Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts.

Selenoproteins containing selenium in the form of selenocysteine are critical for bone remodeling. However, their underlying mechanism of action is not fully understood. Herein, we report the identification of selenoprotein W (SELENOW) through large-scale mRNA profiling of receptor activator of nuclear factor (NF)-κΒ ligand (RANKL)-induced osteoclast differentiation, as a protein that is downregulated via RANKL/RANK/tumour necrosis factor receptor-associated factor 6/p38 signaling. RNA-sequencing analysis revealed that SELENOW regulates osteoclastogenic genes. SELENOW overexpression enhances osteoclastogenesis in vitro via nuclear translocation of NF-κB and nuclear factor of activated T-cells cytoplasmic 1 mediated by 14-3-3γ, whereas its deficiency suppresses osteoclast formation. SELENOW-deficient and SELENOW-overexpressing mice exhibit high bone mass phenotype and osteoporosis, respectively. Ectopic SELENOW expression stimulates cell-cell fusion critical for osteoclast maturation as well as bone resorption. Thus, RANKL-dependent repression of SELENOW regulates osteoclast differentiation and blocks osteoporosis caused by overactive osteoclasts. These findings demonstrate a biological link between selenium and bone metabolism.

Metabolic activities affect femur and lumbar vertebrae remodeling, and anti-resorptive risedronate disturbs femoral cortical bone remodeling.

Metabolic activities are closely correlated with bone remodeling and long-term anti-resorptive bisphosphonate treatment frequently causes atypical femoral fractures through unclear mechanisms. To explore whether metabolic alterations affect bone remodeling in femurs and lumbar vertebrae and whether anti-osteoporotic bisphosphonates perturb their reconstruction, we studied three mouse strains with different fat and lean body masses (BALB/c, C57BL6, and C3H mice). These mice displayed variable physical activity, food and drink intake, energy expenditure, and respiratory quotients. Following intraperitoneal calcein injection, double calcein labeling of the femoral diaphysis, as well as serum levels of the bone-formation marker procollagen type-I N-terminal propeptide and the bone-resorption marker C-terminal telopeptide of type-I collagen, revealed increased bone turnover in mice in the following order: C3H > BALB/c ≥ C57BL6 mice. In addition, bone reconstitution in femurs was distinct from that in lumbar vertebrae in both healthy control and estrogen-deficient osteoporotic mice with metabolic perturbation, particularly in terms of femoral trabecular and cortical bone remodeling in CH3 mice. Interestingly, subcutaneous administration of bisphosphonate risedronate to C3H mice with normal femoral bone density led to enlarged femoral cortical bones with a low bone mineral density, resulting in bone fragility; however, this phenomenon was not observed in mice with ovariectomy-induced femoral cortical bone loss. Together, these results suggest that diverse metabolic activities support various forms of bone remodeling and that femur remodeling differs from lumbar vertebra remodeling. Moreover, our findings imply that the adverse effect of bisphosphonate agents on femoral cortical bone remodeling should be considered when prescribing them to osteoporotic patients.

Sinus floor augmentation using mixture of mineralized cortical bone and cancellous bone allografts: Radiographic and histomorphometric evaluation.

BACKGROUND/PURPOSE: Due to the pneumatization of the maxillary sinus, the sinus floor augmentation is often performed to implant placement in the maxillary posterior region. The aim was to perform radiographic and histomorphometric evaluation after placement of mixed allografts (cortical freeze-dried bone allograft [FDBA] 50%:cancellous FDBA 50%) during sinus floor augmentation. MATERIALS AND METHODS: In 37 patients, anorganic bovine bone (ABB, sites = 16), mineralized cancellous bone allograft (MCBA, sites = 15), and mixed allografts (Mixed AG, sites = 20) were placed during sinus floor elevation via the lateral approach (LSFE), at total 51 sites with residual alveolar bone height (RBH) < 5 mm. Cone-beam computed tomography images were obtained before LSFE (T0), after surgery (T1), and 6 months after surgery (T2) for radiographic analysis. After a 6-month healing period, core biopsies were harvested and histomorphometric analysis was performed. RESULTS: The mean augmented bone height (ABH) of ABB, MCBA, and mixed AG groups after surgery was similar (13.86 ±â€¯4.19 mm, 13.99 ±â€¯4.07 mm, and 14.20 ±â€¯3.12 mm, respectively; P > 0.05). The mean ABH of ABB, MCBA, and mixed AG groups after 6 months was similar (13.72 ±â€¯4.55 mm, 11.83 ±â€¯3.31 mm, and 12.53 ±â€¯2.97 mm, respectively; P > 0.05). In the ABB, MCBA, and mixed AG groups, the proportion of newly formed bone (NB) was similar (36.13 ±â€¯10.01%, 39.26 ±â€¯10.72%, and 31.27 ±â€¯18.31%, respectively; P > 0.05). CONCLUSION: This result demonstrated that mixed AG led to sufficient bone augmentation and histologically comparable NB formation as compared to ABB and MCBA for sinus floor augmentation.

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.

Pisidium coreanum Inhibits Multinucleated Osteoclast Formation and Prevents Estrogen-Deficient Osteoporosis.

Mollusks have served as important sources of human food and medicine for a long time. Raw Pisidium coreanum, a freshwater bivalve of the phylum Mollusca, is used in traditional therapies in parts of Asia. However, the therapeutic effects of Pisidium coreanum on bone diseases are not known. We investigated the functional roles of Pisidium coreanum in osteoporotic bone diseases. Pisidium coreanum inhibited the differentiation of bone marrow-derived monocytic cells into mature osteoclasts in vitro. The ovariectomized mice that received oral administration of Pisidium coreanum showed improvements in both trabecular and cortical bones. This preventive activity of Pisidium coreanum against bone loss was due to limited osteoclast maturation with reduced osteoclast surface extent in trabecular bone tissue. The formation of large multinucleated osteoclasts in vitro was significantly decreased in response to Pisidium coreanum, consistent with the reduced expression levels of osteoclast markers and fusion-related genes, such as NFATc1, p65, integrin αvß3, DC-STAMP, OC-STAMP, Atp6v0d2, FAK, CD44, and MFR. These data suggest that Pisidium coreanum inhibits osteoclast differentiation by negatively regulating the fusion of mononuclear osteoclast precursors. Thus, our data demonstrate the ability of Pisidium coreanum to effectively prevent estrogen-deficient osteoporosis through inhibition of multinucleated osteoclast formation.

Specific targeting of PKCδ suppresses osteoclast differentiation by accelerating proteolysis of membrane-bound macrophage colony-stimulating factor receptor.

c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.

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.

Orthognathic surgery for patients with fibrous dysplasia involved with dentition.

BACKGROUND: Fibrous dysplasia (FD) is characterized by the replacement of normal bone by abnormal fibro-osseous connective tissue and typically treated with surgical contouring of the dysplastic bone. When dysplastic lesions involve occlusion, not only is surgical debulking needed, orthognathic surgery for correction of dentofacial deformity is mandatory. However, the long-term stability of osteotomized, dysplastic bone segments is a major concern because of insufficient screw-to-bone engagement during surgery and the risk of FD lesion re-growth. CASE PRESENTATION: This case report reviewed two patients with non-syndromic FD that presented with maxillary occlusal canting and facial asymmetry. Le Fort I osteotomy with recontouring of the dysplastic zygomaticomaxillary region had been performed. The stability of osseous segments were favorable. However, dysplastic, newly formed bone covered the previous plate fixation site and mild bony expansion was observed, which did not influence the facial profile. Including the current cases, 15 cases of orthognathic surgery for FD with dentition have been reported in the literature. CONCLUSION: The results showed that osteotomy did not appear to significantly reduce the long-term stability of the initial fixation insufficiency of the screw to the dysplastic bone. However, based on our results and those of the others, long-term follow-up and monitoring are needed, even in cases where the osteotomized segment shows stable results.

Suppressive effect of syndecan ectodomains and N-desulfated heparins on osteoclastogenesis via direct binding to macrophage-colony stimulating factor.

Syndecans, a family of cell surface heparan sulfate proteoglycans, regulate cell differentiation via binding of their heparan sulfate chains to growth factors and cytokines and play a role in tumor growth and progression, wound repair, and intestinal mucosal damage. However, the functional and mechanistic roles of syndecans in osteoclast differentiation and bone metabolism are yet unclear. Here, we demonstrated that post-translationally glycosylated ectodomains of syndecan-1 to 4 obtained from mammalian cells efficiently suppressed osteoclast differentiation compared to those obtained from Escherichia coli with no systems for glycosylation. A concomitant decrease in the expression of osteoclast markers such as nuclear factor of activated T cells 1 (NFATc1), c-Fos, and ATP6V0D2 was observed. In addition, heparan sulfate and selectively N-desulfated heparin derivatives with 2-O- and 6-O-sulfate groups and no anticoagulant activity in blood inhibited osteoclast differentiation. The inhibitory effects of syndecan ectodomains, heparan sulfate, and N-desulfated heparin derivatives on osteoclast differentiation were attributed to their direct binding to the macrophage-colony stimulating factor (M-CSF), resulting in the blocking of M-CSF-mediated downstream signals such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice injected with syndecan ectodomains, heparan sulfate, and N-desulfated heparin derivatives into periosteal regions of calvaria showed reduction in the formation of tartrate-resistant acid phosphatase (TRAP)-positive mature osteoclasts on the calvarial bone surface, thereby exhibiting decreased bone resorption. Together, these results revealed a novel role of heparan sulfate chains of syndecan ectodomains in the regulation of osteoclast differentiation.

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.

MacroH2A1.2 inhibits prostate cancer-induced osteoclastogenesis through cooperation with HP1α and H1.2.

Osteoclasts are multinuclear bone-resorbing cells that differentiate from hematopoietic precursor cells. Prostate cancer cells frequently spread to bone and secrete soluble signaling factors to accelerate osteoclast differentiation and bone resorption. However, processes and mechanisms that govern the expression of osteoclastogenic soluble factors secreted by prostate cancer cells are largely unknown. MacroH2A (mH2A) is a histone variant that replaces canonical H2A at designated genomic loci and establishes functionally distinct chromatin regions. Here, we report that mH2A1.2, one of the mH2A isoforms, attenuates prostate cancer-induced osteoclastogenesis by maintaining the inactive state of genes encoding soluble factors in prostate cancer cells. Our functional analyses of soluble factors identify lymphotoxin beta (LTß) as a major stimulator of osteoclastogenesis and an essential mH2A1.2 target for its anti-osteoclastogenic activity. Mechanistically, mH2A1.2 directly interacts with HP1α and H1.2 and requires them to inactivate LTß gene in prostate cancer cells. Consistently, HP1α and H1.2 have an intrinsic ability to inhibit osteoclast differentiation in a mH2A1.2-dependent manner. Together, our data uncover a new and specific role for mH2A1.2 in modulating osteoclastogenic potential of prostate cancer cells and demonstrate how this signaling pathway can be exploited to treat osteolytic bone metastases at the molecular level.

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.

Magnesium phosphate ceramics incorporating a novel indene compound promote osteoblast differentiation in vitro and bone regeneration in vivo.

Incorporating bioactive molecules into synthetic ceramic scaffolds is challenging. In this study, to enhance bone regeneration, a magnesium phosphate (MgP) ceramic scaffold was incorporated with a novel indene compound, KR-34893. KR-34893 induced the deposition of minerals and expression of osteoblast marker genes in primary human bone marrow mesenchymal stem cells (BMSCs) and a mouse osteoblastic MC3T3-E1 cell line. Analysis of the mode of action showed that KR-34893 induced the phosphorylation of MAPK/extracellular signal-regulated kinase and extracellular signal-regulated kinase, and subsequently the expression of bone morphogenetic protein 7, accompanied by SMAD1/5/8 phosphorylation. Accordingly, KR-34893 was incorporated into an MgP scaffold prepared by 3D printing at room temperature, followed by cement reaction. KR-34893-incorporated MgP (KR-MgP) induced the expression of osteoblast differentiation marker genes in vitro. In a rat calvaria defect model, KR-MgP scaffolds enhanced bone regeneration and increased bone volume compared with MgP scaffolds, as assessed by micro-computed tomography and histological analyses. In conclusion, we developed a method for producing osteoinductive MgP scaffolds incorporating a bioactive organic compound, without high temperature sintering. The KR-MgP scaffolds enhanced osteoblast activation in vitro and bone regeneration in vivo.

Fucoidan-induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair.

Osteogenesis and angiogenesis, including cell-cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan-induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan-MSC-CM). Next, the angiogenic activity of Fucoidan-MSC-CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan-induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor-specific binding inhibitor. Furthermore, Fucoidan-MSC-CM increased the phosphorylation of mitogen-activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan-induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair.

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.

Blocking of the Ubiquitin-Proteasome System Prevents Inflammation-Induced Bone Loss by Accelerating M-CSF Receptor c-Fms Degradation in Osteoclast Differentiation.

Anti-osteoporotic activity of a blocker of the ubiquitin-proteasome system, bortezomib, has known to be achieved by directly opposed action in increased bone formation by osteoblasts and in decreased bone destruction by osteoclasts. However, the mechanisms underlying the proteasome blocker inhibition of osteoclast differentiation and function are not fully understood. Here, we observed that proteasome inhibitors, such as MG132 and bortezomib, in osteoclasts accelerated the degradation of c-Fms, a cognate receptor of macrophage colony-stimulating factor (M-CSF), and did not affect the amount of receptor activator of nuclear factor kappa-B (RANK), a receptor of receptor activator of nuclear factor kappa-B ligand (RANKL). c-Fms degradation induced by proteasome inhibitors was controlled by the activation of p38/tumor necrosis factor-alpha converting enzyme (TACE)-mediated regulated intramembrane proteolysis (RIPping). This was validated through the restoration of c-Fms using specific inhibitors of p38 and TACE, and a stimulation of p38-dependent TACE. In addition, c-Fms degradation by proteasome inhibition completely blocked M-CSF-mediated intrinsic signalling and led to the suppression of osteoclast differentiation and bone resorption. In a mouse model with intraperitoneal administration of lipopolysaccharide (LPS) that stimulates osteoclast formation and leads to bone loss, proteasome blockers prevented LPS-induced inflammatory bone resorption due to a decrease in the number of c-Fms-positive osteoclasts. Our study showed that accelerating c-Fms proteolysis by proteasome inhibitors may be a therapeutic option for inflammation-induced bone loss.

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].

The purinergic receptor P2X5 regulates inflammasome activity and hyper-multinucleation of murine osteoclasts.

Excessive bone resorption by osteoclasts (OCs) can result in serious clinical outcomes, including bone loss that may weaken skeletal or periodontal strength. Proper bone homeostasis and skeletal strength are maintained by balancing OC function with the bone-forming function of osteoblasts. Unfortunately, current treatments that broadly inhibit OC differentiation or function may also interfere with coupled bone formation. We therefore identified a factor, the purinergic receptor P2X5 that is highly expressed during the OC maturation phase, and which we show here plays no apparent role in early bone development and homeostasis, but which is required for osteoclast-mediated inflammatory bone loss and hyper-multinucleation of OCs. We further demonstrate that P2X5 is required for ATP-mediated inflammasome activation and IL-1ß production by OCs, and that P2X5-deficient OC maturation is rescued in vitro by addition of exogenous IL-1ß. These findings identify a mechanism by which OCs react to inflammatory stimuli, and may identify purinergic signaling as a therapeutic target for bone loss-related inflammatory conditions.

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.