Induced Osteoclast Differentiation Research Articles

Desoxyrhapontigenin inhibits RANKL‑induced osteoclast formation and prevents inflammation‑mediated bone loss.

Desoxyrhapontigenin (DRG), a stilbene compound from Rheum undulatum, has been found to exhibit various pharmacological activities, however, its impact on osteoclast formation has not been investigated. The present study investigated the effect of DRG on receptor activator of nuclear factor‑κB ligand (RANKL)‑induced osteoclast differentiation in mouse bone marrow macrophages (BMMs) and inflammation‑induced bone loss invivo. BMMs or RAW264.7 cells were treated with DRG, followed by an evaluation of cell viability, RANKL‑induced osteoclast differentiation, actin‑ring formation and resorption pits activity. The effects of DRG on the RANKL‑induced phosphorylation of MAPK and the expression of nuclear factor of activated T cells cytoplasmic1 (NFATc1) and c‑Fos were evaluated using western blot analysis once the BMMs were exposed to RANKL and DRG. The expression levels of osteoclast marker genes were also evaluated using western blot analysis and reverse transcription‑quantitative polymerase chain reaction A lipopolysaccharide (LPS)‑induced murine bone loss model was used to evaluate the protective effect of DRG on inflammation‑induced bone‑loss. The results demonstrated that DRG suppressed the RANKL‑induced differentiation of BMMs into osteoclasts, osteoclast actin‑ring formation and bone resorption activity in a dose‑dependent manner. Furthermore, DRG significantly inhibited LPS‑induced bone loss in a mouse model. At the molecular level, DRG inhibited the RANKL‑induced activation of extracellular signal‑regulated kinase, the expression of c‑Fos, and the induction of NFATc1, a crucial transcription factor for osteoclast formation. DRG decreased the expression levels of osteoclast marker genes, including matrix metalloproteinase‑9, tartrate‑resistant acid phosphatase and cathepsin K. In conclusion, these findings suggested that DRG inhibited the differentiation of BMMs into mature osteoclasts by suppressing the RANKL‑induced activator protein‑1 and NFATc1 signaling pathways, and may be a potential candidate for treating and/or preventing osteoclast‑associated diseases, including osteoporosis.

The Anti-Periodontitis Effects of Ethanol Extract Prepared Using Lactobacillus paracasei subsp. paracasei NTU 101.

Poor oral health and related diseases, including caries, periodontal disease, and oral cancer, are highly prevalent across the world, particularly in the elderly. This study aimed to investigate the anti-periodontitis activity of fermented skim milk produced using the promising probiotic Lactobacillus paracasei subsp. paracasei NTU 101 (NTU101FM). An initial analysis found that an ethanol extract of NTU101FM displayed anti-oxidative activities. Further investigation of pathogen growth inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs) revealed that the NTU101FM ethanol extract also had anti-periodontal pathogen activities. In addition, the NTU101FM ethanol extract significantly decreased the release of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) in RAW 264.7 macrophage cells. Finally, the NTU101FM ethanol extract was found to inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by reducing tartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-positive multinucleated osteoclasts. In summary, our study demonstrated that ethanol extract prepared from NTU101FM has potential use as an anti-periodontitis agent.

Retraction of: A005, a novel inhibitor of phosphatidylinositol 3-kinase/mammalian target of rapamycin, prevents osteosarcoma-induced osteolysis.

Osteosarcoma is the most frequent primary bone tumor in children and adolescents. The phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is an attractive anticancer target because it plays key roles in the regulation of cell growth, division and differentiation. In this study, we demonstrated high expression of PI3K/mTOR signaling pathway-related genes in patients with osteosarcoma. We thus investigated the effects of A005, a newly synthesized dual PI3K/mTOR inhibitor, on osteosarcoma cells and in a mouse xenograft tumor model. The results confirmed that A005 inhibited the proliferation, migration and invasion of human osteosarcoma cells. In addition, A005 also inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation and bone resorption in vitro. Therefore, A005 was further applied to a SaOS-2 osteosarcoma-induced mouse osteolysis model. A005 inhibited tumor growth and prevented osteosarcoma-associated osteolysis via modulation of the PI3K/AKT/mTOR pathway. Overall, our results showed that A005 inhibited osteoclastogenesis and prevented osteosarcoma-induced bone osteolysis by suppressing PI3K/AKT/mTOR signaling. These findings indicated that A005 may be a promising candidate drug for the treatment of human osteosarcoma.

PoligoniMultiflori Radix enhances osteoblast formation and reduces osteoclast differentiation.

Poligoni Multiflori Radix (PMR) is a traditional Korean medicinal herb that is known to have various pharmacological effects, including antihyperlipidemic, anticancer, and anti-inflammatory effects. However, the effects of PMR on bone metabolism have not been elucidated to date. The present study aimed to investigate the in vitro and in vivo effect of PMR water extract on the regulation of osteoblast and osteoclast activity. Effects of PMR water extract on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation and survival of mouse bone marrow macrophages (BMMs) obtained from femurs were investigated by tartrate-acid resistant acid phosphatase (TRAP)-positive cells and XTT assay. Expression of osteoclast-related genes was assayed by western blot analysis and reverse transcription-quantitative polymerase chain reaction. Additionally, the effects of PMR water extract on osteoblastic proliferation and differentiation were investigated by alkaline phosphatase (ALP) activity assay, alizarin red staining, and levels of mRNA encoding known osteoblast markers. Furthermore, the effects of PMR water extract on lipopolysaccharide (LPS)-induced bone loss were examined in a mouse model. PMR inhibited RANKL-induced osteoclast differentiation of BMMs in a dose-dependent manner without significant cytotoxicity, and suppressed expression of the main osteoclast differentiation markers Fos proto-oncogene and nuclear factor of activated T-cell. In addition, PMR decreased the mRNA expression levels of NFATc1 target genes, including TRAP, osteoclast-associated receptor, ATPase H+ transporting, lysosomal 38 kDa V0 subunit d2, and Cathepsin K. These inhibitory effects were mediated by the p38 and extracellular signal-regulated kinase/nuclear factor-κB pathway. Simultaneously, PMR enhanced the differentiation of primary osteoblasts, and increased the mRNA expression of runt-related transcription factor 2, ALP, osterix, and osteocalcin. Notably, PMR improved LPS-induced trabecular bone loss in mice. Collectively, the present findings demonstrated that PMR may regulate bone remodeling by reducing osteoclast differentiation and stimulating osteoblast formation. These results suggest that PMR may be used for the treatment of bone diseases, such as osteoporosis and rheumatoid arthritis.

Tacrolimus regulates endoplasmic reticulum stress-mediated osteoclastogenesis and inflammation: In vitro and collagen-induced arthritis mouse model.

Tacrolimus is an immunosuppressive drug that inhibits the release of inflammatory cytokines involved in rheumatoid arthritis development by blocking T cell activation. "Endoplasmic reticulum stress," an imbalance between protein folding load and capacity leading to the accumulation of unfolded proteins in the endoplasmic reticulum lumen, has been implicated in rheumatoid arthritis and other inflammatory and metabolic diseases. We aimed to investigate the effect of tacrolimus on endoplasmic reticulum stress-mediated osteoclastogenesis and inflammation and elucidate the underlying mechanisms. In vitro studies were performed using mouse bone marrow cells that were cultured with or without interleukin-1β, thapsigargin, or tacrolimus to induce osteoclast differentiation. A mouse model of arthritis was established by immunizing mice with bovine type II collagen. Tacrolimus was orally administered to mice from day 20 to 45 following the initial immunization, and histopathological changes and expression of specific biomarkers of endoplasmic reticulum stress-mediated inflammatory signaling pathways were examined. In vitro, tacrolimus inhibited receptor activator of nuclear factor-κB ligand-mediated osteoclast formation augmented by interleukin-1β, thapsigargin, or both. Furthermore, tacrolimus inhibited glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 (IRE 1), and activating transcription factor 6 (ATF6) augmented by interleukin-1β, thapsigargin, or both. Tacrolimus significantly ameliorated osteolysis and endoplasmic reticulum stress intensity in mice. Simultaneously, it reduced inflammatory cell infiltration, osteoclastogenesis, and inflammatory responses by inhibiting GRP78, IRE 1, and ATF6. These findings suggest that tacrolimus exhibits an anti-inflammation effect in rheumatoid arthritis and might inhibit joint damage progression by inhibiting endoplasmic reticulum stress.

Suppression of RANKL-Induced Osteoclastogenesis by the Metabolites from the Marine Fungus Aspergillus flocculosus Isolated from a Sponge Stylissa sp.

A new α-pyrone merosesquiterpenoid possessing an angular tetracyclic carbon skeleton, ochraceopone F (1), and four known secondary metabolites, aspertetranone D (2), cycloechinulin (3), wasabidienone E (4), and mactanamide (5), were isolated from the marine fungus Aspergillus flocculosus derived from a sponge Stylissa sp. collected in Vietnam. The structures of Compounds 1–5 were elucidated by analysis of 1D and 2D NMR spectra and MS data. All the isolated compounds were evaluated for anti-proliferation activity and their suppression effects on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation using tartate-resisant acid phosphatase (TRAP). Compounds 1–5 had no anti-proliferative effect on human cancer cell lines up to 30 μg/mL. Among these compounds, aspertetranone D (2) and wasabidienone E (4) exhibited weak osteoclast differentiation inhibitory activity at 10 μg/mL. However, mactanamide (5) showed a potent suppression effect of osteoclast differentiation without any evidence of cytotoxicity.

Resolvin E1 Inhibits Osteoclastogenesis and Bone Resorption by Suppressing IL-17-induced RANKL Expression in Osteoblasts and RANKL-induced Osteoclast Differentiation

Resolvin E1 (RvE1) derived from the ω-3 polyunsaturated fatty acid eicosapentaenoic acid is known to be a potent pro-resolving lipid mediator that prevents chronic inflammation and osteoclastogenesis. We investigated the inhibitory effects of RvE1 on osteoclastogenesis and bone resorption to clarify its therapeutic potential for rheumatoid arthritis (RA). Receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was assessed with tartrate-resistant acid phosphatase staining. RANKL-induced bone resorption was assessed by the measurement of pit formation using calcium phosphate-labeled fluorescent polyanionic molecules in RAW264.7 cells as osteoclast precursors. The effects of RvE1 on the RANKL-induced mRNA expression of osteoclast-specific genes and transcriptional factors such as c-fos and nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells were measured by quantitative real-time PCR. The distribution of NFATc1 induced by RANKL was evaluated by immunofluorescence staining in RAW264.7 cells. To analyze the mechanism of the inhibitory effect of RvE1 on osteoclastogenesis, we measured IL-17-induced RANKL mRNA expression in MC3T3-E1 osteoblast cells treated with RvE1 using quantitative real-time PCR and determined the level of prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. RvE1 significantly suppressed RANKL-induced osteoclast differentiation and bone resorption. RvE1 inhibited the RANKL-induced mRNA expression of osteoclast-specific genes along with the transcription factors NFATc1 and c-fos. Moreover, NFATc1 translocation from the cytoplasm to the nucleus of RAW264.7 cells was suppressed following RvE1 treatment. RvE1 also inhibited IL-17-induced RANKL mRNA expression and PGE2 production in MC3T3-E1 cells. RvE1 inhibited osteoclastogenesis and bone resorption by suppressing RANKL-induced NFATc1 and c-fos expression in osteoclasts and IL-17-induced RANKL expression through the autocrine action of PGE2 in osteoblasts. Our data suggest RvE1 as a new therapeutic target of RA.

Does TNF Promote or Restrain Osteoclastogenesis and Inflammatory Bone Resorption?

Chronic inflammation is one of the most evident and common pathological conditions leading to deregulated osteoclastogenesis and bone remodeling. Tumor necrosis factor (TNF) as a pleiotropic cytokine plays a key role, not only in inflammation, but also in bone erosion in diseases associated with bone loss. TNF can stimulate the proliferation of osteoclast precursors and, in most conditions, act together with other cytokines and growth factors such as receptor activator of nuclear factor (NF)-[kappa]B ligand (RANKL), interleukin-6, and transforming growth factor beta to synergistically promote osteoclast formation and bone resorption in vivo. A longstanding enigma in the field is why TNF alone is not able to induce osteoclast differentiation as effectively as the same superfamily member RANKL, a physiological master osteoclastogenic cytokine. Recent studies have highlighted several lines of evidence showing the intrinsic mechanisms through RBP-J, NF-[kappa]B p100/TNF receptor-associated factor 3, or interferon regulatory factor-8 that restrain TNF-induced osteoclast differentiation and bone resorption. These feedback inhibitory mechanisms driven by TNF shed light into the current paradigm of osteoclastogenesis and would provide novel therapeutic implications on controlling inflammatory bone resorption.

Diphlorethohydroxycarmalol from Ishige okamurae Suppresses Osteoclast Differentiation by Downregulating the NF-κB Signaling Pathway.

Marine algae possess a variety of beneficial effects on human health. In this study, we investigated whether diphlorethohydroxycarmalol (DPHC), isolated from Ishige okamurae, a brown alga, suppresses receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. DPHC significantly suppressed RANKL-induced osteoclast differentiation and macrophage-colony stimulating factor (M-CSF) expression in a dose-dependent manner. In addition, it significantly inhibited actin ring formation, the expression of osteoclast marker genes, such as tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1), cathepsin K (Ctsk), and dendritic cell-specific transmembrane protein (Dcstamp), and osteoclast-induced bone resorption. Analysis of the RANKL-mediated signaling pathway showed that the phosphorylation of both IκB and p65 was specifically inhibited by DPHC. These results suggest that DPHC substantially suppresses osteoclastogenesis by downregulating the RANK-NF-κB signaling pathway. Thus, it holds significant potential for the treatment of skeletal diseases associated with an enhanced osteoclast activity.

Water extract of Uncaria sinensis suppresses RANKL-induced bone loss by attenuating osteoclast differentiation and bone resorption

BackgroundThe hooks and stems of Uncaria sinensis have been used to mitigate cardiovascular and central nervous system disorders in Asia traditional medicine. Regulation of osteoclast differentiation and activity is a major target for preventing and treating pathological bone diseases. MethodsTartrate-resistant acid phosphatase (TRAP) activity and the number of TRAP-stained multinucleated cells were used to examine receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. The activation of RANKL-induced signaling pathways and the expression of transcription factors were investigated by western blot analysis and quantitative real-time polymerase chain reaction. The bone resorption activity of osteoclast was studied using a plate coated with hydroxyl-apatite. Trabecular bone destruction was investigated using a RANKL-induced trabecular bone loss mouse model. ResultsWe found that water extract of the hooks and stems of U. sinensis (WEUS) inhibits RANKL-induced differentiation of murine bone marrow macrophages and RAW264.7 cells into osteoclasts. WEUS inhibited the activation of NF-κB and the expression of nuclear factor of activated T-cells, cytoplasmic 1. In addition, WEUS suppressed the bone resorbing activity of mature osteoclasts without affecting their survival. Furthermore, oral administration of WEUS suppressed RANKL-induced bone loss with a significant amelioration of trabecular bone micro-structures. WEUS also reduced RANKL-induced increase in serum TRAP5b activity and C-terminal cross-linked telopeptide of type I collagen levels. ConclusionThe present study demonstrates that WEUS has a pharmacological activity that inhibits osteoclast-mediated bone destruction by suppressing osteoclast differentiation and function. These results suggest that U. sinensis could be a promising herbal candidate for preventing and treating bone diseases such as osteoporosis.

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

Bone regenerative effect of aqueous Cynanchum wilfordii extract in receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation and estrogen deficiency-induced osteoporosis

<p>Osteoporosis increases with age, most frequently in postmenopausal women because of reduced ovarian hormone levels. Furthermore, estrogen deficiency impairs trabecular metaphyseal bone. Although efficacious, long-term hormone replacement therapy (HRT) has estrogen-like side effects including breast and endometrial cancers, and non-hormonal or herbal therapies may be safer alternatives. Therefore, the aim of this study was to investigate the effects of aqueous extracts of <em>Cynanchum wilfordii</em> (CWW) on receptor activator of nuclear factor-κ B (NF-κ B) ligand (RANKL)-induced osteoclast differentiation <em>in vitro</em> and ovariectomy-mediated osteoporosis <em>in vivo</em>. CWW inhibited RANKL-induced osteoclast formation and tartrate-resistant acid phosphatase (TRAP) activity in primary mouse bone marrow-derived cells. We investigated the osteoprotective effect of CWW in an ovariectomized (OVX) Sprague-Dawley rat model treated with vehicle (OVX/vehicle), 17<span style="font-family: 맑은 고딕;">β</span>-estradiol (OVX/E2), or three CWW doses (100, 200, and 400 mg/kg). After a 24-week treatment, the body and uterus weights were not affected except in the OVX/E2 group. Additionally, bone mineral density (BMD) and histological analyses showed that the BMD of the femurs of CWW400-treated rats was significantly higher than that of the OVX/vehicle rats, and comparable to that of the OVX/E2 group rats. Serum levels of bone turnover markers alkaline phosphatase (ALP), osteocalcin, collagen type I C-telopeptide, and TRAP significantly decreased in the CWW400 group. Our results show that compared to the vehicle, CWW had a significant anti-osteoporotic effect in the OVX model. Taken together, CWW exhibited inhibitory effects on osteoclastogenesis <em>in vitro,</em> and we confirmed its in vivo efficacy in the prevention of osteoporosis.</p>

Adrenomedullin inhibits osteoclast differentiation through the suppression of receptor activator of nuclear factor-κB ligand-induced nuclear factor-κB activation in glucocorticoid-induced osteoporosis.

The current study aimed to improve the understanding on the association between adrenomedullin and osteoporosis in mice with glucocorticoid-induced osteoporosis. Bone resorption and osteoporosis-associated indexes, including maximum load, stiffness, energy to failure, ultimate strength, elastic modulus, post-yield displacement and post-yield displacement, in mice with osteoporosis were analyzed in order to evaluate the effect of adrenomedullin. The receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was investigated subsequent to treatment with adrenomedullin in vitro. The results demonstrated that adrenomedullin significantly improved bone mass loss, density, bone strength and osteoporosis disease in the mice with glucocorticoid-induced osteoporosis. In addition, adrenomedullin markedly improved the osteoporosis-associated NFATc1, TRAP, OSCAR and c-Fos expression levels. Furthermore, the current findings indicated that RANKL-mediated osteoclast differentiation was suppressed in vitro and in vivo. Notably, the data revealed that adrenomedullin significantly improved the osteoporotic symptoms through inhibition of RANKL-induced NF-κB activation in glucocorticoid-induced osteoporosis. In conclusion, adrenomedullin serves an essential role in the progression of glucocorticoid-induced osteoporosis, regulating the bone mass loss, density and strength through the NF-κB signaling pathway.

Inflammation induces osteoclast differentiation from peripheral mononuclear cells in chronic kidney disease patients: crosstalk between the immune and bone systems.

Inflammation and immune system alterations contribute to bone damage in many pathologies by inducing the differentiation of osteoclasts (OCs), the bone resorbing cells. This link is largely unexplored in chronic kidney disease (CKD) and haemodialysis (HD) patients, in which reduced renal function is accompanied by an increased inflammatory state and skeletal abnormality. We used ex vivo culture experiments to investigate the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) of CKD and HD patients, focusing on immune cell subsets and inflammatory cytokines such as LIGHT and receptor activator of nuclear factor κB ligand (RANKL). We observed spontaneous osteoclastogenesis with a significant increase in OC formation and bone resorbing activity in late-stage CKD and HD patients when compared with early-stage CKD patients and healthy donors, likely due to an increased expression of RANKL and LIGHT (homologous to Lymphotoxins exhibiting Inducible expression and competing with herpes simplex virus Glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) in PBMCs. Specific inhibition of these cytokines in PBMCs isolated from CKD stages 3b-5 and HD patients induced the reduction of OC formation in vitro. The phenotypic characterization of peripheral blood cells revealed a significant increase of OC precursors (CD14+CD11b+CD51/61+) and CD14+CD16+ monocytes in advanced CKD and HD patients compared with the control group. Our results suggest that circulating inflammatory monocytes from advanced CKD or HD patients trans differentiate into OCs in vitro and play a relevant role in mineral bone disorders and that LIGHT and RANKL represent new potential therapeutic targets in these settings.

G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways.

GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases.

The Alpha 7 Nicotinic Acetylcholine Receptor of Deciduous Dental Pulp Stem Cells Regulates Osteoclastogenesis During Physiological Root Resorption.

The physiological root resorption of deciduous teeth is a normal phenomenon, but the mechanisms underlying this process are still unclear. In this study, deciduous dental pulp stem cells (DDPSCs) and permanent dental pulp stem cells (DPSCs) were derived from deciduous teeth and normal permanent teeth at different stages of resorption. In the middle stage of root resorption, DDPSCs exhibited an increase in the ability to induce osteoclast differentiation. Activation of the alpha 7 nicotinic acetylcholine receptor (α7 nAChR) by secretory mammalian Ly-6 urokinase-type plasminogen activator receptor-associated protein 1 (SLURP-1) caused a significant increase in the expression levels of NF-κB, receptor activator of nuclear factor-kappa B ligand (RANKL), and the ratio of RANKL/osteoprotegerin (OPG). These effects were inhibited by alpha-bungarotoxin (α-BTX). Furthermore, the expression levels of RANKL/OPG were significantly reduced following inhibition of NF-κB. High-strength, dynamic positive pressure increased the expression of SLURP-1 and α7 nAChR in DDPSCs in the stable stage. These data indicated that mechanical stress stimulated the expression of SLURP-1 and α7 nAChR in DDPSCs. Additionally, SLURP-1 activated α7 nAChR, thereby upregulating the expression of NF-κB and enhancing its activity, thus regulating RANKL/OPG expression and affecting the ability of DDPSCs to influence osteoclastogenesis, which likely enhances root resorption and leads to the physiological loss of deciduous teeth.

Amphiregulin contained in NSCLC-exosomes induces osteoclast differentiation through the activation of EGFR pathway

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths worldwide. The majority of patients are diagnosed in advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts and bone-forming osteoblasts activity is lost in bone metastasis, inducing osteoclastogenesis. In NSCLC, the epidermal growth factor receptor (EGFR) pathway is constitutively activated. EGFR binds Amphiregulin (AREG) that is overexpressed in several cancers such as colon, breast and lung. Its levels in plasma of NSCLC patients correlate with poor prognosis and AREG was recently found as a signaling molecule in exosomes derived from cancer cell lines. Exosomes have a key role in the cell-cell communication and they were recently indicated as important actors in metastatic niche preparation. In the present work, we hypothesize a role of AREG carried by exosomes derived from NSCLC in bone metastasis induction. We observed that NSCLC-exosomes, containing AREG, induce EGFR pathway activation in pre-osteoclasts that in turn causes an increased expression of RANKL. RANKL is able to induce the expression of proteolytic enzymes, well-known markers of osteoclastogenesis, triggering a vicious cycle in osteolytic bone metastasis.

長時間作用型NO供与体(NOC-18)による破骨細胞分化誘導(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

長時間作用型NO供与体(NOC-18)による破骨細胞分化誘導(大阪歯科大学大学院歯学研究科博士(歯学)学位論文内容要旨および論文審査結果要旨の公表)

Inhibitory Effects of Water Extracts of Eucommiae Cortex and Psoraleae Semen Alone and in Combination on Osteoclast Differentiation and Bone※

Objectives : The purpose of this study was to evaluate the effects of water extracts of Eucommiae cortex (EC), Psoraleae semen (PS), and their combination on receptor activator of nuclear factor- kappa-B ligand (RANKL)-induced osteoclast differentiation. Methods : We assayed the protein expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, mitogen-activated protein kinases (MAPKs), and β-actin in cell lysates using western blotting. Similarly, mRNA expression levels of NFATc1, c-Fos, tartrateresistant acid phosphate (TRAP), and glyceraldehyde-3-phosphate dehydrogenase, spermatogeni (GAPDHS) from bone marrow macrophages (BMMs) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, we determined the anti-osteoporotic effects of the water extracts of EC, PS, and their combination in a lipopolysaccharide (LPS)-induced bone-loss mouse model. Results : The in vitro data revealed showed that the combination of EC and PS extract showed a more remarkable inhibition of osteoclast differentiation than each herb did alone. The combination downregulated the induction of c-Fos, NFATc1, and TRAP by suppressing the phosphorylation of p38 and c-Jun N-terminal kinases (JNKs) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Lastly, the in vivo data showed that PS reduced the LPS-induced bone erosion. Conclusion : The result of this study suggests that EC and PS could be potential therapeutic agents for bone loss diseases such as osteoporosis.

Staphylococcusaureus Protein A induces osteoclastogenesis via the NF‑κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor-activated (NF)-κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose-dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast-specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA-induced osteoclast differentiation was associated with the degradation of inhibitor of κB-α, phosphorylation of NF-κB p65 and increased expression of nuclear factor of activated T-cells. However, by treatment with JSH-23, an NF-κB inhibitor, the formation of osteoclast-like cells and resorption pits was significantly reduced, and the expression of osteoclast-specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF-κB signaling pathway was involved in this process.