Britanin inhibits titanium wear particle­induced osteolysis and osteoclastogenesis.

Wear particle­induced osteolysis is a serious complication that occurs in individuals with titanium (Ti)­based implants following long­term usage due to loosening of the implants. The control of excessive osteoclast differentiation and inflammation is essential for protecting against wear particle­induced osteolysis. The present study evaluated the effect of britanin, a pseudoguaianolide sesquiterpene isolated from Inula japonica, on osteoclastogenesis in vitro and Ti particle­induced osteolysis in vivo. The effect of britanin was examined in the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) using TRAP staining, RT­PCR, western blotting and immunocytochemistry. The protective effect of britanin was examined in a mouse calvarial osteolysis model and evaluated using micro­CT and histomorphometry. Britanin inhibited osteoclast differentiation and F­actin ring formation in the presence of macrophage colony­stimulating factor and receptor activator of nuclear factor kB ligand in BMMs. The expression of osteoclast­specific marker genes, including tartrate­resistant acid phosphatase, cathepsin K, dendritic cell­specific transmembrane protein, matrix metallopeptidase 9 and nuclear factor of activated T­cells cytoplasmic 1, in the BMMs was significantly reduced by britanin. In addition, britanin reduced the expression of B lymphocyte­induced maturation protein­1, which is a transcriptional repressor of negative osteoclastogenesis regulators, including interferon regulatory factor­8 and B­cell lymphoma 6. Conversely, britanin increased the expression levels of anti­oxidative stress genes, namely nuclear factor erythroid­2­related factor 2, NAD(P)H quinone oxidoreductase 1 and heme oxygenase 1 in the BMMs. Furthermore, the administration of britanin significantly reduced osteolysis in a Ti particle­induced calvarial osteolysis mouse model. Based on these findings, it is suggested that britanin may be a potential therapeutic agent for wear particle­induced osteolysis and osteoclast­associated disease.

Suppressive effects of (-)-tubaic acid on RANKL-induced osteoclast differentiation and bone resorption.

Regulation of osteoclastogenesis and bone-resorbing activity can be an efficacious strategy for treating bone loss diseases because excessive osteoclastic bone resorption leads to the development of such diseases. Here, we investigated the role of (-)-tubaic acid, a thermal degradation product of rotenone, in osteoclast formation and function in an attempt to identify alternative natural compounds. (-)-Tubaic acid significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclast differentiation at both the early and late stages, suggesting that (-)-tubaic acid affects the commitment and differentiation of osteoclast progenitors as well as the cell-cell fusion of mononuclear osteoclasts. (-)-Tubaic acid attenuated the activation of extracellular signal-regulated kinase (ERK) and expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and its target genes in response to RANKL. Furthermore, a pit-formation assay revealed that (-)-tubaic acid significantly impaired the bone-resorbing activity of osteoclasts. Our results demonstrated that (-)-tubaic acid exhibits anti-osteoclastogenic and anti-resorptive effects, indicating its therapeutic potential in the management of osteoclast-related bone diseases.

Recombinant Unique Cartilage Matrix-associated Protein Potentiates Osteogenic Differentiation and Mineralization of MC3T3-E1 Cells.

OBJECTIVE: The relative balance of osteoblasts in bone formation and osteoclasts in bone resorption is crucial for maintaining bone health. With age, this balance between osteoblasts and osteoclasts is broken, resulting in bone loss. Anabolic drugs are continuously being developed to counteract this low bone mass. Recombinant proteins are used as biotherapeutics due to being relatively easy to produce on a large scale and are cost-effective through various expression systems. This study aimed to develop a recombinant protein that would positively impact osteoblast differentiation and mineralized nodule formation using unique cartilage matrix-associated protein (UCMA). METHODS: A recombinant glutathione-S-transferase (GST)-UCMA fusion protein was generated in an E.coli system, and purified by affinity chromatography. MC3T3-E1 osteoblast cells and Osterix (Osx)-knockdown stable cells were cultured for 14 days to investigate osteoblast differentiation and nodule formation in the presence of the recombinant GST-UCMA protein. The differentiated cells were assessed by alizarin red S staining and quantitative PCR of the osteoblast differentiation marker osteocalcin. In addition, cell viability in the presence of the recombinant GST-UCMA protein was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell adhesion assay. RESULTS: The isolation of both purified recombinant GST-only and GST-UCMA proteins were confirmed at 26 kDa and 34 kDa, respectively, by Coomassie staining and western blot analysis. Neither dose-dependent nor time-dependent presence of recombinant GST-UCMA affected MC3T3-E1 cell viability. However, MC3T3-E1 cell adhesion to the recombinant GST-UCMA protein increased dose-dependently. Osteoblast differentiation and nodule formation were promoted in both MC3T3-E1 osteoblast cells and Osxknockdown stable cells when cultured in the presence of recombinant GST-UCMA protein. CONCLUSION: A recombinant GST-UCMA protein induces osteogenic differentiation and mineralization, suggesting its potential use as an anabolic drug to increase low bone mass in osteoporotic patients.

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function.

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.

S100 Calcium-Binding Protein P Secreted from Megakaryocytes Promotes Osteoclast Maturation.

Megakaryocytes (MKs) differentiate from hematopoietic stem cells and produce platelets at the final stage of differentiation. MKs directly interact with bone cells during bone remodeling. However, whether MKs are involved in regulating bone metabolism through indirect regulatory effects on bone cells is unclear. Here, we observed increased osteoclast differentiation of bone marrow-derived macrophages (BMMs) cultured in MK-cultured conditioned medium (MK CM), suggesting that this medium contains factors secreted from MKs that affect osteoclastogenesis. To identify the MK-secreted factor, DNA microarray analysis of the human leukemia cell line K562 and MKs was performed, and S100 calcium-binding protein P (S100P) was selected as a candidate gene affecting osteoclast differentiation. S100P was more highly expressed in MKs than in K562 cells, and showed higher levels in MK CM than in K562-cultured conditioned medium. In BMMs cultured in the presence of recombinant human S100P protein, osteoclast differentiation was promoted and marker gene expression was increased. The resorption area was significantly larger in S100P protein-treated osteoclasts, demonstrating enhanced resorption activity. Overall, S100P secreted from MKs promotes osteoclast differentiation and resorption activity, suggesting that MKs indirectly regulate osteoclast differentiation and activity through the paracrine action of S100P.

PF-3845, a Fatty Acid Amide Hydrolase Inhibitor, Directly Suppresses Osteoclastogenesis through ERK and NF-κB Pathways In Vitro and Alveolar Bone Loss In Vivo.

Alveolar bone loss, the major feature of periodontitis, results from the activation of osteoclasts, which can consequently cause teeth to become loose and fall out; the development of drugs capable of suppressing excessive osteoclast differentiation and function is beneficial for periodontal disease patients. Given the difficulties associated with drug discovery, drug repurposing is an efficient approach for identifying alternative uses of commercially available compounds. Here, we examined the effects of PF-3845, a selective fatty acid amide hydrolase (FAAH) inhibitor, on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis, its function, and the therapeutic potential for the treatment of alveolar bone destruction in experimental periodontitis. PF-3845 significantly suppressed osteoclast differentiation and decreased the induction of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific markers. Actin ring formation and osteoclastic bone resorption were also reduced by PF-3845, and the anti-osteoclastogenic and anti-resorptive activities were mediated by the suppression of phosphorylation of rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase, (ERK) and nuclear factor κB (NF-κB) inhibitor (IκBα). Furthermore, the administration of PF-3845 decreased the number of osteoclasts and the amount of alveolar bone destruction caused by ligature placement in experimental periodontitis in vivo. The present study provides evidence that PF-3845 is able to suppress osteoclastogenesis and prevent alveolar bone loss, and may give new insights into its role as a treatment for osteoclast-related diseases.

Bobby sox homolog regulates tooth root formation through modulation of dentin sialophosphoprotein.

Tooth root development occurs through the interaction of multiple growth factors and transcription factors expressed in Hertwig's epithelial root sheath (HERS) and dental mesenchyme. Previously, we demonstrated that bobby sox homolog (Bbx) regulates odontoblast differentiation of human dental pulp stem cells. Here, we generated Bbx knockout (Bbx-/- ) mice to address the functional role of Bbx in tooth formation. During tooth development, Bbx was expressed in both dental epithelium and mesenchyme. However, molar and incisor morphology in Bbx-/- mice at postnatal Day 0 (P0) exhibited no prominent abnormalities compared with their wild-type (Bbx+/+ ) littermates. Until P28, the crown morphology in Bbx-/- mice was not distinctively different from Bbx+/+ littermates. Meanwhile, the length of the mandibular base in Bbx-/- mice was notably less at P28. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first molar were reduced by 21.33% and 16.28% at P14 and 16.28% and 16.24% at P28, respectively, in Bbx-/- mice. The second molar of Bbx-/- mice also showed 10.16% and 6.4% reductions at P28 in the mesial and distal lengths, compared with Bbx+/+ mice, respectively. The gene expression analysis during early tooth root formation (P13) showed that the expression of dentin sialophosphoprotein (Dspp) was significantly decreased in Bbx-/- mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression.

Secretoneurin, a Neuropeptide, Enhances Bone Regeneration in a Mouse Calvarial Bone Defect Model.

BACKGROUND: This study investigates the effects of a neuropeptide, secretoneurin (SN), on bone regeneration in an experimental mouse model. METHODS: The effects of SN on cell proliferation, osteoblast marker genes expression, and mineralization were evaluated using the CCK-8 assay, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and alizarin red S staining, respectively. To examine the effects of SN on bone regeneration in vivo, bone defects were created in the calvaria of ICR mice, and 0.5 or 1 µg/ml SN was applied. New bone formation was analyzed by micro-computed tomography (micro-CT) and histology. New blood vessel formation was assessed by CD34 immunohistochemistry. RESULTS: SN had no significant effect on proliferation and mineralization of MC3T3-E1 cells. However, SN partially induced the gene expression of osteoblast differentiation markers such as runt-related transcription factor 2, alkaline phosphatase, collagen type I alpha 1, and osteopontin. A significant increase of bone regeneration was observed in SN treated calvarial defects. The bone volume (BV), BV/tissue volume, trabecular thickness and trabecular number values were significantly increased in the collagen sponge plus 0.5 or 1 µg/ml SN group (p < 0.01) compared with the control group. Histologic analysis also revealed increased new bone formation in the SN-treated groups. Immunohistochemical staining of CD34 showed that the SN-treated groups contained more blood vessels compared with control in the calvarial defect area. CONCLUSION: SN increases new bone and blood vessel formation in a calvarial defect site. This study suggests that SN may enhance new bone formation through its potent angiogenic activity.

Inhibitory effect of oolonghomobisflavan B on osteoclastogenesis by suppressing p38 MAPK activation.

Suppression of differentiation and/or function of osteoclasts is considered an effective therapeutic strategy for osteolytic bone diseases such as periodontitis and osteoporosis. Evidence regarding the health benefits of oolong tea consumption is accumulating, and tea polyphenols have various pharmacological properties such as anti-cancer and anti-diabetes effects. In this study, we investigated the effect of oolonghomobisflavan B (OFB), a polyphenolic compound in oolong tea, on osteoclast differentiation. OFB suppressed receptor activator of nuclear factor-κB (RANKL)-induced formation of tartate-resistant acid phosphatase-positive multinuclear cells without cytotoxicity. OFB also significantly attenuated p38 phosphorylation, which is essential for RANKL-induced osteoclastogenesis, and inhibited the expressions of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and osteoclast-specific target genes, including dendritic cell-specific transmembrane protein and cathepsin K. Our findings suggest that OFB exhibits an anti-osteoclastogenic activity by inhibiting RANKL-mediated p38 activation, which is useful for the prevention and treatment of osteolytic bone diseases.

Pentamidine Inhibits Titanium Particle-Induced Osteolysis In Vivo and Receptor Activator of Nuclear Factor-κB Ligand-Mediated Osteoclast Differentiation In Vitro.

Background: Wear debris-induced osteolysis leads to periprosthetic loosening and subsequent prosthetic failure. Since excessive osteoclast formation is closely implicated in periprosthetic osteolysis, identification of agents to suppress osteoclast formation and/or function is crucial for the treatment and prevention of wear particle-induced bone destruction. In this study, we examined the potential effect of pentamidine treatment on titanium (Ti) particle-induced osteolysis, and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Methods: The effect of pentamidine treatment on bone destruction was examined in Ti particle-induced osteolysis mouse model. Ti particles were implanted onto mouse calvaria, and vehicle or pentamidine was administered for 10 days. Then, calvarial bone tissue was analyzed using micro-computed tomography and histology. We performed in vitro osteoclastogenesis assay using bone marrow-derived macrophages (BMMs) to determine the effect of pentamidine on osteoclast formation. BMMs were treated with 20 ng/mL RANKL and 10 ng/mL macrophage colony-stimulating factor in the presence or absence of pentamidine. Osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining, real-time polymerase chain reaction, and immunofluorescence staining. Results: Pentamidine administration decreased Ti particle-induced osteoclast formation significantly and prevented bone destruction compared to the Ti particle group in vivo. Pentamidine also suppressed RANKL-induced osteoclast differentiation and actin ring formation markedly, and inhibited the expression of nuclear factor of activated T cell c1 and osteoclast-specific genes in vitro. Additionally, pentamidine also attenuated RANKL-mediated phosphorylation of IκBα in BMMs. Conclusion: These results indicate that pentamidine is effective in inhibiting osteoclast formation and significantly attenuates wear debris-induced bone loss in mice.

Fermented Oyster Extract Prevents Ovariectomy-Induced Bone Loss and Suppresses Osteoclastogenesis.

There is growing interest in bioactive substances from marine organisms for their potential use against diverse human diseases. Osteoporosis is a skeletal disorder associated with bone loss primarily occurring through enhanced osteoclast differentiation and resorption. Recently, we reported the anti-osteoclastogenic activity of fermented Pacific oyster (Crassostrea gigas) extract (FO) in vitro. The present study focused on investigating the anti-osteoporotic efficacy of FO in bone loss prevention in an experimental animal model of osteoporosis and elucidating the mechanism underlying its effects. Oral administration of FO significantly decreased ovariectomy-induced osteoclast formation and prevented bone loss, with reduced serum levels of bone turnover biomarkers including osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus (CTX). FO significantly suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts and attenuated the induction of osteoclast-specific genes required for osteoclastogenesis and bone resorption. Furthermore, FO inhibited RANKL-mediated IκBα and p65 phosphorylation in BMMs. Taken together, these results demonstrate that FO effectively suppresses osteoclastogenesis in vivo and in vitro, and that FO can be considered as a potential therapeutic option for the treatment of osteoporosis and osteoclast-mediated skeletal diseases.

2-O-digalloyl-1,3,4,6-tetra-O-galloyl-ß-D-glucose isolated from Galla Rhois suppresses osteoclast differentiation and function by inhibiting NF-κB signaling.

Natural compounds isolated from medicinal herbs and plants have immense significance in maintaining bone health. Hydrolysable tannins have been shown to possess a variety of medicinal properties including antiviral, anticancer, and anti-osteoclastogenic activities. As a part of a study on the discovery of alternative agent against skeletal diseases, we isolated a hydrolysable tannin, 2-O-digalloyl-1,3,4,6-tetra-Ogalloyl- ß-D-glucose (DTOGG), from Galla Rhois and examined the effect on osteoclast formation and function. We found that DTOGG significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by downregulating the expression of the key regulator in osteoclastogenesis as well as osteoclast-related genes. Analysis of RANKL/RANK signaling revealed that DTOGG impaired activation of IκBα and p65 in the nuclear factor kappa-lightchain- enhancer of activated B cells (NF-κB) signaling pathway. Furthermore, DTOGG reduced bone resorbing activity of osteoclasts, compared to the vehicle-treated control. These results suggest that DTOGG could be a useful natural compound to manage osteoclast-mediated skeletal diseases. [BMB Reports 2019; 52(6): 409-414].

Inhibitory Effect of KP-A038 on Osteoclastogenesis and Inflammatory Bone Loss Is Associated With Downregulation of Blimp1.

Excessive osteoclastic activity results in pathological bone resorptive diseases, such as osteoporosis, periodontitis, and rheumatoid arthritis. As imidazole-containing compounds possess extensive therapeutic potential for the management of diverse diseases, we synthesized a series of imidazole derivatives and investigated their effects on osteoclast differentiation and function. In the present study, we found that a novel imidazole derivative, KP-A038, suppressed receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis and bone-resorbing activity in vitro and attenuated lipopolysaccharide (LPS)-induced bone destruction in vivo. KP-A038 significantly inhibited the induction of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and the expression of its target genes, including tartrate-resistant acid phosphatase (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), and matrix metallopeptidase 9 (Mmp9). KP-A038 upregulated the expression of negative regulators of osteoclast differentiation, such as interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6). Consistently, KP-A038 downregulated the expression of B lymphocyte-induced maturation protein-1 (Blimp1 encoded by Prdm1), a repressor for Irf8 and Bcl6. Moreover, administration of KP-A038 reduced LPS-induced bone erosion by suppressing osteoclast formation in vivo. Thus, our findings suggest that KP-A038 may serve as an effective therapeutic agent for the treatment and/or prevention of bone loss in pathological bone diseases, including osteoporosis and periodontitis.

A novel benzamide derivative protects ligature-induced alveolar bone erosion by inhibiting NFATc1-mediated osteoclastogenesis.

Since elevated osteoclast formation and/or activity by inhibitory responses against pathogens leads to diverse osteolytic bone diseases including periodontitis, inhibition of osteoclast differentiation and bone resorption has been a primary therapeutic strategy. In this study, we investigated the therapeutic potential of a novel benzamide-linked molecule, OCLI-070, for preventing alveolar bone loss in mice with ligature-induced experimental periodontitis. OCLI-070 inhibited osteoclast formation by acting on both early and late stages of differentiation, and attenuated the induction of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and the expression of osteoclast-specific genes. In addition, OCLI-070 significantly suppressed the formation of actin rings and resorption pits. Analysis of the inhibitory action of OCLI-070 showed that it markedly suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced extracellular signal-regulated kinase (ERK) and NF-κB signaling cascades. Moreover, OCLI-070 prevented ligature-induced alveolar bone erosion in mice by suppressing osteoclast formation. These findings demonstrate that OCLI-070 attenuated osteoclast differentiation and function as well as ligature-induced bone erosion by inhibiting RANKL-mediated ERK and NF-κB signaling pathways.

Inhibitory effects of methyl-3,5-di-O-caffeoyl-epi-quinate on RANKL-induced osteoclast differentiation.

In this study, we have shown that methyl-3,5-di-O-caffeoyl-epi-quinate, a naturally occurring compound isolated from Ainsliaea acerifolia, inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and the expression of osteoclast marker genes. Methyl-3,5-di-O-caffeoyl-epi-quinate also inhibited RANKL-induced activation of p38, Akt and extracellular signal-regulated kinase (ERK) as well as the expression of nuclear factor of activated T-cell (NFATc1), the key regulator of osteoclast differentiation. Negative regulators for osteoclast differentiation was upregulated by methyl-3,5-di-O-caffeoyl-epi-quinate. Collectively, our results suggested that methyl-3,5-di-O-caffeoyl-epi-quinate suppresses osteoclast differentiation via downregulation of RANK signaling pathways and NFATc1.

Inhibitory Effect of Purpurogallin on Osteoclast Differentiation in Vitro through the Downregulation of c-Fos and NFATc1.

Purpurogallin, a benzotropolone-containing natural compound, has been reported to exhibit numerous biological and pharmacological functions, such as antioxidant, anticancer, and anti-inflammatory effects. In this study, we enzymatically synthesized purpurogallin from pyrogallol and investigated its role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Purpurogallin attenuated the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, and suppressed upregulation of osteoclast-specific markers, including TRAP (Acp5), cathepsin K (Ctsk), and dendritic cell-specific transmembrane protein (Dcstamp). However, purpurogallin did not affect the bone resorbing function of mature osteoclasts evident by the resorption pit assay. Activation of mitogen-activated protein kinases, Akt and IkB pathways in RANK signaling were not altered by purpurogallin, whereas the expression of c-Fos and NFATc1, key transcriptional regulators in osteoclastogenesis, was dramatically inhibited by purpurogallin. Purpurogallin also significantly reduced the expression level of B lymphocyte-induced maturation protein-1 (Blimp1) gene (Prdm1). Further, downregulation of Blimp1 led to forced expression of anti-osteoclastogenic genes, including interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6) genes. Taken together, our data suggested that purpurogallin inhibits osteoclast differentiation via downregulation of c-Fos and NFATc1.

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.

Afatinib ameliorates osteoclast differentiation and function through downregulation of RANK signaling pathways.

Non-small-cell lung cancer (NSCLC) is the third most common cancer that spreads to the bone, resulting in osteolytic lesions caused by hyperactivation of osteoclasts. Activating mutations in epidermal growth factor receptor-tyrosine kinase (EGF-TK) are frequently associated with NSCLC, and afatinib is a first-line therapeutic drug, irreversibly targeting EGF-TK. However, the effects of afatinib on osteoclast differentiation and activation as well as the underlying mechanism remain unclear. In this study, afatinib significantly suppressed receptor activator of nuclear factor κB (RANK) ligand (RANKL)-induced osteoclast formation in bone marrow macrophages (BMMs). Consistently, afatinib inhibited the expression of osteoclast marker genes, whereas, it upregulated the expression of negative modulator genes. The bone resorbing activity of osteoclasts was also abrogated by afatinib. In addition, afatinib significantly inhibited RANKL-mediated Akt/protein kinase B and c-Jun N-terminal kinase phosphorylation. These results suggest that afatinib substantially suppresses osteoclastogenesis by downregulating RANK signaling pathways, and thus may reduce osteolysis after bone metastasis. [BMB Reports 2017; 50(3): 150-155].

OCLI-023, a Novel Pyrimidine Compound, Suppresses Osteoclastogenesis In Vitro and Alveolar Bone Resorption In Vivo.

An abnormal increase in osteoclast differentiation and activation results in various bone-resorptive diseases, including periodontitis, rheumatoid arthritis, and osteoporosis. Chemical compounds containing pyrimidine ring have been shown to regulate a variety of biological processes. Therefore, in order to identify an antiresorptive agent, we synthesized a series of pyrimidine ring-containing chemical compounds, and found that OCLI-023 suppressed the differentiation and activation of osteoclasts in vitro. OCLI-023 directly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced differentiation of bone marrow macrophages into osteoclasts, without a cytotoxic response. OCLI-023 also downregulated the RANKL-induced mRNA expression of osteoclast markers as well as inhibited the formation of actin rings and resorption pits. OCLI-023 attenuated the RANKL-induced activation of c-Jun N-terminal kinase and nuclear factor kappa-light-chain-enhancer of activated B cell signaling pathways. In a mouse model of periodontitis, ligature induced an increase of distance between cementoenamel junction (CEJ) and alveolar bone crest (ABC) in the second molar, and OCLI-023 significantly reduced it. Histological analysis showed ligature-induced increase of osteoclast numbers was also significantly reduced by OCLI-023. These data demonstrated the inhibitory effect of OCLI-023 on osteoclast differentiation and activity of osteoclasts in vitro, as well as on ligature-induced bone loss in vivo, and OCLI-023 can be proposed as a novel anti-resorptive compound.

Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative, on Osteoclast Differentiation, Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway.

Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.