RESUMEN
BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.
Asunto(s)
Resorción Ósea/genética , Fuerza Compresiva/fisiología , Osteoclastos/metabolismo , Animales , Resorción Ósea/metabolismo , Anhidrasa Carbónica II/metabolismo , Catepsina K/metabolismo , Diferenciación Celular/genética , Línea Celular , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , FN-kappa B/metabolismo , Osteoclastos/fisiología , Ligando RANK/metabolismo , Células RAW 264.7 , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transducción de SeñalRESUMEN
Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics. In previous in vivo and in vitro studies, LIPUS has been shown to have promising effects on cellular elements in articular cartilage, particularly chondrocytes in patients with osteoarthritis. However, the effects of LIPUS on the cellular mechanisms through which LIPUS alters extracellular matrix (ECM) synthesis in chondrocytes are unclear. In this study, we investigated the effects of the optimal intensity and cellular mechanisms of LIPUS on the regeneration of cartilage matrix in chondrocytes. LIPUS induced collagen synthesis and the remodeling of aggrecan via the activation of ERK1/2. In contrast, MMP13 expression was decreased in chondrocytes. Additionally, chondrocytes responded optimally to LIPUS at an intensity higher than the clinical setting for bone fracture healing. These results suggested that LIPUS induced ECM regeneration via increases in hypertrophic chondrocytes and delayed endochondral ossification in chondrocytes.
Asunto(s)
Cartílago Articular/efectos de la radiación , Condrocitos/metabolismo , Matriz Extracelular/efectos de la radiación , Metaloproteinasa 13 de la Matriz/metabolismo , Ondas Ultrasónicas , Agrecanos/metabolismo , Animales , Colágeno/biosíntesis , Humanos , Osteoartritis/patología , Osteogénesis/efectos de la radiaciónRESUMEN
OBJECTIVE: MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF). MATERIALS AND METHODS: Total RNA and protein were extracted from HGF after stimulation by interleukin-1ß (IL-1ß; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1ß, inhibitor of nuclear factor kappa-B kinaseß (IKKß), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot. RESULTS: IL-1ß and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKß and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKß and ZEB1 and increased E-cadherin mRNA and protein levels in HGF. CONCLUSIONS: These results suggest that miR-200b attenuates inflammatory response via IKKß and ZEB1 in periodontal tissue.
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Fibroblastos/metabolismo , Encía/metabolismo , Quinasa I-kappa B/genética , Interleucina-6/genética , MicroARNs/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/metabolismo , MicroARNs/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Inflammatory cytokines, interleukin (IL)-1, IL-6, and TNF-α, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However, the mechanism underlying effects of LIPUS on LPS-induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS-induced IL-1α production. Mouse calvaria osteoblast-like cells MC3T3-E1 were incubated in the presence or absence of LPS (Porphyromonas gingivalis), and then stimulated with LIPUS for 30 min/day. To investigate the role of LIPUS, we determined the expression of IL-1α stimulated with LIPUS and treated with an angiotensin II receptor type 1 (AT1) antagonist, Losartan. We also investigate to clarify the pathway of LIPUS, we transfected siRNA silencing AT1 (siAT1) in MC3T3-E1. LIPUS inhibited mRNA and protein expression of LPS-induced IL-1α. LIPUS also reduced the nuclear translocation of NF-κB by LPS-induced IL-1α. Losartan and siAT1 blocked all the stimulatory effects of LIPUS on IL-1α production and IL-1α-mediated NF-κB translocation induced by LPS. Furthermore, PLCß inhibitor U73122 recovered NF-κB translocation. These results suggest that LIPUS inhibits LPS-induced IL-1α via AT1-PLCß in osteoblasts. We exhibit that these findings are in part of the signaling pathway of LIPUS on the anti-inflammatory effects of IL-1α expression.
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Interleucina-1alfa/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Ondas Ultrasónicas , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Lipopolisacáridos/farmacología , Ratones , Fosfolipasa C beta/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.
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Adenosina Trifosfato/análogos & derivados , Proteínas de la Matriz Extracelular/biosíntesis , Matriz Extracelular/fisiología , Quinasa 2 de Adhesión Focal/metabolismo , Mecanotransducción Celular/fisiología , Osteoblastos/fisiología , Piranos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Células 3T3 BALB , Matriz Extracelular/efectos de los fármacos , Macrólidos , Mecanotransducción Celular/genética , Ratones , Osteoblastos/efectos de los fármacos , Piranos/agonistasRESUMEN
The association between obesity and inflammation is well documented in epidemiological studies. Proteolysis of extracellular matrix (ECM) proteins is involved in adipose tissue enlargement, and matrix metalloproteinases (MMPs) collectively cleave all ECM proteins. Here, we examined the effects of C-reactive protein (CRP), an inflammatory biomarker, on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs), which are natural inhibitors of MMPs, in adipocyte-differentiated 3T3-L1 cells. We analyzed the expression of Fcγ receptor (FcγR) IIb and FcγRIII, which are candidates for CRP receptors, and the effects of anti-CD16/CD32 antibodies, which can act as FcγRII and FcγRIII blockers on CRP-induced alteration of MMP and TIMP expression. Moreover, we examined the effects of CRP on the activation of mitogen-activated protein kinase (MAPK) signaling, which is involved in MMP and TIMP expression, in the presence or absence of anti-CD16/CD32 antibodies. Stimulation with CRP increased MMP-1, MMP-3, MMP-9, MMP-11, MMP-14, and TIMP-1 expression but did not affect MMP-2, TIMP-2, and TIMP-4 expression; TIMP-3 expression was not detected. Adipocyte-differentiated 3T3-L1cells expressed FcγRIIb and FcγRIII; this expression was upregulated on stimulation with CRP. Anti-CD16/CD32 antibodies inhibited CRP-induced expression of MMPs, except MMP-11, and TIMP-1. CRP induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and p38 MAPK but did not affect SAPK/JNK phosphorylation, and Anti-CD16/CD32 attenuated the CRP-induced phosphorylation of p38 MAPK, but not that of ERK1/2. These results suggest that CRP facilitates ECM turnover in adipose tissue by increasing the production of multiple MMPs and TIMP-1 in adipocytes. Moreover, FcγRIIb and FcγRIII are involved in the CRP-induced expression of MMPs and TIMP-1 and the CRP-induced phosphorylation of p38, whereas the FcγR-independent pathway may regulate the CRP-induced MMP-11 expression and the CRP-induced ERK1/2 phosphorylation.
Asunto(s)
Proteína C-Reactiva/genética , Inflamación/genética , Obesidad/genética , Receptores de IgG/genética , Células 3T3-L1 , Tejido Adiposo/crecimiento & desarrollo , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Inflamación/patología , Metaloproteinasas de la Matriz/clasificación , Metaloproteinasas de la Matriz/genética , Ratones , Obesidad/patología , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Orthodontic tooth movement induces alveolar bone resorption and formation by mechanical stimuli. Force exerted on the traction side promotes bone formation. Adenosine triphosphate (ATP) is one of the key mediators that respond to bone cells by mechanical stimuli. However, the effect of tension force (TF)-induced ATP on osteogenesis is inadequately understood. Accordingly, we investigated the effect of TF on ATP production and osteogenesis in MC3T3-E1 cells. Cells were incubated in the presence or absence of P2X7 receptor antagonist A438079, and then stimulated with or without cyclic TF (6% or 18%) for a maximum of 24 h using Flexercell Strain Unit 3000. TF significantly increased extracellular ATP release compared to control. Six percent TF had maximum effect on ATP release compared to 18% TF and control. Six percent TF induced the expression of Runx2 and Osterix. Six percent TF also increased the expression of extracellular matrix proteins (ECMPs), ALP activity, and the calcium content in ECM. A438079 blocked the stimulatory effect of 6% TF on the expression of Runx2, Osterix and ECMPs, ALP activity, and calcium content in ECM. This study indicated that TF-induced extracellular ATP is released in osteoblasts, suggesting that TF-induced ATP promotes osteogenesis by autocrine action through P2X7 receptor in osteoblasts.
Asunto(s)
Adenosina Trifosfato/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Estrés Mecánico , Animales , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ratones , Osteogénesis/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways. DESIGN: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting. RESULTS: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3. CONCLUSIONS: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Osteoblastos/metabolismo , Estrés Mecánico , Animales , Células Cultivadas , Regulación hacia Abajo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Fosforilación , Regulación hacia ArribaRESUMEN
Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (ß), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kß and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.
Asunto(s)
Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Androstadienos/farmacología , Animales , Resorción Ósea/prevención & control , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Citoesqueleto/efectos de los fármacos , Indazoles/farmacología , Isoenzimas/antagonistas & inhibidores , Morfolinas/farmacología , Osteoclastos/citología , Quinazolinas/farmacología , Ligando RANK/metabolismo , Conejos , Ratas , Sulfonamidas/farmacología , WortmaninaRESUMEN
Visceral adipose tissue-derived serine protease inhibitor (vaspin), an adipokine that was recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. In this study, we investigated whether vaspin inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using two types of osteoclast precursors: RAW264.7 cells and bone marrow cells (BMCs). Vaspin inhibited RANKL-induced osteoclastogenesis in RAW264.7 cells and BMCs. Interestingly, vaspin also inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and BMCs. Furthermore, it inhibited the RANKL-induced upregulation of matrix metalloproteinase-9 and cathepsin K in RAW264.7 cells. Thus, we suggest that vaspin downregulates osteoclastogenesis in part by inhibiting expression of the transcription factor NFATc1.
Asunto(s)
Adipoquinas/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Ligando RANK/farmacología , Serpinas/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Catepsina K/metabolismo , Línea Celular , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Factores de Transcripción NFATC/metabolismo , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , RatasRESUMEN
Advanced glycation end-products (AGEs) are increased under hyperglycemia in vivo and are associated with the onset of diabetes. According to previous studies, AGEs exacerbate inflammatory diseases. However, the mechanism by which AGEs aggravate osteoblast inflammation remains unknown. Therefore, the aim of this study was to determine the effects of AGEs on the production of inflammatory mediators in MC3T3-E1 cells and the underlying molecular mechanisms. Co-stimulation with AGEs and lipopolysaccharide (LPS) was found to increase the mRNA and protein levels of cyclooxygenase 2 (COX2), interleukin-1α (IL-1α), S100 calcium-binding protein A9 (S100A9), and the production of prostaglandin E2 (PGE2) compared to no stimulation (untreated control) or individual stimulation with LPS or AGEs. In contrast, the phospholipase C (PLC) inhibitor, U73122, inhibited these stimulatory effects. Co-stimulation with AGEs and LPS also increased the nuclear translocation of nuclear factor-kappa B (NF-κB) compared to no stimulation (untreated control) or individual stimulation with LPS or AGE. However, this increase was inhibited by U73122. Co-stimulation with AGEs and LPS-induced phosphorylated phospholipase Cγ1 (p-PLCγ1) and phosphorylated c-Jun N-terminal kinase (p-JNK) expression compared to no stimulation or individual stimulation with LPS or AGEs. U73122 inhibited the effects induced by co-stimulation. siPLCγ1 did not increase the expression of p-JNK and the translocation of NF-κB. Overall, co-stimulation with AGEs and LPS may promote inflammation mediators in MC3T3-E1 cells by activating the nuclear translocation of NF-κB via PLCγ1-JNK activation.
Asunto(s)
Lipopolisacáridos , FN-kappa B , Productos Finales de Glicación Avanzada/metabolismo , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Sistema de Señalización de MAP Quinasas , FN-kappa B/metabolismo , Animales , RatonesRESUMEN
Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca(2+)-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca(2+) (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) suppressed Ca(2+) oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca(2+)-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Núcleo Celular/metabolismo , Factores de Transcripción NFATC/inmunología , Oligopéptidos/farmacología , Osteoclastos/metabolismo , Osteopontina/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Señalización del Calcio/fisiología , Bovinos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Oligopéptidos/metabolismo , Osteoclastos/citología , Osteopontina/metabolismo , Conejos , RatasRESUMEN
Lysophosphatidic acid (LPA) is a bioactive phospholipid whose functions are mediated by multiple G protein-coupled receptors. We have shown that osteoblasts produce LPA, raising the possibility that it mediates intercellular signaling among osteoblasts and osteoclasts. Here we investigated the expression, signaling and function of LPA receptors in osteoclasts. Focal application of LPA elicited transient increases in cytosolic calcium concentration ([Ca(2+)](i)), with 50% of osteoclasts responding at approximately 400 nm LPA. LPA-induced elevation of [Ca(2+)](i) was blocked by pertussis toxin or the LPA(1/3) receptor antagonist VPC-32183. LPA caused sustained retraction of osteoclast lamellipodia and disrupted peripheral actin belts. Retraction was insensitive to VPC-32183 or pertussis toxin, indicating involvement of a distinct signaling pathway. In this regard, inhibition of Rho-associated kinase stimulated respreading after LPA-induced retraction. Real-time reverse transcription-PCR revealed transcripts encoding LPA(1) and to a lesser extent LPA(2), LPA(4), and LPA(5) receptor subtypes. LPA induced nuclear translocation of NFATc1 and enhanced osteoclast survival, effects that were blocked by VPC-32183 or by a specific peptide inhibitor of NFAT activation. LPA slightly reduced the resorptive activity of osteoclasts in vitro. Thus, LPA binds to at least two receptor subtypes on osteoclasts: LPA(1), which couples through G(i/o) to elevate [Ca(2+)](i), activate NFATc1, and promote survival, and a second receptor that likely couples through G(12/13) and Rho to evoke and maintain retraction through reorganization of the actin cytoskeleton. These findings reveal a signaling axis in bone through which LPA, produced by osteoblasts, acts on multiple receptor subtypes to induce pleiotropic effects on osteoclast activity and function.
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Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Citosol/metabolismo , Lisofosfolípidos/farmacología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Amidas/farmacología , Animales , Animales Recién Nacidos , Línea Celular , Células Cultivadas , Citosol/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Osteoclastos/citología , Toxina del Pertussis/farmacología , Piridinas/farmacología , Conejos , Ratas , Receptores del Ácido Lisofosfatídico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
PURPOSE: Diabetes causes hyperglycemic disorders due to insufficient activity of insulin, and it also increases blood glucose level. Recent studies have reported the relationship between diabetes and periodontal disease. Periodontitis is advanced by inflammatory cytokines stimulated with LPS. The purpose of this study was to investigate the effects of hyperglycemia on the expression of inflammatory cytokines induced by LPS in osteoblasts. METHODS: Cells were cultured for 7 and 14 days in the presence or absence of LPS and glucose. The expression mRNA level of IL-6, RANKL and OCN was determined using real-time PCR. The protein expression of IL-6 and RANKL was also measured using ELISA. RESULTS: LPS and glucose increased the mRNA expression of IL-6, coupled with a decrease in the mRNA expression of OCN, which is associated with IL-6 and glucose. It also increased the protein expression of IL-6 compared to LPS. However, LPS+Glucose did not affect the mRNA and protein expression of RANKL. Furthermore, GLUT4 inhibitor, WZB117, blocked the stimulatory effect of glucose on LPS-induced IL-6 mRNA expression. WZB117 did not affect LPS-reduced OCN mRNA expression. CONCLUSION: These results suggest that high glucose levels increase LPS-induced IL-6 expression mediated by GLUT4.
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Transportador de Glucosa de Tipo 4/fisiología , Interleucina-6 , Lipopolisacáridos , Glucosa , Proteínas Facilitadoras del Transporte de la Glucosa , Interleucina-6/metabolismo , Osteoblastos/metabolismoRESUMEN
The cause of burning mouth syndrome (BMS) is unknown. Although no effective treatment has been established, BMS patients frequently chew gum to alleviate pain. To identify the cause and new treatments for BMS, this study investigated the psychophysical and pharmacological properties of gum chewing to better understand its pain-relieving effects. In this prospective, blinded study, plasma catecholamine and serotonin levels and Profile of Mood States (POMS) scores were assessed after gum chewing or simulated chewing in 40 women (20 BMS patients and 20 age-matched controls). Visual analogue scale (VAS) scores for pain decreased significantly in BMS patients after gum chewing and simulated chewing. Moreover, resting VAS scores of BMS patients were significantly positively correlated with plasma adrenaline level. Furthermore, gum chewing was significantly correlated with lower plasma adrenaline level, VAS score, and tension-anxiety score. These results suggest that adrenaline is important in the pathogenesis of BMS pain and that the analgesic effect of gum chewing is induced through the potential effects of anxiety reduction, although this effect might not be specific to BMS. In addition, the analgesic effect of gum chewing was not induced solely by chewing motion.
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Síndrome de Boca Ardiente/tratamiento farmacológico , Síndrome de Boca Ardiente/terapia , Analgésicos/uso terapéutico , Goma de Mascar , Femenino , Humanos , Masticación , Estudios ProspectivosRESUMEN
Prostaglandin (PG) E(2), which exerts its actions via the PG receptors EP1-4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1beta induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE(2) in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1beta with or without 1 muM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1-4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE(2) production was determined using ELISA. IL-1beta treatment caused a marked dose- and time-dependent increase in the levels of PGE(2), COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1beta. When celecoxib was also present, IL-1beta failed to stimulate PGE(2) production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1beta increases the production of PGE(2), COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE(2) production.
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Condrocitos/metabolismo , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Interleucina-1beta/fisiología , Receptores de Prostaglandina E/biosíntesis , Celecoxib , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Ciclooxigenasa 1/efectos de los fármacos , Ciclooxigenasa 2/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/agonistas , Humanos , Interleucina-1beta/farmacología , Pirazoles/farmacología , Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E , Sulfonamidas/farmacologíaRESUMEN
AIMS: During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. MAIN METHODS: The cells were continuously stimulated with 0.3, 0.6, or 1.1â¯g/cm2 CF-which was generated by increasing the volume of culture medium in the wells of a 96-well plate-in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4â¯days. KEY FINDINGS: In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1â¯g/cm2 CF as compared to 0.3â¯g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4-another RANKL receptor was downregulated by 0.6 and 1.1â¯g/cm2 CF as compared to 0.3â¯g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1â¯g/cm2 CF in the presence of RANKL. SIGNIFICANCE: Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.
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Fusión Celular , Osteoclastos/fisiología , Ligando RANK/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Resorción Ósea , Proteínas de la Membrana/biosíntesis , Ratones , Factores de Transcripción NFATC/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Estimulación Física , Transporte de Proteínas , Células RAW 264.7 , Fosfatasa Ácida Tartratorresistente/biosíntesisRESUMEN
Interleukin-1 (IL-1) is a proinflammatory cytokine that is a potent stimulator of bone resorption and an inhibitor of bone formation, whereas macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB (RANK) ligand (RANKL) are essential and sufficient for osteoclast differentiation. Recently, we showed that IL-1alpha affects mineralized nodule formation in vitro and halts bone matrix turnover. We also showed that IL-1alpha stimulates osteoclast formation via the interaction of RANKL with RANK by increasing M-CSF and prostaglandin E(2) and decreasing osteoprotegerin. Here, we examined the effects of IL-1alpha or RANKL and/or M-CSF in the presence of IL-1alpha on the expression of carbonic anhydrase II (CAII), cathepsin K, matrix metalloproteinase-9 (MMP-9), RANK, M-CSF receptor (c-fms), and c-fos transcription factor using RAW264.7 cells as osteoclast precursors. Cells were cultured for up to 14 days in 0 or 100 U/ml IL-1alpha and either 50 ng/ml RANKL, 10 ng/ml M-CSF, or 50 ng/ml RANKL+10 ng/ml M-CSF in the presence of 100 U/ml IL-1alpha. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase staining. Expression of the genes coding for the six proteins of interest was determined using real-time PCR, and the expression of the three enzymes was examined using Western blotting or ELISA. In the presence of IL-1alpha, expression of CAII, cathepsin K, and MMP-9 was markedly increased in cells cultured with RANKL or M-CSF+RANKL, whereas expression was difficult to detect in cells cultured with IL-1alpha alone and M-CSF. RANK and c-fos expression was also increased in cells cultured with RANKL or M-CSF+RANKL in the presence of IL-1alpha, whereas c-fms expression did not change. These results indicate that the expression of CAII, cathepsin K, and MMP-9 in RAW264.7 cells is not induced by M-CSF, but by RANKL in the presence of IL-1alpha.
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Anhidrasa Carbónica II/metabolismo , Catepsinas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , FN-kappa B/metabolismo , Osteoclastos/efectos de los fármacos , Ligando RANK/farmacología , Animales , Anhidrasa Carbónica II/genética , Catepsina K , Catepsinas/genética , Línea Celular , Combinación de Medicamentos , Expresión Génica/efectos de los fármacos , Interleucina-1alfa/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Metaloproteinasa 9 de la Matriz/genética , Ratones , FN-kappa B/genética , Osteoclastos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mineral trioxide aggregate (MTA) has excellent biocompatibility as well as bioactivity, including an ability to induce osteoblast differentiation. We examined the effects of the calcium-sensing receptor (CaSR) on osteogenic gene expression induced by MTA. MC3T3-E1 cells were cultured with or without (control) MTA. The expression levels of Runx2, type I collagen, and CaSR genes were analyzed by real-time polymerase chain reaction and their products were measured using enzyme-linked immunosorbent assays. The levels were increased significantly in cells exposed to MTA compared with control. Next, MC3T3-E1 cells were cultured with MTA and EGTA (a calcium chelator), because calcium ions were released continuously from MTA into the culture. Expression levels were decreased to control levels by MTA plus EGTA. NPS2143 (a CaSR antagonist) also reduced MTA-induced gene expression. These results suggest that MTA induced osteogenic gene expressions of Runx2 and type I collagen via CaSR in MC3T3-E1 cells.
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Compuestos de Aluminio , Compuestos de Calcio , Osteogénesis , Óxidos , Receptores Sensibles al Calcio , Silicatos , Animales , Diferenciación Celular , Combinación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Ratones , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Periodontal disease is caused by inflammation induced by Porphyromonas gingivalis (P.g.) lipopolysaccharide (LPS) and involves expression of proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α, and receptor activator of nuclear factor kappa B ligand (RANKL), which are implicated in bone resorption. Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of bone fracture. However, the mechanisms by which LIPUS inhibits LPS-induced inflammatory cytokines are poorly understood. Therefore, we investigated the effects of LIPUS on LPS-induced expression of the proinflammatory cytokines IL-6 and RANKL. MC3T3-E1 cells were incubated in the presence or absence of P.g. LPS and then stimulated with LIPUS for 30 min/day for a maximum of 14 days. LPS increased mRNA and protein expressions of IL-6 and RANKL on day 14. In addition, mRNA expression of COX-2 LPS was higher after 3 and 7 days of LIPUS treatment. PGE2 was induced by LPS after 7 and 14 days of culture. LIPUS suppressed all stimulatory effects of LPS. These results suggest that LIPUS inhibits LPS-induced expression of inflammation cytokines by suppressing PGE2 production and might thus have potential applications in the treatment of periodontitis.