RESUMO
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
Human monocyte chemoattractant protein-1 (MCP-1) in mice has two orthologs, MCP-1 and MCP-5. MCP-1, which is highly expressed in osteoclasts rather than in osteoclast precursor cells, is an important factor in osteoclast differentiation. However, the roles of MCP-5 in osteoclasts are completely unknown. In this study, contrary to MCP-1, MCP-5 was downregulated during receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation and was considered an inhibitory factor in osteoclast differentiation. The inhibitory role of MCP-5 in osteoclast differentiation was closely related to the increase in Ccr5 expression and the inhibition of IκB degradation by RANKL. Transgenic mice expressing MCP-5 controlled by Mx-1 promoter exhibited an increased bone mass because of a decrease in osteoclasts. This result strongly supported that MCP-5 negatively regulated osteoclast differentiation. MCP-5 also prevented severe bone loss caused by RANKL.
Assuntos
Diferenciação Celular , Glicoproteínas de Membrana , Proteínas Quimioatraentes de Monócitos , Osteoclastos , Animais , Humanos , Masculino , Camundongos , Células Cultivadas , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos ICR , Proteínas Quimioatraentes de Monócitos/genética , Proteínas Quimioatraentes de Monócitos/metabolismo , Proteínas Quimioatraentes de Monócitos/farmacologia , NF-kappa B/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/farmacologia , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Regulação para CimaRESUMO
Sestrins are stress-responsive proteins with antioxidant properties. They participate in cellular redox balance and protect against oxidative damage. This study investigated the effects of Sestrin2 (Sesn2) on osteoclast differentiation and function. Overexpressing Sesn2 in osteoclast precursor cells significantly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis. This was assessed as reduced expression of various osteoclast markers, including c-Fos, nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor, tartrate-resistant acid phosphatase, and cathepsin K. Conversely, downregulation of Sesn2 produced the opposite effect. Mechanistically, Sesn2 overexpression enhanced AMPK activation and the nuclear translocation of nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2), promoting antioxidant enzymes. Moreover, azithromycin (Azm) induced Sesn2 expression, which suppressed RANKL-induced osteoclast differentiation. Specifically, Azm treatment reduced RANKL-induced production of reactive oxygen species in osteoclasts. Furthermore, intraperitoneal administration of Azm ameliorated RANKL-induced bone loss by reducing osteoclast activity in mice. Taken together, our results suggested that Azm-induced Sesn2 act as a negative regulator of RANKL-induced osteoclast differentiation through the AMPK/NFATc1 signaling pathway. Concisely, targeting Sesn2 can be a potential pharmacological intervention in osteoporosis.
Assuntos
Osteogênese , Ligante RANK , Animais , Camundongos , Osteogênese/genética , Espécies Reativas de Oxigênio/metabolismo , Ligante RANK/genética , Ligante RANK/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Antioxidantes/farmacologia , Osteoclastos/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Diferenciação CelularRESUMO
CrkII, a member of the adaptor protein family, is known to participate in bone homeostasis via the regulation of osteoclasts and osteoblasts. Therefore, silencing CrkII would beneficially impact the bone microenvironment. In this study, CrkII siRNA encapsulated by a bone-targeting peptide (AspSerSer)6-liposome was evaluated for its therapeutic applications using a receptor activator of nuclear factor kappa-B ligand (RANKL)-induced bone loss model. (AspSerSer)6-liposome-siCrkII maintained its gene-silencing ability in both osteoclasts and osteoblasts in vitro and significantly reduced osteoclast formation while increasing osteoblast differentiation in vitro. Fluorescence image analyses showed that the (AspSerSer)6-liposome-siCrkII was present largely in bone, where it remained present for up to 24 hours and was cleared by 48 hours, even when systemically administrated. Importantly, microcomputed-tomography revealed that bone loss induced by RANKL administration was recovered by systemic administration of (AspSerSer)6-liposome-siCrkII. Collectively, the findings of this study suggest that (AspSerSer)6-liposome-siCrkII is a promising therapeutic strategy for the development of treatments for bone diseases, as it overcomes the adverse effects derived from ubiquitous expression via bone-specific delivery of siRNA.
Assuntos
Doenças Ósseas , Reabsorção Óssea , Humanos , Osteogênese , RNA Interferente Pequeno/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Lipossomos/metabolismo , Osteoclastos , Osteoblastos , Doenças Ósseas/metabolismo , Diferenciação CelularRESUMO
Neurogenin 1 (Ngn1) belongs to the basic helix-loop-helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption.
Assuntos
Osteoclastos , Osteogênese , Diferenciação Celular , Interleucina-17/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genéticaRESUMO
The LIM-homeodomain transcription factor Lmx1b plays a key role in body pattern formation during development. Although Lmx1b is essential for the normal development of multiple tissues, its regulatory mechanism in bone cells remains unclear. Here, we demonstrated that Lmx1b negatively regulates bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation. Overexpressed Lmx1b in the osteoblast precursor cells inhibited alkaline phosphatase (ALP) activity and nodule formation, as well as the expression of osteoblast maker genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alpl), bone sialoprotein (Ibsp), and osteocalcin (Bglap). Conversely, the knockdown of Lmx1b in the osteoblast precursors enhanced the osteoblast differentiation and function. Lmx1b physically interacted with and repressed the transcriptional activity of Runx2 by reducing the recruitment of Runx2 to the promoter region of its target genes. In vivo analysis of BMP2-induced ectopic bone formation revealed that the knockdown of Lmx1b promoted osteogenic differentiation and bone regeneration. Our data demonstrate that Lmx1b negatively regulates osteoblast differentiation and function through regulation of Runx2 and provides a molecular basis for therapeutic targets for bone diseases.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Fatores de Transcrição , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Osteogênese/genética , Fatores de Transcrição/metabolismoRESUMO
Activating transcription factor 3 (ATF3) has been identified as a negative regulator of osteoblast differentiation in in vitro study. However, it was not associated with osteoblast differentiation in in vivo study. To provide an understanding of the discrepancy between the in vivo and in vitro findings regarding the function of ATF3 in osteoblasts, we investigated the unidentified roles of ATF3 in osteoblast biology. ATF3 enhanced osteoprotegerin (OPG) production, not only in osteoblast precursor cells, but also during osteoblast differentiation and osteoblastic adipocyte differentiation. In addition, ATF3 increased nodule formation in immature osteoblasts and decreased osteoblast-dependent osteoclast formation, as well as the transdifferentiation of osteoblasts to adipocytes. However, all these effects were reversed by the OPG neutralizing antibody. Taken together, these results suggest that ATF3 contributes to bone homeostasis by regulating the differentiation of various cell types in the bone microenvironment, including osteoblasts, osteoclasts, and adipocytes via inducing OPG production.
Assuntos
Osteoclastos , Osteoprotegerina , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Adipócitos/metabolismo , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/metabolismoRESUMO
Interferon regulatory factors (IRFs) play roles in various biological processes including cytokine signaling, cell growth regulation and hematopoietic development. Although it has been reported that several IRFs are involved in bone metabolism, the role of IRF2 in bone cells has not been elucidated. Here, we investigated the involvement of IRF2 in RANKL-induced osteoclast differentiation. IRF2 overexpression in osteoclast precursor cells enhanced osteoclast differentiation by regulating the expression of NFATc1, a master regulator of osteoclastogenesis. Conversely, IRF2 knockdown inhibited osteoclast differentiation and decreased the NFATc1 expression. Moreover, IRF2 increased the translocation of NF-κB subunit p65 to the nucleus in response to RANKL and subsequently induced the expression of NFATc1. IRF2 plays an important role in RANKL-induced osteoclast differentiation by regulating NF-κB/NFATc1 signaling pathway. Taken together, we demonstrated the molecular mechanism of IRF2 in osteoclast differentiation, and provide a molecular basis for potential therapeutic targets for the treatment of bone diseases characterized by excessive bone resorption. [BMB Reports 2021; 54(9): 482-487].
Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator Regulador 2 de Interferon/metabolismo , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Núcleo Celular/metabolismo , Fator Regulador 2 de Interferon/antagonistas & inibidores , Fator Regulador 2 de Interferon/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Remodelação Óssea/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genéticaRESUMO
STAT5 is a transcription factor that is activated by various cytokines, hormones, and growth factors. Activated STAT5 is then translocated to the nucleus and regulates the transcription of target genes, affecting several biological processes. Several studies have investigated the role of STAT5 in adipogenesis, but unfortunately, its role in adipogenesis remains controversial. In the present study, we generated adipocyte-specific Stat5 conditional knockout (cKO) (Stat5fl/fl;Apn-cre) mice to investigate the role of STAT5 in the adipogenesis of bone marrow mesenchymal stem cells (BMSCs). BMSC adipogenesis was significantly inhibited upon overexpression of constitutively active STAT5A, while it was enhanced in the absence of Stat5 in vitro. In vivo adipose staining and histological analyses revealed increased adipose volume in the bone marrow of Stat5 cKO mice. ATF3 is the target of STAT5 during STAT5-mediated inhibition of adipogenesis, and its transcription is regulated by the binding of STAT5 to the Atf3 promoter. ATF3 overexpression was sufficient to suppress the enhanced adipogenesis of Stat5-deficient adipocytes, and Atf3 silencing abolished the STAT5-mediated inhibition of adipogenesis. Stat5 cKO mice exhibited reduced bone volume due to an increase in the osteoclast number, and coculture of bone marrow-derived macrophages with Stat5 cKO adipocytes resulted in enhanced osteoclastogenesis, suggesting that an increase in the adipocyte number may contribute to bone loss. In summary, this study shows that STAT5 is a negative regulator of BMSC adipogenesis and contributes to bone homeostasis via direct and indirect regulation of osteoclast differentiation; therefore, it may be a leading target for the treatment of both obesity and bone loss-related diseases.
Assuntos
Adipócitos/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Homeostase , Células-Tronco Mesenquimais/metabolismo , Fator de Transcrição STAT5/metabolismo , Adipócitos/citologia , Adipogenia/genética , Animais , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Osteogênese/genética , Ligação Proteica , Fator de Transcrição STAT5/genética , Transdução de SinaisRESUMO
The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbß, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.
Assuntos
Reabsorção Óssea/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , Animais , Remodelação Óssea , Reabsorção Óssea/genética , Diferenciação Celular , Células Cultivadas , Relógios Circadianos , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The adaptor protein CrkII is involved in several biological activities, including mitogenesis, phagocytosis, and cytoskeleton reorganization. Previously, we demonstrated that CrkII plays an important role in osteoclast differentiation and function through Rac1 activation both in vitro and in vivo. In this study, we investigated whether CrkII also regulates the differentiation and function of another type of bone cells, osteoblasts. Overexpression of CrkII in primary osteoblasts inhibited bone morphogenetic protein (BMP) 2-induced osteoblast differentiation and function, whereas knockdown of CrkII expression exerted the opposite effect. Importantly, CrkII strongly enhanced c-Jun-N-terminal kinase (JNK) phosphorylation, and the CrkII overexpression-mediated attenuation of osteoblast differentiation and function was recovered by JNK inhibitor treatment. Furthermore, transgenic mice overexpressing CrkII under control of the alpha-1 type I collagen promoter exhibited a reduced bone mass phenotype. Together, these results indicate that CrkII negatively regulates osteoblast differentiation and function through JNK phosphorylation. Given that CrkII acts as a negative and positive regulator of osteoblast and osteoclast differentiation, respectively, the regulation of CrkII expression in bone cells may help to develop new strategies to enhance bone formation and inhibit bone resorption.
Assuntos
Reabsorção Óssea/genética , Neuropeptídeos/genética , Osteogênese/genética , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Proteína Morfogenética Óssea 2/genética , Reabsorção Óssea/patologia , Diferenciação Celular/genética , Colágeno Tipo I , Cadeia alfa 1 do Colágeno Tipo I , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Fosforilação , Transdução de Sinais/genéticaRESUMO
Kruppel-like factor 2 (KLF2) has been implicated in the regulation of cell proliferation, differentiation, and survival in a variety of cells. Recently, it has been reported that KLF2 regulates the p65-mediated transactivation of NF-κB. Although the NF-κB pathway plays an important role in the differentiation of osteoclasts and osteoblasts, the role of KLF2 in these bone cells has not yet been fully elucidated. In this study, we demonstrated that KLF2 regulates osteoclast and osteoblast differentiation. The overexpression of KLF2 in osteoclast precursor cells inhibited osteoclast differentiation by downregulating c-Fos, NFATc1, and TRAP expression, while KLF2 overexpression in osteoblasts enhanced osteoblast differentiation and function by upregulating Runx2, ALP, and BSP expression. Conversely, the downregulation of KLF2 with KLF2-specific siRNA increased osteoclast differentiation and inhibited osteoblast differentiation. Moreover, the overexpression of interferon regulatory protein 2-binding protein 2 (IRF2BP2), a regulator of KLF2, suppressed osteoclast differentiation and enhanced osteoblast differentiation and function. These effects were reversed by downregulating KLF2. Collectively, our data provide new insights and evidence to suggest that the IRF2BP2/KLF2 axis mediates osteoclast and osteoblast differentiation, thereby affecting bone homeostasis. [BMB Reports 2019; 52(7): 469-474].
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Fatores de Transcrição/metabolismo , Homeostase , HumanosRESUMO
Anoctamin 5 (Ano5) mutations are responsible for gnathodiaphyseal dysplasia, a rare skeletal syndrome. Despite the close linkage of Ano5 to bone remodeling, the molecular mechanisms underlying the role of Ano5 in bone remodeling remain unknown. In this study, we investigated whether Ano5 regulates osteoblast or osteoclast differentiation to maintain normal bone remodeling. Downregulation of Ano5 expression did not affect osteoblast differentiation and mineralization, while ectopic expression of Ano5 significantly enhanced receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation. Furthermore, Ano5-mediated Akt phosphorylation resulted in nuclear factor of activated T-cells c1 (NFATc1) activation, indicating that Ano5 regulates osteoclast differentiation through activation of the Akt-NFATc1 signaling pathway. Thus, our results suggest a possibility that Ano5 is involved in bone remodeling through regulating the function of osteoclasts rather than that of osteoblasts.
Assuntos
Anoctaminas/genética , Diferenciação Celular , Predisposição Genética para Doença , Osteoblastos/citologia , Osteoclastos/citologia , Osteogênese Imperfeita/genética , Animais , Animais Recém-Nascidos , Anoctaminas/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
The tripartite motif protein 38 (TRIM38), a member of the TRIM family, is involved in various cellular processes such as cell proliferation, differentiation, apoptosis, and antiviral defense. However, the role of TRIM38 in osteoclast and osteoblast differentiation is not yet known. In this study, we report the involvement of TRIM38 in osteoclast and osteoblast differentiation. Overexpression of TRIM38, in osteoclast precursor cells, attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast formation, RANKL-triggered NF-κB activation, and expression of osteoclast marker genes, such as NFATc1, osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP); and down-regulation of TRIM38 expression showed the opposite effects. Ectopic expression of TRIM38 in osteoblast precursors induced increased osteoblast differentiation and function. Elevated expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin was also observed due to blockade of NF-κB activation. Conversely, knockdown of TRIM38 showed the opposite effects. TRIM38 also induced degradation of lysosome-dependent transforming growth factor beta-activated kinase 1 and MAP3K7-binding protein 2 (TAB2), further blocking NF-κB activation. Taken together, our data suggest that TRIM38 plays a critical role in bone remodeling as a negative regulator of NF-κB in both osteoclast and osteoblast differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Remodelação Óssea/fisiologia , Proteínas de Transporte/metabolismo , NF-kappa B/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animais , Diferenciação Celular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Osteoblastos/citologia , Osteoclastos/citologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The non-receptor tyrosine kinase c-Src participates in bone metabolism by regulating the activities of both the bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we investigated whether megakaryocyte-associated tyrosine kinase (Matk), a potent inhibitor of c-Src, affects the functions of murine osteoclasts and osteoblasts. Results revealed that the formation of osteoclasts with actin rings was attenuated by Matk overexpression in osteoclast precursor cells but was enhanced by Matk knockdown. The inhibitory effect of Matk on osteoclasts was closely related with the inhibition of c-Src activity. Intriguingly, Matk overexpression in osteoblasts reduced bone nodule formation. Conversely, Matk knockdown increased osteoblast function. Most importantly, binding of Matk to Runx2 resulted in the inhibition of Runx2 translocation into the nucleus and downregulation of Runx2 target genes. Taken together, our findings demonstrated that Matk plays a critical role in bone metabolism by impairing the functions of osteoclasts and osteoblasts via distinct mechanisms involving inhibition of c-Src-dependent and -independent signaling pathways.
Assuntos
Desenvolvimento Ósseo/fisiologia , Reabsorção Óssea/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Osso e Ossos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologiaRESUMO
Endoplasmic reticulum (ER) stress is triggered by various metabolic factors, such as cholesterol and proinflammatory cytokines. Recent studies have revealed that ER stress is closely related to skeletal disorders, such as osteoporosis. However, the precise mechanism by which ER stress regulates osteoclast differentiation has not been elucidated. In this study, we identified an ER-bound transcription factor, cAMP response element-binding protein H (CREBH), as a downstream effector of ER stress during RANKL-induced osteoclast differentiation. RANKL induced mild ER stress and the simultaneous accumulation of active nuclear CREBH (CREBH-N) in the nucleus during osteoclastogenesis. Overexpression of CREBH-N in osteoclast precursors enhanced RANKL-induced osteoclast formation through NFATc1 upregulation. Inhibiting ER stress using a specific inhibitor attenuated the expression of osteoclast-related genes and CREBH activation. In addition, inhibition of reactive oxygen species using N-acetylcysteine attenuated ER stress, expression of osteoclast-specific marker genes, and RANKL-induced CREBH activation. Furthermore, inhibition of ER stress and CREBH signaling pathways using an ER stress-specific inhibitor or CREBH small interfering RNAs prevented RANKL-induced bone destruction in vivo. Taken together, our results suggest that reactive oxygen species/ER stress signaling-dependent CREBH activation plays an important role in RANKL-induced osteoclastogenesis. Therefore, inactivation of ER stress and CREBH signaling pathways may represent a new treatment strategy for osteoporosis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Osteogênese/fisiologia , Ligante RANK/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologiaRESUMO
Many studies have determined that PI3K-Akt signaling pathways play important roles in osteoclast differentiation and function. In the present study, we investigated the roles of B-cell adaptor for PI3K (BCAP), which is a PI3K binding molecule, in osteoclasts. Overexpression of BCAP in osteoclast precursor cells enhanced osteoclast differentiation induced by tumor necrosis factor alpha (TNF-α) as well as receptor activator of nuclear factor-κB ligand (RANKL). Conversely, osteoclast differentiation mediated by both cytokines was attenuated when BCAP expression was downregulated using small interfering RNA. Notably, BCAP induced Akt activation only upon stimulation by RANKL, but not by TNF-α. However, BCAP activated p38-dependent cAMP response element-binding protein (CREB) phosphorylation induced by both RANKL and TNF-α. Collectively, we showed that BCAP plays an important role in osteoclast differentiation by regulating the p38-dependent CREB signaling pathway, and that BCAP might be a new therapeutic target for bone diseases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Osteoclastos/citologia , Animais , Camundongos , Camundongos Endogâmicos ICR , Osteoclastos/metabolismoRESUMO
Tumor suppressor candidate 2 (Tusc2, also known as Fus1) regulates calcium signaling, and Ca2ï¼-dependent nuclear factor of activated T-cells (NFAT) and nuclear factor kappa B (NF-κB) pathways, which play roles in osteoclast differentiation. However, the role of Tusc2 in osteoclasts remains unknown. Here, we report that Tusc2 positively regulates the differentiation of osteoclasts. Overexpression of Tusc2 in osteoclast precursor cells enhanced receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. In contrast, small interfering RNA-mediated knockdown of Tusc2 strongly inhibited osteoclast differentiation. In addition, Tusc2 induced the activation of RANKL-mediated NF-κB and calcium/calmodulin-dependent kinase IV (CaMKIV)/cAMP-response element (CRE)-binding protein CREB signaling cascades. Taken together, these results suggest that Tusc2 acts as a positive regulator of RANKL-mediated osteoclast differentiation. [BMB Reports 2017; 50(9): 454-459].
Assuntos
NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Camundongos , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , Osteoblastos , Proteínas Supressoras de Tumor/genéticaRESUMO
Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding protein family of transcription factors, has been implicated in the regulation of cell proliferation and differentiation. However, whether ATF3 is involved in osteoclast differentiation and activity has not been well-studied. In the present study, we examined the role of ATF3 in osteoclast differentiation and function. ATF3 expression was down-regulated during RANKL-induced osteoclast differentiation. Overexpression of ATF3 in bone marrow-derived monocyte/macrophage lineage cells (BMMs) promoted osteoclast differentiation and activity and strongly induced the expression of osteoclast genes encoding nuclear factor of activated T-cells c1 (NFATc1) and tartrate-resistant acid phosphatase (TRAP) compared to that in the control group. In contrast, small interfering RNA-mediated knockdown of ATF3 prevented the formation of multinucleated osteoclasts and markedly abrogated the expression of osteoclast marker genes. Mechanistically, ATF3 synergistically enhanced c-Fos- or NFAT-mediated transcriptional activity of the NFATc1 or TRAP promoter, respectively. Furthermore, ATF3 physically interacted with c-Fos and NFATc1 and enhanced the binding affinity of c-Fos and NFATc1 to the promoters. Interestingly, ATF3 is involved in calcium signaling during osteoclastogenesis. Taken together, these results suggest that ATF3 is a new co-factor of c-Fos and NFATc1 to activate osteoclast differentiation and activity.