Ligand-induced Osteoclastogenesis Research Articles

Conditioned Medium From Stem Cells of Human Exfoliated Deciduous Teeth Alleviates Mouse Osteoarthritis by Inducing sFRP1-Expressing M2 Macrophages.

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1β-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.

Irpetones A and B, Anti-Osteoclastic Heptaketides from a Marine Mesophotic Zone Ircinia Sponge-Associated Fungus Irpex sp. NBUF088.

Chemical investigation of Irpex sp. NBUF088, associated with an Ircinia sp. sponge located at an 84 m deep mesophotic zone, led to the discovery of two new heptaketides, named irpetones A (1) and B (2). Their structures were identified by analysis of spectroscopic data and quantum-chemical calculations. Compound 1 exhibited inhibition against the receptor activator of NF-κB ligand-induced osteoclastogenesis in bone marrow monocytes with an IC50 of 6.3 ± 0.2 μM, causing no notable cytotoxicity. It was also determined that 1 inhibited the phosphorylation of ERK1/2-JNK1/2-p38 MAPKs and the nuclear translocation of NF-κB, consequently suppressing the activation of MAPK and NF-κB signaling pathways induced by the NF-κB ligand.

Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

Extracellular Vesicles Derived From Murine Cementoblasts Possess the Potential to Increase Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclastogenesis.

Cementum resorption, unlike bone resorption, is clinically known to occur only with limited pathological stimuli, such as trauma, orthodontic forces, and large apical periodontitis; however, the molecular mechanisms that control osteoclast formation on the cementum surface remain unclear. In this study, we focused on extracellular vesicles (EVs) secreted by cementoblasts and analyzed their effects on osteoclast differentiation. EVs were extracted from the conditioned medium (CM) of the mouse cementoblast cell line OCCM-30. Transmission electron microscopy (TEM) analysis confirmed the presence of EVs with a diameter of approximately 50–200 nm. The effect of the EVs on osteoclast differentiation was examined using the mouse osteoclast progenitor cell line RAW 264.7 with recombinant receptor activator of nuclear factor (NF)-κB ligand (rRANKL) stimulation. EVs enhanced the formation of tartrate-resistant acid phosphatase (TRAP) activity-positive cells upon rRANKL stimulation. EVs also enhanced the induction of osteoclast-associated gene and protein expression in this condition, as determined by real-time PCR and Western blotting, respectively. On the other hand, no enhancing effect of EVs was observed without rRANKL stimulation. A Western blot analysis revealed no expression of receptor activator of NF-κB ligand (RANKL) in EVs themselves. The effect on rRANKL-induced osteoclast differentiation was examined using the CM of cementoblasts in terms of TRAP activity-positive cell formation and osteoclast-associated gene expression. The conditioned medium partly inhibited rRANKL-induced osteoclast differentiation and almost completely suppressed its enhancing effect by EVs. These results indicate that cementoblasts secreted EVs, which enhanced RANKL-induced osteoclast differentiation, and simultaneously produced soluble factors that neutralized this enhancing effect of EVs, implicating this balance in the regulation of cementum absorption. A more detailed understanding of this crosstalk between cementoblasts and osteoclasts will contribute to the development of new therapies for pathological root resorption.

Nitazoxanide, an Antiprotozoal Drug, Reduces Bone Loss in Ovariectomized Mice by Inhibition of RANKL-Induced Osteoclastogenesis.

Nitazoxanide (NTZ) is an FDA-approved anti-parasitic drug with broad-spectrum anti-infective, anti-inflammatory, and antineoplastic potential. However, its regulatory effects on osteoclastogenesis and the underlying mechanisms remain unclear. The present study found that NTZ potently inhibited osteoclast formation at the early stage of receptor activator of NF-κB ligand-induced osteoclastogenesis in a concentration-dependent manner at a non-growth inhibitory concentration. NTZ suppressed actin ring formation and decreased osteoclast marker gene expression, including TRAP, MMP9, and cathepsin K. NTZ significantly impaired the bone resorption activity of osteoclasts. In vivo, ovariectomized mice were treated with 50, 100 and 200 mg/kg/d NTZ for 3 months. NTZ (100 mg/kg/d) administration markedly reduced ovariectomy-induced bone loss by suppressing osteoclast activity. Mechanistically, osteoclastogenesis blockade elicited by NTZ resulted from inhibition of STAT3 phosphorylation, and reduction of the Ca2+ fluorescence intensity and NFATc1 expression. NTZ weakened the binding between STAT3 and the NFATc1 promoter region. Furthermore, enforced NFATc1 expression partly rescued the impaired osteoclast differentiation in NTZ-treated RAW264.7 cells. In summary, NTZ could inhibit osteoclastogenesis and bone loss through modulation of the receptor activator of NF-κB ligand-induced STAT3-NFATc1 signaling pathway, which might be a potential alternative treatment regimen against bone destruction-related diseases including osteoporosis.

The Fungal Metabolite (+)-Terrein Abrogates Ovariectomy-Induced Bone Loss and Receptor Activator of Nuclear Factor-κB Ligand-Induced Osteoclastogenesis by Suppressing Protein Kinase-C α/βII Phosphorylation.

Osteoporosis is a common disease characterized by a systemic impairment of bone mass and microarchitecture that results in fragility fractures. Severe bone loss due to osteoporosis triggers pathological fractures and consequently decreases the daily life activity and quality of life. Therefore, prevention of osteoporosis has become an important issue to be addressed. We have reported that the fungal secondary metabolite (+)-terrein (TER), a natural compound derived from Aspergillus terreus, has shown receptor activator of nuclear factor-κB ligand (RANKL)–induced osteoclast differentiation by suppressing nuclear factor of activated T-cell 1 (NFATc1) expression, a master regulator of osteoclastogenesis. TER has been shown to possess extensive biological and pharmacological benefits; however, its effects on bone metabolism remain unclear. In this study, we investigated the effects of TER on the femoral bone metabolism using a mouse-ovariectomized osteoporosis model (OVX mice) and then on RANKL signal transduction using mouse bone marrow macrophages (mBMMs). In vivo administration of TER significantly improved bone density, bone mass, and trabecular number in OVX mice (p < 0.01). In addition, TER suppressed TRAP and cathepsin-K expression in the tissue sections of OVX mice (p < 0.01). In an in vitro study, TER suppressed RANKL-induced phosphorylation of PKCα/βII, which is involved in the expression of NFATc1 (p < 0.05). The PKC inhibitor, GF109203X, also inhibited RANKL-induced osteoclastogenesis in mBMMs as well as TER. In addition, TER suppressed the expression of osteoclastogenesis-related genes, such as Ocstamp, Dcstamp, Calcr, Atp6v0d2, Oscar, and Itgb3 (p < 0.01). These results provide promising evidence for the potential therapeutic application of TER as a novel treatment compound against osteoporosis.

Celastrol Attenuates RANKL-Induced Osteoclastogenesis in vitro and Reduces Titanium Particle-Induced Osteolysis and Ovariectomy-Induced Bone Loss in vivo.

Excessive bone resorption by osteoclasts contributes significantly to osteoclast-related diseases such as periprosthetic osteolysis and osteoporosis. Osteolysis in a titanium particle-induced calvarial model and bone loss in an ovariectomized mice model occurred similarly to those in humans; thus, these models can be used to evaluate potential therapies for aseptic prosthetic loosening and osteoporosis. Celastrol, which is extracted from the seeds of the genus Tripterygium, has been thoroughly investigated for its anti-inflammatory and anti-cancer pharmacological effects. However, the mechanisms involving bone metabolism by which celastrol inhibits osteoclastogenesis are not yet fully understood. We demonstrated that celastrol inhibited the receptor activator of nuclear factor κB ligand-induced osteoclastogenesis and the bone resorptive function of osteoclasts in vitro by inhibiting the activation of transforming growth factor β-activated kinase 1-mediated NF-κB and mitogen-activated protein kinase signaling pathways and downregulating osteoclastogenesis marker-related genes. Furthermore, celastrol was also shown to be beneficial in both the titanium particle-induced osteolysis calvarial and the murine ovariectomy-induced bone loss. Collectively, our results suggested that celastrol is promising for the prevention of aseptic prosthetic loosening and osteoporosis in the treatment of osteolytic diseases induced by disrupted osteoclast formation and function.

Protocatechualdehyde inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclastogenesis and attenuates lipopolysaccharide-induced inflammatory osteolysis.

Inflammatory osteolysis as a consequence of chronic bacterial infection underlies several lytic bone conditions, such as otitis media, osteomyelitis, septic arthritis, periodontitis, periprosthetic infection, and aseptic loosening of orthopedic implants. In consideration of the lack of effective preventive or treatments options against infectious osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. The present study assessed the effect of protocatechualdehyde (PCA), a natural occurring polyphenolic compound with diverse biological activities including but not limited to antibacterial and antiinflammatory properties, on nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone loss in vivo. In the present study, it was found that PCA potently inhibited RANKL-induced osteoclast formation, fusion, and activation toward bone resorption in a dose-dependent manner via the suppression of the ERK/c-Fos/nuclear factor of activated T-cells, cytoplasmic 1 signaling axis. It was further demonstrated that the in vivo administration of PCA could effectively protect mice against the deleterious effects of LPS-induced calvarial bone destruction by attenuating osteoclast formation and activity in a dose-dependent manner. Collectively, these findings provided evidence for the potential therapeutic application of PCA in the prevention and treatment of infectious osteolytic conditions, and potentially other osteoclast-mediated bone diseases.

4-Hexylresorcinol inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway and reverses bone loss in ovariectomized mice.

4-Hexylresorcinol (4HR) is a small organic compound that is widely used as an antiseptic and antioxidant. In the present study, its role in osteoclastogenesis was investigated. Bone marrow-derived macrophages from mice were used to examine the role of 4HR in osteogenesis. An ovariectomy (OVX) mouse model was constructed to examine the effect of 4HR in vivo, followed by hematoxylin and eosin and tartrate resistant acid phosphatase staining. In the present study, 4HR effectively suppressed receptor activator of NF-κB ligand-induced osteoclastogenesis in a dose-dependent manner. 4HR was also found to significantly suppress the expression of osteoclast (OC)-specific markers, including tartrate-resistant acid phosphatase, cathepsin K, nuclear factor of activated T-cell cytoplasmic 1 and c-Fos in the presence of RANKL in BMMs. Furthermore, 4HR inhibited osteoclastogenesis by inhibiting the activation of the NF-κB signaling pathway in BMMs. Consistent with the in vitro results, 4HR effectively ameliorated OVX-induced bone loss and markedly reduced OC number in the proximal tibia in vivo. In conclusion, the present results suggested that 4HR inhibited osteoclastogenesis in vitro and rescued bone loss in vivo, suggesting that 4HR may serve as a novel therapeutic agent for osteoporosis treatment.

Glycyrrhizin improves bone metabolism in ovariectomized mice via inactivating NF-κB signaling

Objectives Postmenopausal osteoporosis (PMO) is a prevalent metabolic bone disease with high morbidity and serious complications. Here, we studied the effect of glycyrrhizin on bone metabolism using the ovariectomized (OVX) mouse model. Methods Osteoclast-related gene expression and osteoclastic function were evaluated in RAW264.7 cells and bone marrow-derived monocytes (BMMs) by real-time polymerase chain reaction and bone resorption assay. For animal studies, female C57BL/6J mice were randomly divided into sham operated, OVX and OVX with glycyrrhizin groups. Bone mass and trabecular microarchitecture were analyzed by micro-computed tomography, dual X-ray absorptiometry, and histomorphometric analysis. Receptor activator of nuclear factor-κB (NF-κB) ligand-induced osteoclastogenesis and the NF-κB signaling pathway were studied by tartrate-resistant acid phosphatase staining and western blotting, respectively. Results Glycyrrhizin inhibits RANKL-induced expression of Nfatc-1, c-Fos, Trap, Ds-stamp, and Ctsk in RAW264.7 cells. Also, fewer bone resorption pits form when BMMs are incubated in the presence of glycyrrhizin. Glycyrrhizin ameliorates bone loss and improves trabecular bone parameters in OVX mice. BMMs isolated from OVX mice show higher ability of RANKL-induced osteoclastogenesis, which is tremendously reversed by glycyrrhizin. There is significantly higher phosphorylation of IκB-α at Ser32 and NF-κB p65 at Ser536, as well as increased protein levels of c-FOS and NFATc-1 in BMMs of OVX mice, which are all greatly suppressed by glycyrrhizin. Conclusions Our findings imply that glycyrrhizin is a potential efficient adjuvant therapeutic for PMO.

Muscone Ameliorates Ovariectomy-Induced Bone Loss and Receptor Activator of Nuclear Factor-κb Ligand-Induced Osteoclastogenesis by Suppressing TNF Receptor-Associated Factor 6-Mediated Signaling Pathways.

Postmenopausal osteoporosis is caused by the deficiency of estrogen, which breaks bone homeostasis and induces levels of pro-inflammatory cytokines. Muscone is a potent anti-inflammatory agent and is used to treat bone fracture in traditional Chinese medicine. However, its anti-osteoclastogenic effects remain unclear. For in vitro study, morphology tests of osteoclastogenesis were firstly performed. And then, factors in RANK-induced NF-κB and MAPK pathways were examined by RT-PCR and Western blot, and the binding of TNF receptor–associated factor (TRAF)6 to RANK was inspected by coimmunoprecipitation and immunofluorescence staining. For in vivo experiments, C57BL/6 ovariectomized (OVX) mice were used for detection, including H&E staining, TRAP staining, and micro CT. As a result, muscone reduced OVX-induced bone loss in mice and osteoclast differentiation in vitro, by inhibiting TRAF6 binding to RANK, and then suppressed NF-κB and MAPK signaling pathways. The expression of the downstream biomarkers was finally inhibited, including NFATc1, CTR, TRAP, cathepsin K, and MMP-9. The inflammatory factors, TNF-a and IL-6, were also reduced by muscone. Taken together, muscone inhibited the binding of TRAF6 to RANK induced by RANKL, thus blocking NF-kB and MAPK pathways, and down-regulating related gene expression. Finally, muscone inhibited osteoclastogenesis and osteoclast function by blocking RANK-TRAF6 binding, as well as downstream signaling pathways in vitro. Muscone also reduced ovariectomy-induced bone loss in vivo.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2+-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression

BackgroundRosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated.MethodsThe effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2+-oscillation was also investigated.ResultsRMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2+-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2.Conclusions RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Acanthopanax senticosus aqueous extract ameliorates ovariectomy-induced bone loss in middle-aged mice by inhibiting the receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

Acanthopanax senticosus (Ciwujia) has broad-spectrum pharmacological activities, including osteoprotective effects. However, the mechanisms underlying these effects remain unclear. We investigated whether Acanthopanax senticosus aqueous extract (ASAE) ameliorates ovariectomy-induced bone loss in middle-aged mice through inhibition of osteoclastogenesis. In vitro, ASAE significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation and formation of F-actin rings by downregulating the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), c-Fos, and osteoclastogenesis-related marker genes and proteins, including c-Src, tartrate-resistant acid phosphatase (TRAP), cathepsin K, β3-integrin, and matrix metallopeptidase-9 (MMP-9). This was achieved by inhibiting RANK signaling pathways, including p65, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 in osteoclast precursors. In vivo, ASAE markedly ameliorated bone loss in ovariectomized (OVX) middle-aged mice. ASAE significantly inhibited the serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b) and RANKL, whereas it increased those of osteocalcin, procollagen 1 N-terminal peptide (P1NP), and osteoprotegerin in OVX mice. ASAE significantly inhibited the OVX-induced expression of osteoclast-specific proteins and genes in the femur. In conclusion, ASAE prevents ovariectomy-induced bone loss in middle-aged mice by inhibiting RANKL-induced osteoclastogenesis through suppression of RANK signaling pathways and could be potentially used in mediated treatment of osteoclast-related diseases (e.g., osteoporosis).

Xylodon flaviporus-Derived Drimane Sesquiterpenoids That Inhibit Osteoclast Differentiation.

The presence of excessive osteoclasts is a major factor in skeletal diseases. The present study aimed to discover osteoclast differentiation inhibitors from the basidiomycete Xylodon flaviporus. Seven new drimane sesquiterpenoids (1-7) and 7-ketoisodrimenin-5-ene (8) were obtained and characterized by various spectroscopic methods. The isolated compounds were evaluated for their inhibitory effects against receptor activator of nuclear factor-kappa-B ligand-induced osteoclastogenesis in mouse bone marrow macrophages. Compounds 1, 3, and 6 showed potent activities with IC50 values of 1.6, 0.9, and 2.1 μM, respectively, while 4, 5, and 7 exhibited relatively weak activities with IC50 values of 10.7, 10.1, and 8.5 μM, respectively.

Benzylideneacetone Derivatives Inhibit Osteoclastogenesis and Activate Osteoblastogenesis Independently Based on Specific Structure-Activity Relationship.

(E)-3,4-Dihydroxybenzylideneacetone (compound 1) inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis of C57BL/6 bone marrow monocyte/macrophages with IC50 of 7.8 μM (IC50 of alendronate, 3.7 μM) while stimulating the differentiation of MC3T3-E1 osteoblastic cells, accompanied by the induction of Runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. (E)-4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one (compound 2c) showed a dramatically increased osteoclast-inhibitory potency with IC50 of 0.11 μM while sustaining osteoblast-stimulatory activity. (E)-4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (compound 2g) stimulated alkaline phosphatase production 2-fold at 50 μM without changing osteoclast-inhibitory activity, compared with compound 1. Oral administration of compounds 1, 2c, and 2g prevented ovariectomy-induced osteoporosis in ddY mice to a degree proportional to their osteoclastogenesis-inhibitory potencies. The administration of 1 (mg/kg)/d compound 2c ameliorated histomorphometry of osteoporotic bone to a degree comparable with 10 (mg/kg)/d alendronate. Conclusively, the in vitro capacity of a few benzylideneacetone derivatives to inhibit osteoclastogenesis supported by independent osteoblastogenesis activation was convincingly reflected in in vivo management of osteoporosis, suggesting a potential novel therapeutics for osteopenic diseases.

Digoxin Attenuates Receptor Activation of NF-κB Ligand-Induced Osteoclastogenesis in Macrophages

Background: Even though hypoxia-inducible factor-1α (HIF-1α) is among the transcriptional factors demonstrated to contribute to the formation of abdominal aortic aneurysms (AAAs), the precise mechanism has been unclear. Digoxin is known as an inhibitor of HIF-1α, and shows a protective effect against the progression of AAAs. Objectives: We tested the effect of digoxin on osteoclastogenesis (OCG) and examined the pathway through which digoxin exerts inhibition of HIF-1α. Materials and Methods: RAW 264.7 macrophage cells were cultured and stimulated by soluble receptor activator of NF-κB ligand (sRANKL) with or without digoxin. First, we tested the effect of digoxin to attenuate macrophage activation, which led to OCG, characterized by tartrate-resistant acid phosphatase (TRAP)-positive macrophages (TPMs). Results: The activation of TPMs stimulated by sRANKL was attenuated by digoxin treatment. Furthermore, the receptor activator of NF-κB (RANK)/receptor activator of NF-κB ligand (RANKL) complex signaling pathway, which is stimulated by HIF-1α, was downregulated by digoxin treatment. Conclusions: These results show that digoxin attenuates OCG. By inhibition of HIF-1α, digoxin decreases OCG through the downregulation of the RANK/RANKL signaling pathway. Therefore, digoxin is a potential candidate for medical treatment of AAAs.

Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Nuclear protein 1 (NUPR1) is a multifunctional stress-induced protein involved in regulating tumorigenesis, apoptosis, and autophagy. Bone homeostasis is maintained by bone-resorbing osteoclasts and bone-forming osteoblasts and osteocytes. We aimed to determine the role of NUPR1 in bone metabolism. Using microcomputed tomography, we found that mice lacking Nupr1 exhibited increased bone volume. Histologic analysis showed that Nupr1 deficiency decreased osteoclast numbers but increased osteoblast numbers and osteoid formation. In vitro culture of bone marrow macrophages showed that receptor activator of NF-κB ligand-induced osteoclastogenesis was down-regulated in Nupr1-deficient mice. In contrast, primary osteoblasts from Nupr1-deficient mice revealed that proliferation of osteoblasts and expression of bone matrix proteins were markedly enhanced. In addition, expression of autophagy-related genes, formation of autophagosomes, and cell survival were up-regulated in Nupr1-deficient osteoblasts. In contract, deletion of Nupr1 reduced the formation of osteocyte cellular projection, which is an indicator of mature osteocytes. Importantly, we found that the expression of sclerostin (Sost), an inhibitor of bone formation, was down-regulated in the osteoblasts and osteocytes of Nupr1-deficient mice. Conversely, Nupr1 overexpression enhanced Sost expression in primary osteoblasts. Collectively, these results indicate that Nupr1 deficiency increases bone volume by attenuating production of Sost and osteoclastogenesis and enhancing differentiation of osteoblasts.-Shiraki, M., Xu, X., Iovanna, J. L., Kukita, T., Hirata, H., Kamohara, A., Kubota, Y., Miyamoto, H., Mawatari, M., Kukita, A. Deficiency of stress-associated gene Nupr1 increases bone volume by attenuating differentiation of osteoclasts and enhancing differentiation of osteoblasts.

Psoralen accelerates bone fracture healing by activating both osteoclasts and osteoblasts.

Bone fracture healing is a complex, dynamic process that involves various cell types, with osteoclasts and osteoblasts playing indispensable roles. In this study, we found that psoralen, the main active ingredient in Psoralea corylifolia L. fruit extract, enhanced bone fracture healing through activation of osteoclast and osteoblast activity via the ERK signaling pathway. In detail, psoralen promoted receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis, mRNA expression of osteoclast-specific genes, and osteoclastic bone resorption in primary bone marrow-derived macrophages. Meanwhile, psoralen induced osteogenic differentiation by promoting the mRNA expression of the osteoblast differentiation markers alkaline phosphatase, runt-related transcription factor 2, osterix, and osteocalcin. At the molecular level, psoralen preferentially activated ERK1/2 but not JNK or p38 MAPKs. Further experiments revealed that psoralen-induced osteoclast and osteoblast differentiation was abrogated by a specific inhibitor of phosphorylated ERK. In addition, psoralen accelerated bone fracture healing in a rat tibial fracture model, and the numbers of osteoclasts and osteoblasts were increased in psoralen-treated fracture callus. Taken together, our findings indicate that psoralen accelerates bone fracture healing through activation of osteoclasts and osteoblasts via ERK signaling and has potential as a novel drug in the orthopedic clinic for the treatment of bone fractures.-Zhang, T., Han, W., Zhao, K., Yang, W., Lu, X., Jia, Y., Qin, A., Qian, Y. Psoralen accelerates bone fracture healing by activating both osteoclasts and osteoblasts.

Taxifolin Inhibits Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis of Human Bone Marrow-Derived Macrophages in vitro and Prevents Lipopolysaccharide-Induced Bone Loss in vivo

It has been reported that taxifolin inhibit osteoclastogenesis in RAW264.7 cells. In our research, the inhibition effects of taxifolin on the osteoclastogenesis of human bone marrow-derived macrophages (BMMs) induced by receptor activator of NF-κB ligand (RANKL) as well as the protection effects in lipopolysaccharide-induced bone lysis mouse model have been demonstrated. In vitro, taxifolin inhibited RANKL-induced osteoclast differentiation of human BMMs without cytotoxicity. Moreover, taxifolin significantly suppressed RANKL-induced gene expression, including tartrate-resistant acid phosphatase, matrix metalloproteinase-9 nuclear factor of activated T cells 1 and cathepsin K, and F-actin ring formation. Further studies showed that taxifolin inhibit osteoclastogenesis via the suppression of the NF-κB signaling pathway. In vivo, taxifolin prevented bone loss in mouse calvarial osteolysis model. In conclusion, the results suggested that taxifolin has a therapeutic potential for osteoclastogenesis-related diseases such as osteoporosis, osteolysis, and rheumatoid arthritis.

Dihydrodaidzein and 6-hydroxydaidzein mediate the fermentation-induced increase of antiosteoporotic effect of soybeans in ovariectomized mice.

The consumption of soybeans is known to have beneficial effects on osteoporosis in postmenopausal women. However, the effects of soybean fermentation on the bioavailability and the antiosteoporotic effect have not yet been elucidated. To address this question, we fed ovariectomized C57BL/6J mice with a 5% nonfermented raw soybean (RS)- or fermented soybean (FS)-supplemented diet. After 18 wk of treatment, microcomputed tomography showed that FSs significantly increased bone mineral density compared with RSs. This was because of the up-regulation of bone morphogenic protein 2 (Bmp2) and its downstream target osteopontin in bone tissues. We analyzed isoflavone metabolite profiles in the sera of RS- or FS-fed mice and observed that the levels of 19 isoflavone metabolites were significantly increased in the sera of FS-fed mice. Among these metabolites, we observed that both dihydrodaidzein (DHD) and 6-hydroxydaidzein (6-HD) increased osteogenesis via Bmp2 signaling pathway in MC3T3-E1 cells and reduced receptor activator of nuclear factor κ-B ligand-induced osteoclastogenesis in RAW264.7 cells through the inhibition of NF-κB activation and MAPK phosphorylation. These data suggest that improved bioavailability of FSs resulted from the production of active metabolites such as DHD and 6-HD after consumption. DHD and 6-HD can be used as potential therapeutics for the amelioration of osteoporotic bone loss.-Kim, J.-S., Lee, H., Nirmala, F. S., Jung, C. H., Kim, M. J., Jang, Y.-J., Ha, T. Y., Ahn, J. Dihydrodaidzein and 6-hydroxydaidzein mediate the fermentation-induced increase of anti-osteoporotic effect of soybeans in ovariectomized mice.