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The increase of bone-resorbing osteoclast activity in bone remodeling is the major characteristic of various bone diseases. Thus, inhibiting osteoclastogenesis and bone-resorbing function may be an effective therapeutic target for bone diseases. Betulinic acid (BA), a natural plant-derived pentacyclic triterpenoid compound, is known to possess numerous pharmacological and biochemical properties including anti-inflammatory, anticancer, and antiadipogenic activity. However, the effect of BA on osteoclast differentiation and function in bone metabolism has not been demonstrated so far. In this study, we investigated whether BA could suppress RANKL-induced osteoclastogenesis and bone resorption. Interestingly, BA significantly suppressed osteoclastogenesis by decreasing the phosphorylation of Akt and IκB, as well as PLCγ2-Ca2+ signaling, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. The inhibition of these pathways by BA was once more confirmed by retrovirus infection of constitutively active (CA)-Akt and CA-Ikkβ retrovirus and measurement of Ca2+ influx. BA also significantly inhibited the expression of osteoclastogenesis-specific marker genes. Moreover, we found that BA administration restored the bone loss induced through acute lipopolysaccharide injection in mice by a micro-CT and histological analysis. Our findings suggest that BA is a potential therapeutic candidate for bone diseases involving osteoclasts.

Osteoporosis is a systemic metabolic bone disorder that is caused by an imbalance in the functions of osteoclasts and osteoblasts and is characterized by excessive bone resorption by osteoclasts. Targeting osteoclast differentiation and bone resorption is considered a good fundamental solution for overcoming bone diseases. β-boswellic acid (βBA) is a natural compound found in Boswellia serrata, which is an active ingredient with anti-inflammatory, anti-rheumatic, and anti-cancer effects. Here, we explored the anti-resorptive effect of βBA on osteoclastogenesis. βBA significantly inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by receptor activator of nuclear factor-B ligand (RANKL) and suppressed bone resorption without any cytotoxicity. Interestingly, βBA significantly inhibited the phosphorylation of IκB, Btk, and PLCγ2 and the degradation of IκB. Additionally, βBA strongly inhibited the mRNA and protein expression of c-Fos and NFATc1 induced by RANKL and subsequently attenuated the expression of osteoclast marker genes, such as OC-STAMP, DC-STAMP, β3-integrin, MMP9, ATP6v0d2, and CtsK. These results suggest that βBA is a potential therapeutic candidate for the treatment of excessive osteoclast-induced bone diseases such as osteoporosis.

Dietary procyanidin has been shown to be an important bioactive component that regulates various pharmacological activities to maintain metabolic homeostasis. In particular, grape seed proanthocyanidin extract (GSPE) is a commercially available medicine for the treatment of venous and lymphatic dysfunction. This study aimed to investigate whether GSPE protects against lipopolysaccharide (LPS)-induced bone loss in vivo and the related mechanism of action in vitro. The administration of GSPE restored the inflammatory bone loss phenotype stimulated by acute systemic injection of LPS in vivo. GSPE strongly suppressed receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and bone resorption activity of mature osteoclasts by decreasing the RANKL-induced nuclear factor-&kappa, B transcription activity. GSPE mediates this effect through decreased phosphorylation and degradation of NF-&kappa, B inhibitor (I&kappa, B) by I&kappa, B kinase&beta, subsequently inhibiting proto-oncogene cellular Fos and nuclear factor of activated T cells. Additionally, GSPE promotes osteoclast proliferation by increasing the phosphorylation of components of the Akt and mitogen-activated protein kinase signaling pathways and it also inhibits apoptosis by decreasing the activity of caspase-8, caspase-9, and caspase-3, as corroborated by a decrease in the Terminal deoxynucleotidyl transferase dUTP nick end labeling -positive cells. Our study suggests a direct effect of GSPE on the proliferation, differentiation, and apoptosis of osteoclasts and reveals the mechanism responsible for the therapeutic potential of GSPE in osteoclast-associated bone metabolism disease.

Receptor activator of NF-&kappa, B ligand (RANKL) induces generation of intracellular reactive oxygen species (ROS), which act as second messengers in RANKL-mediated osteoclastogenesis. Dual oxidase maturation factor 1 (Duoxa1) has been associated with the maturation of ROS-generating enzymes including dual oxidases (Duox1 and Duox2). In the progression of osteoclast differentiation, we identified that only Duoxa1 showed an effective change upon RANKL stimulation, but not Duox1, Duox2, and Duoxa2. Therefore, we hypothesized that Duoxa1 could independently act as a second messenger for RANKL stimulation and regulate ROS production during osteoclastogenesis. Duoxa1 gradually increased during RANKL-induced osteoclastogenesis. Using siRNA or retrovirus transduction, we found that Duoxa1 regulated RANKL-stimulated osteoclast formation and bone resorption positively. Furthermore, knockdown of Duoxa1 decreased the RANKL-induced ROS production. During Duoxa1-related control of osteoclastogenesis, activation of tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated early signaling molecules including MAPKs, Akt, I&kappa, B, Btk, Src and PLC&gamma, 2 was affected, which sequentially modified the mRNA or protein expression levels of key transcription factors in osteoclast differentiation, such as c-Fos and NFATc1, as well as mRNA expression of osteoclast-specific markers. Overall, our data indicate that Duoxa1 plays a crucial role in osteoclastogenesis via regulating RANKL-induced intracellular ROS production and activating TRAF6-mediated signaling.

Excessive bone resorption plays a central role in the development of inflammatory bone diseases, including osteoporosis and rheumatoid arthritis. Thus, identification of agents that can effectively suppress excessive osteoclast formation and function is crucial for the prevention and treatment of inflammatory bone loss. Umbelliferone (Umb), a derivative of coumarin, is a natural bioactive compound with anti-inflammatory and antioxidant properties. However, the effect of Umb on metabolic bone diseases is unknown. In this study, we found that Umb exhibited a strong inhibitory effect on lipopolysaccharide (LPS)-induced inflammatory bone loss in vivo. Histological analysis confirmed that Umb prevented trabecular bone matrix degradation and osteoclast formation in bone tissue. In addition, Umb suppressed RANKL-induced osteoclast differentiation and abrogated bone resorption. We found that the anti-osteoclastic and anti-resorptive activities of Umb are mediated via suppression of the RANKL-induced Akt-c-Fos-NFATc1 signaling pathway and the attenuation of osteoclast-specific genes, such as TRAP, OSCAR, ATP6v0d2, and CtsK. In particular, Umb downregulated the stability of c-Fos and NFATc1 proteins, but did not suppress the expression of their mRNAs. These results indicate that Umb may be a potential therapeutic agent for inflammatory bone diseases associated with abnormal osteoclast formation and function.

Claudins (Cldns) are well-established components of tight junctions (TJs) that play a pivotal role in the modulation of paracellular permeability. Several studies have explored the physiologic aspects of Cldn family members in bone metabolism. However, the effect of Cldn11, a major component of central nervous system myelin, on bone homeostasis has not been reported. In this study, we demonstrate that Cldn11 is a potential target for bone disease therapeutics as a dual modulator of osteogenesis enhancement and osteoclastogenesis inhibition. We found that Cldn11 played a negative role in the receptor activator of nuclear factor kappa B ligand-induced osteoclast (OC) differentiation and function by downregulating the phosphorylated form of extracellular signal-regulated kinase (ERK), Bruton’s tyrosine kinase, and phospholipase C gamma 2, in turn impeding c-Fos and nuclear factor in activated T cell c1 expression. The enhancement of osteoblast (OB) differentiation by positive feedback of Cldn11 was achieved through the phosphorylation of Smad1/5/8, ERK, and c-Jun amino-terminal kinase. Importantly, this Cldn11-dependent dual event in bone metabolism arose from targeting EphrinB2 ligand reverse signaling in OC and EphB4 receptor forward signaling in OB. In agreement with these in vitro effects, subcutaneous injection of Cldn11 recombinant protein exerted anti-resorbing effects in a lipopolysaccharide-induced calvarial bone loss mouse model and increased osteogenic activity in a calvarial bone formation model. These findings suggest that Cldn11 is a novel regulator in bone homeostasis., Bone metabolism: A new connection Claudin 11 (Cldn11), a protein that helps form leak-proof connections between cells, also regulates bone density. Claudins are part of tight junctions, which connect cells that form barriers, such as skin cells. Some claudins have been reported to also affect bone metabolism. Ju-Young Kim and Myeung Su Lee from Wonkwang University in Iksan, South Korea, and colleagues investigated how Cldn11 affects bone metabolism. Bone undergoes constant remodeling, and the balance between new bone formation and old bone breakdown is critical. The team found that in cultured cells increasing Cldn11 increased the numbers of bone-forming cells and decreased the numbers of bone-degrading cells. In mice with experimentally induced bone weakness, injection of Cldn11 increased new bone formation and reduced bone resorption. Cldn11 provides a new target to study and treat diseases that cause bone loss, such as osteoporosis.

Vigeo is a mixture of fermented extracts of Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK) manufactured using the traditional Korean nuruk fermentation method. Although the bioactive effects of ESM, AJN, and AJK have already been reported, the pharmacological effects of Vigeo have not been proven. Therefore, in this study, we investigated whether Vigeo had inhivitory effects on lipopolysaccharide (LPS)-induced inflammatory bone loss in vivo and receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclastogenesis and the related mechanism in vitro. Vigeo administration conferred effective protection against bone loss induced by excessive inflammatory response and activity of osteoclasts in LPS-induced inflammatory osteoporosis mouse model. In addition, Vigeo significantly suppressed the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by RANKL and inhibited F-actin formation and bone resorbing activity without any cytotoxicity. Moreover, Vigeo significantly inhibited RANKL-induced phosphorylation of p38, ERK, JNK, IκB, and AKT and degradation of IkB. Additionally, Vigeo strongly inhibited the mRNA and protein expression of c-FOS and NFATc1 and subsequently attenuated the expression of osteoclast specific marker genes induced by RANKL. We demonstrated for the first time the anti-osteoporosis effect of Vigeo, suggesting that it could be a potential therapeutic candidate for the treatment of osteoclast-mediated inflammatory bone diseases.

Securinine (Sec) is a naturally derived compound separated from the roots of Securinega suffruticosa, which has long been used as a herbal medicine. Sec is widely known as a GABA receptor antagonist, it is also known as an innate immune cell agonist and has been reported to increase macrophage activity and promote monocyte maturation. On the basis of these studies, we investigated the effect of Sec on osteoclast differentiation and bone resorbing function. We have found that Sec inhibits RANKL-induced osteoclast differentiation, fusion, actin ring formation, and bone resorbing function by the inhibition of gene expression associated with each stage. Moreover, Sec significantly suppressed osteoclastogenesis by decreasing the phosphorylation of p38, Akt, JNK, IκB, and PLCγ2, in pathways involved in early osteoclastogenesis as well as through the subsequent suppression of c-Fos and NFATc1. Finally, Sec effectively protected bone loss induced by the excessive inflammatory responses and activity of osteoclasts in vivo by a micro-CT and histological analysis. In conclusion, our findings suggest that Sec may be a promising drug for bone metabolic diseases such as osteoporosis, which is associated with the excessive activity of osteoclasts.

Background: Anthrax toxin receptor 1 (ANTXR1) has been known to have relationship with extracellular transmembrane protein deeply associated with the process of bone formation and exert important role in angiogenesis. However, there have been no reports to prove the effects of ANTXR1 on bone metabolism mediated by two types of bone cells, osteoclasts and osteoblasts. The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. Objectives: The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. Methods: To determine the effect of ANTXR1 on osteoclastogenesis or osteoblast differentiation, we examined TRAP staining, F-actin staining and Pit assay, or ALP and Alizarin Red-mineralization staining, respectively. The mechanism of ANTXR1 by transfection of retrovirus or siRNA analyzed using real-time PCR and western blot analysis. Also, the effect of ANTXR1 on osteoclast-mediated angiogenesis of endothelial cells assessed by in vitro vascular tubule formation assay of human umbilical vein endothelial cells (HUVECs). Results: Through performing gain- and loss-of-function studies, we found that ANTXR1 positively regulated receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption with no effects on osteoblast differentiation. During ANTXR1-mediated regulation of osteoclastogenesis, phosphorylation of early signal transducers, c-jun N-terminal kinase (JNK), Akt, and inhibitor of kappa B (IκB) was affected, which in turn alteration of mRNA and protein levels of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In addition, genetic manipulation of ANTXR1 in bone marrow macrophages (BMMs) modulated the capillary-like tube formation by HUVECs via two kinds of angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A). These results explained the important role of ANTXR1 in osteoclast differentiation and functional activity, as well as, osteoclast-mediated angiogenesis of endothelial cells. Conclusion: Taken together, it was proposed that ANTXR1 might be a promising candidate for gene therapy related with bone metabolic diseases and further, have potential to be served as an important biomarker in the research fields of bone metastasis associated with vascularization. Disclosure of Interests: None declared

Excessive osteoclast activity, along with relatively weak osteoblast function, is strongly associated with bone disease. Therefore, studies to identify novel anti-osteoporosis candidates with dual actions of inhibiting osteoclastogenesis and increasing osteoblastogenesis may provide an ideal approach for treating osteoporosis. Pitavastatin, an inhibitor of 3-hydroxy-3 methyl-glutaryl coenzyme A reductase, has demonstrated various pharmacological activities, including anti-inflammation, bone anabolic effects, vasodilation, and inhibition of revascularization; however, the precise effects and mechanisms of pitavastatin on the regulation of osteoblast and osteoclast activity need to be comprehensively elucidated. Herein, we demonstrated that pitavastatin is a potential candidate for treating osteoporosis by enhancing osteoblast differentiation and bone growth and inhibiting osteoclast differentiation and bone resorption. Pitavastatin exerted dose-dependent inhibitory effects on receptor activator of nuclear factor kappa-B ligand-induced osteoclast formation, bone resorption, and osteoclast-specific marker gene expression. These inhibitory effects were achieved by inhibiting the Akt, NF-κB, and mitogen-activated protein kinase (p38, ERK, and JNK) signaling pathways, resulting in the downregulation of major transcription factors c-Fos and NFATc1. Furthermore, pitavastatin potentially stimulated osteoblast differentiation by activating alkaline phosphatase (ALP), enhancing mineralization by Alizarin Red S, and increasing the expression of osteoblastogenic marker genes such as runt-related transcription factor 2, ALP, osteocalcin, and collagen type 1 alpha. Furthermore, we evaluated the therapeutic potential of pitavastatin in ovariectomy-induced systematic bone loss based on micro-computed tomography and histological analysis of femurs. Our findings demonstrated a new function and mechanism for pitavastatin in bone remodeling, indicating its potential as a therapeutic candidate in treating osteoporosis by inhibiting osteoclastic resorption and promoting osteoblastic formation.

The adipokine nicotinamide phosphoribosyltransferase (Nampt), also known as pre-B-cell colony-enhancing factor or the insulin-mimetic hormone visfatin, has a crucial role in the conversion of nicotinamide to nicotinamide mononucleotide during biosynthesis of the coenzyme nicotinamide adenine dinucleotide. Previous reports have demonstrated the inhibitory effects of Nampt on osteoclast formation from human peripheral blood mononuclear cells and CD14+ monocytes. However, the effects of Nampt on bone marrow macrophage (BMM)‑derived osteoclastogenesis and its precise role in the process remain unclear. The present in vitro study used recombinant Nampt and BMMs as osteoclast precursors demonstrated that Nampt suppresses receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclastogenesis by decreasing the phosphorylation of various early signal transducers, including c‑Jun N‑terminal kinase, Akt, glycogen synthase kinase‑3 β, Bruton's tyrosine kinase and phospholipase C γ‑2. In addition, western blotting and reverse transcription‑quantitative polymerase chain reaction analysis indicated that Nampt downregulates the mRNA and protein expression levels of c‑Fos and nuclear factor of activated T cells, cytoplasmic 1, leading to a decrease in the expression of osteoclast‑specific genes including tartrate‑resistant acid phosphatase, osteoclast‑associated receptor and cathepsin K. However, the bone‑resorbing activity of mature osteoclasts treated with Nampt was similar to untreated control osteoclasts. This finding indicates that Nampt exerts its anti‑osteoclastogenic activity by targeting osteoclast precursor cells rather than mature osteoclasts. Consequently, the present study demonstrated that Nampt acts as a negative regulator of RANKL‑mediated differentiation of BMMs into osteoclasts, suggesting the potential therapeutic targets to treat bone-related disorders such as osteoporosis.

Anthrax toxin receptor 1 (ANTXR1) is a transmembrane protein with an extracellular domain which is deeply associated with the process of bone formation and plays an important role in angiogenesis. However, there have been no reports investigating the effects of ANTXR1 on bone metabolism mediated by the two types of bone cells, osteoclasts, and osteoblasts. The aim of this study is to reveal the role of ANTXR1 in the differentiation and function of osteoclasts and osteoblasts. We found that ANTXR1 positively regulated the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption with no effects on osteoblast differentiation by performing gain- and loss-of-function studies. During ANTXR1-mediated regulation of osteoclastogenesis, phosphorylation of early signal transducers such as c-Jun N-terminal kinase (JNK), Akt, inhibitor of kappa B (IκB), and phospholipase C gamma 2 (PLCγ2) was affected, which in turn altered the mRNA and protein levels of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). In addition, genetic manipulation of ANTXR1 in bone marrow macrophages (BMMs) modulated the capillary-like tube formation in HUVECs via secretion of two angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor-A (VEGF-A). These results elucidated the importance of ANTXR1 in osteoclast differentiation and functional activity, as well as, osteoclast-mediated angiogenesis of endothelial cells. Taken together, we propose that ANTXR1 might be a promising candidate for gene therapy for bone metabolic diseases and further, might potentially serve as an important biomarker in the field of bone metastasis associated with vascularization.

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. These results indicate that ebselen is a potentially safe drug for treating metabolic bone diseases such as osteoporosis.

Protocatechuic acid (PCA) plays a critical role in nutritional metabolism; it is a major metabolite of anthocyanins, which are flavonoids with a range of health benefits. PCA has a variety of biological activities including anti-oxidant, antiinflammatory, anti-apoptosis, and anti-microbial activities. However, the pharmacological effect of PCA, especially on osteoclastogenesis, remains unknown. We examined the effect of PCA on receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. PCA dose-dependently inhibited RANKL-induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and suppressed the bone-resorbing activity of mature osteoclasts. At the molecular level, PCA suppressed RANKL-induced phosphorylation of JNK among MAPKs only, without significantly affecting the early signaling pathway. PCA also suppressed RANKL-stimulated expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1) at the mRNA and protein levels, without altering c-Fos mRNA expression. Additionally, PCA down-regulated the expression of downstream osteoclastogenesis-related genes including β3-integrin, DC-STAMP, OC-STAMP, Atp6v0d2, CTR, and CtsK. Mice treated with PCA efficiently recovered from lipopolysaccharide-induced bone loss in vivo. Thus, PCA inhibits RANKL-induced osteoclast differentiation and function by suppressing JNK signaling, c-Fos stability, and expression of osteoclastic marker genes. These results suggest that PCA could be useful in treatment of inflammatory bone disorders.

Ingestion of Citrus unshiu Markovich (CUM) has been widely used as an alternative therapy for thousands years in Korean traditional medicine. Both anti-osteoclastic actions of CUM extracts (CUME) and the possibility for development as a healthy food product were investigated. CUME significantly (p

Adipokines derived from adipocytes are important factors that act as circulating regulators of bone metabolism. C1q/tumor necrosis factor (TNF)-related Protein-3 (CTRP3) is a novel adipokine with multiple effects such as lowering glucose levels, inhibiting gluconeogenesis in the liver, and increasing angiogenesis and anti-inflammation. However, the effects and the mechanisms of CTRP3 on bone metabolism, which is regulated by osteoblasts and osteoclasts, have not been investigated. Here, we found that CTRP3 inhibited osteoclast differentiation induced by osteoclastogenic factors in bone marrow cell-osteoblast co-cultures, but did not affect the ratio of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to osteoprotegerin (OPG) induced by osteoclastogenic factors in osteoblasts. We also found that CTRP3 inhibited osteoclast differentiation from mouse bone marrow macrophages (BMMs) induced by RANKL in a dose-dependent manner without cytotoxicity. Functionally, CTRP3 inhibited the F-actin formation and bone resorbing activity of mature osteoclasts. Pretreatment with CTRP3 significantly inhibited RANKL-induced expression of c-Fos and nuclear factor of activated T-cells (NFATc1), essential transcription factors for osteoclast development. Surprisingly, the activation of AMP-activated protein kinase (AMPK) was considerably increased by pretreatment with CTRP3 for 1h. The CTRP3-stimulated AMPK activation was also maintained during RANKL-induced osteoclastogenesis. CTRP3 did not affect RANKL-induced p38, ERK, JNK, Akt, IκB, CREB, and calcium signaling (Btk and PLCγ2). These results suggest that CTRP3 plays an important role as a negative regulator of RANKL-mediated osteoclast differentiation by acting as an inhibitor of NFATc1 activation through the AMPK signaling pathway. Furthermore, CTRP3 treatment reduced RANKL-induced osteoclast formation and bone destruction in mouse calvarial bone in vivo based on micro-CT and histologic analysis. In conclusion, these findings strongly suggest that CTRP3 deserves new evaluation as a potential treatment target in various bone diseases associated with excessive osteoclast differentiation and bone destruction.

The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca(2+) oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption.

Prohibitin-1 (PHB) regulates diverse cellular processes by controlling several signaling pathways. In this study, we investigated the functional involvement of PHB in osteoclast differentiation. PHB expression was time-dependently increased by RANKL in BMMs. However, the retroviral over-expression of PHB strongly inhibited the expression of c-Fos and NFATc1, and activation of p38-Elk-1-SRE signaling pathway. Anti-osteoclastogenic action of PHB was significantly inhibited by constitutively active forms of MKK6, but not Elk-1. Collectively, PHB negatively regulates the formation of mature osteoclasts via inhibition of MKK6 activity that affects the activation of the p38-Elk-1 signaling axis required for the expression of c-Fos and NFATc1.

Drug repositioning is a rational approach for expanding the use of existing drugs or candidate drugs to treat additional disorders. Here we investigated the possibility of using the anticancer p21-activated kinase 4 (PAK4)-targeted inhibitor PF-3758309 to treat osteoclast-mediated disorders. PAK4 was highly expressed in bone marrow cells and was phosphorylated during their differentiation into osteoclasts, and osteoclast differentiation was significantly inhibited by the dominant negative form of PAK4 and by PF-3758309. Specifically, PF-3758309 significantly inhibited the fusion of preosteoclasts, the podosome formation, and the migration of preosteoclasts. PF-3758309 also had in vivo antiresorptive activity in a lipopolysaccharide-induced bone erosion model and in vitro antiosteoclastogenic activity in the differentiation of human bone marrow-derived cells and peripheral blood mononuclear cells into osteoclasts. These data demonstrate the relevance of PAK4 in osteoclast differentiation and the potential of PAK4 inhibitors for treating osteoclast-related disorders.

Purslane (Portulaca oleracea L.) is popular as a potherb in many areas of Europe, Asia, and the Mediterranean region and is widely distributed around the globe. It has a wide range of pharmacological effects, such as antibacterial, anti-aging, anti-inflammatory, and anti-oxidative properties. Although the extract of purslane has numerous beneficial pharmacological effects, its effect on osteoclasts remains unknown. We aimed to investigate the anti-osteoclastogenic activity in vitro and in vivo and to elucidate the underlying mechanism. The effect of purslane on the differentiation and function of bone marrow-derived macrophages (BMMs) into osteoclasts was examined using a phenotype assay such as tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and pit assay and followed by confirmation by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. To address the effect of purslane in vivo, the inflammatory, lipopolysaccharide (LPS)-induced osteolysis mouse model was chosen. Bone volume and bone microarchitecture were evaluated by microcomputed tomography and histologic analysis. Purslane inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-stimulated osteoclast differentiation accompanied by inhibition of Akt/glycogen synthase kinase 3β (GSK3β) signaling, which could underlie purslane-induced downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression levels, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion and resorption-related molecules. Moreover, in vivo studies further verified the bone protection activity of purslane in the LPS-induced osteolysis animal model. Purslane could exhibit its anti-osteoclastogenic activity by inhibiting Akt/GSK3β-c-Fos-NFATc1 signaling cascades. Therefore, purslane is a potential natural medicine for the treatment of osteoclast-related diseases.

The roots of Ostericum koreanum (OK) Maximowicz have traditionally been used to produce an herbal medicine reported to possess anti-inflammatory, anti-oxidant, antimicrobial, and antitumor activities; however, its effect on bone metabolism has not yet been reported. The present study examined the effects of OK extract on lipopolysaccharide (LPS)-induced bone loss in mice by investigating bone structure and the levels of the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in serum and bone marrow fluid (BMF). The effects of OK extract on osteoclastogenesis were also investigated in mouse bone marrow macrophages by examining the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, the actin ring, and bone resorption activity. OK reduced LPS-induced bone destruction in vivo via a decrease in the RANKL/OPG ratio. Furthermore, it suppressed the formation of TRAP-positive cells and the actin ring, and reduced the bone-resorbing activity of mature osteoclasts. OK also significantly down-regulated the expression of various osteoclast-specific genes. However, it did not affect osteoblast differentiation, or the expression of genes involved in this process. These results demonstrated that OK prevented LPS-induced bone loss by decreasing the RANKL/OPG ratio in serum and BMF, and inhibited osteoclast differentiation and function, suggesting that OK represents a potential therapeutic drug for the treatment of osteoclast-associated bone diseases.

Osteoclasts play a critical role in bone resorbing disorders such as osteoporosis, periodontitis, and rheumatoid arthritis. Therefore, discovery of agents capable of suppressing osteoclast differentiation may aid the development of a therapeutic access for the treatment of pathological bone loss. Ampelopsis brevipedunculata has been used as herbal folk medicine to treat liver diseases and inflammation in Asia. However, its effects on osteoclast differentiation are unknown. We were aimed to investigate the anti-osteoclastogenic activity in vitro and in vivo and to elucidate the underlying mechanism of Ampelopsis brevipedunculata extract (ABE). In this study, ABE inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, the formation of filamentous actin rings and the bone resorbing activity of mature osteoclasts. ABE inhibited RANKL-induced p38 and IκB phosphorylation and IκB degradation. Also, ABE suppressed the mRNA and protein expression of nuclear factor of activated T cells c1 (NFATc1) and c-Fos, and the mRNA expression of genes required for cell fusion and bone resorption, such as osteoclast-associated receptor (OSCAR), tartrate resistant acid phosphatase (TRAP), cathepsin K, dendritic cell-specific transmembrane protein (DC-STAMP), β3-integrin and osteoclast stimulatory transmembrane protein (OC-STAMP). Furthermore, results of micro-CT and histologic analysis indicated that ABE remarkably prevented lipopolysaccharide (LPS)-induced bone erosion. These results demonstrate that ABE prevents LPS-induced bone erosion through inhibition of osteoclast differentiation and function, suggesting the promise of ABE as a potential cure for various osteoclast-associated bone diseases.

Translationally-controlled tumor protein (TCTP) plays a role in a number of cellular processes, but there is limited information about its function in cell differentiation. Previous observations of a twofold induction of TCTP mRNA during osteoclast differentiation prompted us to investigate its involvement in osteoclast differentiation. The osteoclastogenicity of TCTP gradually expressed during osteoclast differentiation was confirmed in mouse and human cells using loss-of-function studies and TCTP heterogeneous mice and transgenic mice. Higher expression ratios of TCTP to p21 could represent TCTP-mediated phenotypic induction of osteoclast differentiation accompanied by p21 down-regulation, attenuating the proliferation of osteoclast precursor cells.

Bone remodeling, a physiological process in which new bone is formed by osteoblasts and the preexisting bone matrix is resorbed by osteoclasts, is vital for the maintenance of healthy bone tissue in adult humans. Imbalances in this process can cause various pathological conditions, including osteoporosis. Emodin, a naturally occurring anthraquinone derivative found in Asian herbal medicines, has numerous beneficial pharmacologic effects, including anticancer and antidiabetic activities. However, the effect of emodin on the regulation of osteoblast and osteoclast activity has not yet been investigated. We show here that emodin is a potential target for osteoporosis therapeutics, as treatment with this agent enhances osteoblast differentiation and bone growth and suppresses osteoclast differentiation and bone resorption. In this study, emodin suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation of bone marrow macrophages (BMMs) and the bone-resorbing activity of mature osteoclasts by inhibiting RANKL-induced NF-κB, c-Fos, and NFATc1 expression. Emodin also increased ALP, Alizarin Red-mineralization activity, and the expression of osteoblastogenic gene markers, such as Runx2, osteocalcin (OCN), and ALP in mouse calvarial primary osteoblasts, as well as activated the p38-Runx2 pathway, which enhanced osteoblast differentiation. Moreover, mice treated with emodin showed marked attenuation of lipopolysaccharide (LPS)-induced bone erosion and increased bone-forming activity in a mouse calvarial bone formation model based on micro-computed tomography and histologic analysis of femurs. Our findings reveal a novel function for emodin in bone remodeling, and highlight its potential for use as a therapeutic agent in the treatment of osteoporosis that promotes bone anabolic activity and inhibits osteoclast differentiation.

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of NF-κB ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and IκB, as well as IκB degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption. [BMB Reports 2014; 47(8): 451-456]

(2S)-2'-Methoxykurarinone (MK), a compound isolated from the roots of Sophora flavescens, has various physiological properties, such as anti-inflammatory, antipyretic, antidiabetic, and antineoplastic effects. However, the effect of S. flavescens-derived MK on osteoclastogenesis remains unknown. Therefore, we examined the effect and mechanism of action of MK on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. MK inhibited osteoclast differentiation in bone marrow cell-osteoblast cocultures but did not affect the RANKL-to-osteoprotegerin ratio induced by osteoclastogenic factors in osteoblasts. MK also inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose-dependent manner, without cytotoxicity. Pretreatment with MK significantly suppressed the Akt, p38, c-Jun N terminal kinase (JNK), c-Fos, and nuclear factor of activated T cells c1 (NFATc1) pathways and inhibited the bone-resorbing activity of mature osteoclasts. These results collectively suggest that MK inhibits osteoclast differentiation and bone resorption through RANKL-induced mitogen-activated protein kinases (MAPKs) and c-Fos-NFATc1 signaling pathways.

People over the age of 50 are at risk of osteoporotic fracture, which may lead to increased morbidity and mortality. Osteoclasts are responsible for bone resorption in bone-related disorders. Genipin is a well-known geniposide aglycon derived from Gardenia jasminoides, which has long been used in oriental medicine for controlling diverse conditions such as inflammation and infection. We aimed to evaluate the effects of genipin on RANKL-induced osteoclast differentiation and its mechanism of action. Genipin dose-dependently inhibited early stage RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) during culture. Genipin inhibited RANKL-induced IκB degradation and suppressed the mRNA expression of osteoclastic markers such as NFATc1, TRAP, and OSCAR in RANKL-treated BMMs, but did not affect c-Fos mRNA expression. Interestingly, genipin markedly inhibited c-Fos protein expression in BMMs, which was reversed in the presence of the proteosome inhibitor MG-132. Furthermore, genipin inhibited RANKL-mediated osteoclast differentiation, which was also rescued by overexpression of c-Fos and NFATc1 in BMMs. Taken together, our findings indicate that genipin down-regulated RANKL-induced osteoclast differentiation through inhibition of c-Fos protein proteolysis as well as inhibition of IκB degradation. Our findings indicate that genipin could be a useful drug candidate that lacks toxic side effects for the treatment of osteoporosis. Keywords:: genipin, osteoclast, differentiation, c-Fos, NFATc1

Costunolide, a sesquiterpene lactone, exhibits anti-inflammatory and anti-oxidant properties and mediates apoptosis. However, its effects and mechanism of action in osteoclasts remain unknown. Herein, we found that costunolide significantly inhibited RANKL-induced BMM differentiation into osteoclasts in a dose-dependent manner without affecting cytotoxicity. Costunolide did not regulate the early signaling pathways of RANKL, including the mitogen-activated protein kinase and NF-κB pathways. However, costunolide suppressed nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression via inhibition of c-Fos transcriptional activity without affecting RANKL-induced c-Fos expression. The inhibitory effects of costunolide were rescued by overexpression of constitutively active (CA)-NFATc1. Taken together, our results suggest that costunolide inhibited RANKL-induced osteoclast differentiation by suppressing RANKL-mediated c-Fos transcriptional activity. Copyright © 2013 John Wiley & Sons, Ltd.

Excessive osteoclastic bone resorption plays a critical role in inflammation-induced bone loss such as rheumatoid arthritis and periodontal bone erosion. Therefore, identification of osteoclast targeted-agents may be a therapeutic approach to the treatment of pathological bone loss. In this study, we isolated chlorogenic acid (CGA) from fructus of Gardenia jasminoides to discover anti-bone resorptive agents. CGA is a polyphenol with anti-inflammatory and anti-oxidant activities, however, its effects on osteoclast differentiation is unknown. Thus, we investigated the effect of CGA in receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation and RANKL signaling. CGA dose-dependently inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. CGA inhibited the phosphorylation of p38, Akt, extracellular signal-regulated kinase (ERK), and inhibitor of nuclear factor-kappa B (IκB), and IκB degradation by RANKL treatment. CGA suppressed the mRNA expression of nuclear factor of activated T cells c1 (NFATc1), TRAP and OSCAR in RANKL-treated bone marrow macrophages (BMMs). Also, overexpression of NFATc1 in BMMs blocked the inhibitory effect of CGA on RANKL-mediated osteoclast differentiation. Furthermore, to evaluate the effects of CGA in vivo, lipopolysaccharide (LPS)-induced bone erosion study was carried out. CGA remarkably attenuated LPS-induced bone loss based on micro-computed tomography and histologic analysis of femurs. Taken together, our findings suggest that CGA may be a potential treatment option for osteoclast-related diseases with inflammatory bone destruction.

VapB has been shown to regulate calcium homeostasis in amyotrophic lateral sclerosis. Calcium signaling is also important in metabolic bone diseases, but the role of VapB in the generation of osteoclasts for bone resorption during osteoclastogenesis is not known. Therefore, we investigated the role of VapB in RANKL-induced osteoclast differentiation. Interestingly, VapB is induced during osteoclastogenesis, and regulates osteoclast differentiation by modulating NFATc1. The results also suggest that VapB regulates osteoclastogenesis via PLCγ2-Ca2+-NFAT signaling. The involvement of PLCγ2-Ca2+-NFAT signaling in VapB-regulated osteoclastogenesis was confirmed by a pharmacological study. Taken together, the results indicate that VapB positively regulates RANKL-mediated osteoclastogenesis via PLCγ2-Ca2+-NFAT signaling.

Osteoclasts are multinucleated cells that are formed by the fusion of mononuclear osteoclasts, which is an essential process in bone resorption leading to bone remodeling. Herein we show that GM-CSF promoted the fusion of prefusion osteoclasts (pOCs). The expression of GM-CSF receptor-α was significantly up-regulated at the fusion stage of pOCs induced by RANKL. GM-CSF induced the expression of dendritic cell-specific transmembrane protein (DC-STAMP), which was mediated by inducing NFATc1 via induction of c-Fos. The expression of c-Fos and NFATc1 was regulated by the ERK signaling pathway. Inhibition of ERK and NFATc1 suppressed the expression of DC-STAMP and led to the fusion inhibition of pOC. However, retrovirus-mediated expression of NFATc1 in pOCs rescued the defect in pOC fusion, despite the presence of U0126 and cyclosporin A. GM-CSF-stimulated pOCs had an intact actin ring and could resorb bone. Importantly, pOCs infected with constitutively active MEK adenovirus expressed c-Fos and NFATc1, followed by the binding of NFATc1 to the DC-STAMP promoter, which enables its transcription and expression. Constitutively active MEK-infected pOCs are able to resorb bone by undergoing cell-cell fusion. Taken together, our results demonstrated that GM-CSF induced fusion of pOCs to form multinucleated osteoclasts, making the osteoclast capable of bone resorption.

Angelica tenuissima has been traditionally used in oriental medicine for its therapeutic effects in headache, toothache, and flu symptoms. It also exerts anti-inflammatory activity via the inhibition of the expression of cyclooxygenase-2 (COX-2). However, the effect of Angelica tenuissima on osteoclast differentiation has not been identified until recently. In this study, we first confirmed that Angelica tenuissima water extract (ATWE) significantly interrupted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in a dose-dependent manner without any cytotoxicity. Next, we clarified the underlying mechanisms linking the suppression effects of ATWE on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. At the molecular level, ATWE induced the dephosphorylation of c-Jun N-terminal kinase (JNK) and Akt and decreased the degradation of IκB in RANKL-dependent early signaling pathways. Subsequently, ATWE caused impaired activation of the protein and mRNA levels of c-Fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, the disassembly of filamentous actin (F-actin) ring and anti-resorptive activity of mature osteoclasts were triggered by ATWE treatment. Although ATWE did not enhance osteogenesis in primary osteoblasts, our results showed that ATWE is a potential candidate for anti-resorptive agent in osteoporosis, a common metabolic bone disorder.

Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis.

In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP) staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLC gamma 2-Ca2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca2+ influx intensity (staining with Fluo-3/AM). Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

Homeostatic bone remodeling is vital to maintain healthy bone tissue. Although the receptor activator of nuclear factor κB ligand (RANKL)/RANK axis is considered the master regulator of osteoclastogenesis, the underlying mechanisms including cell fusion remain incompletely defined. Here, we introduce a new axis in the formation of multinucleated cells via RANK signaling: the progranulin (PGRN)/PIRO (PGRN-induced receptor-like gene during osteoclastogenesis) axis. When mouse bone marrow-derived macrophages were stimulated with PGRN in the presence of RANKL, explosive OC formation was observed. PGRN knockdown experiments suggested that endogenous PGRN is an essential component of the RANKL/RANK axis. Our efforts for identifying genes that are induced by PGRN unveiled a remarkably induced (20-fold) gene named PIRO. Substantial PGRN and PIRO expression was detected after 2 and 3 days, respectively, suggesting that their sequential induction. PIRO was predicted to be a five transmembrane domain-containing receptor-like molecule. The tissue distribution of PGRN and PIRO mRNA expression suggested that bone marrow cells are the most suitable niche. Mouse and human PIRO are part of a multigene family. Knockdown experiments suggested that PIRO is a direct target for the formation of multinucleated cells by PGRN. PGRN levels were also substantially higher in ovariectomized mice than in sham control mice. These observations suggest that PGRN and PIRO form a new regulatory axis in osteoclastogenesis that is included in RANK signaling in cell fusion and OC resorption of osteoclastogenesis, which may offer a novel therapeutic modality for osteoporosis and other bone-associated diseases.

Aconitum pseudo-laeve var. erectum (APE) has been widely shown in herbal medicine to have a therapeutic effect on inflammatory conditions. However, there has been no evidence on whether the extract of APE is involved in the biological bone metabolism process, particularly osteoclast-mediated bone resorption. In this study, we confirmed that the administration of APE could restore normal skeletal conditions in a murine model of lipopolysaccharide (LPS)-induced bone loss via a decrease in the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio and osteoclast number. We then investigated the effect of APE on the RANKL-induced formation and function of osteoclasts to elucidate its underlying molecular mechanisms. APE suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells, as well as the bone-resorbing activity of mature osteoclasts. Furthermore, APE attenuated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and c-Fos without affecting any early signal pathway of osteoclastogenesis. Subsequently, APE significantly downregulated the expression of various genes exclusively expressed in osteoclasts. These results demonstrate that APE restores LPS-induced bone loss through a decrease of the serum RANKL/OPG ratio, and inhibits osteoclast differentiation and function, suggesting the promise of APE as a potential cure for various osteoclast-associated bone diseases.

Osteopenic diseases, such as osteoporosis, are characterized by progressive and excessive bone resorption mediated by enhanced receptor activator of nuclear factor-κB ligand (RANKL) signaling. Therefore, downregulation of RANKL downstream signals may be a valuable approach for the treatment of bone loss-associated disorders. In this study, we investigated the effects of the naphthohydroquinone mollugin on osteoclastogenesis and its function in vitro and in vivo. Mollugin efficiently suppressed RANKL-induced osteoclast differentiation of bone marrow macrophages (BMMs) and bone resorbing activity of mature osteoclasts by inhibiting RANKL-induced c-Fos and NFATc1 expression. Mollugin reduced the phosphorylation of signaling pathways activated in the early stages of osteoclast differentiation, including the MAP kinase, Akt, and GSK3β and inhibited the expression of different genes associated with osteoclastogenesis, such as OSCAR, TRAP, DC-STAMP, OC-STAMP, integrin αν, integrin β3, cathepsin K, and ICAM-1. Furthermore, mice treated with mollugin showed significant restoration of lipopolysaccharide (LPS)-induced bone loss as indicated by micro-CT and histological analysis of femurs. Consequently, these results suggested that mollugin could be a novel therapeutic candidate for bone loss-associated disorders including osteoporosis, rheumatoid arthritis, and periodontitis.

1 : Department of Anatomy, Institute for Skeletal Disease, 2 : Imaging Science-based Lung and Bone Diseases Research Center, 3 : Department of Pathology, College of Korean Medicine & Research Center of Traditional Korean Medicine, 4 : Department of Internal Medicine, Division of Rheumatology and Institute of Wonkwang Medical Science College of Medicine, 5 : Institute for Environmental Science, Wonkwang University

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 ofChrysanthemum indicumextract (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.

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.

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.

Background Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. Objectives We investigated the effects of ebselen on RANKL-induced differentiation of osteoclasts and their functions and the underlying molecular mechanisms. Furthermore, we determined the effects of ebselen on LPS-induced bone erosion in vivo . Methods To generate osteoclasts from BMMs, we cultured BMMs for 4 days in the condition of M-CSF and RANKL pretreated with ebselen. The cells were then stained with TRAP solution, rhodamine-conjugated phalloidin for F-actin ring labeling and DAPI solution to detect apoptotic body formation. The change of F-actin ring on mature osteoclasts induced by ebselen was quantified by calculating the ratio of actin ring positive (AR+) osteoclasts versus actin ring negative (AR-) osteoclasts. to detect the formation of apoptotic osteoclasts, we performed TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay. Primary calvaria osteoblasts and BMCs were co-cultured and were re-seeded in hydroxyapatite-coated plates or dentin slices with or without ebselen. ICR mice were divided into 4 experimental groups comprising 5 mice each: phosphate-buffered saline-treated (control) group, ebselen only-treated group, LPS only-treated group, and LPS- and ebselen-treated group. Ebselen (10 mg/kg) or PBS was administered orally every 8 days, and LPS was injected intraperitoneally on days 1 and 4. Micro-computed tomography (μ-CT) data containing 3D images and bone parameters and histological data were acquired Results Ebselen suppressed the formation of TRAP-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of RANKL/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its μ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate receptor. Conclusions Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. Disclosure of Interest None declared

Receptor activator of NF-kappa B ligand (RANKL) triggers the differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) of hernatopoietic origin into osteoclasts through the activation of mitogen-activated protein (MAP) kinases and transcription factors. Recently, reactive oxygen species (ROS) and antioxidant enzymes were shown to be closely associated with RANKL-mediated osteoclast differentiation. Although glutaredoxin2 (Glrx2) plays a role in cellular redox homeostasis, its role in RANKL-mediated osteoclastogenesis is unclear. We found that Glrx2 isoforrn b (Glrx2b) expression is induced during RANKL-mediated osteoclastogenesis. Over-expression of Glrx2b strongly enhanced RANKL- mediated osteoclastogenesis. In addition, Glrx2b-transduced BMMs enhanced the expression of key transcription factors c-Fos and NFATc1, but pre-treatment with SB203580, a p38-specific inhibitor, completely blocked this enhancement Conversely, down-regulation of Glrx2b decreased RANKL- mediated osteoclastogenesis and the expression of c-Fos and NFATc1 proteins. Also, Glrx2b down-regulation attenuated the RANKL-induced activation of p38. Taken together, these results suggest that Glrx2b enhances RANKL-induced osteoclastogenesis via p38 activation. [BMB reports 2012; 45(3): 171-176]

Interleukin-3 (IL-3) is produced under various pathological conditions and is thought to be involved in the pathogenesis of inflammatory diseases; however, its function in bone homeostasis under normal conditions or nature of the downstream molecular targets remains unknown. Here we examined the effect of IL-3 on osteoclast differentiation from mouse and human bone marrow-derived macrophages (BMMs). Although IL-3 can induce osteoclast differentiation of multiple myeloma bone marrow cells, IL-3 greatly inhibited osteoclast differentiation of human BMMs isolated from healthy donors. These inhibitory effects of IL-3 were only observed at early time points (days 0 and 1). IL-3 inhibited the expression of c-Fos and NFATc1 in BMMs treated with RANKL. However, IL-3-mediated inhibition of osteoclast differentiation was not completely reversed by ectopic expression of c-Fos or NFATc1. Importantly, IL-3 induced inhibitor of DNA binding/differentiation (Id)1 in hBMMs, while Id2 were sustained during osteoclast differentiation of mBMMs treated with IL-3. Ectopic expression of NFATc1 in Id2-deficient BMMs completely reversed the inhibitory effect of IL-3 on osteoclast differentiation. Furthermore, inflammation-induced bone erosion was markedly inhibited by IL-3 administration. Taken together, our results suggest that IL-3 plays an inhibitory role in osteoclast differentiation by regulating c-Fos and Ids, and also exerts anti-bone erosion effects.

Risedronate, a nitrogen-containing bisphosphonate, is widely used in the clinical field for the treatment of osteoporosis. Risedronate is known to exert its effects through binding to hydroxyapatite in bone tissue, inhibiting osteoclastic activity, and inducing apoptosis of osteoclasts. The purpose of this study was to determine the effects of risedronate on osteoclast differentiation in vitro and on an inflammatory bone loss model in vivo. Risedronate inhibited osteoclast differentiation in co-culture of bone marrow cells (BMCs) and osteoblasts, and suppressed receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from bone marrow-derived macrophages (BMMs) in a dose-dependent manner without toxicity. Risedronate significantly inhibited expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 induced by RANKL. To examine the effect of risedronate on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice administered with risedronate. Taken together, we conclude that risedronate exerts beneficial effects on osteoporosis by inhibiting osteoclast differentiation both directly and indirectly. In infectious conditions, the inhibitory effect of risedronate on bone erosion was excellent. Thus risedronate could be a treatment option for osteoporosis caused by inflammatory and infectious conditions.

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-kappaB 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.

It has been reported that Janus tyrosine kinase (JAK)-dependent signaling pathways play a critical role in the pathogenesis of numerous malignancies and immune reactions, and inhibition of JAK has been implicated in cell growth inhibition. The role which JAK has on osteoclast differentiation and anti-bone resorptive activity is not well understood. In this study, we investigated the effects of a pan-JAK inhibitor, pyridone 6, on osteoclast differentiation and bone-resorption in vitro and ex vivo. Pyridone 6 inhibited osteoclast differentiation in mouse bone marrow macrophage (BMM) cultures stimulated by the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and co-cultures of bone marrow cells and osteoblasts. Pyridone 6 suppressed the expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 in BMMs. It also inhibited the bone resorptive activity of mature osteoclasts that was accompanied by disruption of actin rings. Pyridone 6 also suppressed I-kappaB degradation and extracellular signal-regulated kinase (ERK) in mature osteoclasts, suggesting that these are the key molecules that pyridone 6 targets in the inhibition of osteoclast function. These results demonstrate inhibition of JAK may be useful for the treatment of bone-resorptive diseases, such as osteoporosis.

Osteoclasts rapidly undergo spontaneous apoptosis when deprived of survival factors. Regulation of osteoclast survival is important to treat bone-related diseases, such as osteoporosis. In this study, we found that the proteasome inhibitors, MG132 and ALLN, significantly inhibited osteoclast apoptosis induced by etoposide, as well as under conditions of survival factor deprivation. MG132 and ALLN inhibited the release of cytochrome c from mitochondria into the cytosol in the absence of survival factors and suppressed the cleavage of pro-caspase-9 and -3 to its active forms induced by etoposide. In addition, MG132 and ALLN enhanced the phosphorylation of Akt and ERK in osteoclasts. However, MG132 and ALLN did not inhibit the cleavage of caspase-9 and -3 in the presence of the phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, while the inhibitory effect of MG132 and ALLN were intact in presence of the MEK1/2 inhibitor, U0126. LY294002 inhibited the survival of osteoclasts induced by MG132 and ALLN. Taken together, our results have demonstrated that proteasome inhibitors suppressed osteoclast apoptosis under conditions of survival factors deprivation through activation of the PI-3K/Akt pathway.

Background 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis. They have been reported to have anti-inflammatory and/or immunomodulatory as well as prophylactic and therapeutic effects in collagen-induced arthritis models. Bone erosion is a central feature of rheumatoid arthritis and is associated with disease severity and poor functional outcome. Objectives This study was designed to define the effect of statins on IL-1β-induced osteoclastogenesis and to elucidate the underlying mechanisms. Methods Bone marrow cells (BMCs) were obtained from 5-week-old male ICR mice and cultured to differentiate them into osteoclasts with M-CSF and RANKL in the presence or absence of IL-1β or atorvastatin. The formation of osteoclasts was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and resorption pit assay with dentine slice. The molecular mechanisms of the above effects of atorvastatin on osteoclastogenesis were investigated using RT-PCR and immunoblotting for osteoclast specific molecules. Results Atorvastatin significantly reduced the number of TRAP-positive multinucleated cells as well as the bone resorption area. Atorvastatin also downregulated the expression of NFATc1 mRNA and inhibited the expression of osteoclast-specific genes. A possible underlying mechanism may be that atorvastatin suppresses the degradation of the inhibitors of NF-κB and blocks the activation of c-JUN N-terminal kinase, extracellular signal-regulated kinase, and p38, thus implicating the NF-κB and MAPK pathway in this process. ![Figure][1] Conclusions These results indicate that atorvastatin is a strong inhibitor of inflammation-induced osteoclastogenesis in inflammatory joint diseases Disclosure of Interest None declared [1]: pending:yes

Background WHI-131/JANEX-1 (4-(49Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline), quinazoline-type small molecule compound is well known a JAK3 inhibitor that demonstrated as potent therapeutic agent about anti-inflammatory, anti-cancer, and anti-leukemia in several animal models. However, has not been fully investigated for regulatory effects on osteoblast and osteoclast activity by WHI-131. Objectives Therefore, in this study, we examined the effects of WHI-131 in bone remodeling during osteoblast and osteoclast differentiation. Results WHI-131 decreased RANKL-induced osteoclast differentiation on the bone marrow derived macrophages cultures and reduced the resorbing activity of mature osteoclasts. WHI-131 suppressed the protein and mRNA expression of c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and down-regulated mRNA expression levels of TRAP, osteoclast-associated receptor (OSCAR), dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), d2 isoform of vacuolar H + ATPase V(0) domain (ATP6v0d2), and Cathepsin K. WHI-131 diminished the phosphorylation of Akt and degradation of I-kB. In addition, WHI-131 suppressed Ca 2+ oscillation by inhibiting phosphorylation of the c-Src-Btk-PLCg2 pathway. WHI-131 expression appeared to increase based on alkaline phosphate and alizarin red staining in relation to increased activity of the differentiation of primary osteoblast cells. WHI-131 increased the mRNA expression of genes related to osteoblast differentiation, including runt-related transcription factor 2 (Runx2), ALP, collagen type 1a (Col1a), osteocalcin (OCN), and OPN and induced the phosphorylation of Akt, p38, and Smad 1/5/8. Interestingly, WHI-131 had a notable anti-resorbing effects in the LPS-induced calvaria bone loss model in vivo . WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Conclusions Thus, WHI-131 may be a useful pharmacological agent that acts as a safe and effective dual-action therapeutic against osteoporosis, promoting robust bone growth while inhibiting resorption. Disclosure of Interest None declared