RESUMEN
Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB.
Asunto(s)
Antineoplásicos/farmacología , Citocinas/biosíntesis , Mediadores de Inflamación/metabolismo , Metotrexato/farmacología , FN-kappa B/metabolismo , Osteoclastos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Citocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Osteoclastos/citología , Osteoclastos/fisiología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Resorción Ósea , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Paclitaxel/farmacología , Ligando RANK/antagonistas & inhibidores , Animales , Neoplasias Óseas/patología , Línea Celular , Citoesqueleto/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Tumor Óseo de Células Gigantes/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Osteoclastos/ultraestructura , Osteólisis , Ligando RANK/farmacologíaRESUMEN
Receptor activator NF-kappaB ligand (RANKL)-activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF-kappaB inhibitor from honeybee propolis has been shown to have anti-tumor and anti-inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL-induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 microM) dose dependently inhibited RANKL-induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL-induced NF-kappaB and NFAT activation, concomitant with delayed IkappaBalpha degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF-kappaB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases.
Asunto(s)
Resorción Ósea/patología , Ácidos Cafeicos/farmacología , Diferenciación Celular/efectos de los fármacos , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/citología , Ligando RANK/farmacología , Fosfatasa Ácida/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Ácidos Cafeicos/administración & dosificación , Caspasa 3/metabolismo , Línea Celular Tumoral , Células Cultivadas , Humanos , Proteínas I-kappa B/metabolismo , Isoenzimas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , Factores de Transcripción NFATC/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Alcohol Feniletílico/análogos & derivados , Própolis/química , Fosfatasa Ácida Tartratorresistente , Factor de Transcripción ReIA/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: This study aims to investigate, utilising micro-computed tomography (micro-CT) and histology, whether the topical application of nerve growth factor (NGF) and/or epidermal growth factor (EGF) can enhance periodontal, alveolar bone, root and pulpal tissue regeneration while minimising the risk of pulpal necrosis, root resorption and ankylosis of replanted molars in a rat model. METHODS: Twelve four-week-old male Sprague-Dawley rats were divided into four groups: sham, collagen, EGF and NGF. The maxillary right first molar was elevated and replanted with or without a collagen membrane impregnated with either the growth factors EGF or NGF, or a saline solution. Four weeks after replantation, the animals were sacrificed and the posterior maxilla was assessed using histological and micro-CT analysis. The maxillary left first molar served as the control for the corresponding right first molar. RESULTS: Micro-CT analysis revealed a tendency for all replanted molars to have reduced root length, root volume, alveolar bone height and inter-radicular alveolar bone volume. It appears that the use of the collagen membrane had a negative effect while no positive effect was noted with the incorporation of EGF or NGF. Histologically, the incorporation of the collagen membrane was found to negatively affect pulpal, root, periodontal and alveolar bone healing with pulpal inflammation and hard tissue formation, extensive root resorption and alveolar bone fragmentation. The incorporation of EGF and NGF did not improve root, periodontal or alveolar bone healing. However, EGF was found to improve pulp vascularisation while NGF-improved pulpal architecture and cell organisation, although not to the level of the control group. CONCLUSIONS: Results indicate a possible benefit on pulpal vascularisation and pulpal cell organisation following the incorporation of EGF and NGF, respectively, into the alveolar socket of replanted molars in the rat model. No potential benefit of EGF and NGF was detected in periodontal or root healing, while the use of a collagen membrane carrier was found to have a negative effect on the healing response.
Asunto(s)
Proceso Alveolar/efectos de los fármacos , Pulpa Dental/efectos de los fármacos , Factor de Crecimiento Epidérmico/uso terapéutico , Diente Molar/efectos de los fármacos , Factor de Crecimiento Nervioso/uso terapéutico , Periodoncio/efectos de los fármacos , Reimplante Dental/métodos , Raíz del Diente/efectos de los fármacos , Proceso Alveolar/patología , Animales , Colágeno , Pulpa Dental/irrigación sanguínea , Pulpa Dental/patología , Necrosis de la Pulpa Dental/prevención & control , Modelos Animales de Enfermedad , Masculino , Maxilar/efectos de los fármacos , Maxilar/patología , Membranas Artificiales , Diente Molar/patología , Neovascularización Fisiológica/efectos de los fármacos , Periodoncio/patología , Pulpitis/etiología , Distribución Aleatoria , Ratas Sprague-Dawley , Regeneración/efectos de los fármacos , Resorción Radicular/prevención & control , Anquilosis del Diente/prevención & control , Raíz del Diente/patología , Cicatrización de Heridas/efectos de los fármacos , Microtomografía por Rayos X/métodosRESUMEN
Osteolytic bone diseases including osteoporosis are commonly accompanied with enhanced osteoclast formation and bone resorption. Naringin, a natural occurring flavonoid has been found to protect against retinoic acid-induced osteoporosis and improve bone quality in rats. Here, we showed that naringin perturbs osteoclast formation and bone resorption by inhibiting RANK-mediated NF-κB and ERK signaling. Naringin suppressed gene expression of key osteoclast marker genes. Naringin was found to inhibit RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. In addition, naringin inhibited RANKL-induced phosphorylation of ERK. This study identifies naringin as an inhibitor for osteoclast formation and bone resorption, and provides evidence that natural compounds such as naringin might be beneficial as an alternative medicine for the prevention and treatment of osteolysis.
Asunto(s)
Resorción Ósea/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavanonas/farmacología , FN-kappa B/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos , Ligando RANK/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Resorción Ósea/genética , Línea Celular , Activación Enzimática/efectos de los fármacos , Ratones , Microscopía Confocal , Osteoclastos/citología , Osteogénesis/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vesicular transport along microtubules must be strictly regulated to sustain the unique structural and functional polarization of bone-resorbing osteoclasts. However, the molecular mechanisms bridging these vesicle-microtubule interactions remain largely obscure. Rab3D, a member of the Rab3 subfamily (Rab3A/B/C/D) of small exocytotic GTPases, represents a core component of the osteoclastic vesicle transport machinery. Here, we identify a new Rab3D-interacting partner, Tctex-1, a light chain of the cytoplasmic dynein microtubule motor complex, by a yeast two-hybrid screen. We demonstrate that Tctex-1 binds specifically to Rab3D in a GTP-dependent manner and co-occupies Rab3D-bearing vesicles in bone-resorbing osteoclasts. Furthermore, we provide evidence that Tctex-1 and Rab3D intimately associate with the dynein motor complex and microtubules in osteoclasts. Finally, targeted disruption of Tctex-1 by RNA interference significantly impairs bone resorption capacity and mislocalizes Rab3D vesicles in osteoclasts, attesting to the notion that components of the Rab3D-trafficking pathway contribute to the maintenance of osteoclastic resorptive function.
Asunto(s)
Resorción Ósea/metabolismo , Dineínas/metabolismo , Osteoclastos/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Resorción Ósea/patología , Línea Celular , Dineínas/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Osteoclastos/patología , Osteogénesis , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rab3/químicaRESUMEN
Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-kappaB is a set of nuclear factors that bind to consensus DNA sequences called kappaB sites, and is essential for osteoclast formation and survival. NF-kappaB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-alpha and IL-1, which differentially regulate classical and/or alternative NF-kappaB pathways in osteoclastic cells. These pathways are also modulated by NF-kappaB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin-proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-kappaB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Paget's disease of bone, and periodontitis. NF-kappaB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-kappaB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.
Asunto(s)
FN-kappa B/fisiología , Animales , Artritis Reumatoide/metabolismo , ADN/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación , Ratones , Modelos Biológicos , Modelos Químicos , FN-kappa B/metabolismo , Osteoartritis/metabolismo , Osteoclastos/metabolismo , Periodontitis/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Calcium/calmodulin-dependent protein kinase (CaMK) is a major down stream mediator of Ca(2+) signaling in a wide range of cellular functions, including ion channel and cell cycle regulation and neurotransmitter synthesis and release. Here we have investigated the role of the CaMK signaling pathway in osteoclast differentiation and bone resorption. We observed that the CaMKI, CaMKII gamma isoforms were present in both bone-marrow derived macrophages and RAW264.7 murine macrophage cell line, and that expression persisted during osteoclast differentiation in the presence of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL). RANKL-induced differentiation was accompanied by increased cyclic AMP response element transcriptional activity, and ERK phosphorylation, which are both downstream targets of CaMK. Two selective inhibitors of CaMKs, KN-93 and KN-62, inhibited osteoclastogenesis in a time and concentration-dependent manner. This was accompanied by suppression of cathepsin K expression and osteoclastic bone resorption, which are markers for differentiated osteoclast function. KN-93 and KN-62 both inhibited RANKL-induced ERK phosphorylation and CREB transcriptional activity. These findings imply a role for CaMK in osteoclast differentiation and bone resorption.