RESUMEN
INTRODUCTION: Increasing evidence support the regulatory role of leptin in osteoarthritis (OA). As high circulating concentrations of leptin disrupt the physiological function of the adipokine in obese individuals, the current study has been undertaken to determine whether the elevated levels of leptin found in the joint from obese OA patients also induce changes in the chondrocyte response to leptin. METHODS: Chondrocytes isolated from OA patients with various body mass index (BMI) were treated with 20, 100 or 500 ng/ml of leptin. The expression of cartilage-specific components (aggrecan, type 2 collagen), as well as regulatory (IGF-1, TGFbeta, MMP-13, TIMP 2) or inflammatory (COX-2, iNOS, IL-1) factors was investigated by real-time PCR to evaluate chondrocyte responsiveness to leptin. Furthermore, the effect of body mass index (BMI) on leptin signalling pathways was analyzed with an enzyme-linked immunosorbent assay for STATs activation. RESULTS: Leptin at 20 ng/ml was unable to modulate gene expression in chondrocytes, except for MMP-13 in obese OA patients. Higher leptin levels induced the expression of IGF-1, type 2 collagen, TIMP-2 and MMP-13. However, the activity of the adipokine was shown to be critically dependent on both the concentration and the BMI of the patients with a negative association between the activation of regulated genes and BMI for 100 ng/ml of adipokine, but a positive association between chondrocyte responsiveness and BMI for the highest leptin dose. In addition, the gene encoding MMP-13 was identified as a target of leptin for chondrocytes originated from obese patients while mRNA level of TIMP-2 was increased in leptin-treated chondrocytes collected from normal or overweight patients. The adipokine at 500 ng/ml triggered signal transduction through a STAT-dependent pathway while 100 ng/ml of leptin failed to activate STAT 3 but induced STAT 1alpha phosphorylation in chondrocytes obtained from obese patients. CONCLUSIONS: The current study clearly showed that characteristics of OA patients and more especially obesity may affect the responsiveness of cultured chondrocytes to leptin. In addition, the BMI-dependent effect of leptin for the expression of TIMP-2 and MMP-13 may explain why obesity is associated with an increased risk for OA.
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Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Leptina/farmacología , Obesidad/metabolismo , Osteoartritis de la Rodilla/metabolismo , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Células Cultivadas , Condrocitos/patología , Colágeno Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Persona de Mediana Edad , Osteoartritis de la Rodilla/patología , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.
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Cartílago Articular/citología , Condrocitos/metabolismo , PPAR gamma/agonistas , Prostaglandinas/biosíntesis , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Dinoprostona/biosíntesis , Interleucina-1/farmacología , Masculino , FN-kappa B/fisiología , PPAR gamma/fisiología , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Ratas , Ratas WistarRESUMEN
Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.
Asunto(s)
Condrocitos/metabolismo , Citoprotección , Terapia Genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Osteoartritis/genética , Osteoartritis/patología , Animales , Apoptosis , Células Cultivadas , Condrocitos/patología , Regulación de la Expresión Génica , Miembro Posterior , Articulaciones/patología , Masculino , Osteoartritis/metabolismo , Ratas , Ratas Wistar , TransfecciónRESUMEN
In addition to aging, obesity is one of the most common underlying causes of osteoarthritis (OA). Mechanical loading, together with biochemical and systemic factors linked to altered lipid metabolism, are thought to contribute to the onset of OA. It has been suggested that OA is a systemic metabolic disease associated with lipid disorders affecting joint homeostasis. These gradual changes may be due to the local effect of adipokines, and especially leptin. Indeed, their relative levels in joints differ from that found in plasma. In particular, leptin levels are increased and adiponectin and resistin levels are reduced This hypothesis is supported by--leptin overexpression in OA cartilage and its correlation with the degree of cartilage destruction,--abundant leptin synthesis by osteophytes, and--the high leptin levels found in OA joints from female patients. This link between OA and adipokines provides new leads regarding the prevention of OA and the identification of new drug targets.
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Leptina/análisis , Obesidad/complicaciones , Osteoartritis/etiología , Adipocitos/metabolismo , Adiponectina/sangre , Adiponectina/metabolismo , Tejido Adiposo/metabolismo , Factores de Edad , Anciano , Animales , Cartílago Articular/química , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Interpretación Estadística de Datos , Modelos Animales de Enfermedad , Femenino , Humanos , Leptina/sangre , Masculino , Obesidad/metabolismo , Osteoartritis/metabolismo , Osteoartritis/prevención & control , Osteoartritis de la Rodilla/etiología , Osteoartritis de la Rodilla/metabolismo , Ratas , Ratas Wistar , Investigación , Resistina/análisis , Resistina/sangre , Factores Sexuales , Líquido Sinovial/química , Membrana Sinovial/química , Membrana Sinovial/metabolismoRESUMEN
Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.
Asunto(s)
Condrocitos/efectos de los fármacos , Factores Inmunológicos/farmacología , Interleucina-1/farmacología , Oxidorreductasas Intramoleculares/metabolismo , PPAR gamma/agonistas , Prostaglandina D2/análogos & derivados , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Combinación de Medicamentos , Expresión Génica/efectos de los fármacos , Oxidorreductasas Intramoleculares/genética , Masculino , Prostaglandina D2/farmacología , Prostaglandina-E Sintasas , ARN Mensajero/metabolismo , Ratas , Rosiglitazona , Tiazolidinedionas/farmacología , TransfecciónRESUMEN
OBJECTIVE: To study the potency of 2 peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-deoxy-PGJ(2)) and rosiglitazone, to modulate the expression of interleukin-1 receptor antagonist (IL-1Ra) in rat synovial fibroblasts. METHODS: Levels of messenger RNA for IL-1Ra and PPAR isotypes (alpha, beta/delta, gamma) were assessed by real-time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL-1beta. PPAR levels were assessed by Western blotting and secreted IL-1Ra levels by immunoassay. The potency of PPARgamma agonists and the PPARbeta/delta agonist GW-501516 on IL-1Ra levels was tested in the range of 1-10 microM and at 100 pM, respectively. The contribution of PPARgamma to the effects of rosiglitazone on IL-1Ra secretion was examined either by its overexpression or by inhibition using wild-type or dominant-negative constructs and the antagonist GW-9662 (10 microM), respectively. The dominant-negative strategy was also performed to investigate the possible contribution of PPARbeta/delta and NF-kappaB activation. RESULTS: IL-1beta-induced IL-1Ra production was increased by 10 microM rosiglitazone but was reduced dose-dependently by 15-deoxy-PGJ(2). Both agonists lowered IL-1beta secretion, but rosiglitazone alone reduced the imbalance of IL-1beta/IL-1Ra toward basal levels. Enhancement of IL-1beta-induced IL-1Ra production by rosiglitazone was not affected by PPARgamma overexpression or by its inhibition with dominant-negative PPARgamma or GW-9662. Inhibition of NF-kappaB was also ineffective against rosiglitazone but abolished the stimulating effect of IL-1beta on IL-1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARgamma decreased dramatically upon IL-1beta exposure, whereas PPARbeta/delta remained stable. Dominant-negative PPARbeta/delta abolished the enhancement of IL-1Ra by rosiglitazone, whereas GW-501516 reproduced the effect of rosiglitazone on IL-1Ra secretion. CONCLUSION: Rosiglitazone stimulates IL-1Ra production by a PPARbeta/delta mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.
Asunto(s)
Factores Inmunológicos/farmacología , Receptores Activados del Proliferador del Peroxisoma/agonistas , Prostaglandina D2/análogos & derivados , Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/metabolismo , Tiazolidinedionas/farmacología , Animales , Técnicas de Cultivo de Célula , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Factores Inmunológicos/inmunología , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/inmunología , Articulación de la Rodilla , Masculino , Modelos Animales , PPAR delta/agonistas , PPAR delta/metabolismo , PPAR gamma/agonistas , PPAR gamma/metabolismo , PPAR-beta/agonistas , PPAR-beta/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Prostaglandina D2/inmunología , Prostaglandina D2/farmacología , Ratas , Ratas Wistar , Receptores de Interleucina-1/efectos de los fármacos , Rosiglitazona , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/inmunología , Tiazolidinedionas/inmunologíaRESUMEN
The aim of this work was to determine whether Hsp70 overexpression via proteasome inhibitor MG132 was able to protect chondrocytes towards mono-iodoacetate (MIA) cytotoxicity both in vitro and in vivo. In vitro, overexpression of Hsp70 via MG132 was significantly able to protect chondrocytes from MIA toxicity (MTT/LDH analyses). Hsp70 essentially mediated this chondroprotective effect as demonstrated by antisense strategy. In vivo, chondrocytic overexpression of Hsp70, after a preventive intra-articular injection of MG132 in rat knee, was sufficient to decrease the severity of OA-induced MIA lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of Hsp70, through proteasome inhibition, could be an interesting tool in protecting chondrocytes from cellular injuries, either necrotic or apoptotic in nature, and thus might be a novel chondroprotective modality in rat experimental OA.
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Cartílago Articular , Condrocitos/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Leupeptinas/farmacología , Inhibidores de Proteasas/farmacología , Animales , Muerte Celular , Células Cultivadas , Condrocitos/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Miembro Posterior , Masculino , Oligonucleótidos Antisentido/farmacología , Ratas , Ratas WistarRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.
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Condrocitos/metabolismo , Proteínas I-kappa B/metabolismo , Interleucina-2/farmacología , Prostaglandina D2/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Antiinflamatorios/farmacología , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Dinoprostona/metabolismo , Femenino , Quinasa I-kappa B , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Prostaglandina D2/análogos & derivados , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/efectos de los fármacos , Activación TranscripcionalRESUMEN
OBJECTIVE: To evaluate the contribution of leptin (an adipose tissue-derived hormone) to the pathophysiology of osteoarthritis (OA), by determining the level of leptin in both synovial fluid (SF) and cartilage specimens obtained from human joints. We also investigated the effect of leptin on cartilage, using intraarticular injections of leptin in rats. METHODS: Leptin levels in SF samples obtained from OA patients undergoing either knee replacement surgery or knee arthroscopy were measured by enzyme-linked immunosorbent assay. In addition, histologic sections of articular cartilage and osteophytes obtained during surgery for total knee replacement were graded using the Mankin score, and were immunostained using antibodies to leptin, transforming growth factor beta (TGFbeta), and insulin-like growth factor 1 (IGF-1). For experimental studies, various doses of leptin (10, 30, 100, and 300 microg) were injected into the knee joints of rats. Tibial plateaus were collected and processed for proteoglycan synthesis by radiolabeled sulfate incorporation, and for expression of leptin, its receptor (Ob-Rb), and growth factors by reverse transcriptase-polymerase chain reaction and immunohistochemical analysis. RESULTS: Leptin was observed in SF obtained from human OA-affected joints, and leptin concentrations correlated with the body mass index. Marked expression of the protein was observed in OA cartilage and in osteophytes, while in normal cartilage, few chondrocytes produced leptin. Furthermore, the pattern and level of leptin expression were related to the grade of cartilage destruction and paralleled those of growth factors (IGF-1 and TGFbeta1). Animal studies showed that leptin strongly stimulated anabolic functions of chondrocytes and induced the synthesis of IGF-1 and TGFbeta1 in cartilage at both the messenger RNA and the protein levels. CONCLUSION: These findings suggest a new peripheral function of leptin as a key regulator of chondrocyte metabolism, and indicate that leptin may play an important role in the pathophysiology of OA.
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Cartílago Articular/metabolismo , Leptina/metabolismo , Osteoartritis/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Inyecciones Intraarticulares , Factor I del Crecimiento Similar a la Insulina/metabolismo , Articulación de la Rodilla , Leptina/genética , Leptina/farmacología , Masculino , Persona de Mediana Edad , Osteoartritis/fisiopatología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Líquido Sinovial/metabolismo , Tibia/química , Tibia/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
OBJECTIVE: To compare the potency of 2 cytokines, interleukin 17 (IL-17) and IL-1beta, on rat cartilage proteoglycan synthesis with special attention to nitric oxide (NO) and peroxynitrite formation. METHODS: Chondrocytes in alginate beads were stimulated with human recombinant (rh) IL-17 (0.03 to 300.0 ng/ml) and/or rhIL-1beta (0.25 to 25.0 ng/ml) in the presence or not of L-NMMA or CuDips. Alternatively, rats were injected with either IL-17 (10.0 micro g) or IL-1beta (1.0 micro g) into each knee joint. NO concentrations were determined by a spectrofluorimetric assay, proteoglycan synthesis by 35SO4-2 incorporation, peroxynitrite generation by immunostaining for 3-nitrotyrosine, and IL-1beta mRNA expression by reverse transcription-polymerase chain reaction. RESULTS: IL-17 inhibited proteoglycan synthesis and increased NO production, both in vitro and in vivo, without inducing expression of IL-1beta mRNA in cartilage. Additive effects were observed when IL-17 was combined with low concentrations of IL-1. Surprisingly, a similar NO synthesis between IL-1 and IL-17 led to a less suppressive effect of IL-17 on cartilage anabolism than with IL-1. Both in vitro and in vivo, peroxynitrite formation was extensive with IL-1beta, but negligible or nonexistent with IL-17. L-NMMA and CuDips completely corrected the suppressive effect of IL-1beta on proteoglycan synthesis, unlike with IL-17. CONCLUSION: These data showed that NO is weakly involved in the IL-17 mediated inhibition of proteoglycan synthesis in rat. NO overload may not be predictive of any inhibitory effect on cartilage anabolism, but instead superoxide is a key regulator of NO contribution to chondrocyte dysfunction. Since IL-17 is a NO-producing cytokine with additive effects when combined with IL-1, it may play a pivotal role in cartilage destruction during rheumatoid arthritis, for which infiltrating cells produce high levels of superoxide and proinflammatory cytokines.
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Cartílago Articular/efectos de los fármacos , Interleucina-17/farmacología , Óxido Nítrico/biosíntesis , Ácido Peroxinitroso/biosíntesis , Proteoglicanos/biosíntesis , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Miembro Posterior/efectos de los fármacos , Inyecciones Intraarticulares , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-1/farmacología , Articulaciones/efectos de los fármacos , Masculino , NG-Nitroarginina Metil Éster/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Salicilatos/farmacologíaRESUMEN
Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.
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Cartílago Articular/metabolismo , Condrocitos/metabolismo , Interleucina-1/farmacología , Óxido Nítrico/metabolismo , Proteoglicanos/biosíntesis , Animales , Enfermedades de los Cartílagos/inmunología , Enfermedades de los Cartílagos/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Depresión Química , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Humanos , Interleucina-17/farmacología , Masculino , Modelos Animales , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Wistar , Salicilatos/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
This work demonstrated the constitutive expression of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-alpha in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-gamma expression induced by 10 microg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-alpha mRNA expression was not modified. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (-80%) and inducible nitric oxide synthase (iNOS) mRNA expression (-80%), whereas troglitazone (10 microM) only inhibited iNOS mRNA expression (-50%). 15d-PGJ(2) decreased LPS-induced interleukin (IL)-1 beta (-25%) and tumor necrosis factor (TNF)-alpha (-40%) expression. Interestingly, troglitazone strongly decreased TNF-alpha expression (-50%) but had no significant effect on IL-1 beta expression. 15d-PGJ(2) was able to inhibit DNA-binding activity of both nuclear factor (NF)-kappa B and AP-1. Troglitazone had no effect on NF-kappa B activation and was shown to increase LPS-induced AP-1 activation. 15d-PGJ(2) and troglitazone modulated the expression of LPS-induced iNOS, COX-2, and proinflammatory cytokines differently. Indeed, troglitazone seems to specifically target TNF-alpha and iNOS pathways. These results offer new insights in regard to the anti-inflammatory potential of the PPAR-gamma ligands and underline different mechanisms of action of 15d-PGJ(2) and troglitazone in synovial fibroblasts.