RESUMEN
Receptor activator of NF-κB ligand (RANKL) is a multifunctional cytokine known to affect immune and skeletal systems, as well as oncogenesis and metastasis1-4. RANKL is synthesized as a membrane-bound molecule, and cleaved into its soluble form by proteases5-7. As the soluble form of RANKL does not contribute greatly to bone remodelling or ovariectomy-induced bone loss8, whether soluble RANKL has a role in pathological settings remains unclear. Here we show that soluble RANKL promotes the formation of tumour metastases in bone. Mice that selectively lack soluble RANKL (Tnfsf11ΔS/ΔS)5-7,9 have normal bone homoeostasis and develop a normal immune system but display markedly reduced numbers of bone metastases after intracardiac injection of RANK-expressing melanoma and breast cancer cells. Deletion of soluble RANKL does not affect osteoclast numbers in metastatic lesions or tumour metastasis to non-skeletal tissues. Therefore, soluble RANKL is dispensable for physiological regulation of bone and immune systems, but has a distinct and pivotal role in the promotion of bone metastases.
Asunto(s)
Neoplasias Óseas/secundario , Neoplasias/patología , Receptor Activador del Factor Nuclear kappa-B/fisiología , Animales , Remodelación Ósea/fisiología , Diferenciación Celular/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Osteoclastos/citología , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
TAK1 (encoded by Map3k7) is a mitogen-activated protein kinase kinase kinase (MAP3K), which activates the transcription factors AP-1 and NF-κB in response to receptor activator of NF-κB ligand (RANKL) stimulation, thus constituting a key regulator of osteoclast differentiation. Here we report the functional relevance of the kinase activity of TAK1 in the late stage of osteoclast differentiation in vivo using Ctsk-Cre mice and TAK1 mutant mice in which the TAK1 kinase domain was flanked by loxP. The Map3k7(flox/kd)Ctsk(Cre/+) mice displayed a severe osteopetrotic phenotype due to a marked decrease in osteoclast number. RANKL-induced activation of MAPK and NF-κB was impaired in the late stage of osteoclast differentiation. The absence of suppressive effect of an administered NF-κB inhibitor on the late stage of osteoclastogenesis led us to investigate unknown TAK1 targets in osteoclast differentiation. We performed a phosphoproteomic analysis of RANKL-stimulated osteoclast precursor cells from Map3k7(flox/kd)Ctsk(Cre/+) mice, revealing multiple targets regulated by TAK1 during osteoclastogenesis. Thus, TAK1 functions as a critical regulator of the phosophorylation status of various cellular proteins that govern osteoclastogenesis.
Asunto(s)
Diferenciación Celular , Quinasas Quinasa Quinasa PAM/metabolismo , Osteoclastos/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Secuencia de Aminoácidos , Animales , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Osteoclastos/citología , Fosfoproteínas/químicaRESUMEN
Autoantibody production and immune complex (IC) formation are frequently observed in autoimmune diseases associated with bone loss. However, it has been poorly understood whether ICs regulate bone metabolism directly. Here we show that the level of osteoclastogenesis is determined by the strength of FcRγ signalling, which is dependent on the relative expression of positive and negative FcγRs (FcγRI/III/IV and IIB, respectively) as well as the availability of their ligands, ICs. Under physiological conditions, unexpectedly, FcγRIII inhibits osteoclastogenesis by depriving other osteoclastogenic Ig-like receptors of FcRγ. Fcgr2b(-/-) mice lose bone upon the onset of a hypergammaglobulinemia or the administration of IgG1 ICs, which act mainly through FcγRIII. The IgG2 IC activates osteoclastogenesis by binding to FcγRI and FcγRIV, which is induced under inflammatory conditions. These results demonstrate a link between the adaptive immunity and bone, suggesting a regulatory role for ICs in bone resorption in general, and not only in inflammatory diseases.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Enfermedades Autoinmunes/inmunología , Resorción Ósea/inmunología , Huesos/inmunología , Osteoclastos/metabolismo , Receptores de IgG/inmunología , Animales , Complejo Antígeno-Anticuerpo/metabolismo , Autoanticuerpos/inmunología , Resorción Ósea/genética , Huesos/metabolismo , Hipergammaglobulinemia/inmunología , Hipergammaglobulinemia/metabolismo , Inmunoglobulina G/inmunología , Ratones , Ratones Noqueados , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMEN
The immune and skeletal systems share a number of regulatory molecules including cytokines, signaling molecules, transcription factors and membrane receptors, in common. Consequently, the physiology and pathology of one system may very well affect the other. Research into the cartilage and bone destruction associated with rheumatoid arthritis (RA) has highlighted the importance of the interplay between the immune and skeletal systems. This interdisciplinary field called osteoimmunology has attracted much attention in recent years. Recently, animal models deficient in immunomodulatory molecules have been found frequently to develop an unexpected skeletal phenotype. Receptor activator of NF-kappaB ligand (RANKL) is an essential factor for the induction of osteoclastogenesis that links the immune and skeletal systems. Thus, osteoimmunology is becoming increasingly important for understanding the pathogenesis of bone destruction in RA and for developing new therapeutic strategies for diseases affecting both systems. Here we summarize recent advances on the study of the regulation of cartilage and bone destruction by the immune system.
Asunto(s)
Artritis Reumatoide/inmunología , Cartílago/fisiopatología , Osteoclastos/fisiología , Linfocitos T/inmunología , Humanos , Ligando RANK/fisiología , Transducción de Señal/fisiologíaRESUMEN
Finding a means to ameliorate and prevent bone destruction is one of the urgent issues in the treatment of rheumatoid arthritis. Recent studies revealed bone-resorbing osteoclasts to be essential for arthritic bone destruction, but to date there has been scarce experimental evidence for the underlying mechanism of the bone-protective effect of antirheumatic drugs. Here we examined the effects of one or a combination of disease-modifying antirheumatic drugs (DMARDs) on osteoclast differentiation to provide a cellular and molecular basis for their efficacy against bone destruction. The effects on osteoclast precursor cells and osteoclastogenesis-supporting cells were distinguished by two in vitro osteoclast culture systems. Methotrexate (MTX), bucillamine (Buc) and salazosulphapyridine (SASP) inhibited osteoclastogenesis by acting on osteoclast precursor cells and interfering with receptor activator of NF-kappaB ligand (RANKL)-mediated induction of the nuclear factor of activated T cells (NFAT) c1. MTX and SASP also suppressed RANKL expression on osteoclastogenesis-supporting mesenchymal cells. Interestingly, the combination of three antirheumatic drugs exerted a marked inhibitory effect on osteoclastogenesis even at a low dose at which there was much less of an effect when administered individually. These results are consistent with the reported efficacy of combined DMARDs therapy in humans and suggest that osteoclast culture systems are useful tools to provide an experimental basis for the bone-protective effects of antirheumatic drugs.
Asunto(s)
Antirreumáticos/farmacología , Artritis Reumatoide/complicaciones , Artritis Reumatoide/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/etiología , Diferenciación Celular/efectos de los fármacos , Animales , Resorción Ósea/inmunología , Células Cultivadas , Técnicas de Cocultivo , Cisteína/análogos & derivados , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Metotrexato/farmacología , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/efectos de los fármacos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Ligando RANK/efectos de los fármacos , Ligando RANK/metabolismo , Sulfasalazina/farmacologíaRESUMEN
In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.
Asunto(s)
Resorción Ósea/inmunología , Huesos/inmunología , Huesos/patología , Activación de Linfocitos/inmunología , Osteoclastos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Resorción Ósea/patología , Diferenciación Celular/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/patología , Linfocitos T Colaboradores-Inductores/patologíaRESUMEN
Calcium (Ca(2+)) signaling is essential for a variety of cellular responses and higher biological functions. Ca(2+)/calmodulin-dependent kinases (CaMKs) and the phosphatase calcineurin activate distinct downstream pathways that are mediated by the transcription factors cAMP response element (CRE)-binding protein (CREB) and nuclear factor of activated T cells (NFAT), respectively. The importance of the calcineurin-NFAT pathway in bone metabolism has been demonstrated in osteoclasts, osteoblasts and chondrocytes. However, the contribution of the CaMK-CREB pathway is poorly understood, partly because of the difficulty of dissecting the functions of homologous family members. Here we show that the CaMKIV-CREB pathway is crucial for osteoclast differentiation and function. Pharmacological inhibition of CaMKs as well as the genetic ablation of Camk4 reduced CREB phosphorylation and downregulated the expression of c-Fos, which is required for the induction of NFATc1 (the master transcription factor for osteoclastogenesis) that is activated by receptor activator of NF-kappaB ligand (RANKL). Furthermore, CREB together with NFATc1 induced the expression of specific genes expressed by differentiated osteoclasts. Thus, the CaMK-CREB pathway biphasically functions to regulate the transcriptional program of osteoclastic bone resorption, by not only enhancing induction of NFATc1 but also facilitating NFATc1-dependent gene regulation once its expression is induced. This provides a molecular basis for a new therapeutic strategy for bone diseases.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Osteoclastos/fisiología , Animales , Resorción Ósea/genética , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Datos de Secuencia Molecular , Factores de Transcripción NFATC/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ligando RANK/farmacología , Transducción de SeñalRESUMEN
Costimulatory signals are required for activation of immune cells, but it is not known whether they contribute to other biological systems. The development and homeostasis of the skeletal system depend on the balance between bone formation and resorption. Receptor activator of NF-kappaB ligand (RANKL) regulates the differentiation of bone-resorbing cells, osteoclasts, in the presence of macrophage-colony stimulating factor (M-CSF). But it remains unclear how RANKL activates the calcium signals that lead to induction of nuclear factor of activated T cells c1, a key transcription factor for osteoclastogenesis. Here we show that mice lacking immunoreceptor tyrosine-based activation motif (ITAM)-harbouring adaptors, Fc receptor common gamma subunit (FcRgamma) and DNAX-activating protein (DAP)12, exhibit severe osteopetrosis owing to impaired osteoclast differentiation. In osteoclast precursor cells, FcRgamma and DAP12 associate with multiple immunoreceptors and activate calcium signalling through phospholipase Cgamma. Thus, ITAM-dependent costimulatory signals activated by multiple immunoreceptors are essential for the maintenance of bone homeostasis. These results reveal that RANKL and M-CSF are not sufficient to activate the signals required for osteoclastogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Huesos/fisiología , Proteínas Portadoras/metabolismo , Homeostasis , Glicoproteínas de Membrana/metabolismo , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencias de Aminoácidos , Animales , Resorción Ósea , Huesos/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Eliminación de Gen , Homeostasis/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteogénesis/fisiología , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores de Superficie Celular/metabolismo , Receptores de IgG/química , Receptores de IgG/genética , Receptores Inmunológicos/química , Receptores Inmunológicos/genéticaRESUMEN
The most reliable explanation for decreasing bone mass in elderly women is an imbalance of osteoclastic resorption and osteoblastic formation resulting from a relative increase in osteoclastic resorption. However, it is not clear whether an increase in osteoclastic bone resorption with age is due to increased osteoclast formation or to osteoclastic bone resorption activity. In this study, using a human bone marrow culture system, we attempt to clarify the increase in osteoclast formation with age. The mononuclear cell-rich fraction from bone marrow, obtained from the proximal region of the femur from female elderly patients with fracture, were cultured for 14 days in the presence of 1,25 dihydroxyvitamin D(3). Tartrate-resistant acid phosphatase-positive multinucleated cells were counted as osteoclasts. In our investigation, human osteoclast formation in the bone marrow culture increased with age in elderly women (age 64-96 years). The osteoclast formation was positively correlated with macrophage-colony stimulation factor and prostaglandin E(2) production in bone marrow culture. Also, osteoclast formation ex vivo was negatively correlated with bone mineral density of the lumbar spine (L2-L4). The above results indicate that the osteoclastogenic potential of bone marrow cells increases with aging in elderly women with fracture, and suggest that a decrease in bone mass of elderly women may be due to an increase in osteoclast population associated with aging.