecting that of p38 MAP kinase or JNK. In addition, the phosphorylation of p44/p42 MAP kinase by PDGF-BB was reduced by geldanamycin. Taken together, these results strongly suggest that HSP90 inhibitors suppress the PDGF-BB-induced osteoblast migration through the attenuation of p44/p42 MAP kinase activity.
Asunto(s)
Becaplermina/farmacología , Benzamidas/farmacología , Benzoquinonas/farmacología , Movimiento Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoindoles/farmacología , Lactamas Macrocíclicas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoblastos/metabolismo , Animales , Línea Celular , Proteínas HSP90 de Choque Térmico/metabolismo , Ratones , Fosforilación/efectos de los fármacosRESUMEN
Heat shock protein (HSP) 90 that is ubiquitously expressed in various tissues is a major molecular chaperone. We have previously demonstrated that prostaglandin D2 (PGD2), a bone remodeling factor, elicits the expression of HSP27, a small HSP, through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of HSP90 in the PGD2-stimulated HSP27 induction and the underlying mechanism in MC3T3-E1 cells. Onalespib, an inhibitor of HSP90, significantly enhanced the PGD2-stimulated HSP27 induction. In addition, geldanamycin, another HSP90 inhibitor, potentiated the HSP27 induction. Both onalespib and geldanamycin markedly amplified the PGD2-induced phosphorylation of SAPK/JNK and p38 MAP kinase. SP600125, an inhibitor of SAPK/JNK, and SB203580, an inhibitor of p38 MAP kinase, suppressed the amplification by onalespib of the PGD2-stimulated HSP27 induction. These results strongly suggest that HSP90 plays a negative role in the HSP27 induction stimulated by PGD2 in osteoblasts, and that the inhibitory effect of HSP90 is mediated through the regulation of SAPK/JNK and p38 MAP kinase.
Asunto(s)
Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Prostaglandina D2/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Células 3T3 , Animales , Antracenos/farmacología , Benzamidas/farmacología , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Isoindoles/farmacología , Ratones , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacologíaRESUMEN
BACKGROUND: We have recently reported that epidermal growth factor (EGF) induces migration of osteoblast-like MC3T3-E1 cells through the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, stress-activated protein kinase/ c-Jun N-terminal kinase (SAPK/JNK) and Akt. Furthermore, we demonstrated that heat shock protein 70 (HSP70) down-regulates the transforming growth factor-ß- stimulated vascular endothelial growth factor synthesis via suppression of p38 MAP kinase in osteoblast-like MC3T3-E1 cells. However, the exact role of HSP70 underlying osteoblast migration is not fully elucidated. OBJECTIVE: The aim of this study is to investigate the effects of HSP70 inhibitors on the EGF-stimulated osteoblast migration, and the underlying mechanism. METHODS: Osteoblast-like MC3T3-E1 cells were treated with two types of HSP70 inhibitors, VER-155008 or YM-08. Transwell cell migration assay and wound-healing assay were analyzed for osteoblast migration. The expression levels of HSP70 and the phosphorylation of p38 MAP kinase, p44/p42 MAP kinase, SAPK/JNK or Akt were evaluated by a Western blot analysis. RESULTS: EGF hardly affected the expression levels of HSP70 at the present or absent of VER-155008. EGF-stimulated migration was significantly reduced by both HSP70 inhibitors, VER-155008 and YM-08, determined by a transwell cell migration assay. The suppressive effects of both HSP70 inhibitors on the migration stimulated by EGF were also observed by a wound-healing assay. VER-155008 inhibited the EGF-induced phosphorylation of p44/p42 MAP kinase and AKT, but not p38 MAP kinase or SAPK/JNK. CONCLUSION: This study provides new evidence that HSP70 inhibitors reduce the EGFstimulated migration of osteoblasts through the suppression of p44/p42 MAP kinase and Akt.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Proteínas HSP70 de Choque Térmico/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Osteoblastos/metabolismo , Animales , Benzotiazoles/farmacología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HSP70 de Choque Térmico/análisis , Ratones , Osteoblastos/citología , Nucleósidos de Purina/farmacología , Tiazolidinas/farmacologíaRESUMEN
Heat shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone in a variety of cells including osteoblasts. We previously showed that insulin-like growth factor-I (IGF-I) elicits migration of osteoblast-like MC3T3-E1 cells through the activation of phosphatidylinositol 3-kinase/Akt and p44/p42 mitogen-activated protein (MAP) kinase. In the present study, we investigated the effects of HSP70 inhibitors on the IGF-I-elicited migration of these cells and the mechanism involved. The IGF-I-stimulated osteoblast migration evaluated by a wound-healing assay and by a transwell cell migration was significantly reduced by VER-155008 and YM-08, which are both HSP70 inhibitors. VER-155008 markedly suppressed the IGF-I-induced phosphorylation of p44/p42 MAP kinase without affecting that of Akt. In conclusion, our results strongly suggest that the HSP70 inhibitor reduces the IGF-I-elicited migration of osteoblasts via the p44/p42 MAP kinase.
RESUMEN
Phenolic compounds provide health benefits in humans. A previous study by our group has indicated that the epidermal growth factor (EGF)induced migration of osteoblastlike MC3T3E1 cells is mediated by the phosphorylation of p44/p42 mitogenactivated protein (MAPK), p38 MAPK, stressactivated protein kinase (SAPK)/cJun Nterminal kinase (JNK) and Akt, and that resveratrol, a major polyphenol in grape skin, suppresses the EGFinduced migration by attenuating Akt and SAPK/JNK activation. In the present study, the effects of chlorogenic acid, a major phenolic acid in coffee, and ()epigallocatechin gallate (EGCG), a major flavonoid in green tea, on the EGFinduced migration of MC3T3E1 cells were investigated. EGCG significantly reduced the EGFinduced migration as evaluated by a Transwell migration assay and by a wound healing assay. However, chlorogenic acid failed to affect the EGFinduced migration. The phosphorylation of p38 MAPK induced by EGF was significantly suppressed by EGCG; however, the EGFinduced phosphorylation of p44/p42 MAP kinase, SAPK/JNK or Akt was not affected by EGCG. These results suggest that EGCG, but not chlorogenic acid, suppresses EGFinduced osteoblast migration through inhibiting p38 MAPK activation.
Asunto(s)
Catequina/análogos & derivados , Movimiento Celular/efectos de los fármacos , Ácido Clorogénico/farmacología , Factor de Crecimiento Epidérmico/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Catequina/farmacología , Línea Celular , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , RatonesRESUMEN
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.
RESUMEN
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 interleukin6 (IL6) synthesis via p44/p42 mitogenactivated protein kinase (MAPK) and p38 MAPK in osteoblastlike MC3T3E1 cells, and that Rhokinase 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 thrombinstimulated synthesis of IL6 and examined the mechanism by which HSP90 is involved in MC3T3E1 cells. Cultured cells were stimulated by treatment with thrombin. IL6 concentrations in MC3T3E1 cells were determined using an ELISA assay, and levels of phosphorylated p38 MAPK, p44/p42 MAPK and myosin phosphatase targeting subunit, a substrate of Rhokinase; were analyzed by western blotting. The 17allylamino17demethoxygeldanamycin (17AAG) and 17dimethylaminoethylamino17demethoxygeldanamycin (17DMAG) HSP90 inhibitors significantly enhanced the thrombinstimulated release of IL6. Geldanamycin, another inhibitor of HSP90, also upregulated the release and mRNA expression of IL6. 17AAG and geldanamycin markedly potentiated the thrombininduced phosphorylation of p38 MAPK without affecting the phosphorylation of p44/p42 MAPK or myosin phosphatase targeting subunit, a substrate of Rhokinase. Additionally, the enhancement by 17AAG of the thrombinstimulated release of IL6 was significantly reduced by SB203580, an inhibitor of p38 MAPK. These results suggested that the thrombinstimulated synthesis of IL6 was limited by HSP90 in osteoblasts, and that the effects of HSP90 were exerted at the point between Rhokinase and p38 MAPK.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Osteoclastos/metabolismo , Trombina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Ratones , Trombina/metabolismoRESUMEN
It has been previously reported that endothelin1 (ET1) stimulates the induction of heat shock protein (HSP) 27 through the activation of p38 mitogenactivated protein (MAP) kinase and stressactivated protein kinase/cJun Nterminal kinase (SAPK/JNK) in osteoblastlike MC3T3E1 cells. The present study investigated whether HSP90, a highmolecularweight HSP, was implicated in the ET1stimulated HSP27 induction in MC3T3E1 cells. The effects of HSP90 inhibitors on the induction of HSP27 were examined. The HSP90 inhibitors geldanamycin and 17demethoxygeldanamycin (17DMAG) significantly amplified HSP27 induction stimulated by ET1 in a dosedependent manner. In addition, onalespib (another HSP90 inhibitor) significantly strengthened the ET1induced HSP27 protein levels. The ET1stimulated phosphorylation of p38 MAP kinase was minimally affected by geldanamycin, 17DMAG or onalespib. Onalespib and 17DMAG significantly enhanced the ET1induced phosphorylation of SAPK/JNK. In addition, SP600125, a SAPK/JNK inhibitor, notably reduced the amplification by onalespib of ET1induced HSP27. These results suggest that HSP90 limits ET1stimulated 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.
Asunto(s)
Endotelina-1/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Osteoblastos/metabolismo , Animales , Benzoquinonas/farmacología , Línea Celular , Humanos , Lactamas Macrocíclicas/farmacología , Ratones , Fosforilación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Heat shock protein 22 (HSP22) is ubiquitously expressed in various types of cells including in osteoblasts. We previously reported that tumor necrosis factor (TNF)-α stimulates interleukin (IL)-6 synthesis via p44/p42 MAPK in osteoblast-like MC3T3-E1 cells and that mTOR/p70 S6 kinase (p70 S6K) negatively regulates the IL-6 synthesis. In this study, we investigated the involvement of HSP22 in TNF-α-stimulated-IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. HSP22 knockdown reduces TNF-α-stimulated release of IL-6. In addition, HSP22 knockdown strengthens TNF-α-induced phosphorylation of p70 S6K but suppresses that of p44/p42 MAPK. HSP22 coimmunoprecipitates with mTOR. HSP22 knockdown increases the basal levels of phosphorylated mTOR. These results strongly suggest that HSP22 interacts with mTOR and regulates TNF-α-induced IL-6 synthesis in osteoblasts.
Asunto(s)
Proteínas del Choque Térmico HSP20/metabolismo , Interleucina-6/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Musculares/metabolismo , Osteoblastos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Proteínas del Choque Térmico HSP20/genética , Proteínas de Choque Térmico , Interleucina-6/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Chaperonas Moleculares , Proteínas Musculares/genética , Osteoblastos/citología , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/genéticaRESUMEN
BACKGROUND/AIMS: We previously demonstrated that transforming growth factor-ß (TGF-ß) 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-ß-stimulated VEGF synthesis and the underlying mechanism in these cells. METHODS: Culture MC3T3-E1 cells were stimulated by TGF-ß. 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-ß-stimulated VEGF release. In addition, the expression level of VEGF mRNA induced by TGF-ß was enhanced by VER-155008. These inhibitors markedly strengthened the TGF-ß-induced phosphorylation of p38 MAP kinase. The TGF-ß-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-ß-induced VEGF release. CONCLUSION: These results strongly suggest that HSP70 acts as a negative regulator in the TGF-ß-stimulated VEGF synthesis in osteoblasts, and that the inhibitory effect of HSP70 is exerted at a point upstream of p38 MAP kinase.