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.

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.

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.

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.

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.

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.

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.

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.

Secretion of a truncated osteopetrosis-associated transmembrane protein 1 (OSTM1) mutant inhibits osteoclastogenesis through down-regulation of the B lymphocyte-induced maturation protein 1 (BLIMP1)-nuclear factor of activated T cells c1 (NFATc1) axis.

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1ß but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1ß in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1ß were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.

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.

Localization and osteoblastic differentiation potential of neural crest-derived cells in oral tissues of adult mice.

In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.

Planar catechin increases bone mass by regulating differentiation of osteoclasts in mice.

OBJECTIVES: While catechins have been reported to exhibit potential to benefit osteoporosis patients, the effects of planar catechin (PCat), synthesized during the development of drugs for Alzheimer's disease, have not been clearly elucidated. Here, we examined the effects of PCat on mouse bone metabolism both in vivo and in vitro. METHODS: Six week old female mice were orally administered PCat (30 mg/kg) every other day for four weeks, and their femurs were analyzed using micro-computed tomography imaging. Osteoclasts and osteoblasts were collected from mice and cultured with PCat. Subsequently, osteoclast formation and differentiation and osteoblast differentiation were observed. RESULTS: Mice orally administered PCat displayed significantly increased femur bone mass compared to the control group. Quantitative polymerase chain reaction findings indicated that PCat addition to osteoclast progenitor cultures suppressed osteoclast formation and decreased osteoclast marker expression without affecting the proliferative potential of the osteoclast progenitor cells. Addition of PCat to osteoblast cultures increased osteoblast marker expression. CONCLUSIONS: PCat inhibits osteoclast differentiation and promotes osteoblast differentiation, resulting in increased bone mass in mice. These results suggest that PCat administration is a promising treatment option for conditions associated with bone loss, including osteoporosis.

Tacrolimus, FK506, promotes bone formation in bone defect mouse model.

OBJECTIVES: Some studies have reported that tacrolimus (FK506), an immunosuppressant, may have positive effects on bone formation. However, the precise effects of FK506 on bone repair or osteoblasts remain inadequately elucidated, and limited research has explored the outcomes of its use in an in vivo mouse model. This study aims to examine the effects of FK506 on bone repair and osteoblast functions using bone defect and BMP-2-induced ectopic ossification mouse models, as well as cultured primary mouse osteoblasts treated with FK506. METHODS: We established mouse models of femur bone defect and BMP-2-induced ectopic ossification to evaluate the effect of FK506 on new bone formation, respectively. Additionally, primary mouse osteoblasts were cultured with FK506 and examined for gene expressions related to osteoblast differentiation. RESULTS: While FK506 promoted the repair of bone defect areas in the femur of the bone defect mouse model, it also led to widespread abnormal bone formation outside the intended area. Additionally, following the implantation of a collagen sponge containing BMP-2 into mouse muscle tissue, FK506 was found to promote ectopic ossification and enhance BMP-2-induced osteoblast differentiation in vitro. Our findings also revealed that FK506 increased the number of immature osteoblasts in the absence of BMP-2 without affecting osteoblast differentiation. Furthermore, direct effects were observed, reducing the ability of osteoblasts to support osteoclastogenesis. CONCLUSIONS: These results indicate that FK506 increases new bone formation during bone repair and influences the proliferation of immature osteoblasts, as well as osteoblast-supported osteoclastogenesis.

Abnormal bone regeneration induced by FK506 in medaka fin revealed by in vivo imaging.

OBJECTIVES: Bone tissue in bony fish demonstrates a remarkable ability to regenerate, particularly evident following induction of extensive bone defects, such as fin amputation. This regenerative capacity has been reported to be promoted by the immunosuppressant FK506, yet its precise effects on bone cells during fin regeneration remains insufficiently elucidated. This study aims to investigate the effects of FK506 treatment on bone morphology, osteoblasts, and osteoclasts in the bony fin rays of osterix promoter-DsRed/TRAP promoter-EGFP double transgenic (Tg) medaka. METHODS: The caudal fin of double Tg medaka was amputated, followed by a 20-day treatment with FK506 (1.0 µg/ml) to observe its effects on fin regeneration. Additionally, the regenerated caudal fin area underwent evaluation using genetic analysis and cell proliferation assays. RESULTS: FK506 treatment significantly increased osterix-positive osteoblast formation, resulting in both a significantly longer fin length and fewer joints in the bony fin rays formed during fin regeneration. Notably, TRAP-positive osteoclast formation and bone resorption were observed to occur primarily during the latter stages of fin regeneration. Furthermore, while the expression levels of osteoblast-related genes in the regenerated area remained unchanged following FK506 treatment, a heightened cell proliferation was observed at the tip of the fin. CONCLUSIONS: Our findings suggest that treatment with FK506 promotes bone regeneration by increasing the number of osteoblasts in the amputated area of the fin. However, long-term treatment disrupts regular bone metabolism by inducing abnormal osteoclast formation.

Octacalcium phosphate suppresses chondrogenic differentiation of ATDC5 cells.

Implantation of octacalcium phosphate (OCP), a hydroxyapatite precursor, has been reported to induce chondrogenesis in vivo. In this study, we examined the effects of OCP on the chondrogenic differentiation of mouse chondroblastic ATDC5 cells in vitro. Contrary to our expectation, chondrogenic differentiation of ATDC5 cells evaluated by the mRNA expression of Col2a1, Acan and Col10a1 was suppressed by OCP. Among Sox9, Sox5 and Sox6, essential transcription factors for chondrogenesis, the expression of Sox6 mRNA was markedly lowered by OCP. Whereas ATDC5 cells dissolved OCP to liberate calcium and inorganic phosphorus, increased calcium or phosphate in the medium had little effect on the differentiation of these cells. Direct contact of ATDC5 cells with OCP was required to suppress the expression of Col2a1 and Sox6 mRNAs, whereas the introduction of Sox6 short interfering RNA lowered the expression of Col2a1 mRNA. On the other hand, the forced expression of Sox6 protein partially but significantly, restored the expression of Col2a1 mRNA suppressed by OCP. These results indicate that OCP suppresses the chondrogenic differentiation of ATDC5 cells, at least in part, at the Sox6 transcription level.

Effects of anti-RANKL antibodies administered to pregnant mice on bone and tooth development in neonates.

OBJECTIVES: This study examined how the anti-bone resorptive agent denosumab, which comprises anti-receptor activator of nuclear factor kappa B ligand (anti-RANKL) monoclonal antibodies, administered during pregnancy affected neonatal development. Anti-RANKL antibodies, which are known to bind to mouse RANKL and inhibit osteoclast formation, were administered to pregnant mice. Following this, the survival, growth, bone mineralization, and tooth development of their neonates were analyzed. METHODS: Anti-RANKL antibodies (5 mg/kg) were injected into pregnant mice on day 17 of gestation. After parturition, their neonatal offspring underwent microcomputed tomography at 24 h and at 2, 4, and 6 weeks after birth. Three-dimensional bone and teeth images were subjected to histological analysis. RESULTS: Approximately 70% of the neonatal mice born to mice who received anti-RANKL antibodies died within 6 weeks after birth. These mice had a significantly lower body weight and significantly higher bone mass compared with the control group. Furthermore, delayed tooth eruption and abnormal tooth morphology (eruption length, enamel surface, and cusps) were observed. Conversely, while the tooth germ shape and mothers against decapentaplegic homolog 1/5/8 expression remained unchanged at 24 h after birth in the neonatal mice born to mice that received anti-RANKL antibodies, osteoclasts were not formed. CONCLUSIONS: These results suggest that anti-RANKL antibodies administered to mice in the late stage of pregnancy results in adverse events in their neonatal offspring. Thus, it is speculated that administering denosumab to pregnant humans will affect fetal development and growth after birth.

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.

Monocarboxylate transporter-1 is required for cell death in mouse chondrocytic ATDC5 cells exposed to interleukin-1beta via late phase activation of nuclear factor kappaB and expression of phagocyte-type NADPH oxidase.

Interleukin-1ß (IL-1ß) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1ß-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1ß-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1ß-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1ß-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1ß was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1ß, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1ß. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1ß, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1ß.

D-chiro-inositol negatively regulates the formation of multinucleated osteoclasts by down-regulating NFATc1.

PURPOSE: Osteoclasts (OCs) are multinucleated giant cells that resorb bone matrix. Accelerated bone destruction by OCs might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. D-pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In this study, we analyzed the effect of D-chiro-inositol on OC differentiation. METHODS: To analyze the role of D-chiro-inositol on OC differentiation, we examined OC differentiation by the three types of osteoclastogenesis cultures with tartrate-resistant acid phosphatase (TRAP) staining and solution assay. Then, we carried out cell fusion assay with purified TRAP(+) mononuclear OC precursors. Finally, we analyzed the effect of D-chiro-inositol on OC maker expression in response to the regulation of nuclear factor of activated T cells c1 (NFATc1). RESULTS: We demonstrated that D-chiro-inositol acts as an inhibitor of receptor activator of NF-κB ligand-induced OC differentiation. The formation of multinucleated OCs by cell-cell fusion is reduced by treatment with D-chiro-inositol in a dose-dependent manner. In addition, we demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. CONCLUSIONS: We have shown that D-chiro-inositol is negatively involved in osteoclastogenesis through the inhibition of multinucleated OC formation by cell-cell fusion. The expression of NFATc1 was significantly down-regulated by D-chiro-inositol in OCs and consequently, the expression of OC marker genes was significantly reduced. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.