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Gallein is known as an inhibitor of Gβγ subunits, but roles of gallein in bone metabolism have not been reported. Fibroblast growth factor 2 (FGF-2) increases angiogenesis and promotes bone regeneration during the early stages of fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in the suppression of bone resorption. Our previous report demonstrated that FGF-2 activates the phosphorylation of p38 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (JNK), and p44/p42 MAPK in osteoblast-like MC3T3-E1 cells. Additionally, FGF-2-activated phosphorylation of p38 MAPK and JNK but not p44/p42 MAPK is positively involved in OPG synthesis in these cells. This work aimed to investigate the effects of gallein on the FGF-2-elicited OPG synthesis in osteoblast-like MC3T3-E1 cells and the mechanism. Our findings demonstrated that gallein significantly increased the FGF-2-elicited OPG synthesis in MC3T3-E1 cells. By contrast, fluorescein, gallein-like compound that does not bind Gβγ, did not affect the FGF-2-elicited OPG synthesis. Gallein significantly enhanced the FGF-2-induced OPG mRNA expression levels. Gallein did not affect the FGF-2-activated phosphorylation of p38 MAPK and p44/p42 MAPK, but significantly increased the FGF-2-activated phosphorylation of JNK, while fluorescein did not affect JNK phosphorylation. SP600125, a specific JNK inhibitor, strongly inhibited gallein-induced enhancement of FGF-2-induced OPG synthesis and mRNA expression levels. Our results indicated that gallein increases the FGF-2-induced OPG synthesis due to the JNK activation in the osteoblast., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Evidence is accumulating that osteal macrophages, in addition to bone-resorbing osteoclasts and bone-forming osteoblasts, participate vitally in bone remodeling process. Oncostatin M (OSM), an inflammatory cytokine belonging to interleukin-6 superfamily, is recognized as an essential factor secreted by osteal macrophages to orchestrate bone remodeling. Osteoprotegerin (OPG) produced by osteoblasts regulates osteoclastogenesis. We have reported that bone morphogenetic protein-4 (BMP-4) stimulates OPG synthesis in MC3T3-E1 osteoblast-like cells, and that SMAD1/5/8(9), p38 mitogen-activated protein kinase (MAPK), and p70 S6 kinase are involved in the OPG synthesis. The present study aims to investigate the effect of OSM on the synthesis of OPG stimulated by BMP-4 in osteoblasts. OSM suppressed the release and the mRNA expression of OPG upregulated by BMP-4 in MC3T3-E1 cells. Neither the BMP-4-induced phosphorylation of SMAD1/5/9 nor that of p38 MAPK was affected by OSM. On the other hand, the phosphorylation of p70 S6 kinase stimulated by BMP-4 was considerably suppressed by OSM. These results strongly suggest that OSM suppresses the BMP-4-stimulated OPG synthesis via inhibition of the p70 S6 kinase-mediated pathway in osteoblast-like cells., (© 2024 The Author(s). Cell Biochemistry and Function published by John Wiley & Sons Ltd.)

Gallein, a small molecule related to fluorescein, is established as an inhibitor of Gβγ subunits to inhibit G protein (Gs) signaling. This agent is providing a potential therapeutic strategy to ameliorate organ dysfunctions especially involved in inflammation, however; the effects on bone metabolism have not yet been clarified. Prostaglandins (PGs) play important roles as autacoids including osteoblasts, and d-type prostanoid (DP) receptor, a member of G protein-coupled receptor specific to PGD 2 , is expressed on osteoblasts. We previously reported that prostaglandin D 2 (PGD 2 ) induces the syntheses of osteoprotegerin (OPG) and interleukin-6 (IL-6), essential factors in bone remodelling process, and p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/p42 MAPK are involved in the signal transduction of PGD 2 in osteoblast-like MC3T3-E1 cells. Thus, we investigated in this study that the effect and the underlying mechanism of gallein, an inhibitor Gβɤ subunits, on the syntheses of OPG and IL-6 induced by PGD 2 in these cells. The cultured cells were treated with gallein or fluorescein, a structurally related compound inactive to Gβɤ subunits, and subsequently stimulated with PGD 2 . Not fluorescein but gallein amplified the PGD 2 -stimulated releases of OPG and IL-6. Gallein enhanced the PGD 2 -upregulated mRNA expression levels of OPG and IL-6. Regarding the signaling mechanism, gallein did not affect the PGD 2 -induced phosphorylation of p38 MAPK, JNK, or p42 MAPK. In conclusion, gallein upregulates the PGD 2 -stimulated syntheses of OPG and IL-6 by the specific effect to inhibit Gβγ subunits in osteoblasts, but the effect is not exerted at the upstream of p38 MAPK, JNK, or p44/p42 MAPK activation., Competing Interests: Declaration of competing interest The authors have declared that there is no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 F 2α (PGF 2α ) 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 PGF 2α -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 PGF 2α -induced synthesis of IL-6 in osteoblast-like MC3T3-E1 cells. In addition, GIP and GLP-1 significantly enhanced the PGF 2α -induced mRNA expression levels of IL-6. On the other hand, GIP and GLP-1 markedly stimulated the PGF 2α -induced synthesis of osteoprotegerin. However, the phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, or JNK induced by PGF 2α was not affected by GIP or GLP-1. Therefore, these results strongly suggest that incretins enhance the PGF 2α -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., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

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.

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 2 (PGD 2 ) 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 PGD 2 -induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD 2 -induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD 2 -stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD 2 -induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD 2 -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 PGD 2 -stimulated osteoprotegerin release. Tramadol enhanced the PGE 2 -induced osteoprotegerin release as well as PGD 2 . These results suggest that tramadol amplifies the PGD 2 -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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2020 John Wiley & Sons Ltd.)

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 2 and PGF 2α induce osteoprotegerin synthesis through p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of acetaminophen on the osteoprotegerin synthesis induced by PGE 2 and PGF 2α in MC3T3-E1 cells. Acetaminophen significantly suppressed the osteoprotegerin release stimulated by PGE 2 and PGF 2α . The PGE 2 -induced expression of osteoprotegerin mRNA was also reduced by acetaminophen. Acetaminophen markedly downregulated the phosphorylation of SAPK/JNK stimulated by PGE 2 and PGF 2α , but not those of p38 MAPK or p44/p42 MAPK. SP600125, an inhibitor of SAPK/JNK, suppressed the levels of PGE 2 - and PGF 2α -upregulated osteoprotegerin mRNA expression. Taken together, these results strongly suggest that acetaminophen reduces the PGE 2 - and PGF 2α -stimulated synthesis of osteoprotegerin in osteoblasts, and that the suppressive effect is exerted via attenuation of SAPK/JNK. These findings provide a molecular basis for the possible effect of acetaminophen on bone tissue metabolism.

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 1 (PGE 1 ) 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 PGE 1 in these cells. Duloxetine enhanced the release from MC3T3-E1 cells of both OPG and IL-6 stimulated by PGE 1 . However, reboxetine, a selective and specific inhibitor of norepinephrine reuptake, failed to affect the PGE 1 -induced release of OPG or IL-6. Oppositely, fluvoxamine and sertraline, agents belonging to the class of selective serotonin reuptake inhibitor, upregulated the PGE 1 -stimulated release of both OPG and IL-6. Duloxetine amplified the expression of OPG mRNA and IL-6 mRNA stimulated by PGE 1 . Duloxetine strengthened the PGE 1 -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 PGE 1 -stimulated release of OPG and IL-6. These results strongly suggest that duloxetine could strengthen osteoblast activation by PGE 1 through the upregulation of p38 MAP kinase, leading to increasing the synthesis of OPG and IL-6., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.

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.