RESUMEN
The nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome mediates caspase-1 activation and IL-1ß processing and is implicated in autoinflammatory as well as other chronic inflammatory diseases. Recent studies have demonstrated that xanthine oxidoreductase (XOR) inhibition attenuated IL-1ß secretion in activated macrophages, but the detailed mechanism of inhibition remains unclear. In this study, we report that febuxostat, an inhibitor of XOR, suppressed NLRP3 inflammasome-mediated IL-1ß secretion and cell death by two mechanisms: in a mitochondrial ROS (mitoROS)-dependent and mitoROS-independent manner. MitoROS-independent effects of febuxostat were mediated by an increase of intracellular ATP and improved mitochondrial energetics via the activation of purine salvage pathway. Our findings suggest that cellular bioenergetics are important in regulating NLRP3 activation, and XOR inhibition may be clinically relevant in NLRP3-related inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Febuxostat/farmacología , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Xantina Deshidrogenasa/antagonistas & inhibidores , Animales , Antiinflamatorios/uso terapéutico , Células Cultivadas , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/inmunología , Febuxostat/uso terapéutico , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamación/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Cultivo Primario de Células , Purinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Xantina Deshidrogenasa/metabolismoRESUMEN
Xanthine oxidase (XO) is an enzyme responsible for the production of uric acid. XO produces considerable amount of oxidative stress throughout the body. To date, however, its pathophysiologic role in hypertension and endothelial dysfunction still remains controversial. To explore the possible involvement of XO-derived oxidative stress in the pathophysiology of vascular dysfunction, by use of a selective XO inhibitor, febuxostat, we investigated the impact of pharmacological inhibition of XO on hypertension and vascular endothelial dysfunction in spontaneously hypertensive rats (SHRs). Sixteen-week-old SHR and normotensive Wistar-Kyoto (WKY) rats were treated with tap water (control) or water containing febuxostat (3 mg/kg/day) for 6 weeks. Systolic blood pressure (SBP) in febuxostat-treated SHR (220 ± 3 mmHg) was significantly (P < 0.05) decreased compared with the control SHR (236 ± 4 mmHg) while SBP in febuxostat-treated WKY was constant. Acetylcholine-induced endothelium-dependent relaxation in aortas from febuxostat-treated SHR was significantly (P < 0.05) improved compared with the control SHR, whereas relaxation in response to sodium nitroprusside was not changed. Vascular XO activity and tissue nitrotyrosine level, a representative indicator of local oxidative stress, were considerably elevated in the control SHR compared with the control WKY, and this increment was abolished by febuxostat. Our results suggest that exaggerated XO activity and resultant increase in oxidative stress in this experimental model contribute to the hypertension and endothelial dysfunction, thereby supporting a notion that pharmacological inhibition of XO is valuable not only for hyperuricemia but also for treating hypertension and related endothelial dysfunction in human clinics.
Asunto(s)
Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Febuxostat/farmacología , Hipertensión/tratamiento farmacológico , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/enzimología , Endotelio Vascular/fisiopatología , Hipertensión/enzimología , Hipertensión/fisiopatología , Masculino , Estrés Oxidativo/efectos de los fármacos , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Factores de Tiempo , Tirosina/análogos & derivados , Tirosina/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1ß processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1ß secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1ß secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Inflamación/inmunología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Proteínas del Tejido Nervioso/metabolismo , Nigericina/metabolismo , Ácido Úrico/metabolismo , Animales , Caspasa 1/metabolismo , Células Cultivadas , Desoxiglucosa/metabolismo , Humanos , Inflamasomas/inmunología , Espacio Intracelular , Glicoproteínas de Membrana , Ratones Endogámicos C57BLRESUMEN
Activation of the NLRP3 inflammasome by microbial ligands or tissue damage requires intracellular generation of reactive oxygen species (ROS). We present evidence that macrophage secretion of IL1ß upon stimulation with ATP, crystals or LPS is mediated by a rapid increase in the activity of xanthine oxidase (XO), the oxidized form of xanthine dehydrogenase, resulting in the formation of uric acid as well as ROS. We show that XO-derived ROS, but not uric acid, is the trigger for IL1ß release and that XO blockade results in impaired IL1ß and caspase1 secretion. XO is localized to both cytoplasmic and mitochondrial compartments and acts upstream to the PI3K-AKT signalling pathway that results in mitochondrial ROS generation. This pathway represents a mechanism for regulating NLRP3 inflammasome activation that may have therapeutic implications in inflammatory diseases.
Asunto(s)
Proteínas Portadoras/inmunología , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/inmunología , Xantina Deshidrogenasa/genética , Xantina Oxidasa/inmunología , Animales , Autofagia , Western Blotting , Calcio/metabolismo , Fosfatos de Calcio/farmacología , Proteínas Portadoras/efectos de los fármacos , Caspasa 1/inmunología , Técnicas de Silenciamiento del Gen , Técnicas In Vitro , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos , Proteína con Dominio Pirina 3 de la Familia NLR , Peritonitis/inmunología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Ácido Úrico/inmunología , Xantina Deshidrogenasa/antagonistas & inhibidores , Xantina Deshidrogenasa/inmunologíaRESUMEN
Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Placa Aterosclerótica/patología , Especies Reactivas de Oxígeno/metabolismo , Tiazoles/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Animales , Aorta/citología , Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/patología , Peso Corporal , Colesterol/sangre , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Febuxostat , Supresores de la Gota/farmacología , Inflamación/tratamiento farmacológico , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Xantina Oxidasa/biosíntesisRESUMEN
Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.
Asunto(s)
Quimiocina CCL2/metabolismo , Fosfatasa 1 de Especificidad Dual/metabolismo , Lipopolisacáridos/farmacología , MAP Quinasa Quinasa 4/metabolismo , Tiazoles/farmacología , Xantina Oxidasa/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Quimiocina CCL2/genética , Fosfatasa 1 de Especificidad Dual/genética , Febuxostat , Femenino , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , MAP Quinasa Quinasa 4/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Fosforilación/genética , ARN Mensajero/genética , Especies Reactivas de Oxígeno/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Aquaporin-5 (AQP5) is expressed in a cell type-specific manner. Here, we show that the AQP5 gene is regulated by CpG methylation. The AQP5 promoter containing a putative CpG island was highly methylated in NIH-3T3 or freshly isolated alveolar epithelial cells, correlating with the repression of this gene in these cells. In contrast, the AQP5 promoter was hypo-methylated in MLE-12 or cultured alveolar epithelial cells, which express high levels of AQP5. Repression of AQP5 transcription in NIH-3T3 cells could be relieved with 5-azacytidine, and in vitro methylation of the AQP5 promoter resulted in inhibition of transcription of the reporter gene in MLE-12 cells. Chromatin immunoprecipitation assays showed that endogenous Sp1 bound to the hypo-methylated, but not highly methylated, AQP5 promoter region. These results demonstrate that the hypo-methylated state of the AQP5 promoter leading to increased Sp1 binding may play a role in regulation of cell type-specific expression of the AQP5 gene.
Asunto(s)
Acuaporina 5/genética , Islas de CpG/genética , Metilación de ADN , Perfilación de la Expresión Génica , Animales , Acuaporina 5/metabolismo , Azacitidina/farmacología , Secuencia de Bases , Western Blotting , Línea Celular Transformada , Células Cultivadas , Relación Dosis-Respuesta a Droga , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/metabolismo , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability.
Asunto(s)
Acuaporina 5/metabolismo , Membrana Celular/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Tretinoina/farmacología , Agua/metabolismo , Animales , Acuaporina 5/genética , Membrana Celular/metabolismo , Células Cultivadas , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Ratones , Mutación , Permeabilidad , Regiones Promotoras Genéticas , ARN Mensajero/metabolismoRESUMEN
Royal jelly (RJ) has diverse physiological and pharmacological functions. We observed its weak estrogenic activity in the previous study. RJ stimulated the proliferation of mouse osteoblast-like MC3T3-E1 cells at 0.1 mg/ml, and the effect was blocked by the specific estrogen receptor antagonist ICI 182,780. The addition of 0.1-1.0 mg/ml RJ enhanced collagen production in culture medium. Oral administration of RJ to normal female mice for 9 weeks increased the ash content of their tibiae. DNA microarray analysis revealed significant changes in gene expression related to extracellular matrix formation when the femurs of mice fed RJ were analyzed. Quantitative reverse transcriptase-PCR (RT-PCR) confirmed up-regulation of procollagen I alpha1 gene expression. These data suggest that RJ as a whole or some of its individual components stimulates production of type I collagen and other activities for bone formation through action on osteoblasts.
Asunto(s)
Ácidos Grasos/farmacología , Osteogénesis/efectos de los fármacos , Animales , Línea Celular , Colágeno Tipo I/biosíntesis , Colágeno Tipo I/genética , Matriz Extracelular/genética , Ácidos Grasos/administración & dosificación , Femenino , Perfilación de la Expresión Génica , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Tibia , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5-2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.