RANKL+ senescent cells under mechanical stress: a therapeutic target for orthodontic root resorption using senolytics.

In dentistry, orthodontic root resorption is a long-lasting issue with no effective treatment strategy, and its mechanisms, especially those related to senescent cells, remain largely unknown. Here, we used an orthodontic intrusion tooth movement model with an L-loop in rats to demonstrate that mechanical stress-induced senescent cells aggravate apical root resorption, which was prevented by administering senolytics (a dasatinib and quercetin cocktail). Our results indicated that cementoblasts and periodontal ligament cells underwent cellular senescence (p21+ or p16+) and strongly expressed receptor activator of nuclear factor-kappa B (RANKL) from day three, subsequently inducing tartrate-resistant acid phosphatase (TRAP)-positive odontoclasts and provoking apical root resorption. More p21+ senescent cells expressed RANKL than p16+ senescent cells. We observed only minor changes in the number of RANKL+ non-senescent cells, whereas RANKL+ senescent cells markedly increased from day seven. Intriguingly, we also found cathepsin K+p21+p16+ cells in the root resorption fossa, suggesting senescent odontoclasts. Oral administration of dasatinib and quercetin markedly reduced these senescent cells and TRAP+ cells, eventually alleviating root resorption. Altogether, these results unveil those aberrant stimuli in orthodontic intrusive tooth movement induced RANKL+ early senescent cells, which have a pivotal role in odontoclastogenesis and subsequent root resorption. These findings offer a new therapeutic target to prevent root resorption during orthodontic tooth movement.

Double-Stranded RNA Induces Mortality in an MDA5-Mediated Type I Interferonopathy Model.

Gain-of-function mutations in the viral dsRNA sensor melanoma differentiation-associated protein 5 (MDA5) lead to autoimmune IFNopathies, including Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome. However, much remains unclear regarding the mechanism of disease progression and how external factors such as infection or immune stimulation with vaccination can affect the immune response. With this aim, we generated mice with human MDA5 bearing the SMS-associated mutation R822Q (hM-R822Q). hM-R822Q transgenic (Tg) mice developed SMS-like heart fibrosis, aortic valve enlargement, and aortic calcification with a systemic IFN-stimulated gene signature resulting in the activation of the adaptive immune response. Although administration of the viral dsRNA mimic polyinosinic-polycytidylic acid [poly(I:C)] did not have remarkable effects on the cardiac phenotype, dramatic inflammation was observed in the intestines where IFN production was most elevated. Poly(I:C)-injected hM-R822Q Tg mice also developed lethal hypercytokinemia marked by massive IL-6 levels in the serum. Interrupting the IFN signaling through mitochondrial antiviral signaling protein or IFN-α/ß receptor alleviated hM-R822Q-induced inflammation. Furthermore, inhibition of JAK signaling with tofacitinib reduced cytokine production and ameliorated mucosal damage, enabling the survival of poly(I:C)-injected hM-R822Q Tg mice. These findings demonstrate that the MDA5 R822Q mutant introduces a critical risk factor for uncontrollable inflammation on viral infection or vaccination.

Effects of Anti-Receptor Activator of Nuclear Factor Kappa B Ligand Antibody and Zoledronic Acid on Periapical Lesion Development in Mice.

INTRODUCTION: Antiresorptive drugs are widely used to treat osteoporosis and other systemic bone diseases, although their efficacy for local bone resorption after localized inflammation has not been fully elucidated. We examined the effects of an anti-receptor activator of nuclear factor kappa B ligand (RANKL) antibody and the bisphosphonate zoledronic acid (ZOL) on periapical lesion (PL) development in mice. METHODS: Dental pulp of lower first molars in mice was removed, with the exposed dental pulp chambers left open to the oral environment to induce apical periodontitis. An anti-RANKL antibody or ZOL was intraperitoneally injected once per week until postoperative day 21, and then micro-computed tomographic imaging and histologic analyses were performed. RESULTS: PL enlargement was inhibited by both the anti-RANKL antibody and ZOL in a dose-dependent manner, and a reduction of inflammatory cell infiltration in apical tissues inhibited periapical bone resorption. The anti-RANKL antibody decreased the number of osteoclasts in periapical tissues, whereas ZOL suppressed periapical bone resorption with osteoclast numbers maintained. Although the administration of each of the antiresorptive drugs increased femoral bone mass, femoral bone mineral density in the PL group was lower compared with the sham-operated group. CONCLUSIONS: These results suggest that an antiresorptive drug administered systemically is distributed to areas of local inflammation in the jaw can prevent PL development.

Neural crest-derived cells in nasal conchae of adult mice contribute to bone regeneration.

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.

Pax5 Negatively Regulates Osteoclastogenesis through Downregulation of Blimp1.

Paired box protein 5 (Pax5) is a crucial transcription factor responsible for B-cell lineage specification and commitment. In this study, we identified a negative role of Pax5 in osteoclastogenesis. The expression of Pax5 was time-dependently downregulated by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) stimulation in osteoclastogenesis. Osteoclast (OC) differentiation and bone resorption were inhibited (68.9% and 48% reductions, respectively) by forced expression of Pax5 in OC lineage cells. Pax5 led to the induction of antiosteoclastogenic factors through downregulation of B lymphocyte-induced maturation protein 1 (Blimp1). To examine the negative role of Pax5 in vivo, we generated Pax5 transgenic (Pax5Tg) mice expressing the human Pax5 transgene under the control of the tartrate-resistant acid phosphatase (TRAP) promoter, which is expressed mainly in OC lineage cells. OC differentiation and bone resorption were inhibited (54.2-76.9% and 24.0-26.2% reductions, respectively) in Pax5Tg mice, thereby contributing to the osteopetrotic-like bone phenotype characterized by increased bone mineral density (13.0-13.6% higher), trabecular bone volume fraction (32.5-38.1% higher), trabecular thickness (8.4-9.0% higher), and trabecular number (25.5-26.7% higher) and decreased trabecular spacing (9.3-10.4% lower) compared to wild-type control mice. Furthermore, the number of OCs was decreased (48.8-65.3% reduction) in Pax5Tg mice. These findings indicate that Pax5 plays a negative role in OC lineage specification and commitment through Blimp1 downregulation. Thus, our data suggest that the Pax5-Blimp1 axis is crucial for the regulation of RANKL-induced osteoclastogenesis.

Inhibition of hepatocyte growth factor/c-Met signalling abrogates joint destruction by suppressing monocyte migration in rheumatoid arthritis.

OBJECTIVES: To determine the expression of hepatocyte growth factor (HGF) in RA biological fluids, the role of HGF in monocyte migration and the therapeutic effect of the c-Met inhibitor savolitinib in an arthritis model mice. METHODS: HGF/c-Met expression in serum, SF and synovial tissues (STs) obtained from RA patients and controls, as well as RA fibroblast-like synoviocytes (FLSs), was evaluated by ELISA and immunostaining. To determine the function of HGF in RA SF, we preincubated RA SF with a neutralizing anti-HGF antibody and measured the chemotactic ability of a human acute monocytic leukaemia cell line (THP-1). Additionally, examinations were conducted of SKG mice treated with savolitinib for 4 weeks. RESULTS: HGF levels in serum from RA patients were significantly higher than those in the controls and were decreased by drug treatment for 24 weeks. Additionally, the HGF level in SF from RA patients was higher than that in SF from OA patients. HGF and c-Met expression was also noted in RA STs. Stimulation of RA FLSs with TNF-α increased HGF/c-Met expression in a concentration-dependent manner, and c-Met signal inhibition suppressed production of fractalkine/CX3CL1 and macrophage inflammatory protein-1α/CCL3. When HGF was removed by immunoprecipitation, migration of THP-1 in RA SF was suppressed. In SKG mice, savolitinib significantly suppressed ankle bone destruction on µCT, with an associated reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. CONCLUSION: HGF produced by inflammation in synovium of RA patients activates monocyte migration to synovium and promotes bone destruction via a chemotactic effect and enhanced chemokine production.

Novel gene Merlot inhibits differentiation and promotes apoptosis of osteoclasts.

Extended osteoclast longevity is deeply involved in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis, though the mechanisms that determine osteoclast lifespan are not fully understood. Here we present findings indicating that the newly characterized gene Merlot, which encodes a highly conserved yet uncharacterized protein in vertebrates, is an important regulator for termination of osteoclastogenesis via induction of apoptosis. Mice lacking Merlot exhibited low bone mass due to increased osteoclast and bone resorption. Furthermore, osteoclast precursors overexpressing Merlot failed to differentiate into mature osteoclasts, while Merlot deficiency led to hyper-nucleation and prolonged survival of osteoclasts, accompanied by sustained nuclear localization of nuclear factor of activated T cell c1 (NFATc1) and derepression of glycogen synthase kinase-3ß (GSK3ß) activity, known to regulate NFATc1 activity and induce apoptosis. Merlot-deficient osteoclasts were found to represent suppression of caspase-3-mediated apoptosis and Merlot deficiency caused transcriptional downregulation of a proapoptotic cascade, including Bax, Bak, Noxa, and Bim, as well as the executor caspase members Casp-3, -6, and -7, and upregulation of anti-apoptotic Bcl2, resulting in a low apoptotic threshold. Thus, Merlot regulates osteoclast lifespan by inhibition of differentiation and simultaneous induction of apoptosis via regulation of the NFATc1-GSK3ß axis.

Biological Effects of Anti-RANKL Antibody and Zoledronic Acid on Growth and Tooth Eruption in Growing Mice.

The anti-bone resorptive drugs denosumab, an anti-human-RANKL antibody, and zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, have recently been applied for treatment of pediatric patients with bone diseases, though details regarding their effects in growing children have yet to be fully elucidated. In the present study, we administered these anti-resorptive drugs to mice from the age of 1 week and continued once-weekly injections for a total of 7 times. Mice that received the anti-RANKL antibody displayed normal growth and tooth eruption, though osteopetrotic bone volume gain in long and alveolar bones was noted, while there were nearly no osteoclasts and a normal of number osteoblasts observed. In contrast, ZOL significantly delayed body growth, tooth root formation, and tooth eruption, with increased osteoclast and decreased osteoblast numbers. These findings suggest regulation of tooth eruption via osteoblast differentiation by some types of anti-resorptive drugs.

Roles of monocarboxylate transporter subtypes in promotion and suppression of osteoclast differentiation and survival on bone.

Monocarboxylate transporters (MCTs) provide transmembrane transport of monocarboxylates such as lactate and pyruvate. The present results showed that α-cyano-4-hydroxycinnamic acid (CHC), an inhibitor of MCTs, promoted osteoclast differentiation from macrophages at lower concentrations (0.1-0.3 mM) and suppressed that at a higher concentration (1.0 mM). On the other hand, CHC reduced the number of mature osteoclasts on the surface of dentin in a concentration-dependent manner. Additionally, macrophages and osteoclasts were found to express the Mct1, Mct2, and Mct4 genes, with Mct1 and Mct4 expression higher in macrophages, and that of Mct2 higher in osteoclasts. Although Mct1 gene knockdown in macrophages enhanced osteoclast formation induced by RANKL, Mct2 gene knockdown suppressed that. Finally, Mct2 gene silencing in mature osteoclasts decreased their number and, thereby, bone resorption. These results suggest that MCT1 is a negative regulator and MCT2 a positive regulator of osteoclast differentiation, while MCT2 is required for bone resorption by osteoclasts.

Singleton-Merten Syndrome-like Skeletal Abnormalities in Mice with Constitutively Activated MDA5.

Singleton-Merten syndrome (SMS) is a type I interferonopathy characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, and psoriasis. A missense mutation in IFIH1 encoding a cytoplasmic viral RNA sensor MDA5 has recently been identified in the SMS patients as well as in patients with a monogenic form of lupus. We previously reported that Ifih1gs/+ mice express a constitutively active MDA5 and spontaneously develop lupus-like nephritis. In this study, we demonstrate that the Ifih1gs/+ mice also exhibit SMS-like bone abnormalities, including decreased bone mineral density and thin cortical bone. Histological analysis revealed a low number of osteoclasts, low bone formation rate, and abnormal development of growth plate cartilages in Ifih1gs/+ mice. These abnormalities were not observed in Ifih1gs/+ ・Mavs-/- and Ifih1gs/+ ・Ifnar1-/- mice, indicating the critical role of type I IFNs induced by MDA5/MAVS-dependent signaling in the bone pathogenesis of Ifih1gs/+ mice, affecting bone turnover. Taken together, our findings suggest the inhibition of type I IFN signaling as a possible effective therapeutic strategy for bone disorders in SMS patients.

In Vitro Study of the Effects of Denosumab on Giant Cell Tumor of Bone: Comparison with Zoledronic Acid.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that contains numerous osteoclasts formed from marrow-derived precursors through receptor activator of nuclear factor κ-B ligand (RANKL), an osteoclast differentiation factor expressed in neoplastic cells of GCTB. Denosumab, a fully human monoclonal antibody targeting RANKL, has recently been used for the treatment of GCTB, and superior treatment effects have been reported. The aim of this work was to elucidate the mechanism of action of denosumab, and the differences between denosumab and zoledronic acid at the level of GCTB cells. We isolated GCTB cells from 3 patients and separated them into osteoclasts, osteoclast precursors and proliferating spindle-shaped stromal cells (the true neoplastic component), and examined the action of denosumab on differentiation, survival and bone resorption activity of osteoclasts. Denosumab and zoledronic acid inhibited osteoclast differentiation from mononuclear cells containing osteoclast precursors. Zoledronic acid inhibited osteoclast survival, whereas an inhibitory effect of denosumab on osteoclast survival was not observed. The inhibitory effect on bone resorption by both agents was confirmed in culture on dentin slices. Furthermore, zoledronic acid showed dose-dependent inhibition of cell growth of neoplastic cells whereas denosumab had no inhibitory effect on these cells. Denosumab has an inhibitory effect on osteoclast differentiation, but no inhibitory effects on survival of osteoclasts or growth of neoplastic cells in GCTBs.

The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages.

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.

Anti-mouse RANKL Antibodies Inhibit Alveolar Bone Destruction in Periodontitis Model Mice.

Denosumab is an anti-bone resorptive drug consisting of complete human monoclonal antibodies that targets receptor activator of nuclear factor κB ligand (RANKL), which is responsible for osteoclast formation. The drug has been adapted for bone diseases, such as osteoporosis and bone metastasis related to cancer, but is not used for alveolar bone destruction related to periodontitis. In the present study, we aimed to clarify whether denosumab prevents bone destruction associated with lipopolysaccharide (LPS)-induced calvaria inflammation and experimental periodontitis in model mice. Denosumab does not bind to mouse RANKL, thus we used anti-mouse monoclonal RANKL antibodies. We also examined the inhibitory effects toward bone destruction of another anti-bone resorptive drug zoledronate, a nitrogen-containing bisphosphonate. Local administration of anti- RANKL antibodies into the calvaria area inhibited LPS-induced osteoclast formation and bone destruction, while zoledronate inhibited bone destruction but not osteoclast formation due to its different action mechanism. In periodontitis model mice, in which the second molars were ligated with a silk suture to induce inflammation, intraperitoneal administration of anti-RANKL antibodies significantly inhibited alveolar bone destruction and tooth root exposure. On the other hand, zoledronate only weakly repressed alveolar bone destruction and failed to inhibit root exposure. These results suggest that denosumab is a promising candidate to prevent alveolar bone destruction associated with periodontitis.

Biological effects of anti-RANKL antibody administration in pregnant mice and their newborns.

Denosumab, a fully human monoclonal antibody that neutralizes receptor activator of nuclear factor-κB ligand (RANKL) and blocks osteoclast differentiation, has received approval in Japan for use as an anti-resorptive drug for osteoporosis and skeletal-related events (SREs) in patients with solid cancer. Denosumab is contraindicated during pregnancy, though the effects of blocking RANKL activity on pregnant mothers and their newborns are unclear. We used mice to investigate the effects of an anti-RANKL antibody on maternal and newborn health. Mothers injected with the anti-RANKL antibody had increased bone mass as compared with the controls, while osteoclast number and the level of tartrate-resistant acid phosphatase (TRAP) in serum were increased at the end of pregnancy. Newborn mice exposed to the antibody in utero were normally born, but showed increased bone mass and died within 48 h after birth. None of the newborns were found to have milk in their stomachs, suggesting that they died due to a maternal defect in lactation. Consistent with this, anti-RANKL antibody-injected mothers displayed impaired mammary gland development. However, fostering by healthy surrogate mothers rescued only 33% of the antibody-exposed newborns, suggesting that neonatal mortality was due, at least in part, to an intrinsic defect in the newborns. Our findings show that anti-RANKL antibody administration during pregnancy results in not only an undesirable increase in bone mass, but also has harmful effects on newborn survival.

Induction of osteoblastic differentiation of neural crest-derived stem cells from hair follicles.

The neural crest (NC) arises near the neural tube during embryo development. NC cells migrate throughout the embryo and have potential to differentiate into multiple cell types, such as peripheral nerves, glial, cardiac smooth muscle, endocrine, and pigment cells, and craniofacial bone. In the present study, we induced osteoblast-like cells using whisker follicles obtained from the NC of mice. Hair follicle cells derived from the NC labeled with enhanced green fluorescent protein (EGFP) were collected from protein zero-Cre/floxed-EGFP double transgenic mice and cultured, then treated and cultured in stem cell growth medium. After growth for 14 days, results of flow cytometry analysis showed that 95% of the EGFP-positive (EGFP+) hair follicle cells derived from the NC had proliferated and 76.2% of those expressed mesenchymal stem cells markers, such as platelet-derived growth factor α and stem cell antigen-1, and also showed constitutive expression of Runx2 mRNA. Cells stimulated with bone morphogenetic protein-2 expressed osteocalcin, osterix, and alkaline phosphatase mRNA, resulting in production of mineralized matrices, which were detected by von Kossa and alizarin red staining. Moreover, EGFP+ hair follicle cells consistently expressed macrophage colony-stimulating factor and osteoprotegerin (OPG). Addition of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (10-8 M) to the cultures suppressed OPG expression and induced RANKL production in the cells. Furthermore, multinucleated osteoclasts appeared within 6 days after starting co-cultures of bone marrow cells with EGFP+ cells in the presence of 1,25(OH)2D3 and PGE2. These results suggest that NC-derived hair follicle cells possess a capacity for osteoblastic differentiation and may be useful for developing new bone regenerative medicine therapies.

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/-) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.

Smad4 is required to inhibit osteoclastogenesis and maintain bone mass.

Bone homeostasis is maintained as a delicate balance between bone-resorption and bone-formation, which are coupled to maintain appropriate bone mass. A critical question is how bone-resorption is terminated to allow bone-formation to occur. Here, we show that TGFßs inhibit osteoclastogenesis and maintain bone-mass through Smad4 activity in osteoclasts. We found that latent-TGFß1 was activated by osteoclasts to inhibit osteoclastogenesis. Osteoclast-specific Smad4 conditional knockout mice (Smad4-cKO) exhibited significantly reduced bone-mass and elevated osteoclast formation relative to controls. TGFß1-activation induced expression of Irf8 and Bcl6, both of which encode factors inhibiting osteoclastogenesis, by blocking their negative regulator, Prdm1, in osteoclasts in a Smad4-dependent manner. Reduced bone-mass and accelerated osteoclastogenesis seen in Smad4-cKO were abrogated by Prdm1 deletion. Administration of latent-TGFß1-Fc to wild-type mice antagonized LPS-induced bone destruction in a model of activated osteoclast-mediated bone destruction. Thus, latent-TGFß1-Fc could serve as a promising new therapeutic agent in bone diseases marked by excessive resorption.

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis.

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.

Bropirimine inhibits osteoclast differentiation through production of interferon-ß.

Bropirimine is a synthetic agonist for toll-like receptor 7 (TLR7). In this study, we investigated the effects of bropirimine on differentiation and bone-resorbing activity of osteoclasts in vitro. Bropirimine inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) induced by receptor activator of nuclear factor κB ligand (RANKL) in a concentration-dependent manner. Furthermore, it suppressed the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor for osteoclast differentiation, without affecting BMM viability. Bropirimine also inhibited osteoclast differentiation induced in co-cultures of mouse bone marrow cells (BMCs) and mouse osteoblastic UAMS-32 cells in the presence of activated vitamin D3. Bropirimine partially suppressed the expression of RANKL mRNA in UAMS-32 cells induced by activated vitamin D3. Finally, the anti-interferon-ß (IFN-ß) antibody restored RANKL-dependent differentiation of BMMs into osteoclasts suppressed by bropirimine. These results suggest that bropirimine inhibits differentiation of osteoclast precursor cells into osteoclasts via TLR7-mediated production of IFN-ß.

Expression of nephronectin is inhibited by oncostatin M via both JAK/STAT and MAPK pathways.

Nephronectin (Npnt), also called POEM, is an extracellular matrix protein considered to play critical roles as an adhesion molecule in the development and functions of various tissues, such as the kidneys, liver, and bones. In the present study, we examined the molecular mechanism of Npnt gene expression and found that oncostatin M (OSM) strongly inhibited Npnt mRNA expression in MC3T3-E1 cells from a mouse osteoblastic cell line. OSM also induced a decrease in Npnt expression in both time- and dose-dependent manners via both the JAK/STAT and MAPK pathways. In addition, OSM-induced inhibition of osteoblast differentiation was recovered by over-expression of Npnt. These results suggest that OSM inhibits Npnt expression via the JAK/STAT and MAPK pathways, while down-regulation of Npnt by OSM influences inhibition of osteoblast differentiation.