RESUMEN
Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volumedependent manner. A coculture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrateresistant acid phosphatase (TRAP)positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAPpositive multinuclear cells (≥8 nuclei) was increased, in a media volumedependent manner. There were also no differences in receptor activator of nuclear factorκB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volumedependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.
Asunto(s)
Resorción Ósea , Calcificación Fisiológica , Técnicas de Cultivo de Célula , Medios de Cultivo , Osteoblastos/fisiología , Osteoclastos/fisiología , Animales , Biomarcadores , Línea Celular , RatonesRESUMEN
Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclastassociated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.
Asunto(s)
Resorción Ósea , Osteoclastos/fisiología , Estrés Mecánico , Animales , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , ARN Mensajero/genéticaRESUMEN
The present study investigated the effect of the natural polyphenols, rosmarinic acid and arbutin, on osteoclast differentiation in RAW 264.7 cells. Rosmarinic acid and arbutin suppressed osteoclast differentiation and had no cytotoxic effect on osteoclast precursor cells. Rosmarinic acid and arbutin inhibited superoxide production in a dose-dependent manner. mRNA expression of the master regulator of osteoclastogenesis, nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and the osteoclast marker genes, matrix metalloproteinase-9, tartrate-resistant acid phosphatase and cathepsin-K, decreased following treatments with rosmarinic acid and arbutin. Furthermore, resorption activity decreased with the number of osteoclasts. These results suggest that rosmarinic acid and arbutin may be useful for the prevention and treatment of bone diseases, such as osteoporosis, through mechanisms involving inhibition of superoxide and downregulation of NFATc1.
RESUMEN
Mechanical stress is an important factor in bone homeostasis, which is maintained by a balance between bone resorption by osteoclasts and bone formation by osteoblasts. However, little is known about the effects of mechanical stress on osteoclast differentiation. In this study, we examined the effects of short-term mechanical stress on osteoclastogenesis by applying tensile force to RAW264.7 cells stimulated with receptor activator of nuclear factor-κB ligand (RANKL) using a Flexercell tension system. We counted the number of osteoclasts that were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (two or more nuclei) with or without application of mechanical stress for 24 h. Osteoclast number was lower after mechanical stress compared with no mechanical stress. Furthermore, mechanical stress for up to 24 h caused downregulation of osteoclast-specific gene expression and fusion-related molecule [dendritic cell specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), E-cadherin, Integrin αV and Integrin ß3] mRNA levels. Protein expression of DC-STAMP decreased with mechanical stress for 24 h compared to the control without mechanical stress, whereas the expression of E-cadherin, Integrin αV and Integrin ß3 was slightly decreased. Nuclear factor of activated T cells c1 (NFATc1) mRNA levels were decreased at 6 h and increased at 12 and 24 h compared with the control. The levels of NFATc2, NFATc3 mRNA did not change compared with the control group. By contrast, mechanical stress for 24 h significantly enhanced NFAT transcriptional activity compared with the control, despite a decrease in DC-STAMP mRNA and protein levels. These results suggest that short-term mechanical stress strongly inhibits osteoclastogenesis through the downregulation of DC-STAMP and other fusion-related molecules and that short-term mechanical stress induces a negative regulatory mechanism that cancels the enhancement of NFAT transcriptional activity.
Asunto(s)
Diferenciación Celular , Regulación hacia Abajo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Osteoclastos/citología , Estrés Mecánico , Animales , Cadherinas/genética , Cadherinas/metabolismo , Recuento de Células , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Integrina alfaV/genética , Integrina alfaV/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Osteoclastos/metabolismo , Ligando RANK/metabolismo , ARN Mensajero/genéticaRESUMEN
Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.
Asunto(s)
Difosfonatos/farmacología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Bioensayo , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Difosfonatos/química , Electroforesis en Gel de Agar , Regulación de la Expresión Génica/efectos de los fármacos , Células Gigantes/citología , Células Gigantes/efectos de los fármacos , Isoenzimas/metabolismo , Ratones , Osteoclastos/enzimología , Osteogénesis/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fosfatasa Ácida Tartratorresistente , Factores de TiempoRESUMEN
Flagellin, the ligand of Toll like receptor 5, is the major subunit of bacterial flagella. Flagellin stimulates various cells to release chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family that is involved in monocyte infiltration in inflammatory diseases. It has been reported that serum MCP-1 levels increase proportionally with the severity of periodontal disease. Inflammatory mediators induce MCP-1 production in various cells, including osteoblasts. However, it remains unclear whether MCP-1 is released from osteoblasts in response to flagellin. In the present study, we investigated the effects of flagellin on the expression of MCP-1 in the mouse osteoblastic cell line, MC3T3-E1 (E1) cells. Flagellin markedly increased MCP-1 mRNA level in a dose-dependent manner. The effect of flagellin on MCP-1 mRNA expression in E1 cells was transient, with a peak at 1 h. Concomitant with MCP-1 mRNA expression, MCP-1 protein levels were clearly elevated at 3 h after flagellin exposure. In addition, we revealed that JNK and MEK-ERK1/2 are involved in flagellin-induced MCP-1 expression in E1 cells. These results indicated that bacterial flagellin may play an important role in the progression of periodontitis. Results of further studies will provide more clues to the prevention of periodontal diseases.
Asunto(s)
Quimiocina CCL2/metabolismo , Flagelina/farmacología , Osteoblastos/citología , Osteoblastos/metabolismo , Animales , Línea Celular , Quimiocina CCL2/genética , Ratones , Enfermedades Periodontales/tratamiento farmacológico , ARN Mensajero/genética , Transducción de Señal , Receptor Toll-Like 5/metabolismoRESUMEN
Recent research has shown that platinum nanoparticles (nano-Pt) efficiently quench reactive oxygen species (ROS) as a reducing catalyst. ROS have been suggested to regulate receptor activator of NF-κB ligand (RANKL)-stimulated osteoclast differentiation. In the present study, we examined the direct effects of platinum nano-Pt on RANKL-induced osteoclast differentiation of murine pre-osteoclastic RAW 264.7 cells. The effect of the nano-Pt on the number of osteoclasts was measured and their effect on the mRNA expression for osteoclast differentiation was assayed using real-time PCR. Nano-Pt appeared to have a ROS-scavenging activity. Nano-Pt decreased the number of osteoclasts (2+ nuclei) and large osteoclasts (8+ nuclei) in a dose-dependent manner without affecting cell viability. In addition, this agent significantly blocked RANKL-induced mRNA expression of osteoclastic differentiation genes such as c-fms, NFATc1, NFATc2, and DC-STAMP as well as that of osteoclast-specific marker genes including MMP-9, Cath-K, CLC7, ATP6i, CTR, and TRAP. Although nano-Pt attenuated expression of the ROS-producing NOX-family oxidases, Nox1 and Nox4, they up-regulated expression of Nox2, the major Nox enzyme in macrophages. These findings suggest that the nano-Pt inhibit RANKL-stimulated osteoclast differentiation via their ROS scavenging property. The use of nano-Pt as scavengers of ROS that is generated by RANKL may be a novel and innovative therapy for bone diseases.
Asunto(s)
Nanopartículas , Osteoclastos/efectos de los fármacos , Platino (Metal)/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Osteoclastos/metabolismo , Platino (Metal)/administración & dosificación , Ligando RANK/antagonistas & inhibidores , ARN Mensajero/metabolismoRESUMEN
The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.
Asunto(s)
Diferenciación Celular , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico/metabolismo , Osteoclastos/fisiología , Estrés Mecánico , Fosfatasa Ácida/genética , Fosfatasa Ácida/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Catepsina K/genética , Catepsina K/fisiología , Línea Celular , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Expresión Génica/genética , Expresión Génica/fisiología , Isoenzimas/genética , Isoenzimas/fisiología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/fisiología , Osteoclastos/citología , Presión , Receptores de Calcitonina/genética , Receptores de Calcitonina/fisiología , Fosfatasa Ácida Tartratorresistente , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/fisiologíaRESUMEN
INTRODUCTION: The purpose of this study was to evaluate the cytotoxicity of mineral trioxide aggregate (MTA) and its potential detoxification by an antioxidant amino acid, N-acetylcysteine (NAC). METHODS: Rat dental pulp cells extracted from rat maxillary incisors were directly cultured on MTA with or without NAC in culture medium. The number of cells and their spreading behavior were both assessed 24 hours after seeding. The intracellular levels of reactive oxygen species (ROS) and glutathione (GSH) were also assessed after 24 hours of culture. RESULTS: The number of cells attached to MTA was 60% greater when NAC was added to the culture medium. In addition, the area and perimeter of the cells were found to be 2-fold greater in the culture containing NAC. Cells cultured on MTA alone showed large ROS concentrations, which disappeared when the medium was supplemented with NAC. The intracellular GSH level, however, increased 3.5-fold with NAC addition. CONCLUSIONS: This study demonstrated that the presence of NAC in environments can substantially improve attachment and spreading behaviors of dental pulp cells on MTA. This biological effect was associated with an improvement in the cellular redox system by NAC and warrants further exploration of NAC for determining its therapeutic value in improving the biocompatibility of MTA.
Asunto(s)
Acetilcisteína/farmacología , Compuestos de Aluminio/farmacología , Antioxidantes/farmacología , Compuestos de Calcio/farmacología , Pulpa Dental/efectos de los fármacos , Óxidos/farmacología , Materiales de Obturación del Conducto Radicular/farmacología , Silicatos/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Recuento de Células , Movimiento Celular/efectos de los fármacos , Forma de la Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorimetría , Medios de Cultivo , Pulpa Dental/citología , Combinación de Medicamentos , Colorantes Fluorescentes , Glutatión/análisis , Indicadores y Reactivos , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/análisis , Sales de Tetrazolio , Factores de TiempoRESUMEN
Glass ionomer cements (GICs) are widely used for the operative restoration of dental caries. However, it has been reported that the components of GICs cause pulpal inflammatory responses. Recently, GICs containing tannin-fluoride preparation (HY agent) were developed. In this study, we investigated the effect of HY agent on prostaglandin E2 (PGE2) release from GIC-stimulated rat dental pulp cells (RPC-C2A). Extracts derived from GIC disks were used with HY(+) and without HY(-) agent. After treatment with GIC extracts, ATP contents, COX-2 mRNA and protein expression in RPC-C2A cells, and PGE2 production in culture media were analyzed. HY agent suppressed HY(-)-stimulated PGE2 release from RPC-C2A cells, as well as COX-2 mRNA and protein expression. Moreover, tannic acid attenuated COX-2 mRNA induced by HY(-) extract in a dose-dependent manner. Taken together, these results suggest that tannic acid in HY agent may suppress GIC-induced production of PGE2 by inhibition of COX-2 expression in dental pulp cells.
Asunto(s)
Pulpa Dental/metabolismo , Dinoprostona/metabolismo , Fluoruros/farmacología , Taninos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Pulpa Dental/citología , Regulación hacia Abajo , Cementos de Ionómero Vítreo/farmacología , RatasRESUMEN
Current dental restorative materials are only used to fill the defect of hard tissues, such as dentin and enamel, because of their cytotoxicity. Therefore, exposed dental pulp tissues in deep cavities must be first covered by a pulp capping material like calcium hydroxide to form a layer of mineralized tissue. However, this tissue mineralization is based on pathological reaction and triggers long-lasting inflammation, often causing clinical problems. This study tested the ability of N-acetyl cysteine (NAC), amino acid derivative, to reduce cytotoxicity and induce mineralized tissue conductivity in resin-modified glass ionomer (RMGI), a widely used dental restorative material having dual cure mechanism. Rat dental pulp cells were cultured on untreated or NAC-supplemented RMGI. NAC supplementation substantially increased the percentage of viable cells from 46.7 to 73.3% after 24-h incubation. Cell attachment, spreading, proliferative activity, and odontoblast-related gene and protein expressions increased significantly on NAC-supplemented RMGI. The mineralization capability of cells, which was nearly suppressed on untreated RMGI, was induced on NAC-supplemented RMGI. These improved behaviors and functions of dental pulp cells on NAC-supplemented RMGI were associated with a considerable reduction in the production of intracellular reactive oxygen species and with the increased level of intracellular glutathione reserves. These results demonstrated that NAC could detoxify and functionalize RMGIs via two different mechanisms involving in situ material detoxification and antioxidant cell protection. We believe that this study provides a new approach for developing dental restorative materials that enables mineralized tissue regeneration.
Asunto(s)
Acetilcisteína/metabolismo , Calcificación Fisiológica , Recubrimiento de la Pulpa Dental/instrumentación , Pulpa Dental/citología , Pulpa Dental/fisiología , Cementos de Ionómero Vítreo/metabolismo , Acetilcisteína/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Supervivencia Celular , Células Cultivadas , Citocinas/inmunología , Recubrimiento de la Pulpa Dental/métodos , Cementos de Ionómero Vítreo/química , Glutatión/metabolismo , Masculino , Ensayo de Materiales , Fenotipo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Regeneración/fisiologíaRESUMEN
Modulation of the physiologically influential Na(+),K(+)-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min-24 h) with those PTP inhibitors (0-100 microM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na(+),K(+)-ATPase activity in both human and pig samples. The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na(+),K(+)-ATPase levels. Despite rigorous activity recovery attempts, the PTP inhibitors' effects were sustained on Na(+),K(+)-ATPase activity. Western blotting experiments revealed the expression of both alpha(1)- and beta(1)-subunits in both human and pig tissues. alpha(1)-Subunits possessed higher tyrosine phosphorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both alpha(1)- and beta(1)-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we provide evidence that Na(+),K(+)-ATPase can be regulated through tyrosine phosphorylation of primarily their alpha(1)-subunits, using PTP inhibitors.
Asunto(s)
Riñón/enzimología , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Catecolaminas/farmacología , Línea Celular Tumoral , Citometría de Flujo , Humanos , Hidroquinonas/farmacología , Compuestos Nitrosos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , PorcinosRESUMEN
Proteomic and genomic studies commonly involve the assessment of mRNA levels using reverse transcription-polymerase chain reaction (PCR) and real-time quantitative PCR. An internal standard RNA is fundamentally analyzed along with the investigated mRNA to document the specificity of the effect(s) on mRNA and to correct for inter-sample variations. In our studies implementing estrogen treatments on different cell lines, we initially used glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal standard. However, the results of PCR amplification demonstrated that 17ß-estradiol enhanced the expression of the G3PDH gene, rendering it impossible to use G3PDH as an unbiased comparative control.
RESUMEN
We investigated the possible roles of estrogen on plasma membrane Ca(2+)-ATPase (PMCA) in human fibroblast-like synovial cells (HFLS) and mouse macrophage-like cells (RAW 264.7). Western blots revealed the expression of PMCA 2 and 4 in both cells. In vitro treatments with 17beta-estradiol for 24 hours resulted in a concentration dependent decrease in PMCA expression. Moreover, Ca(2+)-ATPase specific activity was similarly decreased with estrogen treatments. However, treatments for 1 hour in the presence or absence of cycloheximide demonstrated non-significant effects. These results suggest that estrogen has a modulatory role on Ca(2+) homeostasis through decreasing PMCA expression and abating their activity.
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Estradiol/farmacología , Macrófagos/efectos de los fármacos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Línea Celular , Cicloheximida/farmacología , Humanos , Macrófagos/citología , Macrófagos/enzimología , Ratones , Inhibidores de la Síntesis de la Proteína/farmacología , Líquido Sinovial/citología , Líquido Sinovial/efectos de los fármacos , Líquido Sinovial/enzimologíaRESUMEN
INTRODUCTION: Recently, mineral trioxide aggregate (MTA) has been routinely used for endodontic treatment. It is well-known that MTA induced secondary dentin formation in pulp cavity when it was applied to dentin, whereas its cytotoxicities were unclear. The purpose of this study was to evaluate the effect of MTA on rat clonal dental pulp cells, RPC-C2A. METHODS: This study was conducted to observe the response of RPC-C2A cells on MTA with reverse-transcriptase polymerase chain reaction, Western blot analysis, and enzyme immunoassay. Data were compared by analysis of variance. Statistical significance was established at P <.01. RESULTS: MTA significantly caused an up-regulation of cyclooxygenase-2 (COX-2) and inducible form of nitric oxide synthase (iNOS) mRNA expression. Furthermore, MTA caused inhibitory kappa B (IkappaB) phosphorylation and translocation of nuclear factor-kappa B (NF-kappaB) subunits to nucleus. Curucumin, an inhibitor of NF-kappaB activation, suppressed MTA-induced COX-2 and iNOS mRNA expressions. In addition, MTA increased the production of prostaglandin E(2) in comparison with the controls. CONCLUSIONS: MTA induces inflammation via NF-kappaB signaling system.
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Compuestos de Aluminio/toxicidad , Compuestos de Calcio/toxicidad , Ciclooxigenasa 2/biosíntesis , Pulpa Dental/efectos de los fármacos , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxidos/toxicidad , Materiales de Obturación del Conducto Radicular/toxicidad , Silicatos/toxicidad , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Células Cultivadas , Inhibidores de la Ciclooxigenasa 2/farmacología , Pulpa Dental/metabolismo , Dinoprostona/metabolismo , Combinación de Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas I-kappa B/química , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fosforilación , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: Cisplatin (CDDP) is one of the major chemotherapeutic drugs, but tumor cells' acquired resistance to CDDP limits its therapeutic potentials. One of the main reasons of resistance is reduced drug accumulation. The mechanism by which tumor cells accumulate reduced CDDP is not well elucidated yet. The aim of this study was to investigate what regulates intracellular CDDP accumulation. METHODS: Six different types of oral squamous carcinoma cells were used in this study. Assessment of CDDP sensitivity was determined by measuring the ATP level of the cells. Intracellular CDDP and copper (Cu) accumulation were measured and CDDP efflux study was conducted. Assessment of Na(+),K(+)-ATPase alpha and beta subunits, ATP7A and ATP7B was done by western blotting. Specific activities of Na(+),K(+)-ATPase and copper-transporting P-type ATPase (Cu(2+)-ATPase) were detected and a role of Na(+),K(+)-ATPase inhibitor in intracellular CDDP accumulation was examined. RESULTS: Among the cells HSC-3 and BHY cells were found most CDDP-sensitive and CDDP-resistant, respectively. The most CDDP-sensitive HSC-3 cells exhibited an increased intracellular cisplatin accumulation, high Na(+),K(+)-ATPase activity and over-expressed Na(+),K(+)-ATPase alpha and beta subunits, ATP7A and ATP7B, compared to the most CDDP-resistant BHY cells, but there were no such differences between the two in the CDDP efflux level or Cu(2+)-ATPase activity. Moreover, pretreatment with Na(+),K(+)-ATPase inhibitor markedly reduced intracellular cisplatin accumulation. CONCLUSIONS: Na(+),K(+)-ATPase activity is responsible for regulating intracellular CDDP accumulation in oral squamous carcinoma cells rather than Cu(2+)-ATPase.
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Adenosina Trifosfatasas/metabolismo , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Proteínas de Transporte de Catión/metabolismo , Cisplatino/farmacología , Neoplasias de la Boca/tratamiento farmacológico , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Western Blotting , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/patología , Cobre/metabolismo , ATPasas Transportadoras de Cobre , Resistencia a Antineoplásicos , Humanos , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/patología , Células Tumorales CultivadasRESUMEN
Although it has been reported that vascular endothelial growth factor (VEGF) promotes not only angiogenesis but also osteoclast and osteoblast differentiation, few reports exist regarding VEGF/VEGF receptor (VEGFR) signaling in osteoblasts, which regulate osteoclast differentiation and generate VEGF. This study examined the expression of the bone remodeling factor VEGF-A and its receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), in murine osteoblastic MC3T3-E1 cells with the application of mechanical stress. The protein concentration of VEGF-A in the mechanical stress group increased markedly compared with the control group, while that of macrophage colony-stimulating factor in the mechanical stress group was lower than in the control group. VEGFR-2 mRNA expression was not detected in osteoblastic MC3T3-E1 cells. Mechanical stress up-regulated VEGF-A, VEGFR-1 and the receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression. In particular, VEGF-A and RANKL mRNA expression increased immediately after mechanical stress. We examined the VEGF/VEGFR system on anti-mouse VEGF neutralizing antibody in osteoblasts with mechanical stress. Neutralizing antibody to VEGF partially inhibited the increase of VEGF-A and RANKL mRNA expression compared with the non-mechanical stress group. VEGFR-1 mRNA expression was completely suppressed to control levels by the neutralizing antibody to VEGF. These findings suggest that mechanical stress up-regulates RANKL expression via the VEGF/VEGFR-1 autocrine pathway in osteoblastic MC3T3-E1 cells, indicating the possibility that, in response to mechanical stress, osteoblasts increase bone resorption by an autocrine up-regulation of VEGF/VEGFR-1 and RANKL expression.
RESUMEN
Matrix metalloproteinases (MMPs) play important roles in the invasion and metastasis to soft tissues of carcinomas including, oral squamous cell carcinomas (SCCs). Although, osteoclastic bone resorption is an important step in bone involvement in a variety of malignancies, the mechanism of bone involvement of oral SCC remains unclear. Once cancer cells arrest in bone, the bone is a storehouse of a variety of cytokines and growth factors and thus provides an extremely fertile environment for cell growth. The bone-invasive oral cancer cell line, BHY, transcriptionally expressed detectable levels of TGF-beta, IL-1beta, IL-8, parathyroid hormone-related protein (PTHrP) and vascular endothelial growth factor (VEGF) mRNAs and failed to express GM-CSF, IL-6, and TNF-alpha. Furthermore, the BHY-conditioned medium greatly upregulated IL-6 and RANKL/ODF mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. Seven out of eleven patients with carcinomas of the lower alveolus and gingiva showing infiltrative bone involvement expressed PTHrP mRNA. These data suggest that the occurrence of PTHrP may be an indication of developing oral malignant carcinomas.
Asunto(s)
Carcinoma de Células Escamosas/patología , Diferenciación Celular , Neoplasias de la Boca/patología , Osteoblastos/citología , Osteoclastos/citología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Animales , Células de la Médula Ósea , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Expresión Génica , Neoplasias Gingivales/genética , Neoplasias Gingivales/metabolismo , Neoplasias Gingivales/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Boca/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Several epidemiological studies have reported that temporomandibular disorder is more prevalent in women, which suggests the involvement of sex hormones, such as estrogen, in the pathogenesis of this disease. PCR amplification and Western blotting were employed to target the expression of estrogen receptors (ERs) in human fibroblast-like synovial and ATDC5 cells. The effect of estrogen was investigated through the expression of RANKL, osteoprotegerin (OPG), M-CSF/CSF-1 and c-fms. We showed expression of M-CSF/ CSF-1 and c-fms, with time-dependent increase in both after the addition of estrogen. Based on previous studies reporting that M-CSF/CSF-1 regulates the proliferation and differentiation of hemopoietic progenitor cells into mature macrophages, we put forward a new hypothesis based on the increased inflammation and tendency of females to suffer more from temporomandibular disorder (TMD) in the presence of external exacerbating factors. Detection of RANKL and OPG in ATDC5 and expression of both in HFLS was confirmed with complete disappearance of the RANKL band, and marked increase in the expression of OPG after 1 h from the addition of estrogen.
Asunto(s)
Estrógenos/farmacología , Fibroblastos/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Anciano , Animales , Western Blotting , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/prevención & control , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/farmacología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/prevención & control , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligando RANK/genética , Ligando RANK/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B/análisis , Receptor Activador del Factor Nuclear kappa-B/efectos de los fármacos , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Articulación Temporomandibular/efectos de los fármacos , Articulación Temporomandibular/metabolismo , Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/genética , Trastornos de la Articulación Temporomandibular/metabolismo , Trastornos de la Articulación Temporomandibular/prevención & controlRESUMEN
Although it is known that mechanical stress to osteoblast and periodontal ligament cells suppresses osteoclast differentiation, little is known about the direct effect of mechanical stress on osteoclast differentiation. In this study, we examined the role of mechanical stress on osteoclast differentiation using murine pre-osteoclastic RAW264.7 cells treated with receptor activator of nuclear factor-kappaB ligand (RANKL). RAW cells were cultured with RANKL, and mechanical stress was applied for a given period. We counted the number of osteoclast cells which were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (2 nuclei or more), and measured mRNA by RT-PCR. There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of nuclei per osteoclast also decreased compared to the number of nuclei per osteoclast cultured with the application of mechanical stress. As the cells were cultured for a period of 1-7 days and/or for different periods of mechanical stress application, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated with the number of osteoclasts. Inducible nitric oxide synthase mRNA which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and NFATc2 mRNA expression increased with mechanical stress. These findings indicate that mechanical stress directly suppresses osteoclast differentiation and increases NFATc1 and NFATc2 suggesting delayed differentiation.