RANKL-stimulated Bone Marrow Macrophages Research Articles

Glycogen Synthase Kinase-3 Beta (GSK3β) as a Potential Drug Target in Regulating Osteoclastogenesis: An Updated Review on Current Evidence.

Glycogen synthase kinase 3-beta (GSK3β) is a highly conserved protein kinase originally involved in glucose metabolism, insulin activity, and energy homeostasis. Recent scientific evidence demonstrated the significant role of GSK3β in regulating bone remodelling through involvement in multiple signalling networks. Specifically, the inhibition of GSK3β enhances the conversion of osteoclast progenitors into mature osteoclasts. GSK3β is recognised as a pivotal regulator for the receptor activator of nuclear factor-kappa B (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), nuclear factor-kappa B (NF-κB), nuclear factor-erythroid 2-related factor 2 (NRF2)/Kelch-like ECH-associated protein 1 (KEAP1), canonical Wnt/beta (β)-catenin, and protein kinase C (PKC) signalling pathways during osteoclastogenesis. Conversely, the inhibition of GSK3β has been shown to prevent bone loss in animal models with complex physiology, suggesting that the role of GSK3β may be more significant in bone formation than bone resorption. Divergent findings have been reported regarding the efficacy of GSK3β inhibitors as bone-protecting agents. Some studies demonstrated that GSK3β inhibitors reduced osteoclast formation, while one study indicated an increase in osteoclast formation in RANKL-stimulated bone marrow macrophages (BMMs). Given the discrepancies observed in the accumulated evidence, further research is warranted, particularly regarding the use of GSK3β silencing or overexpression models. Such efforts will provide valuable insights into the direct impact of GSK3β on osteoclastogenesis and bone resorption.

A natural compound (LCA) isolated from Litsea cubeba inhibits RANKL-induced osteoclast differentiation by suppressing Akt and MAPK pathways in mouse bone marrow macrophages

Ethnopharmacological relevanceLitsea cubeba (Lour.) Pers. has been traditionally used as a folk prescription for treating rheumatic diseases in China. Aim of the studyThis study aimed to investigate the effects and underlying mechanism of LCA, a new type of dibenzyl butane lignin compound extracted from L. cubeba, on macrophage colony stimulating factor (M-CSF) plus receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in mouse-derived bone marrow macrophages (BMMs). Material and methodsTRAP staining, TRAP enzyme activity assay and actin ring staining were applied to identify the effects of LCA on osteoclast differentiation. Protein expression of NFATc1, c-Fos and MMP-9, and phosphorylation of p65, Akt, JNK, ERK and p38 in RANKL-induced osteoclasts was determined using western blotting to investigate the underlying mechanism. ResultsLCA significantly suppressed RANKL-induced osteoclast differentiation by inhibiting TRAP activity, decreasing the number of TRAP+ multinuclear osteoclasts and reducing the formation of F-actin ring without obvious cytotoxicity in BMMs. Moreover, LCA treatment strongly reduced protein expression of NFATc1, c-Fos and MMP-9, and attenuated the phosphorylation of p65, Akt, JNK, ERK and p38 in RANKL-stimulated BMMs. ConclusionsLCA ameliorated RANKL-induced osteoclast differentiation via inhibition of Akt and MAPK signalings in BMMs, and may serve as a potential pro-drug for bone destruction prevention.

Gypenoside inhibits RANKL-induced osteoclastogenesis by regulating NF-κB, AKT, and MAPK signaling pathways.

Gypenoside (GP) is one of the most pharmacologically active components in Gynostemma pentaphyllum and possesses neuroprotective, anticancer, anti-oxidant, anti-inflammatory, anti-diabetic, and anti-osteoarthritis effects. However, the involvement of GP the osteoclast differentiation has not yet been investigated. In the present study, we examined the effect of GP on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Our results demonstrated that GP significantly inhibited the formation of osteoclast, as well as suppressed the expression of osteoclastogenesis-related marker proteins in RANKL-stimulated bone marrow macrophages (BMMs). For molecular mechanisms, GP inhibited RANKL-induced NF-κB and MAPK activation and AKT phosphorylation in BMMs. Collectively, these findings suggest that GP inhibits RANKL-induced osteoclastogenesis via regulating NF-κB, AKT, and MAPK signaling pathways. Therefore, GP may be a potential agent in the treatment of osteoclast-related diseases such as osteoporosis.

Gentiopicroside inhibits RANKL-induced osteoclastogenesis by regulating NF-κB and JNK signaling pathways

Gentiopicroside, a main active component from the traditional Chinese herb medicine Gentiana manshurica Kitag, has been shown to possess anti-arthritis effect. However, the molecular mechanism of gentiopicroside on the osteoclast formation remains unclear. The present study was designed to investigate the effects and mechanisms of gentiopicroside on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. The results showed that pre-treatment with gentiopicroside significantly inhibited RANKL-induced osteoclast formation from mouse bone marrow macrophages (BMMs). In addition, we observed that gentiopicroside efficiently suppressed osteoclastogenesis-related marker genes expression in RANKL-stimulated BMMs. Mechanistically, gentiopicroside suppressed RANKL-induced the activation of JNK and NF-κB signaling pathways in BMMs. Taken together, the present study demonstrated that gentiopicroside inhibits RANKL-induced osteoclastogenesis through the inactivation of JNK and NF-κB signaling pathways. Thus, gentiopicroside may be a promising agent for the treatment of osteoporosis.

Methanol Extract of Holarrhena antidysenterica Inhibits the Growth of Human Oral Squamous Cell Carcinoma Cells and Osteoclastogenesis of Bone Marrow Macrophages.

Oral squamous cell carcinoma (OSCC) frequently invades mandibular bone, and outcomes for treatment with surgical resection are typically poor, ultimately resulting in death. Holarrhena antidysenterica L. (Apocynaceae), distributed throughout Sri Lanka and India, has been used as a folk remedy to treat various diseases. Treatment with methanol extract of H. antidysenterica bark (HABE) inhibited cell viability and BrdU incorporation and induced apoptotic cell death in Ca9-22 gingival and HSC-3 tongue SCC cells. Flow cytometric analysis indicated that HABE treatment preferentially induces apoptotic cell death via increasing the sub-G1 peak in Ca9-22 cells and cell cycle arrest at the G1 phase in HSC-3 cells. HABE treatment in the presence of zVAD-fmk, a pan-caspase inhibitor, rescued cell viabilities in both OSCC cell lines. The ratio of Bax to Bcl-2 increased with reductions in the Bcl-2 protein expression, and the activation of caspase 3 and subsequent cleavage of PARP was detected in HABE-treated Ca9-22 and HSC-3 cells. Furthermore, HABE treatment at noncytotoxic concentrations inhibited osteoclast formation in RANKL-stimulated bone marrow macrophages. Taken together, HABE possesses the inhibitory activity on the growth of OSCC cells and antiosteoclastogenic activity. Therefore, HABE may be a promising alternative and complementary agent for preventing and treating OSCC.

Prostaglandin-mediated inhibition of PTH-stimulated β-catenin signaling in osteoblasts by bone marrow macrophages

Bone marrow macrophages (BMMs), in the presence of cyclooxygenase-2 (Cox2) produced PGE2, secrete an inhibitory factor in response to Rankl that blocks PTH-stimulated osteoblastic differentiation. This study was to determine if the inhibitory factor also blocks PTH-stimulated Wnt signaling. Primary calvarial osteoblasts (POBs) were co-cultured with conditioned medium (CM) from Rankl-treated wild type (WT) BMMs, which make the inhibitory factor, and Cox2 knockout (KO) BMMs, which do not. PTH induced cAMP production was blocked by WT CM but not by KO CM. In the presence of KO CM, PTH induced phosphorylation at β-catenin serine sites, ser552 and ser675, previously shown to be phosphorylated by protein kinase A (PKA). Phosphorylation was blocked by WT CM and by H89, a PKA inhibitor. PTH did not increase total β-catenin. PTH-stimulated transcription factor/lymphoid enhancer-binding factor response element activity in POBs was blocked by WT CM and by serum amyloid A (SAA), the human recombinant analog of murine Saa3, which has recently been shown to be the inhibitory factor. In POBs cultured with Cox2 KO CM, PTH increased expression of multiple genes associated with the anabolic actions of PTH and decreased expression of Wnt antagonists. This differential regulation of gene expression was not seen in POBs cultured with WT CM. These data highlight the ability of PTH to phosphorylate β-catenin directly via PKA and demonstrate the ability of a Cox2-dependent inhibitory factor, secreted by Rankl-stimulated BMMs, to abrogate PTH stimulated β-catenin signaling. Our results suggest that PTH can stimulate a novel negative feedback of its anabolic actions by stimulating Rankl and Cox2 expression.

Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts

Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RANKL-treated WT, but not KO, BMMs blocked PTH-stimulated cAMP production in POBs. Inhibition was reversed by pertussis toxin (PTX), which blocks Gαi/o activation. Saa3 was the most highly differentially expressed gene in a microarray comparison of RANKL-treated WT versus Cox2 KO BMMs, and RANKL induced Saa3 protein secretion only from WT BMMs. CM from RANKL-stimulated BMMs with Saa3 knockdown did not inhibit PTH-stimulated responses in POBs. SAA added to POBs inhibited PTH-stimulated cAMP responses, which was reversed by PTX. Selective agonists and antagonists of formyl peptide receptor 2 (Fpr2) suggested that Fpr2 mediated the inhibitory actions of Saa3 on osteoblasts. In BMMs committed to become osteoclasts by RANKL treatment, Saa3 expression peaked prior to appearance of multinucleated cells. Flow sorting of WT marrow revealed that Saa3 was secreted only from the RANKL-stimulated B220(-) CD3(-)CD11b(-/low) CD115(+) preosteoclast population. We conclude that Saa3 secretion from preosteoclasts, induced by RANKL in a Cox2-dependent manner, inhibits PTH-stimulated cAMP signaling and osteoblast differentiation via Gαi/o signaling. The induction of Saa3 by PTH may explain the suppression of bone formation when PTH is applied continuously and may be a new therapeutic target for osteoporosis.

Angelica sinensis extract inhibits RANKL-mediated osteoclastogenesis by down-regulated the expression of NFATc1 in mouse bone marrow cells.

BackgroundDestructive erosion of bone or osteolysis is a major complication of inflammatory conditions such as rheumatoid arthritis (RA), periodontal disease, and periprosthetic osteolysis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease.MethodsThe effect of Angelica sinensis (AS) extract on RANKL-induced osteoclast differentiation was examined in this study. The osteoclast precursor cell line bone marrow macrophages (BMMs) was cultured and stimulated with RANKL followed by treatment with AS at several doses. Gene expression profiles of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR were sequentially evaluated.ResultsAS extract inhibited RANKL-mediated osteoclast differentiation in BMMs in a dose-dependent manner without any evidence of cytotoxicity. AS extract strongly inhibited p38, ERK, JNK, p65 phosphorylation and I-κB degradation in RANKL-stimulated BMMs. AS extract also inhibited the mRNA expression of c-Fos, c-Jun, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs. Moreover, RANKL-induced c-Fos, c-Jun and NFATc1 protein expression was suppressed by AS extract.ConclusionsThese results collectively suggested that AS extract demonstrated inhibitory effects on RANKL-mediated osteoclast differentiation in bone marrow macrophages in vitro, indicating that AS may therefore serve as a useful drug in the prevention of bone loss.

Hypericin suppresses osteoclast formation and wear particle-induced osteolysis via modulating ERK signalling pathway

Osteoclast-induced bone resorption and wear-particle-induced osteolysis leads to prosthetic loosening, one of the most common causes of joint implant failure, resulting in revision surgery. Thus, inhibition of osteoclastic bone resorption, which further prevents wear particle-induced osteolysis, is a potential treatment strategy for prosthetic loosening. Here, we examined the therapeutic effect of hypericin (HP), which was photosensitive, on osteoclastogenesis and wear particle-induced osteolysis in the absence of visible light. HP inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) and RAW264.7 cell line without any evidence of cytotoxicity. The bone-resorbing activity of mature osteoclasts was significantly inhibited by HP. As HP has been previously reported to inhibit signalling pathway such as ERK and NF-κB in other cells, which is also important in osteoclast differentiation. We thus examined the molecular mechanism and showed that HP significantly inhibited the ERK/mitogen-activated protein kinase (MAPK) signalling pathway without affecting nuclear factor kappaB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 signalling in RANKL-stimulated BMMs. Further in vivo studies revealed HP attenuated osteoclast formation and subsequently prevented wear particle-induced bone erosion. Taken together, the results suggest that HP inhibits RANKL-mediated osteoclastogenesis via affecting ERK signalling in vitro and suppresses wear particle-induced osteolysis in vivo. We therefore conclude that HP may be an innovative and safe alternative treatment for osteoclast-related prosthetic loosening.

9-Hydroxy-6,7-dimethoxydalbergiquinol inhibits osteoclast differentiation through down-regulation of Akt, c-Fos and NFATc1

Recently, natural plant-derived products have been recognized as one of the main sources for drug discovery and development in human disease. 9-Hydroxy-6,7-dimethoxydalbergiquinol (HDDQ) isolated from the heart wood of Dalbergia odorifera is widely used in oriental medicine, however, the pharmacological effect of HDDQ in osteoclast-associated diseases remains unknown. In this study, HDDQ dose-dependently inhibited the early stage of RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity. HDDQ strongly inhibited Akt phosphorylation in RANKL-stimulated BMMs and did not show any effects on p38, JNK, and IκB phosphorylation and IκB degradation. Interestingly, we found that HDDQ down-regulated the induction by RANKL of c-Fos protein by suppressing its translation. Also, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by HDDQ. Furthermore, the Akt/c-Fos/NFATc1-regulated expression of genes required for osteoclastogenesis, such as OSCAR and TRAP, was inhibited by HDDQ. These findings suggest that HDDQ prevents osteoclast differentiation via down-regulation of Akt, c-Fos, and NFATc1 signaling molecules, suggesting a potential therapeutic value of HDDQ for bone disorders associated with increased bone resorption.

Dual Effect ofChrysanthemum indicumExtract to Stimulate Osteoblast Differentiation and Inhibit Osteoclast Formation and ResorptionIn Vitro

The risk of bone-related diseases increases due to the imbalance between bone resorption and bone formation by osteoclasts and osteoblasts, respectively. The goal in the development of antiosteoporotic treatments is an agent that will improve bone through simultaneous osteoblast stimulation and osteoclast inhibition without undesirable side effects. To achieve this goal, numerous studies have been performed to identify novel approaches using natural oriental herbs to treat bone metabolic diseases. In the present study, we investigated the effect of Chrysanthemum indicum extract (CIE) on the differentiation of osteoclastic and osteoblastic cells. CIE inhibited the formation of TRAP-positive mature osteoclasts and of filamentous-actin rings and disrupted the bone-resorbing activity of mature osteoclasts in a dose-dependent manner. CIE strongly inhibited Akt, GSK3β, and IκB phosphorylation in RANKL-stimulated bone marrow macrophages and did not show any effects on MAP kinases, including p38, ERK, and JNK. Interestingly, CIE also enhanced primary osteoblast differentiation via upregulation of the expression of alkaline phosphatase and the level of extracellular calcium concentrations during the early and terminal stages of differentiation, respectively. Our results revealed that CIE could have a potential therapeutic role in bone-related disorders through its dual effects on osteoclast and osteoblast differentiation.

Oleanolic acid acetate inhibits osteoclast differentiation by downregulating PLCγ2–Ca2 +-NFATc1 signaling, and suppresses bone loss in mice

Owing to their potential pharmacological activities in human disease, natural plant-derived compounds have recently become the focus of increased research interest. In this study, we first isolated oleanolic acid acetate (OAA), a triterpenoid compound, from Vigna angularis (azuki bean) to discover anti-bone resorptive agents. Many studies have identified and described the various medicinal effects of V. angularis extract. However, the pharmacological effect of OAA-derived V. angularis extract, particularly the effect on osteoclastogenesis, is not known. Therefore, we investigated the effect and mechanism of OAA in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. OAA inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. Interestingly, OAA significantly inhibited Btk phosphorylation, phospholipase Cγ2 (PLCγ2) phosphorylation, calcium ion (Ca2+) oscillation, and nuclear factor of activated T cell c1 (NFATc1) expression in RANKL-stimulated BMMs, but did not affect RANKL-induced mitogen-activated protein kinase. OAA also inhibited the bone-resorbing activity of mature osteoclasts. Furthermore, mice treated with OAA demonstrated marked attenuation of lipopolysaccharide-induced bone erosion based on micro-computed tomography and histologic analysis of femurs. Taken together, the results suggested that OAA inhibited RANKL-mediated osteoclastogenesis via PLCγ2-Ca2+-NFATc1 signaling in vitro and suppressed inflammatory bone loss in vivo.

Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro

Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE2) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE2 receptors (Ptger4 and Ptger2 KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE2 to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE2 but also BMMs. Sufficient PGE2 to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE2 was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger4, but not Ptger2, in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE2 also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE2, acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This suppression of OB differentiation could contribute to the bone loss seen with continuous PTH in vivo.

Inhibition of osteoclast differentiation and bone resorption by rotenone, through down-regulation of RANKL-induced c-Fos and NFATc1 expression

Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. The effect of rotenone in RANKL-induced osteoclast differentiation was examined in this study. Rotenone inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs was inhibited by rotenone treatment. Rotenone strongly inhibited p38 and ERK phosphorylation and I-κB degradation in RANKL-stimulated BMMs, and did not inhibit JNK phosphorylation. Further, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by rotenone. Rotenone additionally inhibited the bone resorptive activity of differentiated osteoclasts. A lipopolysaccharide (LPS)-induced bone erosion study was also performed to assess the effects of rotenone in vivo. Mice treated with rotenone demonstrated marked attenuation of bone erosion based on Micro CT and histologic analysis of femurs. These results collectively suggested that rotenone demonstrated inhibitory effects on osteoclast differentiation in vitro and suppressed inflammatory bone loss in vivo. Rotenone may therefore serve as a useful drug in the prevention of bone loss.

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Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. The effect of rotenone in RANKL-induced osteoclast differentiation was examined in this study. Rotenone inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs was inhibited by rotenone treatment. Rotenone strongly inhibited p38 and ERK phosphorylation and I-κB degradation in RANKL-stimulated BMMs, and did not inhibit JNK phosphorylation. Further, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by rotenone. Rotenone additionally inhibited the bone resorptive activity of differentiated osteoclasts. A lipopolysaccharide (LPS)-induced bone erosion study was also performed to assess the effects of rotenone in vivo. Mice treated with rotenone demonstrated marked attenuation of bone erosion based on Micro CT and histologic analysis of femurs. These results collectively suggested that rotenone demonstrated inhibitory effects on osteoclast differentiation in vitro and suppressed inflammatory bone loss in vivo. Rotenone may therefore serve as a useful drug in the prevention of bone loss.