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Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

Oncostatin M produced by osteal macrophages has important roles in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to RANK ligand (RANKL) as a decoy receptor, and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. IL-6 is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. We also demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through the MAP kinase activation. The osteoblast-like MC3T3-E1 cells were treated with oncostatin M and then stimulated with PGD2. OPG and IL-6 synthesis were measured using an OPG and IL-6 enzyme-linked immunosorbent assay kit. OPG and IL-6 mRNA expression levels were evaluated by real-time RT-PCR. The phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase were evaluated by Western blotting. As a result, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affect bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

Bone fracture is an important trauma frequently encountered into emergency medicine as well as orthopedics reflecting an aging society. Oncostatin M, an inflammatory cytokine produced by osteal macrophages, has been considered to play a crucial role in fracture healing. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts is essential in osteoclastgenesis, and the secretion is stimulated by transforming growth factor-β (TGF-β). The aim of this study is to elucidate the effects of oncostatin M on the TGF-β-induced M-CSF synthesis in osteoblast-like MC3T3-E1 cells and the underlying mechanisms. Oncostatin M attenuated the TGF-β-stimulated M-CSF release and the mRNA expressions. SMAD3 inhibitor SIS3, p38 MAP kinase inhibitor SB203580, MEK1/2 inhibitor PD98059, and SAPK/JNK inhibitor SP600125 significantly suppressed the M-CSF release. Oncostatin M suppressed the TGF-β-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK, but failed to affect the phosphorylation of SMAD3 and p38 MAP kinase. Oncostatin M attenuated the TGF-β-stimulated vascular endothelial growth factor (VEGF) release and the TGF-β-induced mRNA expressions of VEGF. These results strongly suggest that oncostatin M downregulates TGF-β signaling upstream of p44/p42 MAP kinase and SAPK/JNK, but not SMAD 2/3 and p38 MAP kinase, in osteoblasts, leading to the attenuation of M-CSF synthesis. Our findings might provide a new therapeutic strategy for the acceleration of fracture healing process.

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 β-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.

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-β (TGF-β), 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-β-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-β. 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-β-induced HSP27 expression. TGF-β inhibitor SB431542 reduced the enhancement by 17-DMAG or onalespib of the TGF-β-induced HSP27 expression levels. HSP90 inhibitors, geldanamycin, onalespib, and 17-DMAG did not affect the TGF-β-stimulated phosphorylation of Smad2. Geldanamycin did not affect the TGF-β-stimulated phosphorylation of p44/p42 MAPK or p38 MAPK but significantly enhanced the TGF-β-stimulated phosphorylation of SAPK/JNK. Onalespib also increased the TGF-β-stimulated phosphorylation of SAPK/JNK. Furthermore, SP600125, a specific inhibitor for SAPK/JNK, significantly suppressed onalespib or geldanamycin’s enhancing effect of the TGF-β-induced HSP27 expression levels. Conclusion Our results strongly suggest that HSP90 inhibitors upregulated the TGF-β-induced HSP27 expression and that these effects of HSP90 inhibitors were mediated through SAPK/JNK pathway in osteoblasts.

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.

Tramadol, a weak μ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D

Acetaminophen is one of the most widely used analgesic and antipyretic medicines, whose long-period use has reportedly been associated with an increased risk of bone fracture. However, the mechanism underlying this undesired effect remains to be investigated. The homeostatic control of bone tissue depends on the interaction between osteoblasts and osteoclasts. Osteoprotegerin produced by osteoblasts is known to play an essential role in suppressing osteoclast induction. We have previously reported that prostaglandin (PG) E

Incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are hormones secreted from small intestine accompanied with oral intake. We previously showed that transforming growth factor (TGF)-α stimulates the migration of hepatocellular carcinoma (HCC) cells via mitogen-activated protein (MAP) kinases, AKT and Rho-kinase. However, it remains to be elucidated whether incretins affect HCC cell functions. In the present study, therefore, we investigated whether incretins affect the migration of HCC cells using human HCC-derived HuH7 cells. GLP-1, but not GIP, reduced both TGF-α- and hepatocyte growth factor (HGF)-induced cell migration. IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, enhanced the suppressive effect of GLP-1. GLP-1 attenuated the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) by TGF-α and HGF. Our results strongly suggest that GLP-1 suppresses TGF-α- and HGF-induced migration of HCC cells through inhibiting the SAPK/JNK signaling pathway, and that the inhibition by GLP-1 is due to cAMP production.

Duloxetine, a serotonin-norepinephrine reuptake inhibitor, is currently recommended as a useful medicine to chronic pain including low back pain. However, as the analogy of classical selective serotonin reuptake inhibitors, there is a concern to deteriorate osteoporosis with remaining to clarify the exact mechanism of duloxetine in bone metabolism. We have previously reported that prostaglandin E

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.

Background Olive oil polyphenols, which possess cytoprotective activities like anti-oxidant and anti-inflammatory effects, could modulate osteoblast functions. The aim of this study is to elucidate the effects and the underlying mechanisms of hydroxytyrosol and oleuropein on the tumor necrosis factor-α (TNF-α)-induced macrophage colony-stimulating factor (M-CSF) and interleukin-6 (IL-6) synthesis in osteoblasts. Methods Osteoblast-like MC3T3-E1 cells were pretreated with hydroxytyrosol, oleuropein, deguelin, PD98059 or wedelolactone, and then stimulated by TNF-α. The levels of M-CSF and IL-6 in the conditioned medium were determined with ELISA. The mRNA expression levels of M-CSF or IL-6 were determined with real-time RT-PCR. The phosphorylation levels of Akt, p44/p42 mitogen-activated protein (MAP) kinase or NF-κB in the cell lysates were determined with Western blot analysis. Results Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated M-CSF release. Deguelin, an inhibitor of Akt, significantly suppressed the TNF-α-stimulated M-CSF release, which failed to be affected by the MEK1/2 inhibitor PD98059 or the IκB inhibitor wedelolactone. Hydroxytyrosol and oleuropein suppressed the TNF-α-induced phosphorylation of Akt and p44/p42 MAP kinase. Hydroxytyrosol and oleuropein attenuated the TNF-α-stimulated IL-6 release. Hydroxytyrosol suppressed the TNF-α-induced mRNA expressions of M-CSF and IL-6. Hydroxytyrosol or oleuropein failed to affect the cell viability. Conclusion Our present findings strongly suggest that olive oil polyphenols hydroxytyrosol and oleuropein down-regulates TNF-α signaling at the points upstream of Akt and p44/p42 MAP kinase in osteoblasts, leading to the attenuation of M-CSF and IL-6 synthesis.

Tramadol, a weak μ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D2 (PGD2) induces osteoprotegerin synthesis whereby p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) are involved in osteoblast-like MC3T3-E1 cells. Herein, we investigated the mechanism underlying the effect of tramadol on the PGD2-induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD2-induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD2-stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD2-induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD2-stimulated phosphorylation of p38 MAP kinase and SAPK/JNK, but not p44/p42 MAP kinase. Both SB203580 and SP600125 suppressed the tramadol effect to enhance the PGD2-stimulated osteoprotegerin release. Tramadol enhanced the PGE2-induced osteoprotegerin release as well as PGD2. These results suggest that tramadol amplifies the PGD2-induced osteoprotegerin synthesis at the upstream of p38 MAP kinase and SAPK/JNK in the involvement of both MOR and 5-HT transporter in osteoblasts.

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.

Duloxetine, a serotonin-norepinephrine reuptake inhibitor, is currently recommended as a useful medicine to chronic pain including low back pain. However, as the analogy of classical selective serotonin reuptake inhibitors, there is a concern to deteriorate osteoporosis with remaining to clarify the exact mechanism of duloxetine in bone metabolism. We have previously reported that prostaglandin E1 (PGE1) induces the synthesis of both osteoprotegerin (OPG) and interleukin-6 (IL-6), essential regulators of bone metabolism, in osteoblast-like MC3T3-E1 cells. Based upon them, we herein investigated the mechanism whereby the effect of duloxetine on the synthesis of OPG and IL-6 induced by PGE1 in these cells. Duloxetine enhanced the release from MC3T3-E1 cells of both OPG and IL-6 stimulated by PGE1. However, reboxetine, a selective and specific inhibitor of norepinephrine reuptake, failed to affect the PGE1-induced release of OPG or IL-6. Oppositely, fluvoxamine and sertraline, agents belonging to the class of selective serotonin reuptake inhibitor, upregulated the PGE1-stimulated release of both OPG and IL-6. Duloxetine amplified the expression of OPG mRNA and IL-6 mRNA stimulated by PGE1. Duloxetine strengthened the PGE1-induced p38 MAP kinase phosphorylation, which was amplified by fluvoxamine as well. SB203880, an inhibitor of p38 MAP kinase, suppressed the amplifying effects by duloxetine or fluvoxamine on the PGE1-stimulated release of OPG and IL-6. These results strongly suggest that duloxetine could strengthen osteoblast activation by PGE1 through the upregulation of p38 MAP kinase, leading to increasing the synthesis of OPG and IL-6.

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., Tramadol; μ-opioid receptor; PGF2α; osteoprotegerin; MAP kinase; osteoblast.; Pharmaceutical chemistry; Cell biology; Pharmaceutical Science; Biochemistry; Bone; Pain Management; Pharmacology

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.