RESUMO
Clonal hematopoiesis of indeterminate potential (CHIP) arises from aging-associated acquired mutations in hematopoietic progenitors, which display clonal expansion and produce phenotypically altered leukocytes. We associated CHIP-DNMT3A mutations with a higher prevalence of periodontitis and gingival inflammation among 4,946 community-dwelling adults. To model DNMT3A-driven CHIP, we used mice with the heterozygous loss-of-function mutation R878H, equivalent to the human hotspot mutation R882H. Partial transplantation with Dnmt3aR878H/+ bone marrow (BM) cells resulted in clonal expansion of mutant cells into both myeloid and lymphoid lineages and an elevated abundance of osteoclast precursors in the BM and osteoclastogenic macrophages in the periphery. DNMT3A-driven clonal hematopoiesis in recipient mice promoted naturally occurring periodontitis and aggravated experimentally induced periodontitis and arthritis, associated with enhanced osteoclastogenesis, IL-17-dependent inflammation and neutrophil responses, and impaired regulatory T cell immunosuppressive activity. DNMT3A-driven clonal hematopoiesis and, subsequently, periodontitis were suppressed by rapamycin treatment. DNMT3A-driven CHIP represents a treatable state of maladaptive hematopoiesis promoting inflammatory bone loss.
Assuntos
Hematopoiese Clonal , DNA (Citosina-5-)-Metiltransferases , DNA Metiltransferase 3A , Periodontite , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Camundongos , Hematopoiese Clonal/genética , Humanos , Periodontite/genética , Periodontite/patologia , Mutação , Masculino , Feminino , Inflamação/genética , Inflamação/patologia , Osteoclastos/metabolismo , Camundongos Endogâmicos C57BL , Adulto , Interleucina-17/metabolismo , Interleucina-17/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Hematopoese/genética , Osteogênese/genética , Células-Tronco Hematopoéticas/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Pessoa de Meia-IdadeRESUMO
Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κß ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and YwhazKO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.
Assuntos
Proteínas 14-3-3 , Osteoclastos , Ligante RANK , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Animais , Camundongos , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Macrófagos/metabolismo , Camundongos Knockout , Osteoclastos/metabolismo , Osteoclastos/citologia , Estabilidade Proteica , Ligante RANK/metabolismo , Ligante RANK/genética , Células RAW 264.7 , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , UbiquitinaçãoRESUMO
Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.
Assuntos
Trifosfato de Adenosina , Diferenciação Celular , Osteoclastos , Osteoprotegerina , Osteoprotegerina/metabolismo , Osteoprotegerina/genética , Osteoclastos/metabolismo , Osteoclastos/citologia , Animais , Trifosfato de Adenosina/metabolismo , Camundongos , Conexina 43/metabolismo , Conexina 43/genética , Fusão Celular , Antígeno CD47/metabolismo , Antígeno CD47/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Transdução de Sinais , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas do Tecido NervosoRESUMO
Bone diseases are increasing with aging populations and it is important to identify clues to develop innovative treatments. Vasn, which encodes vasorin (Vasn), a transmembrane protein involved in the pathophysiology of several organs, is expressed during the development in intramembranous and endochondral ossification zones. Here, we studied the impact of Vasn deletion on the osteoblast and osteoclast dialog through a cell Coculture model. In addition, we explored the bone phenotype of Vasn KO mice, either constitutive or tamoxifen-inducible, or with an osteoclast-specific deletion. First, we show that both osteoblasts and osteoclasts express Vasn. Second, we report that, in both KO mouse models but not in osteoclast-targeted KO mice, Vasn deficiency was associated with an osteopenic bone phenotype, due to an imbalance in favor of osteoclastic resorption. Finally, through the Coculture experiments, we identify a dysregulation of the Wnt/ß-catenin pathway together with an increase in RANKL release by osteoblasts, which led to an enhanced osteoclast activity. This study unravels a direct role of Vasn in bone turnover, introducing a new biomarker or potential therapeutic target for bone pathologies.
Assuntos
Remodelação Óssea , Técnicas de Cocultura , Osteoblastos , Osteoclastos , Via de Sinalização Wnt , Animais , Camundongos , Osso e Ossos/metabolismo , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , Remodelação Óssea/fisiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Ligante RANK/metabolismo , Ligante RANK/genéticaRESUMO
Several members of the transforming growth factor beta (TGF-ß) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-ß superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.
Assuntos
Diferenciação Celular , Proteína Nodal , Osteoclastos , Fator de Transcrição STAT1 , Animais , Camundongos , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição NFATC/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Fosforilação , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Ligante RANK/metabolismo , Transdução de Sinais , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Masculino , Camundongos Endogâmicos ICR , Proteína Nodal/genética , Proteína Nodal/metabolismo , Proteína Nodal/farmacologiaRESUMO
Abnormalities in osteoclastic generation or activity disrupt bone homeostasis and are highly involved in many pathologic bone-related diseases, including rheumatoid arthritis, osteopetrosis, and osteoporosis. Control of osteoclast-mediated bone resorption is crucial for treating these bone diseases. However, the mechanisms of control of osteoclastogenesis are incompletely understood. In this study, we identified that inosine 5'-monophosphate dehydrogenase type II (Impdh2) positively regulates bone resorption. By histomorphometric analysis, Impdh2 deletion in mouse myeloid lineage cells (Impdh2LysM-/- mice) showed a high bone mass due to the reduced osteoclast number. qPCR and western blotting results demonstrated that the expression of osteoclast marker genes, including Nfatc1, Ctsk, Calcr, Acp5, Dcstamp, and Atp6v0d2, was significantly decreased in the Impdh2LysM-/- mice. Furthermore, the Impdh inhibitor MPA treatment inhibited osteoclast differentiation and induced Impdh2-cytoophidia formation. The ability of osteoclast differentiation was recovered after MPA deprivation. Interestingly, genome-wide analysis revealed that the osteoclastic mitochondrial biogenesis and functions, such as oxidative phosphorylation, were impaired in the Impdh2LysM-/- mice. Moreover, the deletion of Impdh2 alleviated ovariectomy-induced bone loss. In conclusion, our findings revealed a previously unrecognized function of Impdh2, suggesting that Impdh2-mediated mechanisms represent therapeutic targets for osteolytic diseases.
Assuntos
IMP Desidrogenase , Mitocôndrias , Osteoclastos , Osteogênese , Osteoporose , Ovariectomia , Fosforilação Oxidativa , Animais , Feminino , Camundongos , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Reabsorção Óssea/etiologia , Diferenciação Celular , IMP Desidrogenase/metabolismo , IMP Desidrogenase/genética , IMP Desidrogenase/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/metabolismo , Osteoporose/etiologia , Osteoporose/genética , Osteoporose/patologiaRESUMO
OBJECTIVE: Osteoporosis is a global health issue characterized by decreased bone mass and microstructural degradation, leading to an increased risk of fractures. This study aims to explore the molecular mechanism by which P2X7 receptors influence osteoclast formation and bone resorption through the PI3K-Akt-GSK3ß signaling pathway. METHODS: An osteoporosis mouse model was generated through ovariectomy (OVX) in normal C57BL/6 and P2X7f/f; LysM-cre mice. Osteoclasts were isolated for transcriptomic analysis, and differentially expressed genes were selected for functional enrichment analysis. Metabolite analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and multivariate statistical analysis and pattern recognition were used to identify differential lipid metabolism markers and their distribution. Bioinformatics analyses were conducted using the Encyclopedia of Genes and Genomes database and the MetaboAnalyst database to assess potential biomarkers and create a metabolic pathway map. Osteoclast precursor cells were used for in vitro cell experiments, evaluating cell viability and proliferation using the Cell Counting Kit 8 (CCK-8) assay. Osteoclast precursor cells were induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-beta ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) staining was performed to compare differentiation morphology, size, and quantity between different groups. Western blot analysis was used to assess the expression of differentiation markers, fusion gene markers, and bone resorption ability markers in osteoclasts. Immunofluorescence staining was employed to examine the spatial distribution and quantity of osteoclast cell skeletons, P2X7 protein, and cell nuclei, while pit assay was used to evaluate osteoclast bone resorption ability. Finally, in vivo animal experiments, including micro computed tomography (micro-CT), hematoxylin and eosin (HE) staining, TRAP staining, and immunohistochemistry, were conducted to observe bone tissue morphology, osteoclast differentiation, and the phosphorylation level of the PI3K-Akt-GSK3ß signaling pathway. RESULTS: Transcriptomic and metabolomic data collectively reveal that the P2X7 receptor can impact the pathogenesis of osteoporosis through the PI3K-Akt-GSK3ß signaling pathway. Subsequent in vitro experiments showed that cells in the Sh-P2X7 + Recilisib group exhibited increased proliferative activity (1.15 versus 0.59), higher absorbance levels (0.68 versus 0.34), and a significant increase in resorption pit area (13.94 versus 3.50). Expression levels of osteoclast differentiation-related proteins MMP-9, CK, and NFATc1 were markedly elevated (MMP-9: 1.72 versus 0.96; CK: 2.54 versus 0.95; NFATc1: 3.05 versus 0.95), along with increased fluorescent intensity of F-actin rings. In contrast, the OE-P2X7 + LY294002 group showed decreased proliferative activity (0.64 versus 1.29), reduced absorbance (0.34 versus 0.82), and a significant decrease in resorption pit area (5.01 versus 14.96), accompanied by weakened expression of MMP-9, CK, and NFATc1 (MMP-9: 1.14 versus 1.79; CK: 1.26 versus 2.75; NFATc1: 1.17 versus 2.90) and decreased F-actin fluorescent intensity. Furthermore, in vivo animal experiments demonstrated that compared with the wild type (WT) + Sham group, mice in the WT + OVX group exhibited significantly increased levels of CTX and NTX in serum (CTX: 587.17 versus 129.33; NTX: 386.00 versus 98.83), a notable decrease in calcium deposition (19.67 versus 53.83), significant reduction in bone density, increased trabecular separation, and lowered bone mineral density (BMD). When compared with the KO + OVX group, mice in the KO + OVX + recilisib group showed a substantial increase in CTX and NTX levels in serum (CTX: 503.50 versus 209.83; NTX: 339.83 versus 127.00), further reduction in calcium deposition (29.67 versus 45.33), as well as decreased bone density, increased trabecular separation, and reduced BMD. CONCLUSION: P2X7 receptors positively regulate osteoclast formation and bone resorption by activating the PI3K-Akt-GSK3ß signaling pathway.
Assuntos
Reabsorção Óssea , Diferenciação Celular , Glicogênio Sintase Quinase 3 beta , Camundongos Endogâmicos C57BL , Osteoclastos , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Receptores Purinérgicos P2X7 , Transdução de Sinais , Animais , Feminino , Camundongos , Reabsorção Óssea/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Osteoclastos/metabolismo , Osteoporose/metabolismo , Osteoporose/genética , Osteoporose/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Ligante RANK/metabolismo , Ligante RANK/genética , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genéticaRESUMO
Long noncoding RNAs (lncRNAs) are important regulators of bone metabolism. In this study, lncRNA microarray analysis was used to identify differentially expressed lncRNAs in differentiated osteoclasts. lncRNA-Gm5532 is highly expressed during osteoclast differentiation. lncRNA-Gm5532 knockdown impairs osteoclast formation and bone resorption. Mechanistic experiments show that lncRNA-Gm5532 functions as a competing endogenous RNA (ceRNA) and acts as a sponge for miR-125a-3p, which promotes TNF receptor-associated factor 6 (TRAF6) expression. miR-125a-3p mimics suppress osteoclast differentiation and TAK1/NF-κB/MAPK signaling. The miR-125a-3p inhibitor reverses the negative effects of siGm5532 on osteoclast differentiation. In summary, our study reveals that lncRNA-Gm5532 functions as an activator in osteoclast differentiation by targeting the miR-125a-3p/TRAF6 axis, making it a novel biomarker and potential therapeutic target for osteoporosis.
Assuntos
Reabsorção Óssea , MicroRNAs , RNA Longo não Codificante , Humanos , MicroRNAs/metabolismo , Osteoclastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismoRESUMO
Globally, osteoporosis is the most common systemic skeletal disease. There are many factors that influence osteoporosis' development and progression. During the pathogenesis of this disease, bone turnover is imbalanced between resorption and the formation of bone tissue. A growing interest has been devoted to the role that microRNA (miRNA) plays in osteoporosis regulation. A microRNA (miRNA) is a group of small single-stranded RNA molecules involved in regulating gene expression in eukaryotic organisms. As microRNAs (miRNAs) are key regulators of gene expression and can modulate processes related to bone metabolism, they have become increasingly important for studying osteoporosis pathogenesis. The available research suggests that miRNAs play an important role in regulating processes associated with bone metabolism, especially by influencing bone resorption and synthesis. Furthermore, microRNAs can also serve as potential therapeutic targets for osteoporosis, besides being a rapid and specific biomarker.
Assuntos
MicroRNAs , Osteoporose , Osteoporose/genética , Osteoporose/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Animais , Regulação da Expressão Gênica , Biomarcadores , Osso e Ossos/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismoRESUMO
Bcl2l1 (Bcl-XL) belongs to the Bcl-2 family, Bcl2 and Bcl2-XL are major anti-apoptotic proteins, and the apoptosis of osteoblasts is a key event for bone homeostasis. As the functions of Bcl2l1 in osteoblasts and bone homeostasis remain unclear, we generated osteoblast-specific Bcl2l1-deficient (Bcl2l1fl/flCre) mice using 2.3-kb Col1a1 Cre. Trabecular bone volume and the trabecular number were lower in Bcl2l1fl/flCre mice of both sexes than in Bcl2l1fl/fl mice. In bone histomorphometric analysis, osteoclast parameters were increased in Bcl2l1fl/flCre mice, whereas osteoblast parameters and the bone formation rate were similar to those in Bcl2l1fl/fl mice. TUNEL-positive osteoblastic cells and serum TRAP5b levels were increased in Bcl2l1fl/flCre mice. The deletion of Bcl2l1 in osteoblasts induced Tnfsf11 expression, whereas the overexpression of Bcl-XL had no effect. In a co-culture of Bcl2l1-deficient primary osteoblasts and wild-type bone-marrow-derived monocyte/macrophage lineage cells, the numbers of multinucleated TRAP-positive cells and resorption pits increased. Furthermore, serum deprivation or the deletion of Bcl2l1 in primary osteoblasts increased apoptosis and ATP levels in the medium. Therefore, the reduction in trabecular bone in Bcl2l1fl/flCre mice may be due to enhanced bone resorption through osteoblast apoptosis and the release of ATP from apoptotic osteoblasts, and Bcl2l1 may inhibit bone resorption by preventing osteoblast apoptosis.
Assuntos
Reabsorção Óssea , Osteogênese , Animais , Feminino , Masculino , Camundongos , Trifosfato de Adenosina/metabolismo , Apoptose/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osso Esponjoso/metabolismo , Diferenciação Celular , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (knockout [KO]), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low-calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.
Assuntos
Fibronectinas , Camundongos Knockout , Osteócitos , Animais , Feminino , Osteócitos/metabolismo , Masculino , Camundongos , Fibronectinas/metabolismo , Fibronectinas/genética , Fatores Sexuais , Reabsorção Óssea/genéticaRESUMO
Bone remodeling is essential for the repair and replacement of damaged or aging bones. Continuous remodeling is necessary to prevent the accumulation of bone damage and to maintain bone strength and calcium balance. As bones age, the coupling mechanism between bone formation and absorption becomes dysregulated, and bone loss becomes dominant. Bone development and repair rely on interaction and communication between osteoclasts and surrounding cells. Osteoclasts are specialized cells that are accountable for bone resorption and degradation, and any abnormalities in their activity can result in notable alterations in bone structure and worsen disease symptoms. Recent findings from transgenic mouse models and bone analysis have greatly enhanced our understanding of the origin, differentiation pathway, and activation stages of osteoclasts. In this review, we explore osteoclasts and discuss the cellular and molecular events that drive their generation, focusing on intracellular oxidative and antioxidant signaling. This knowledge can help develop targeted therapies for diseases associated with osteoclast activation.
Assuntos
Reabsorção Óssea , Osteoclastos , Camundongos , Animais , Osteoclastos/metabolismo , Antioxidantes/metabolismo , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular , Camundongos Transgênicos , OxirreduçãoRESUMO
Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for â¼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation.
Assuntos
Reabsorção Óssea , Osteoclastos , Animais , Camundongos , Osteoclastos/metabolismo , Kisspeptinas/genética , Kisspeptinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Camundongos Knockout , Reabsorção Óssea/genética , Receptores de Kisspeptina-1RESUMO
Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.
Assuntos
Reabsorção Óssea , Fatores Reguladores de Interferon , Reabsorção da Raiz , Animais , Humanos , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Mutação , Fatores de Transcrição NFATC/genética , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Reabsorção da Raiz/genética , Reabsorção da Raiz/metabolismoRESUMO
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by synovitis, bone and cartilage destruction, and increased fracture risk with bone loss. Although disease-modifying antirheumatic drugs have dramatically improved clinical outcomes, these therapies are not universally effective in all patients because of the heterogeneity of RA pathogenesis. Therefore, it is necessary to elucidate the molecular mechanisms underlying RA pathogenesis, including associated bone loss, in order to identify novel therapeutic targets. In this study, we found that Budding uninhibited by benzimidazoles 1 (BUB1) was highly expressed in RA patients' synovium and murine ankle tissue with arthritis. As CD45+CD11b+ myeloid cells are a Bub1 highly expressing population among synovial cells in mice, myeloid cell-specific Bub1 conditional knockout (Bub1ΔLysM) mice were generated. Bub1ΔLysM mice exhibited reduced femoral bone mineral density when compared with control (Ctrl) mice under K/BxN serum-transfer arthritis, with no significant differences in joint inflammation or bone erosion based on a semi-quantitative erosion score and histological analysis. Bone histomorphometry revealed that femoral bone mass of Bub1ΔLysM under arthritis was reduced by increased osteoclastic bone resorption. RNA-seq and subsequent Gene Set Enrichment Analysis demonstrated a significantly enriched nuclear factor-kappa B pathway among upregulated genes in receptor activator of nuclear factor kappa B ligand (RANKL)-stimulated bone marrow-derived macrophages (BMMs) obtained from Bub1ΔLysM mice. Indeed, osteoclastogenesis using BMMs derived from Bub1ΔLysM was enhanced by RANKL and tumor necrosis factor-α or RANKL and IL-1ß treatment compared with Ctrl. Finally, osteoclastogenesis was increased by Bub1 inhibitor BAY1816032 treatment in BMMs derived from wildtype mice. These data suggest that Bub1 expressed in macrophages plays a protective role against inflammatory arthritis-associated bone loss through inhibition of inflammation-mediated osteoclastogenesis.
Rheumatoid arthritis (RA) is a disease caused by an abnormal immune system, resulting in inflammation, swelling, and bone destruction in the joints, along with systemic bone loss. While new medications have dramatically improved treatment efficacy, these therapies are not universally effective for all patients. Therefore, we need to understand the regulatory mechanisms behind RA, including associated bone loss, to develop better therapies. In this study, we found that Budding uninhibited by benzimidazoles 1 (Bub1) was highly expressed in inflamed joints, especially in myeloid cells, which are a type of immune cells. To explore its role, we created myeloid cellspecific Bub1 conditional knockout (cKO) mice and induced arthritis to analyze its role during arthritis. The cKO mice exhibited lower bone mineral density when compared with control mice under inflammatory arthritis because of increased osteoclastic bone resorption, without significant differences in joint inflammation or bone erosion. Further investigation showed that Bub1 prevents excessive osteoclast differentiation induced by inflammation in bone marrow macrophages. These data suggest that Bub1 in macrophages protects against bone loss caused by inflammatory arthritis, offering potential insights for developing treatments that focus on bone health.
Assuntos
Artrite Experimental , Artrite Reumatoide , Doenças Ósseas Metabólicas , Reabsorção Óssea , Animais , Humanos , Camundongos , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Doenças Ósseas Metabólicas/patologia , Reabsorção Óssea/genética , Inflamação/patologia , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.
Assuntos
Reabsorção Óssea , Periodontite , Receptor EphB2 , Receptor EphB4 , Animais , Ratos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/microbiologia , Osteoclastos/metabolismo , Periodontite/microbiologia , Porphyromonas gingivalis/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Transdução de Sinais , Receptor EphB2/metabolismo , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/microbiologiaRESUMO
Histone methylation plays a crucial role in various cellular processes. We previously reported the in vitro function of histone lysine demethylase 7 A (KDM7A) in osteoblast and adipocyte differentiation. The current study was undertaken to investigate the physiological role of KDM7A in bone homeostasis and elucidate the underlying mechanisms. A conditional strategy was employed to delete the Kdm7a gene specifically in osterix-expressing osteoprogenitor cells in mice. The resulting mutant mice exhibited a significant increase in cancellous bone mass, accompanied by an increase in osteoblasts and bone formation, as well as a reduction in osteoclasts, marrow adipocytes and bone resorption. The bone marrow stromal cells (BMSCs) and calvarial pre-osteoblastic cells derived from the mutant mice exhibited enhanced osteogenic differentiation and suppressed adipogenic differentiation. Additionally, osteoclastic precursor cells from the mutant mice exhibited impaired osteoclast differentiation. Co-culturing BMSCs from the mutant mice with wild-type osteoclast precursor cells resulted in the inhibition of osteoclast differentiation. Mechanistic investigation revealed that KDM7A was able to upregulate the expression of fibroblast activation protein α (FAP) and receptor activator of nuclear factor κB ligand (RANKL) in BMSCs through removing repressive di-methylation marks of H3K9 and H3K27 from Fap and Rankl promoters. Moreover, recombinant FAP attenuated the dysregulation of osteoblast and adipocyte differentiation in BMSCs from Kdm7a deficient mice. Finally, Kdm7a deficiency prevented ovariectomy-induced bone loss in mice. This study establish the role of KDM7A in bone homeostasis through its epigenetic regulation of osteoblast and osteoclast differentiation. Consequently, inhibiting KDM7A may prove beneficial in ameliorating osteoporosis. KDM7A suppresses osteoblast differentiation and bone formation through. upregulating FAP expression and inactivating canonical Wnt signaling, and conversely promotes osteoclast differentiation and bone resorption through upregulating RANKL expression. These are based on its epigenetic removal of the repressive H3K9me2 and H3K27me2 marks from Fap and Rankl promoters. As a result, the expression of KDM7A in osteoprogenitor cells tends to negatively modulate bone mass.
Assuntos
Reabsorção Óssea , Histona Desmetilases com o Domínio Jumonji , Osteoclastos , Animais , Feminino , Camundongos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Epigênese Genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Homeostase , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Ligante RANK/genética , Ligante RANK/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismoRESUMO
Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.
Assuntos
Reabsorção Óssea , Osteoporose , Feminino , Camundongos , Animais , Humanos , Osteoclastos/metabolismo , Microtomografia por Raio-X , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osteoporose/tratamento farmacológico , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Diferenciação Celular , NF-kappa B/genética , NF-kappa B/metabolismo , OvariectomiaRESUMO
Declining estrogen production in postmenopausal females causes osteoporosis in which the resorption of bone exceeds the increase in bone formation. Although clinical drugs are currently available for the treatment of osteoporosis, sustained medication use is accompanied by serious side effects. Corydalis bungeana Herba, a famous traditional Chinese herb listed in the Chinese Pharmacopoeia Commission, constitutes various traditional Chinese Medicine prescriptions, which date back to thousands of years. One of the primary active components of C. bungeana Turcz. is Corynoline (Cor), a plant isoquinoline alkaloid derived from the Corydalis species, which possesses bone metabolism disease therapeutic potential. The study aimed at exploring the effects as well as mechanisms of Cor on osteoclast formation and bone resorption. TRAcP staining, F-actin belt formation, and pit formation were employed for assessing the osteoclast function. Western blot, qPCR, network pharmacology, and docking analyses were used for analyzing the expression of osteoclast-associated genes and related signaling pathways. The study focused on investigating how Cor affected OVX-induced trabecular bone loss by using a mouse model. Cor could weaken osteoclast formation and function by affecting the biological receptor activators of NF-κB and its ligand at various concentrations. Mechanistically, Cor inhibited the NF-κB activation, and the MAPKs pathway stimulated by RANKL. Besides, Cor enhanced the protein stability of the Nrf2, which effectively abolished the RANKL-stimulated ROS generation. According to an OVX mouse model, Cor functions in restoring bone mass, improving microarchitecture, and reducing the ROS levels in the distal femurs, which corroborated with its in vitro antiosteoclastogenic effect. The present study indicates that Cor may restrain osteoclast formation and bone loss by modulating NF-κB/MAPKs and Nrf2 signaling pathways. Cor was shown to be a potential drug candidate that can be utilized for the treatment of osteoporosis.
Assuntos
Alcaloides de Berberina , Reabsorção Óssea , Osteoporose , Feminino , Humanos , Osteogênese , NF-kappa B/genética , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Osteoclastos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/genética , Osteoporose/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Diferenciação CelularRESUMO
Mechanical loading is required for bone homeostasis, but the underlying mechanism is still unclear. Our previous studies revealed that the mechanical protein polycystin-1 (PC1, encoded by Pkd1) is critical for bone formation. However, the role of PC1 in bone resorption is unknown. Here, we found that PC1 directly regulates osteoclastogenesis and bone resorption. The conditional deletion of Pkd1 in the osteoclast lineage resulted in a reduced number of osteoclasts, decreased bone resorption, and increased bone mass. A cohort study of 32,500 patients further revealed that autosomal dominant polycystic kidney disease, which is mainly caused by loss-of-function mutation of the PKD1 gene, is associated with a lower risk of hip fracture than those with other chronic kidney diseases. Moreover, mice with osteoclast-specific knockout of Pkd1 showed complete resistance to unloading-induced bone loss. A mechanistic study revealed that PC1 facilitated TAZ nuclear translocation via the C-terminal tail-TAZ complex and that conditional deletion of Taz in the osteoclast lineage resulted in reduced osteoclastogenesis and increased bone mass. Pharmacological regulation of the PC1-TAZ axis alleviated unloading- and estrogen deficiency- induced bone loss. Thus, the PC1-TAZ axis may be a potential therapeutic target for osteoclast-related osteoporosis.