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1.
Int J Mol Med ; 47(4): 1-9, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33604678

RESUMO

Glucosamine (GlcN) functions as a building block of the cartilage matrix, and its multifaceted roles in promoting joint health have been extensively investigated. However, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. As a result, the benefit of GlcN application in bone disorders remains controversial. In order to further elucidate the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, the effect of GlcN treatment was investigated in human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were cultured with two­dimensional (2D) traditional methods or co­cultured in a 3D dynamic system more closely resembling the in vivo bone microenvironment. Under these conditions, osteoclastogenesis was supported by hOBs and sizeable self­assembling aggregates were obtained. The differentiated hOCs were evaluated using tartrate­resistant acid phosphatase assays and osteogenic differentiation was monitored by analyzing mineral matrix deposition via Alizarin Red staining, with expression of specific osteogenic markers determined via reverse transcription­quantitative PCR. It was found that crystalline GlcN sulfate was effective in decreasing osteoclastic cell differentiation and function. hOCs isolated from patients with OA were more sensitive compared with those from healthy donors. Additionally, GlcN exhibited anabolic effects on hOCs both in 2D conventional cell culture and in hOC/hOB 3D dynamic co­culture. The present study demonstrated for the first time the effectiveness of a 3D dynamic co­culture system for characterizing the spectrum of action of GlcN on the bone microenvironment, which may pave the way for more fully determining the potential applications of a compound such as GlcN, which is positioned between pharmaceuticals and nutraceuticals. Based on the present findings, it is hypothesized that GlcN may have potential benefits in the treatment of osteopenic diseases such as osteoporosis, as well as in bone maintenance.


Assuntos
Osso e Ossos/metabolismo , Glucosamina/farmacologia , Osteoblastos/citologia , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Adulto , Idoso , Apoptose/efeitos dos fármacos , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoporose/tratamento farmacológico
2.
Int J Mol Sci ; 22(1)2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33406741

RESUMO

BACKGROUND: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK-RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis.; Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. RESULTS: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3ß phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models; and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.


Assuntos
Proteínas Mutantes/metabolismo , Mutação , Osteoclastos/citologia , Osteogênese , Ligante RANK/metabolismo , Animais , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/administração & dosagem , Proteínas Mutantes/genética , Osteoclastos/metabolismo , Ligante RANK/genética , Transdução de Sinais
3.
J Bone Miner Metab ; 39(1): 13-18, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33385253

RESUMO

RANKL, the essential cue for osteoclast differentiation, is the membrane-bound factor expressed by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes. In vivo evidence indicates that RANKL functions as the indispensable and irreplaceable in the program of osteoclast differentiation. The reason why RANKL plays a critical role in osteoclastogenesis is discussed from the viewpoint of the distinct signaling pathways mediated by co-stimulatory receptors and the key transcription factor NFATc1.


Assuntos
Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Animais , Humanos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteogênese , Transdução de Sinais
4.
Arch Oral Biol ; 122: 105029, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33387850

RESUMO

OBJECTIVE: The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN: Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. RESULTS: CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. CONCLUSION: Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.


Assuntos
Reabsorção Óssea , Diferenciação Celular/efeitos dos fármacos , Chrysanthemum/química , Osteoclastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ligante RANK/metabolismo , Animais , Células da Medula Óssea , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo
5.
Nat Commun ; 12(1): 496, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479228

RESUMO

Chronic inflammation during many diseases is associated with bone loss. While interferons (IFNs) are often inhibitory to osteoclast formation, the complex role that IFN and interferon-stimulated genes (ISGs) play in osteoimmunology during inflammatory diseases is still poorly understood. We show that mice deficient in IFN signaling components including IFN alpha and beta receptor 1 (IFNAR1), interferon regulatory factor 1 (IRF1), IRF9, and STAT1 each have reduced bone density and increased osteoclastogenesis compared to wild type mice. The IFN-inducible guanylate-binding proteins (GBPs) on mouse chromosome 3 (GBP1, GBP2, GBP3, GBP5, GBP7) are required to negatively regulate age-associated bone loss and osteoclastogenesis. Mechanistically, GBP2 and GBP5 both negatively regulate in vitro osteoclast differentiation, and loss of GBP5, but not GBP2, results in greater age-associated bone loss in mice. Moreover, mice deficient in GBP5 or chromosome 3 GBPs have greater LPS-mediated inflammatory bone loss compared to wild type mice. Overall, we find that GBP5 contributes to restricting age-associated and inflammation-induced bone loss by negatively regulating osteoclastogenesis.


Assuntos
Reabsorção Óssea/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Interferons/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Fatores Etários , Animais , Reabsorção Óssea/genética , Diferenciação Celular/genética , Fusão Celular , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoclastos/citologia , Osteogênese/genética , Transdução de Sinais/genética
6.
Cell Prolif ; 54(2): e12973, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33382502

RESUMO

OBJECTIVES: NLRP3 inflammasome is a critical part of the innate immune system and plays an important role in a variety of inflammatory diseases. However, the effects of NLRP3 inflammasome on periodontitis have not been fully studied. MATERIALS AND METHODS: We used ligature-induced periodontitis models of NLRP3 knockout mice (NLRP3KO ) and their wildtype (WT) littermates to compare their alveolar bone phenotypes. We further used Lysm-Cre/RosanTnG mouse to trace the changes of Lysm-Cre+ osteoclast precursors in ligature-induced periodontitis with or without MCC950 treatment. At last, we explored MCC950 as a potential drug for the treatment of periodontitis in vivo and in vitro. RESULTS: Here, we showed that the number of osteoclast precursors, osteoclast differentiation and alveolar bone loss were reduced in NLRP3KO mice compared with WT littermates, by using ligature-induced periodontitis model. Next, MCC950, a specific inhibitor of the NLRP3 inflammasome, was used to inhibit osteoclast precursors differentiation into osteoclast. Further, we used Lysm-Cre/RosanTnG mice to demonstrate that MCC950 decreases the number of Lysm-Cre+ osteoclast precursors in ligature-induced periodontitis. At last, treatment with MCC950 significantly suppressed alveolar bone loss with reduced IL-1ß activation and osteoclast differentiation in ligature-induced periodontitis. CONCLUSION: Our findings reveal that NLRP3 regulates alveolar bone loss in ligature-induced periodontitis by promoting osteoclastic differentiation.


Assuntos
Perda do Osso Alveolar/patologia , Diferenciação Celular , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteoclastos/citologia , Periodontite/patologia , Perda do Osso Alveolar/metabolismo , Perda do Osso Alveolar/prevenção & controle , Animais , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Periodontite/tratamento farmacológico , Periodontite/etiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Sulfonas/farmacologia , Sulfonas/uso terapêutico
7.
Int J Nanomedicine ; 15: 6355-6372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922006

RESUMO

Background: Cerium oxide nanoparticles (CeO2NPs) are potent scavengers of cellular reactive oxygen species (ROS). Their antioxidant properties make CeO2NPs promising therapeutic agents for bone diseases and bone tissue engineering. However, the effects of CeO2NPs on intracellular ROS production in osteoclasts (OCs) are still unclear. Numerous studies have reported that intracellular ROS are essential for osteoclastogenesis. The aim of this study was to explore the effects of CeO2NPs on osteoclast differentiation and the potential underlying mechanisms. Methods: The bidirectional modulation of osteoclast differentiation by CeO2NPs was explored by different methods, such as fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. The cytotoxic and proapoptotic effects of CeO2NPs were detected by cell counting kit (CCK-8) assay, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and flow cytometry. Results: The results of this study demonstrated that although CeO2NPs were capable of scavenging ROS in acellular environments, they facilitated the production of ROS in the acidic cellular environment during receptor activator of nuclear factor kappa-Β ligand (RANKL)-dependent osteoclast differentiation of bone marrow-derived macrophages (BMMs). CeO2NPs at lower concentrations (4.0 µg/mL to 8.0 µg/mL) promoted osteoclast formation, as shown by increased expression of Nfatc1 and C-Fos, F-actin ring formation and bone resorption. However, at higher concentrations (greater than 16.0 µg/mL), CeO2NPs inhibited osteoclast differentiation and promoted apoptosis of BMMs by reducing Bcl2 expression and increasing the expression of cleaved caspase-3, which may be due to the overproduction of ROS. Conclusion: This study demonstrates that CeO2NPs facilitate osteoclast formation at lower concentrations while inhibiting osteoclastogenesis in vitro by inducing the apoptosis of BMMs at higher concentrations by modulating cellular ROS levels.


Assuntos
Diferenciação Celular , Cério/química , Osteoclastos/citologia , Espécies Reativas de Oxigênio/metabolismo , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nanopartículas/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
8.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32953088

RESUMO

Obesity and osteoporosis are both common conditions with high rates of morbidity and mortality. There is a relationship between obesity and bone. There are multiple factors that influence the risk of fracture, including the quality of bone, the risk of falls, and the padding around the bone. These multiple factors partly explain the finding that obesity protects against fractures in some sites while increasing the risk in other parts of the body. While it is well known that increased weight builds bone, there are several mechanisms related to the obese state that make the bone more fragile. These include the increased production of bone marrow fat cells at the expense of bone-forming osteoblasts, an increase in inflammatory cytokines leading to the activation of bone-resorbing osteoclasts, mutations in the FTO gene, and obesity-induced increased osteoblast senescence. Surprisingly, the relationship between bone and obesity is not unidirectional; there is now evidence that osteocytes are able to regulate body weight by acting as weighing machines.


Assuntos
Osso e Ossos/patologia , Obesidade/fisiopatologia , Osteoblastos/citologia , Osteoclastos/citologia , Osteócitos/citologia , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Senescência Celular , Humanos
9.
Chem Biol Interact ; 331: 109235, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971123

RESUMO

Ellagic acid (EA) is a naturally occurring polyphenolic compound that has been shown to exhibit diverse beneficial pharmacological activities including anti-osteoclastogenesis effect. However, the molecular mechanism by which EA inhibits osteoclastogenesis remains to be elucidated. The protein-protein interaction between receptor activator of nuclear factor (NF)-κB ligand (RANKL) and its receptor RANK contributes to osteoclast differentiation and activation in bone remodeling, and is regarded as an important therapeutic target for the treatment of osteoporosis. The current study is focused on investigating whether EA can directly bind to RANKL and/or RANK and block the interaction between RANKL and RANK, thereby inhibiting downstream signaling pathways. Interestingly, we found that EA had strong affinities to RANK and RANKL, with the estimated equilibrium dissociation constants (KD) of 2.485 × 10-11 and 1.688 × 10-9 M, respectively, and could disrupt the interaction between RANKL and RANK, thereby inhibiting RANKL-induced canonical RANK signaling pathways (p65, JNK, ERK, and p38) and expression of downstream master transcriptional factors (NFATc1 and c-Fos) and osteoclast-specific genes and proteins (TRAP, c-Src, and cathepsin K), which could ultimately suppress RANKL-induced osteoclast differentiation and F-actin ring formation. Taken together, our results revealed that EA could block RANKL-RANK interaction and suppress RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways in RAW 264.7 murine macrophages.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Elágico/farmacologia , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Cinética , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/farmacologia , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismo
10.
Cell Prolif ; 53(10): e12912, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32964544

RESUMO

OBJECTIVES: Mechanical force plays an important role in modulating stem cell fate and behaviours. However, how periodontal ligament stem cells (PDLSCs) perceive mechanical stimulus and transfer it into biological signals, and thereby promote alveolar bone remodelling, is unclear. MATERIALS AND METHODS: An animal model of force-induced tooth movement and a compressive force in vitro was used. After force application, tooth movement distance, mesenchymal stem cell and osteoclast number, and proinflammatory cytokine expression were detected in periodontal tissues. Then, rat primary PDLSCs with or without force loading were isolated, and their stem cell characteristics including clonogenicity, proliferation, multipotent differentiation and immunoregulatory properties were evaluated. Under compressive force in vitro, the effects of the ERK signalling pathway on PDLSC characteristics were evaluated by Western blotting. RESULTS: Mechanical force in vivo induced PDLSC proliferation, which was accompanied with inflammatory cytokine accumulation, osteoclast differentiation and TRPV4 activation; the force-stimulated PDLSCs showed greater clonogenicity and proliferation, reduced differentiation ability, improved induction of macrophage migration, osteoclast differentiation and proinflammatory factor expression. The biological changes induced by mechanical force could be partially suppressed by TRPV4 inhibition. Mechanistically, force-induced activation of TRPV4 in PDLSCs regulated osteoclast differentiation by affecting the RANKL/OPG system via ERK signalling. CONCLUSIONS: Taken together, we show here that TRPV4 activation in PDLSCs under mechanical force contributes to changing their stem cell characteristics and modulates bone remodelling during tooth movement.


Assuntos
Remodelação Óssea , Ligamento Periodontal/citologia , Células-Tronco/citologia , Canais de Cátion TRPV/metabolismo , Animais , Fenômenos Biomecânicos , Proliferação de Células , Células Cultivadas , Humanos , Masculino , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligamento Periodontal/metabolismo , Ratos , Ratos Sprague-Dawley , Células-Tronco/metabolismo , Estresse Mecânico
11.
Cell Prolif ; 53(10): e12882, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32871020

RESUMO

OBJECTIVES: Intracellular reactive oxygen species (ROS) induced by receptor activator of NF-kB ligand (RANKL) has been proven to be a critical factor in the development of osteoclasts. This study aimed to prove that schisandrin A (Sch), a novel anti-oxidant compound, is able to suppress osteoclastogenesis and prevent bone loss in ovariectomized (OVX) mice by suppressing ROS via nuclear factor erythroid 2-related factor (Nrf2). MATERIAL AND METHODS: Micro-CT was used to detect bone formation. The effects of Sch on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS) were measured by dihydroethidium (DHE) staining in vivo and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in vitro. Immunofluorescence staining was used to detect the expression of Nrf2 in vivo. siRNA was used to evaluate the effect of Nrf2 in osteoclastogenesis. RESULTS: Sch suppresses RANKL-induced ROS production by regulating nuclear factor erythroid 2-related factor (Nrf2) in vitro and vivo. Mechanistically, Sch enhances the expression of Nrf2 by regulating the degradation of Nrf2. Further, Sch suppresses phosphorylation of P65 and its nuclear translocation, as well as the degradation of IκBα. Collectively, our findings reveal that Sch protects against OVX-induced bone loss by suppressing ROS via Nrf2. CONCLUSIONS: Our results showed the potential of anti-oxidant compound schisandrin A in the treatment of osteoporosis, highlighting Nrf2 as a novel promising target in osteoclast-related disease.


Assuntos
Ciclo-Octanos/farmacologia , Lignanas/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Osteogênese/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Feminino , Fêmur/diagnóstico por imagem , Fêmur/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Regulação para Cima/efeitos dos fármacos
12.
PLoS One ; 15(8): e0237354, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790690

RESUMO

The TCIRG1 gene encodes the a3 isoform of vacuolar H+-ATPase (V-ATPase), which forms a proton transport channel in osteoclasts. Defects in this gene lead to functional impairment of osteoclasts and increased bone mass; however, the molecular mechanisms of TCIRG1 loss have not been fully elucidated. In the current study, we transfected mouse bone marrow-derived monocytes with control or Tcirg1-knockdown lentiviruses to further investigate the mechanisms of TCIRG1. Our results demonstrate that knockdown of Tcirg1 inhibits large-osteoclast (>100 µm) generation by decreasing the expression of nuclear factor of activated T-cells 1 (NFATc1) and inositol-1,4,5-trisphosphate receptor 2 (IP3R2). The decreased IP3R2 reduces intracellular calcium levels, which limits the nuclear translocation of NFATc1 in RANKL-induced mouse bone marrow-derived monocytes. These findings provide a mechanism to explain the effects of TCIRG1 impairment, with potential implications for the development of therapies for osteopetrosis.


Assuntos
Regulação para Baixo , Receptores de Inositol 1,4,5-Trifosfato/genética , Fatores de Transcrição NFATC/genética , Osteogênese , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(32): 19276-19286, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719141

RESUMO

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka (Oryzias latipes) osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits mpeg1-positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in cxcr3.2 prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.


Assuntos
Matriz Óssea/metabolismo , Quimiocina CXCL9/metabolismo , Proteínas de Peixes/metabolismo , Oryzias/metabolismo , Osteoclastos/metabolismo , Osteoporose/metabolismo , Receptores CXCR3/metabolismo , Células-Tronco/metabolismo , Animais , Matriz Óssea/crescimento & desenvolvimento , Diferenciação Celular , Quimiocina CXCL9/genética , Modelos Animais de Doenças , Proteínas de Peixes/genética , Humanos , Macrófagos/metabolismo , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoporose/genética , Osteoporose/fisiopatologia , Ligação Proteica , Receptores CXCR3/genética , Células-Tronco/citologia
14.
Int J Nanomedicine ; 15: 4705-4716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636626

RESUMO

Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease. Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot. Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs. Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.


Assuntos
Nanopartículas Metálicas/química , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Muramidase/química , Muramidase/metabolismo , Fatores de Transcrição NFATC/metabolismo , Nanomedicina/métodos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7
15.
Life Sci ; 258: 118093, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32673666

RESUMO

BACKGROUND: Nuclear factor-kappa B (NF-κB) is an important nuclear transcription factor in cells, involving in a series of processes such as cell proliferation, apoptosis, and differentiation. In this study, we explored the specific mechanism of NF-κB on the differentiation of osteoclasts. METHODS: MicroRNAs (miRNAs) expression microarray data GSE105027 related to osteoarthritis was obtained to screen out the differentially expressed miRNA. Phorbol-12-myristate-13-acetate (PMA) was used to induce THP-1 cells to differentiate into macrophages, followed by induction to osteoclasts using macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). ELISA and RT-qPCR were conducted to examine IL-6 and IL-1ß expression. The binding of NF-κB to the miR-1276 promoter region was demonstrated by ChIP assay, and targeting relationship between miR-1276 and MITF was verified by dual luciferase reporter assay. KK, iKBα, NF-kB, p-IKK, p-iKBα, p-NF-kB expression was analyzed by western blot. NF-κB and miR-1276 expression in osteoclasts was examined later. After gain- and less-of-function study, the effects on osteoclast differentiation were detected by TRAP-positive osteoclasts, TRAP activity, TRAP-5b content, F-Actin expression, as well as osteoclast differentiation marker genes expression. RESULTS: NF-κB was activated in osteoclasts, and down-regulation of NF-κB inhibited osteoclast differentiation. Next, miR-1276 was downregulated in osteoclasts after differentiation from monocytes. Meanwhile, NF-κB decreased the expression of miR-1276 by binding to the miR-1276 promoter, thereby elevating MITF expression, thereby promoting osteoclast differentiation. CONCLUSION: In summary, NF-κB promoted osteoclast differentiation through downregulating miR-1276 to upregulate MITF.


Assuntos
Diferenciação Celular , Regulação para Baixo/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Inativação Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Células THP-1
16.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510503

RESUMO

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.


Assuntos
Remodelação Óssea , Dispositivos Lab-On-A-Chip , Mecanotransdução Celular , Osteócitos/citologia , Animais , Humanos , Osteoblastos/citologia , Osteoclastos/citologia , Osteogênese
17.
Life Sci ; 255: 117827, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32450170

RESUMO

AIMS: Data suggest pharmacological treatment of depression with selective serotonin reuptake inhibitors (SSRI) may impair bone health. Our group has previously modeled compromised craniofacial healing after treatment with sertraline, a commonly prescribed SSRI, and hypothesized potential culprits: alterations in bone cells, collagen, and/or inflammation. Here we interrogate bone lineage cell alterations due to sertraline treatment as a potential cause of the noted compromised bone healing. MAIN METHODS: Murine pre-osteoblast, pre-osteoclast, osteoblast, and osteoclast cells were treated with clinically relevant concentrations of the SSRI. Studies focused on serotonin pathway targets, cell viability, apoptosis, differentiation, and the osteoblast/osteoclast feedback loop. KEY FINDINGS: All cells studied express neurotransmitters (e.g. serotonin transporter, SLC6A4, SSRI target) and G-protein-coupled receptors associated with the serotonin pathway. Osteoclasts presented the greatest native expression of Slc6a4 with all cell types exhibiting decreases in Slc6a4 expression after SSRI treatment. Pre-osteoclasts exhibited alteration to their differentiation pathway after treatment. Pre-osteoblasts and osteoclasts showed reduced apoptosis after treatment but showed no significant differences in functional assays. RANKL: OPG mRNA and protein ratios were decreased in the osteoblast lineage. Osteoclast lineage cells treated with sertraline demonstrated diminished TRAP positive cells when pre-exposed to sertraline prior to RANKL-induced differentiation. SIGNIFICANCE: These data suggest osteoclasts are a likely target of bone homeostasis disruption due to sertraline treatment, most potently through the osteoblast/clast feedback loop.


Assuntos
Osso e Ossos/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Inibidores de Captação de Serotonina/toxicidade , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Osso e Ossos/citologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Osteoblastos/citologia , Osteoclastos/citologia , Ligante RANK/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo
18.
Cell Prolif ; 53(6): e12827, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32406154

RESUMO

OBJECTIVES: Previously, we found that by regulating T helper (Th) cell polarization, calcitriol intervention inhibited lipopolysaccharide (LPS)-induced alveolar bone loss in an animal periodontitis model, but the underlying cellular events remain unknown. MATERIALS AND METHODS: In this study, mouse Th cells were incubated in an inflammatory environment in the presence of dendritic cells (DCs) and LPS. Then, the potential of the Th cells to undergo Th2/Th17 polarization, the RANKL expression of the polarized Th cells and the subsequent influences of the polarized Th cells on RAW264.7 cell osteoclastogenesis in response to calcitriol administration were assessed. Finally, the effects of calcitriol on antigen presentation by DCs during these cellular events were evaluated. RESULTS: In response to calcitriol administration, Th cells in an inflammatory environment exhibited an enhanced potential for Th2 polarization along with a decreased potential for Th17 polarization. In addition, RANKL expression in Th17-polarized cells was largely inhibited. Furthermore, inflammation-induced osteoclastogenesis in RAW264.7 cells was suppressed following coculture with calcitriol-treated Th cells. During these cellular events, increased expression of Th2 promoters (such as OX-40L and CCL17) and decreased expression of Th17 promoters (such as IL-23 and IL-6) were found in DCs. CONCLUSIONS: Calcitriol can inhibit osteoclastogenesis in an inflammatory environment by changing the proportion and function of Th cell subsets. Our findings suggest that calcitriol may be an effective therapeutic agent for treating periodontitis.


Assuntos
Calcitriol/farmacologia , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Células Cultivadas , Células Dendríticas/imunologia , Inflamação , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Ligante RANK/metabolismo , Células RAW 264.7 , Células Th17/imunologia , Células Th2/imunologia
19.
Actual. osteol ; 16(1): 35-46, Ene - abr. 2020. graf, ilus
Artigo em Espanhol | LILACS | ID: biblio-1139966

RESUMO

La erupción dental es un proceso estrictamente regulado y programado espacial y temporalmente. El objetivo del trabajo fue estudiar el efecto de la exposición prenatal a fluoruro de sodio (NaF) sobre los eventos morfológicos y celulares que ocurren en el hueso supracoronal del primer molar de crías de rata durante la etapa preeruptiva. Se emplearon crías (n=6-8 por grupo) provenientes de madres que bebieron crónicamente agua con diferentes concentraciones de F- en forma de NaF durante la gestación y lactancia: control y NaF (50 mg/L). En cortes histológicos de la mandíbula de crías de 3 y 10 días se analizaron parámetros de histomorfometría estática en la zona supracoronal de la canastilla ósea a la altura del primer molar inferior: volumen óseo trabecular [BV/TV (%)], número de osteoclastos por milímetro (N.Oc/mm) y las variables indirectas: número de trabéculas [Tb.N (1/mm)], espesor [Tb.Th (µm)] y separación trabecular [Tb.Sp (µm)]. En crías de 15 días se midió el grado de erupción [TED (µm)] del primer molar inferior. Los resultados se analizaron con el test "t" de Student considerando diferencias significativas a p<0,05. El análisis histomorfométrico demostró un incremento en el BV/TV (%) del hueso supracoronal (p<0,01) asociado con disminución del N.Oc/mm (p<0,01) en crías de 3 y 10 días expuestas prenatalmente al F-. El grado de erupción dental fue menor en animales expuestos prenatalmente al F- en comparación con los controles (p<0,01). En conclusión, los resultados observados en la mandíbula de crías expuestas durante la etapa prenatal y posnatal temprana al F- sugieren un efecto disruptivo sobre la actividad resortiva necesaria para formación del canal eruptivo. (AU)


Tooth eruption is a tightly regulated and spatially and temporally programmed process. The aim of this study was to examine the effect of prenatal NaF exposure on the morphological and cellular events that occur in the supracoronal area of bony crypt of the first rat molar during the preeruptive stage. Offspring from two groups of rats were used (6-8 per group): Control and 50 mg/L NaF. The treatment was performed during pregnancy and lactation. Suckling pups were euthanized at 3-, 10- and 15-days-old by cervical dislocation. Mandibles were removed and histologically processed to obtain buccolingual sections stained with H&E. In sections of first mandibular molar of 3- and 10-days-old pups, the following static histomorphometric parameters were evaluated: trabecular bone volume [BV/TV (%)] and number of osteoclasts (N.Oc/mm). Also, indirect parameters were obtained: trabecular number [Tb.N (1/mm)], trabecular thickness [Tb.Th (µm)], and trabecular separation [Tb.Sp (µm)]. The degree of tooth eruption [TED (µm)] was determined. Results are expressed as mean ± SE and analyzed by Student t-test. Histomorphometric analysis showed an increase in the BV/TV (%) of the bone crypt of 3- and 10- days-old pups exposed to NaF (p <0.01); this increase was associated with a decrease in the N.Oc/mm (p <0.01). TED of mandibular first molar was lower in prenatal NaF exposed group than in control group (p<0.01). In conclusion, the increased BV/TV and the lower N.Oc observed in the bone crypt of 3- and 10- days-old pups from mothers treated with NaF suggested a disruptive effect triggered by F- on the formation events of the eruptive pathway in the offspring. (AU)


Assuntos
Humanos , Animais , Masculino , Feminino , Lactente , Pré-Escolar , Ratos , Fluoreto de Sódio/efeitos adversos , Erupção Dentária , Osteoclastos/citologia , Efeitos Tardios da Exposição Pré-Natal , Fluoreto de Sódio/administração & dosagem , Fluoreto de Sódio/metabolismo , Fluoreto de Sódio/urina , Fluoreto de Sódio/síntese química , Ratos Wistar , Mandíbula/anatomia & histologia , Dente Molar/crescimento & desenvolvimento , Fluorose Dentária/diagnóstico
20.
Int J Mol Sci ; 21(6)2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32197293

RESUMO

Interleukin (IL)-35 is an immunosuppressive cytokine mainly produced by regulatory T cells. IL-35 mediates immunological functions by suppressing the inflammatory immune response. However, the role of IL-35 in bone-destructive diseases remains unclear, especially in terms of osteoclastogenesis. Therefore, the current study investigated the synergistic effect of IL-35 on osteoclastogenesis that is involved the pathogeneses of periodontitis and rheumatoid arthritis. Osteoclastic differentiation and osteoclastogenesis of RAW264 (RAW) cells induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) and IL-35 were evaluated by tartrate-resistant acid phosphate staining, hydroxyapatite resorption assays, and quantitative polymerase chain reaction. The effect of IL-35 on RANKL-stimulated signaling pathways was assessed by Western blot analysis. Costimulation of RAW cells by RANKL and IL-35 induced osteoclastogenesis significantly compared with stimulation by RANKL alone. Phosphorylations of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase tended to be increased by RANKL and IL-35 compared with RANKL or IL-35 alone. Additionally, the osteoclastogenesis induced by RANKL and IL-35 was suppressed by inhibition of ERK. In this study, IL-35 and RANKL induced osteoclastogenesis synergistically. Previous reports have shown that IL-35 suppresses the differentiation of osteoclasts. Therefore, IL-35 might play dual roles of destruction and protection in osteoclastogenesis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Interleucinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/metabolismo , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Animais , Interleucinas/agonistas , Camundongos , Monócitos/citologia , Osteoclastos/citologia , Ligante RANK/agonistas , Células RAW 264.7
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