RESUMEN
TET1 mediates demethylation in tumors, but its role in diabetic nephropathy (DN), a prevalent diabetic complication, is unclear. We attempted to probe the possible mechanism of TET1 in DN. A DN rat model was established and verified by marker detection and histopathological observation. The in vitro model was established on human mesangial cells (HMCs) induced by high glucose (HG), and verified by evaluation of fibrosis and inflammation. The differentially expressed mRNA was screened out by microarray analysis. The most differentially expressed mRNA (TET1) was reduced in DN rats and HG-HMCs. The upstream and downstream factors of TET1 were verified, and their roles in DN were analyzed by gain- and loss-function assays. TET1 was decreased in DN rats and HG-HMCs. High expression of TET1 decreased biochemical indexes and renal injury of DN rats and hampered the activity, fibrosis, and inflammation of HG-HMCs. Ap1 lowered TET1 expression, and enhanced inflammation in HG-HMCs, and accentuated renal injury in DN rats. TET1 overexpression inhibited the effect of Ap1 on DN. TET1 promoted the transcription of Nrf2. The Ap1/TET1 axis mediated the Nrf2/ARE pathway activity. Overall, TET1 overexpression weakened the inhibitory effect of Ap1 on the Nrf2/ARE pathway, thus alleviating inflammation and renal injury in DN.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Dioxigenasas/biosíntesis , Factor 2 Relacionado con NF-E2/biosíntesis , Transducción de Señal/fisiología , Factor de Transcripción AP-1/biosíntesis , Animales , Células Cultivadas , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/patología , Dioxigenasas/antagonistas & inhibidores , Humanos , Masculino , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Extensive phytochemical analysis of the CHCl3-soluble part of an ethanolic extract of branches and twigs of Broussonetia papyrifera led to the isolation of fourteen compounds, including a novel 5,11-dioxabenzo[b]fluoren-10-one derivative named broussofluorenone C (12). The isolated compounds 1-14 were characterized based on their NMR and HRMS data, and examined for their anti-inflammatory activities in LPS-stimulated THP-1 cells as well as for their cellular antioxidant effects. Compounds 7-10 and 12 showed inhibitory effects on NF-κB/AP-1 activation and compounds 7-9 were subsequently confirmed to suppress the secretion of both IL-1ß and TNF-α in LPS-stimulated THP-1 cells more significantly than the prednisone used as a positive control. In the CAA assay, compound 10 exhibited the greatest antioxidant effect, greater than that of the quercetin used as a positive control. The results show possible beneficial effects and utilization of B. papyrifera wood in the treatment of inflammatory diseases as well as oxidative stress.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Broussonetia/química , Antiinflamatorios/química , Antiinflamatorios/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Estructura Molecular , FN-kappa B/análisis , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Relación Estructura-Actividad , Células THP-1 , Factor de Transcripción AP-1/análisis , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/biosíntesis , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
As one of the most popular anesthetics, sevoflurane is widely used in pediatric anesthesia. Unfortunately, an increasing number of studies have demonstrated that sevoflurane has potential neurotoxic effects on the developing brain and cognition, even in adolescence. Connexin 43 (Cx43) has been documented to contribute to cognitive dysfunction. The present study hypothesized that Cx43 may participate in sevoflurane-induced neuroinjury and investigated the underlying mechanisms in young Sprague Dawley (SD) rats. Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h. The levels of Cx43,mitogen-activated protein kinase (MAPK) signaling pathway components(including total and phosphorylated p38, extracellular signal-regulated kinase (ERK), and c-Jun n-terminal kinase (JNK) and activator protein 1(AP-1) transcription factors (including total and phosphorylated c-Fos, and c-Jun) were assessed by Western blot analysis. Neuronal apoptosis was detected using immunohistochemistry (IHC). The Morris water maze (MWM) was performed to evaluate cognitive function from P28 to P33. The results showed that anesthesia with 3% sevoflurane for 4 h increased Cx43 levels in the rat hippocampus from 6 h to 3 d, and compared with sevoflurane exposure in the control group rats, exposure in P7 SD rats also increased the ratios of phosphorylated JNK to JNK and, phosphorylated c-Jun to c-Jun in the hippocampus from 6 h to 3 d. All these effects could be alleviated by pretreatment with the JNK inhibitor SP600125 (10 mg/kg). Neuroapoptosis was similarly increased from 6 h to 1 d after inhaled sevoflurane exposure. Finally, the MWM indicated that sevoflurane could increase the escape latency and, decrease the number of platform crossings from P28 to P33. Overall, our findings suggested that sevoflurane increased Cx43 expression and induced to apoptosis by activating the JNK/c-Jun signaling pathway in the hippocampus of P7 rats. This finding may reveal a new strategy for preventing sevoflurane-induced neuronal dysfunction.
Asunto(s)
Conexina 43/biosíntesis , Hipocampo/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Sevoflurano/toxicidad , Factor de Transcripción AP-1/biosíntesis , Animales , Animales Recién Nacidos , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Inhibidores de Agregación Plaquetaria/toxicidad , Ratas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Pentalinon andrieuxii Muell Arg is a Mexican-Central American plant anciently used by local people to treat cutaneous leishmaniasis. We evaluated a hexane extract of the root we called PAE for its chemical content and for its immunochemical and in vitro activity against Leishmania donovani and healing of experimental Kala-azar. Chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS) identified hexadecanoic acid, hexadecanoic acid ethyl ester, 9, 12-octadecadienoic acid ethyl ester, octadecanoic acid ethyl ester, 9-octadecenoic acid ethyl ester and diethyl phthalate as the main compounds present in PAE. We also demonstrated PAE kills promastigotes and amastigotes in vitro and significantly reduces parasite loads in liver and spleen of infected Balb/c mice. PAE induces expression of NFkB/AP-1 transcription factors and production of IL-2 and IFN-γ by spleen cells of PAE treated but not in the untreated control mice. Furthermore, there were not IL-6, IL-10 nor TNF production in macrophages treated in vitro with PAE. We developed an affordable extract of P. andrieuxii effective to treat experimental Kala-azar in Balb/c mice.
Asunto(s)
Apocynaceae/química , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Animales , Ácidos Grasos Insaturados/análisis , Cromatografía de Gases y Espectrometría de Masas , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Leishmaniasis Cutánea/tratamiento farmacológico , Hígado/parasitología , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , FN-kappa B/biosíntesis , Ácido Oléico/análisis , Ácidos Oléicos , Carga de Parásitos , Ácidos Ftálicos/análisis , Bazo/parasitología , Estearatos/análisis , Factor de Transcripción AP-1/biosíntesisRESUMEN
Within the testis, connexin43 encoded by Gja1 plays an important role in cell-to-cell communication between Leydig cells as well as between Sertoli cells and spermatogonia. In the adult male, Leydig cells are the principal producers of testosterone sustaining spermatogenesis, while Sertoli cells nourish, protect and support the differentiating germ cells. It has been shown previously that members of the AP-1 family regulate Gja1 expression in myometrial cells, suggesting that such regulatory mechanism may also be relevant within the testis. Thus, we performed cotransfections of AP-1 expression plasmids with different mouse Gja1 promoter/luciferase reporter constructs within TM3 Leydig and TM4 Sertoli cells. We showed that a functional cooperation between cJun and cFos activates Gja1 expression and requires an AP-1 DNA regulatory element located between -132 and -26 bp. In addition, such synergy relies on the recruitment of cFos to this region of the mouse Gja1 promoter. Hence, our data indicate that AP-1 members are important for optimal expression of Gja1 within Sertoli and Leydig cells from the testis.
Asunto(s)
Comunicación Celular/genética , Conexina 43/genética , Proteínas Oncogénicas v-fos/genética , Factor de Transcripción AP-1/genética , Animales , Conexina 43/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Proteínas Oncogénicas v-fos/biosíntesis , Regiones Promotoras Genéticas , Células de Sertoli/metabolismo , Espermatogénesis/genética , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Testosterona/genética , Factor de Transcripción AP-1/biosíntesisRESUMEN
Anthraquinone-2-carboxlic acid (9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid, AQCA) was identified as one of the major anthraquinones in Brazilian taheebo. Since there was no report explaining its immunopharmacological actions, in this study, we aimed to investigate the molecular mechanism of AQCA-mediated anti-inflammatory activity using reporter gene assays, kinase assays, immunoblot analyses, and overexpression strategies with lipopolysaccharide (LPS)-treated macrophages. AQCA was found to suppress the release of nitric oxide (NO) and prostaglandin (PG) E2 from LPS-treated peritoneal macrophages without displaying any toxic side effects. Molecular analysis revealed that AQCA was able to inhibit the activation of the nuclear factor (NF)-κB and activator protein (AP)-1 pathways by direct suppression of upstream signaling enzymes including interleukin-1 receptor-associated kinase 1 (IRAK1) and spleen tyrosine kinase (Syk). Therefore, our data strongly suggest that AQCA-mediated suppression of inflammatory responses could be managed by a direct interference of signaling cascades including IRAK and Syk, linked to the activation of NF-κB and AP-1.
Asunto(s)
Antraquinonas/administración & dosificación , Inflamación/tratamiento farmacológico , Quinasas Asociadas a Receptores de Interleucina-1/biosíntesis , Quinasa Syk/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Antraquinonas/química , Antiinflamatorios/administración & dosificación , Antiinflamatorios/química , Brasil , Humanos , Inflamación/patología , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/patología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , Óxido Nítrico/metabolismo , Prostaglandinas/metabolismo , Quinasa Syk/antagonistas & inhibidores , Tabebuia/química , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
AIMS: Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes. METHODS AND RESULTS: We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively. CONCLUSIONS: LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes.
Asunto(s)
Lipoproteínas LDL/sangre , Monocitos/metabolismo , Receptor Toll-Like 4/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Citocinas/biosíntesis , Citocinas/genética , Elafina/genética , Humanos , Lipoproteínas LDL/biosíntesis , FN-kappa B/biosíntesis , FN-kappa B/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesisRESUMEN
AIMS: Gastroesophageal reflux disease is considered to be a major risk in the development of esophageal adenocarcinoma. Nuclear factor-kappa B (NF-κB) plays important roles in the regulation of several genes coding for cytokines, cell proliferation, and apoptosis. To understand the role of bile and acid in the causation of esophageal cancer, we have examined the effects of bile acids and acid on NF-κB activation in the esophageal epithelial cells OE33 and SKGT-4 qualitatively and quantitatively. MATERIALS AND METHODS: Analysis of NF-κB activation in esophageal epithelial cells in response to bile acids and acid was performed by electrophoretic mobility shift assay, Western blotting and the translocation NF-κB was assessed by high content analysis (HCA). Cyclooxygenase-2 (COX-2) promoter activity was assessed by transient transfection assays. RESULTS: This study demonstrated that bile acids and acid activated NF-κB in a dose- and time-dependent manner. HCA analysis was an invaluable method in quantifying NF-κB translocation at the single cell population level following bile or acid treatment. Furthermore, deoxycholic acid (DCA) and acid-induced COX-2 promoter activity, and a mutation in the NF-κB and activator protein-1 (AP-1) binding sites remarkably reduced the reporter gene activity induced by DCA or acid. CONCLUSIONS: Our data demonstrate that bile and acid induce NF-κB activation in esophageal cells qualitatively and quantitatively. The induction of COX-2 promoter activity by DCA and acid was mediated via NF-κB and AP-1 transcription. The activation of NF-κB signaling pathway in esophageal cells may contribute to the development of esophageal cancer, and, therefore, modulating of NF-κB pathway may uncover new therapeutic strategies.
Asunto(s)
Adenocarcinoma/genética , Ciclooxigenasa 2/genética , Neoplasias Esofágicas/genética , FN-kappa B/genética , Factor de Transcripción AP-1/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/administración & dosificación , Sitios de Unión/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Ácido Desoxicólico/administración & dosificación , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Humanos , FN-kappa B/biosíntesis , Regiones Promotoras Genéticas/efectos de los fármacos , Factor de Transcripción AP-1/biosíntesis , Activación Transcripcional/efectos de los fármacosRESUMEN
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.
Asunto(s)
Hepacivirus/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Citoplasma/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Hepacivirus/genética , Hepatitis C/virología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Interacciones Huésped-Patógeno , Humanos , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Replicón/fisiología , Análisis de Secuencia de Proteína , Eliminación de Secuencia , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genética , Replicación Viral/fisiologíaRESUMEN
PURPOSE: In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as ß-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of ß-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. MATERIALS AND METHODS: Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. RESULTS: H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (ß-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. CONCLUSION: H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , FN-kappa B/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Western Blotting , Ácidos Cafeicos , Línea Celular Tumoral , Proliferación Celular , ADN Bacteriano/análisis , ADN Bacteriano/genética , Mucosa Gástrica/patología , Gastritis/patología , Regulación Bacteriana de la Expresión Génica , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Infecciones por Helicobacter/fisiopatología , Helicobacter pylori/patogenicidad , Helicobacter pylori/fisiología , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fragmentos de Péptidos , Alcohol Feniletílico/análogos & derivados , Proteínas Proto-Oncogénicas c-jun , Proteínas RepresorasRESUMEN
BACKGROUND: Tumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined. METHODS: The expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment. RESULTS: TNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo. CONCLUSION: Taken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.
Asunto(s)
Neoplasias de la Vesícula Biliar/genética , Linfangiogénesis/genética , Factor de Transcripción AP-1/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Neoplasias de la Vesícula Biliar/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Interferencia de ARN , Factor de Transcripción AP-1/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor D de Crecimiento Endotelial Vascular/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Coenzyme Q (CoQ) analogs with variable number of isoprenoid units have been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study we used CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains), a novel quinone derivative, and investigated its molecular actions against LPS-induced inflammation and redox imbalance in murine RAW264.7 macrophages and mice. In LPS-stimulated macrophages, non-cytotoxic concentrations of CoQ0 (2.5-10 µM) inhibited iNOS/COX-2 protein expressions with subsequent reductions of NO, PGE2, TNF-α and IL-1ß secretions. This inhibition was reasoned by suppression of NFκB (p65) activation, and inhibition of AP-1 (c-Jun., c-Fos, ATF2) translocation. Our findings indicated that IKKα-mediated I-κB degradation and MAPK-signaling are involved in regulation of NFκB/AP-1 activation. Furthermore, CoQ0 triggered HO-1 and NQO-1 genes through increased Nrf2 nuclear translocation and Nrf2/ARE-signaling. This phenomenon was confirmed by diminished CoQ0 protective effects in Nrf2 knockdown cells, where LPS-induced NO, PGE2, TNF-α and IL-1ß productions remained high. Molecular evidence revealed that CoQ0 enhanced Nrf2 steady-state level at both transcriptional and translational levels. CoQ0-induced Nrf2 activation appears to be regulated by ROS-JNK-signaling cascades, as evidenced by suppressed Nrf2 activation upon treatment with pharmacological inhibitors of ROS (N-acetylcysteine) and JNK (SP600125). Besides, oral administration of CoQ0 (5 mg/kg) suppressed LPS-induced (1 mg/kg) induction of iNOS/COX-2 and TNF-α/IL-1ß through tight regulation of NFκB/Nrf2 signaling in mice liver and spleen. Our findings conclude that pharmacological actions of CoQ0 are mediated via inhibition of NFκB/AP-1 activation and induction of Nrf2/ARE-signaling. Owing to its potent anti-inflammatory and antioxidant properties, CoQ0 could be a promising candidate to treat inflammatory disorders.
Asunto(s)
Benzoquinonas/administración & dosificación , Inflamación/genética , Factor 2 Relacionado con NF-E2/genética , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción ReIA/genética , Ubiquinona/administración & dosificación , Animales , Ciclooxigenasa 2/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hemo-Oxigenasa 1/genética , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipopolisacáridos/toxicidad , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Factor 2 Relacionado con NF-E2/biosíntesis , FN-kappa B/genética , Óxido Nítrico Sintasa de Tipo II/genética , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción AP-1/genética , Factor de Transcripción ReIA/biosíntesis , Ubiquinona/análogos & derivadosRESUMEN
Toll-like receptor 4 (TLR4) is an important sensor of Gram-negative bacteria and can trigger activation of the innate immune system. Increased activation of TLR4 can lead to the induction of oxidative stress. Herein, the pathway whereby TLR4 affects antioxidant activity was studied. In TLR4-overexpressing sheep, TLR4 expression was found to be related to the integration copy number when monocytes were challenged with lipopolysaccharide (LPS). Consequently, production of malondialdehyde (MDA) was increased, which could increase the activation of prooxidative stress enzymes. Meanwhile, activation of an antioxidative enzyme, glutathione peroxidase (GSH-Px), was increased. Real-time PCR showed that expression of activating protein-1 (AP-1) and the antioxidative-related genes was increased. By contrast, the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) were reduced. In transgenic sheep, glutathione (GSH) levels were dramatically reduced. Furthermore, transgenic sheep were intradermally injected with LPS in each ear. The amounts of inflammatory infiltrates were correlated with the number of TLR4 copies that were integrated in the genome. Additionally, the translation of γ-glutamylcysteine synthetase (γ-GCS) was increased. Our findings indicated that overexpression of TLR4 in sheep could ameliorate oxidative injury through GSH secretion that was induced by LPS stimulation. Furthermore, TLR4 promoted γ-GCS translation through the AP-1 pathway, which was essential for GSH synthesis.
Asunto(s)
Glutatión/metabolismo , Lipopolisacáridos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Animales , Animales Modificados Genéticamente , Regulación de la Expresión Génica , Glutamato-Cisteína Ligasa/biosíntesis , Glutamato-Cisteína Ligasa/genética , Glutatión/genética , Glutatión Peroxidasa/biosíntesis , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Ovinos , Receptor Toll-Like 4/genética , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/genéticaRESUMEN
Nuclear steroid hormone receptors are ubiquitously expressed transcription factors whose activity can be altered by post-translational modifications, such as phosphorylation. The consequences of post-translational modifications have been described for several members of the nuclear steroid hormone receptor superfamily; however, little is known about the effects of oestrogen receptor (ER)ß phosphorylation in the brain. Moreover, to our knowledge, the presence of phosphorylated ERß has not been detected in the brain of any species to date. Oestrogen receptor ß is highly expressed in several regions of the brain and in vitro studies have demonstrated that it can be phosphorylated at two serine residues (S87 and S105) in the N-terminal AF-1 region. The present study aimed to determine whether phosphorylated ERß is detectable in the hippocampus of aged female rats, as well as the functional consequences of ERß S87 and S105 phosphorylation on transcriptional activity in neuronal cells. First, we used a novel PhosTag(™) approach to detect phosphorylated forms of ERß in the dorsal hippocampus of aged female rats. The data obtained demonstrated abundant forms of phosphorylated ERß in the dorsal hippocampus, suggesting that this post-translational modification might be an important regulator of ERß function. To assess the functional consequences of ERß phosphorylation in neuronal cells, we created phospho-mimetic (S87E, S105E) and phospho-null (S87A, S105A) ERß receptors that were transiently transfected in a hippocampal-derived cell line. Collectively, our results showed that phosphorylation of S87 and S105 altered both ligand-independent and ligand-dependent ERß transcriptional regulation. Overall, these data demonstrate that phosphorylated forms of ERß are present in the brain of aged female rats and that phosphorylation of ERß could differentially alter ERß-mediated gene expression.
Asunto(s)
Receptor beta de Estrógeno/química , Receptor beta de Estrógeno/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Animales , Línea Celular , Estradiol/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Mutagénesis Sitio-Dirigida , Neuronas/efectos de los fármacos , Fosforilación , Ratas , Elementos de Respuesta/genética , Tamoxifeno/farmacología , Factor de Transcripción AP-1/biosíntesis , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: Dipeptidyl peptidase 4 (DPP4) is an aminopeptidase that is widely expressed in different cell types. Recent studies suggested that DPP4 plays an important role in tumour progression in several human malignancies. Here we have examined the mechanisms by which up-regulation of DPP4 expression causes epithelial transformation and mammary tumourigenesis. EXPERIMENTAL APPROACH: Expression of DPP4 and the peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), and the cytotoxic effects of combined treatment with sitagliptin and juglone were investigated by immunohistochemistry, immunoblotting, real-time PCR, TUNEL and soft agar assays, using MCF7 cells. The effects of sitagliptin on tumour development in vivo were studied in the syngeneic 4T1 metastatic breast cancer model. KEY RESULTS: Activity of the transcription factor E2F1 induced by EGF was enhanced by DPP4, thus increasing PIN1 expression. Furthermore, DPP4 enhanced MEK/ERK and JNK/c-Jun signalling induced by EGF, inducing AP-1 activity and epithelial cell transformation. In contrast, DPP4 silencing or DPP4 inhibition in MCF7 cells inhibited PIN1 expression via E2F1 activity induced by EGF, decreasing colony formation and inducing DNA fragmentation. In the syngeneic 4T1 metastatic breast cancer model, DPP4 overexpression increased tumour development, whereas treatment with sitagliptin and/or juglone suppressed it. Consistent with these observations, DPP4 levels were positively correlated with PIN1 expression in human breast cancer. CONCLUSIONS AND IMPLICATIONS: DPP4 promoted EGF-induced epithelial cell transformation and mammary tumourigenesis via induction of PIN1 expression, suggesting that sitagliptin targeting of DPP4 could be a treatment strategy in patients with breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Dipeptidil Peptidasa 4/metabolismo , Isomerasa de Peptidilprolil/genética , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Ciclina D1/genética , Factor de Crecimiento Epidérmico/farmacología , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Peptidilprolil Isomerasa de Interacción con NIMA , Proteínas Quinasas/metabolismo , Transducción de Señal , Fosfato de Sitagliptina/farmacología , Factor de Transcripción AP-1/biosíntesis , Regulación hacia ArribaRESUMEN
An imbalance in pro- and anti-inflammation is an important mechanism of steroid resistance in UC (ulcerative colitis), and miRNAs may participate in this process. The present study aimed to explore whether miRNAs play a role in the steroid resistance of UC by regulating gene expression of the inflammation signal pathway. SS (steroid-sensitive) patients, SR (steroid-resistant) patients and healthy individuals were recruited. In vivo miRNA profiles of serum samples showed that miR-195 was decreased significantly in the SR group compared with the SS group (P<0.05). This result was confirmed by qPCR (quantitative real-time PCR) and miRNA ISH (in situ hybridization) in serum and colon tissue samples. Online software was used to identify Smad7 mRNA as a potential target of miR-195. The direct interaction of miR-195 and Smad7 mRNA was investigated using a biotinylated miR-195 pull-down assay. Overexpression of a miR-195 precursor lowered cellular levels of Smad7 protein; conversely, antagonism of miR-195 enhanced Smad7 translation without disturbing Smad7 mRNA levels. A luciferase reporter assay revealed a repressive effect of miR-195 via a single Smad7 3'-UTR target site, and point mutation of this site prevented miR-195-induced repression of Smad7 translation. Furthermore, increased levels of miR-195 led to a decrease in c-Jun and p65 expression. In contrast, transfection with anti-miR-195 led to increased levels of c-Jun and p65 protein. The decrease in miR-195 led to an increase in Smad7 expression and corresponding up-regulation of p65 and the AP-1 (activator protein 1) pathway, which might explain the mechanism of steroid resistance in UC patients.
Asunto(s)
Colitis Ulcerosa/genética , Resistencia a Medicamentos/genética , MicroARNs/genética , Proteína smad7/genética , Esteroides/uso terapéutico , Regiones no Traducidas 3' , Adulto , Células CACO-2 , Colitis Ulcerosa/sangre , Colitis Ulcerosa/tratamiento farmacológico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , MicroARNs/biosíntesis , MicroARNs/sangre , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Proto-Oncogénicas c-jun/sangre , Transducción de Señal/genética , Proteína smad7/biosíntesis , Proteína smad7/sangre , Factor de Transcripción AP-1/biosíntesis , Factor de Transcripción AP-1/sangre , eIF-2 Quinasa/biosíntesis , eIF-2 Quinasa/sangreRESUMEN
OBJECTIVES: Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored. METHODS: SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis. RESULTS: Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1. CONCLUSIONS: Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.
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Interleucina-1alfa/biosíntesis , Metaloproteinasa 13 de la Matriz/biosíntesis , Osteoartritis/genética , Serpina E2/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Sistema de Señalización de MAP Quinasas/genética , FN-kappa B/genética , Osteoartritis/fisiopatología , Cultivo Primario de Células , Serpina E2/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.
Asunto(s)
Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Péptidos/toxicidad , Venenos de Escorpión/toxicidad , Factor de Transcripción AP-1/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/antagonistas & inhibidores , Calor , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
Spermatogenic cells express cell-specific molecules with the potential to be seen as "foreign" by the immune system. Owing to the time difference between their appearance in puberty and the editing of the lymphocyte repertoire around birth, local adaptations of the immune system coined immune privilege are required to confer protection from autoattack. Testicular macrophages (TM) play an important role in maintaining testicular immune privilege and display reduced proinflammatory capacity compared with other macrophages. However, the molecular mechanism underlying this macrophage phenotype remained elusive. We demonstrate that TM have a lower constitutive expression of TLR pathway-specific genes compared with peritoneal macrophages. Moreover, in TM stimulated with LPS, the NF-κB signaling pathway is blocked due to lack of IκBα ubiquitination and, hence, degradation. Instead, challenge of TM with LPS or polyinosinic-polycytidylic acid induces MAPK, AP-1, and CREB signaling pathways, which leads to production of proinflammatory cytokines such as TNF-α, although at much lower levels than in peritoneal macrophages. Pretreatment of TM with inhibitors for MAPKs p38 and ERK1/2 suppresses activation of AP-1 and CREB signaling pathways and attenuates LPS-induced TNF-α and IL-10 secretion. High levels of IL-10 production and activation of STAT3 by LPS stimulation in TM indicate a regulatory macrophage phenotype. Our results suggest that TM maintain testicular immune privilege by inhibiting NF-κB signaling through impairment of IκBα ubiquitination and a general reduction of TLR cascade gene expression. However, TM do maintain some capacity for innate immune responses through AP-1 and CREB signaling pathways.
Asunto(s)
Proteínas I-kappa B/metabolismo , Inflamación/inmunología , Macrófagos/inmunología , FN-kappa B/antagonistas & inhibidores , Testículo/inmunología , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Tolerancia Inmunológica/inmunología , Inmunidad Innata/inmunología , Interleucina-10/biosíntesis , Interleucina-10/metabolismo , Lipopolisacáridos , Sistema de Señalización de MAP Quinasas/inmunología , Masculino , Inhibidor NF-kappaB alfa , Poli I-C , Ratas , Ratas Wistar , Factor de Transcripción STAT3/metabolismo , Testículo/citología , Receptores Toll-Like/inmunología , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Dermatophytes cause superficial and cutaneous fungal infections in immunocompetent hosts and invasive disease in immunocompromised hosts. However, the host mechanisms that regulate innate immune responses against these fungi are largely unknown. Here, we utilized commercially available epidermal tissues and primary keratinocytes to assess (i) damage induction by anthropophilic, geophilic, and zoophilic dermatophyte strains and (ii) the keratinocyte signaling pathways, transcription factors, and proinflammatory responses induced by a representative dermatophyte, Trichophyton equinum. Initially, five dermatophyte species were tested for their ability to invade, cause tissue damage, and induce cytokines, with Microsporum gypseum inducing the greatest level of damage and cytokine release. Using T. equinum as a representative dermatophyte, we found that the mitogen-activated protein kinase (MAPK) pathways were predominantly affected, with increased levels of phospho-p38 and phospho-Jun N-terminal protein kinase (JNK) but decreased levels of phospho-extracellular signal-regulated kinases 1 and 2 (ERK1/2). Notably, the NF-κB and PI3K pathways were largely unaffected. T. equinum also significantly increased expression of the AP-1-associated transcription factor, c-Fos, and the MAPK regulatory phosphatase, MKP1. Importantly, the ability of T. equinum to invade, cause tissue damage, activate signaling and transcription factors, and induce proinflammatory responses correlated with germination, indicating that germination may be important for dermatophyte virulence and host immune activation.