RESUMEN
Transient receptor potential vanilloid 1 (TRPV1) channels were originally described as the receptors of capsaicin, the main constituent of hot chili pepper. The biological functions of TRPV1 channels include pain sensation and inflammatory thermal hyperalgesia. Here, we show that stimulation of HEK293 cells expressing TRPV1 channels (H2C1 cells) with capsaicin or the TRPV1 ligand resiniferatoxin activated transcription mediated by the transcription factor AP-1. No cell death was occurring under these experimental conditions. The AP-1 activity was not altered in capsaicin or resiniferatoxin-stimulated HEK293 cells lacking TRPV1. We identified the AP-1 DNA binding site as the capsaicin/resiniferatoxin-responsive element. Stimulation with the TRPV1 ligand N-arachidonoyldopamine increased AP-1 activity in a TRPV1-dependent and TRPV1-independent manner. Stimulation of TRPV1 channels induced an influx of Ca2+ into the cells and this rise in intracellular Ca2+ was essential for activating AP-1 in capsaicin or resiniferatoxin-stimulated cells. N-arachidonoyldopamine stimulation induced a rise in intracellular Ca2+ in a TRPV-1 dependent and independent manner. AP-1 is a dimeric transcription factor, composed of proteins of the c-Jun, c-Fos and ATF families. Stimulation of TRPV1 channels with capsaicin increased c-Jun and c-Fos biosynthesis in H2C1 cells. The signal transduction of capsaicin, leading to enhanced AP-1-mediated transcription, required extracellular signal-regulated protein kinase ERK1/2 as a signal transducer and the activation of the transcription factors c-Jun and ternary complex factor. Together, these data suggest that the intracellular functions of TRPV1 stimulation may rely on the activation of a stimulus-regulated protein kinase and stimulus-responsive transcription factors.
Asunto(s)
Capsaicina/farmacología , Canales Catiónicos TRPV/metabolismo , Factor de Transcripción AP-1/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Fármacos del Sistema Sensorial/farmacología , Canales Catiónicos TRPV/agonistas , Factor de Transcripción AP-1/agonistasRESUMEN
Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that ß2â1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether ß2â1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 ß2â1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by ß2â1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain ß2â1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain ß2â1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with ß2â1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that ß2â1-fructans are specific ligands for TLR2. To conclude, ß2â1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of ß2â1-fructan-mediated health effects.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Colon/metabolismo , Fructanos/metabolismo , Mucosa Intestinal/metabolismo , Sustancias Protectoras/metabolismo , Uniones Estrechas/metabolismo , Receptor Toll-Like 2/agonistas , Antiinflamatorios no Esteroideos/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/química , Anticuerpos Bloqueadores/farmacología , Línea Celular , Colon/efectos de los fármacos , Colon/inmunología , Diglicéridos/farmacología , Fructanos/antagonistas & inhibidores , Fructanos/química , Fármacos Gastrointestinales/antagonistas & inhibidores , Fármacos Gastrointestinales/química , Fármacos Gastrointestinales/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/metabolismo , Ligandos , Moduladores del Transporte de Membrana/antagonistas & inhibidores , Moduladores del Transporte de Membrana/toxicidad , Estructura Molecular , FN-kappa B/agonistas , FN-kappa B/metabolismo , Oligopéptidos/farmacología , Prebióticos/análisis , Sustancias Protectoras/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/toxicidad , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/inmunología , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismoRESUMEN
Steroid hormones exert genotropic effects through members of the nuclear hormone receptor family. In the present study, we examined the effects of 17ß-estradiol (E2) on nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation and investigated the mechanisms in mouse bone marrow-derived macrophages (BMMs). E2 alone did not affect NO production. In contrast, E2 inhibited LPS-induced production of NO in BMMs. Using a cell-impermeable E2 conjugated to BSA (E2-BSA), which has been used to investigate the nongenomic effects of estrogen, we found that the increase in NO production induced by LPS was also attenuated. In addition, the intracellular estrogen receptor blocker, ICI 182780, only partially antagonized the total effects of E2 on LPS-stimulated NO production capacity. E2 also attenuated the LPS activation of p38 mitogen-activated protein kinase (MAPK) but not that of extracellular-regulated protein kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK). This attenuation was not abrogated by ICI 182780. Moreover, the p38 inhibitor, SB 203580, greatly reduced the LPS-induced NO production, and the remaining NO levels were no longer regulated by E2. Additionally, E2-BSA inhibited LPS-mediated changes in p38 MAPK activation to the same extent as E2. Moreover, E2 and E2-BSA inhibited LPS-induced activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). This inhibitory effect of E2 was only partially antagonized by ICI 182780. Taken together, these results suggest that E2 has an inhibitory effect on LPS-induced NO production in BMMs through inhibition of p38 MAPK phosphorylation, and blockade of NF-κB and AP-1 activation. These effects are mediated at least in part via a nongenomic pathway.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Estradiol/farmacología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Óxido Nítrico/antagonistas & inhibidores , Albúmina Sérica Bovina/farmacología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Estradiol/análogos & derivados , Moduladores de los Receptores de Estrógeno/farmacología , Fulvestrant , Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/agonistas , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Óxido Nítrico/biosíntesis , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The sympathetic nervous system can regulate both osteoblast and chondrocyte growth and activity through ß(2)-adrenergic receptors (ß(2)-AR). We have shown previously that ß(2)-AR activate both adenylyl cyclase and mitogen-activated protein kinases ERK1/2 in growth plate chondrocytes prepared from ribs of embryonic E18.5 mice. Here we examined ß(2)-AR inhibition of collagen type II (Col II) expression in growth plate chondrocytes and the molecular pathways involved. Stimulation of ß(2)-AR by isoproterenol inhibited Col II mRNA and protein levels by â¼50% beginning at 2 h, with both remaining suppressed over 24 h. This inhibition was blocked by propranolol and inhibitors of either MEK1 or PKA. Isoproterenol stimulated an AP-1-luciferase reporter and increased the expression of AP-1 factors c-Fos, Fra-1, Fra-2, c-Jun, and Jun-B but had no effect on Jun-D. Stimulation of AP-1 activity was blocked by inhibitors of MEK1 or PKA. siRNA inhibition of AP-1 factors showed that depletion of only Jun-B attenuated isoproterenol-mediated inhibition of Col II. Transfection with jun-B or c-fos showed selective inhibition of Col II mRNA and a Col II luciferase reporter construct by jun-B. Isoproterenol as well as jun-B overexpression in the chondrocytes also inhibited the expression of Sox-6 mRNA and protein, and depletion of Jun-B abrogated ß(2)-AR inhibition of Sox-6. Collectively, these findings demonstrate regulation of chondrocyte differentiation through ß(2)-AR mediated by ERK1/2 and PKA stimulation of the AP-1 factor Jun-B that inhibits the expression of Sox-6 and Col II.
Asunto(s)
Condrocitos/metabolismo , Colágeno Tipo II/genética , Placa de Crecimiento/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores Adrenérgicos beta 2/fisiología , Factor de Transcripción AP-1/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Células Cultivadas , Condrocitos/efectos de los fármacos , Colágeno Tipo II/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Placa de Crecimiento/citología , Placa de Crecimiento/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Ratones , Embarazo , Proteínas Proto-Oncogénicas c-jun/agonistas , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Factores de Transcripción SOXD/genética , Factores de Transcripción SOXD/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/antagonistas & inhibidoresRESUMEN
Beta-glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel beta-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the beta-glucan significantly increased luciferase activity in cells transfected with NFkappaB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFkappaB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the beta-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by beta-glucan induced phosphorylation of IkappaB and the consequent translocation of NFkappaB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that beta-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFkappaB signaling pathway.
Asunto(s)
Activación de Macrófagos , Macrófagos/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/agonistas , Paenibacillus/química , beta-Glucanos/farmacología , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Núcleo Celular/metabolismo , Proteínas I-kappa B/metabolismo , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Fosforilación , Factor de Transcripción STAT1/agonistas , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismo , beta-Glucanos/aislamiento & purificaciónRESUMEN
Thioredoxin (Trx) is a small ubiquitous protein, which has been shown to be involved in redox-dependent cellular functions. In this article, we demonstrate that the increased level of Trx induces AP-1 DNA binding in a redox-dependent manner by activating JNK subgroup of MAPKs. The majority of AP-1 DNA binding complex was found to be composed of cJun, JunB, and Fra-1. Increased expression of Trx resulted in phosphorylation of cJun, Jun B, and Fra-1. Further, increased expression of Trx induced the phosphorylation of MKK4 and MKK7 which are upstream kinases of the JNK signaling cascade. In co-transfection studies, AP-1-dependent luciferase reporter vector and pcDNA3-Trx increased luciferase activity demonstrating that increased expression of Trx increases AP-1 transactivation. In addition, dominant-negative JNK kinase (dnJNK/MKK4) or dominant-negative JNK (dnJNK) inhibited Trx-mediated AP-1 transactivation, as well as AP-1 DNA binding. Furthermore, transfection of kinase-dead MEKK1, an initiating kinase of the JNK pathway inhibited Trx-mediated AP-1 transactivation and DNA binding, suggesting that MEKK1 may mediate Trx-induced AP-1 activation. In contrast, wild-type MEKK1 overexpression did not inhibit Trx-mediated AP-1 activation. Taken together, our data demonstrate that increased expression of Trx induces MKK4/MKK7-dependent JNK activation, resulting in enhanced DNA binding, and transactivation of AP-1 transcription factor.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Tiorredoxinas/farmacología , Factor de Transcripción AP-1/agonistas , ADN/metabolismo , Genes Reporteros/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas Quinasa Quinasa PAM/fisiología , Fosforilación/efectos de los fármacos , Fosforilación/genética , Unión Proteica/efectos de los fármacos , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Transfección , Células Tumorales CultivadasRESUMEN
We have previously reported that a disintegrin inhibits solid tumor growth and metastasis in mouse model [I.C. Kang, Y.D. Lee, D.S. Kim, A novel disintegrin salmosin inhibits tumor angiogenesis, Cancer Res. 59 (1999) 3754-3760; S.I. Kim, K.S. Kim, H.S. Kim, D.S. Kim, Y. Jang, K.H. Chung, Y.S. Park, Inhibitory effect of the salmosin gene transferred by cationic liposomes on the progression of B16BL6 tumors, Cancer Res. 63 (2003) 6458-462]. In this study, we have investigated the modulatory effect of a disintegrin, saxatilin, on the balance between MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human ovarian cancer cell line MDAH 2774. Functional mechanism of the disintegrin-mediated transcriptional regulation of MMP-9 and TIMP-1 was examined in the ovarian cancer cell line. Saxatilin strongly induced TIMP-1 expression in dose- and time-dependent manners, while the disintegrin suppressed MMP-9 expression. Further analyses clearly indicated that interaction of the disintegrin and integrin alphavbeta3 results in the TIMP-1 promoter activation via c-fos to suppress TNF-alpha-induced cancer cell invasion. These results demonstrate that integrin alphavbeta3-mediated transcriptional regulation of MMP-9 and TIMP-1 is critical for suppressing the ovarian cancer cell invasion.
Asunto(s)
Desintegrinas/farmacología , Regulación Neoplásica de la Expresión Génica , Integrina alfaVbeta3/metabolismo , Neoplasias Ováricas/patología , Inhibidor Tisular de Metaloproteinasa-1/genética , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Femenino , Humanos , Integrina alfaVbeta3/genética , Metaloproteinasa 9 de la Matriz/genética , Invasividad Neoplásica , Neoplasias Ováricas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
Asunto(s)
Herpesvirus Saimiriino 2/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Línea Celular , Detergentes , Humanos , Iones , FN-kappa B/agonistas , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Factor de Transcripción STAT3/metabolismo , Solubilidad , Factor de Transcripción AP-1/agonistasRESUMEN
Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif 10PQENDE15 in STP-A11 reveals that Glu (E)12 residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.
Asunto(s)
Humanos , Transcripción Genética , Factor de Transcripción AP-1/agonistas , Factor 6 Asociado a Receptor de TNF/metabolismo , Solubilidad , Factor de Transcripción STAT3/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Unión Proteica , Proteínas Oncogénicas Virales/metabolismo , FN-kappa B/agonistas , Iones , Herpesvirus Saimiriino 2/metabolismo , Detergentes , Línea CelularRESUMEN
Density-dependent growth inhibition secures tissue homeostasis. Dysfunction of the mechanisms, which regulate this type of growth control is a major cause of neoplasia. In confluent normal rat kidney (NRK) fibroblasts, epidermal growth factor (EGF) receptor levels decline, ultimately rendering these cells irresponsive to EGF. Using an activator protein (AP)-1 sensitive reporter construct, we show that AP-1 activity is strongly decreased in density-arrested NRK cells, but is restored after relaxation of densitydependent growth inhibition by removing neighboring cells. EGF could not induce AP-1 activity or S-phase entry in density-arrested cells, but could do so after pretreatment with retinoic acid, which enhances EGF receptor expression. Our results support a model in which the EGF receptor regulates density-dependent growth control in NRK fibroblasts, which is reflected by EGF-induced mitogenic signaling and consequent AP-1 activity.
Asunto(s)
Proliferación Celular , Receptores ErbB/fisiología , Fibroblastos/citología , Factor de Transcripción AP-1/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Fibroblastos/efectos de los fármacos , Riñón/citología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Ratas , Transducción de Señal , Factor de Transcripción AP-1/agonistas , Tretinoina/farmacologíaRESUMEN
AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis and its activation during T cell receptor stimulation has recently been reported. In this study, we examined the role of AMPK in interleukin (IL)-2 production in T cells. Inhibition of AMPK by compound C, a specific inhibitor of AMPK or small interfering RNA of AMPKalpha1 suppressed IL-2 production in Jurkat T cells and peripheral blood lymphocytes stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti-CD3 plus anti-CD28. We then showed that AMPK inhibition reduced PMA/Io-induced IL-2 mRNA expression and IL-2 promoter activation. Moreover, inhibition of AMPK suppressed transcriptional activation of NF-AT and AP-1, but not NF-kappaB, in PMA/Io-activated Jurkat cells. Finally, we found that compound C inhibited PMA/Io-induced phosphorylation of p38, JNK, and GSK-3beta but not of ERK. These results suggest that AMPK mediates IL-2 production by regulating NF-AT and AP-1activation during T cell stimulation.
Asunto(s)
Interleucina-2/metabolismo , Complejos Multienzimáticos/fisiología , Factores de Transcripción NFATC/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Linfocitos T/inmunología , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas Activadas por AMP , Regulación hacia Abajo , Humanos , Interleucina-2/genética , Células Jurkat , Complejos Multienzimáticos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Factor de Transcripción AP-1/agonistas , Activación TranscripcionalRESUMEN
The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.
Asunto(s)
Curcumina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo , Animales , Antioxidantes/farmacología , Línea Celular , Curcumina/análogos & derivados , Ciclooxigenasa 2/metabolismo , Humanos , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/antagonistas & inhibidores , Indoles/farmacología , Neoplasias de la Mama/genética , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Estrógenos/farmacología , Humanos , Regiones Promotoras Genéticas , Receptores de Progesterona/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Tannins are plant-derived water-soluble polyphenols with wide-ranging biological activities. The mechanisms underlying the anti-inflammatory effect of tannins are not fully understood and may be the result of inhibition of poly(ADP-ribose) (PAR) glycohydrolase (PARG), the main catabolic enzyme of PAR metabolism. Therefore, we set out to investigate the mechanism of the anti-inflammatory effect of gallotannin (GT) in A549 cells with special regard to the role of poly(ADP-ribosyl)ation. Using an inflammation-focused low-density array and reverse transcription-polymerase chain reaction, we found that GT suppressed the expression of most cytokines and chemokines in cytokine-stimulated A549 cells, whereas the PARP inhibitor PJ-34 only inhibited few transcripts. Activation of the transcription factors, nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), was blocked by GT, whereas PJ-34 only suppressed NF-kappaB activation but not AP-1 activation. GT also inhibited IkappaB phosphorylation and nuclear translocation of NF-kappaB, but PJ-34 had no effect on these upstream events. In the AP-1 pathway, GT treatment, even in the absence of cytokines, caused maximal phosphorylation of c-Jun N-terminal kinase and c-Jun. GT also caused a low-level phosphorylation of p38, extracellular signal-regulated kinases 1 and 2, activating transcription factor2, and cAMP-response element-binding protein but inhibited cytokine-induced phosphorylation of these kinases and transcription factors. GT inhibited protein phosphatases 1 and 2A, which may explain the increased phosphorylation of mitogen-activated protein kinase and their substrates. GT exerted potent antioxidant effect but failed to cause PAR accumulation. In summary, the potent inhibitory effects of GT on the transcription of cytokine and chemokine genes are probably not related to PARG inhibition. Inhibition of AP-1 activation and upstream signaling events may be responsible for the effects of GT.
Asunto(s)
Antioxidantes/farmacología , Quimiocinas/antagonistas & inhibidores , Citocinas/antagonistas & inhibidores , Taninos Hidrolizables/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Secuencia de Bases , Línea Celular , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Cartilla de ADN , Humanos , Mediadores de Inflamación/metabolismo , Fenantrenos , Poli Adenosina Difosfato Ribosa/metabolismo , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/metabolismoRESUMEN
Mitogen-activated protein kinases (MAP kinases), including extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38, play a central role in cellular responses by various stress stimuli such as cell proliferation, apoptosis, migration, or gene expression. Furthermore, activator protein-1 (AP-1), a transcription factor which can be activated by MAP kinases, also is involved in a variety of celllar responses, as well as MAP kinases. MAP kinases and AP-1 are significantly activated in vascular tissues by hypertension, angiotensin II, or balloon injury. We have made dominant negative mutants of MAP kinases or c-Jun, to specifically inhibit in vivo activation of MAP kinases or AP-1. Vascular gene transfer of each dominant negative mutant of MAP kinases or c-Jun prevents intimal hyperplasia after balloon injury, which is associated with the inhibition of smooth muscle cell proliferation in the intima and the media and probably also associated with inhibition of smooth muscle cell migration. However, in vitro findings on cultured vascular smooth muscle cells suggest that the molecular mechanism underlying inhibition of intimal hyperplasia may be different among each dominant negative mutant of MAP kinases and c-Jun. MAP kinases and c-Jun seem to be the promising therapeutic target for vascular remodeling.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Factor de Transcripción AP-1/metabolismo , Animales , Técnicas de Transferencia de Gen , Hiperplasia , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/agonistas , Factor de Transcripción AP-1/genéticaRESUMEN
Monocytic differentiation of HL60 cells induced by 1,25-dihydroxyvitamin D(3) (1,25 D(3)) has been recently shown (Exp Cell Res 258, 425, 2000) to be enhanced by an exposure to SB203580 or to SB202190, specific inhibitors of p38MAP kinase, with concomitant up-regulation of the c-jun N terminal kinase (JNK) pathway. In the present study we inquired if this enhancement and the JNK up-regulation are limited to 1,25 D(3)-induced differentiation, or if they occur more generally in HL60 cell differentiation. We found that dimethylsulfoxide (DMSO)-induced differentiation, and to a lesser extent tetradecanoylphorbol acetate (TPA)-induced macrophage differentiation were also potentiated by the p38MAPK inhibitors, but that granulocytic differentiation in response to all-trans retinoic acid (RA) was not. The enhancement of differentiation by p38MAPK inhibitors was accompanied by an activation of the JNK MAPK pathway, as shown by the phosphorylation levels of these kinases and by AP-1 binding, but only in 1,25 D(3)-treated cells. This shows that an up-regulation of the JNK stress pathway during 1,25 D(3)-induced monocytic differentiation occurs selectively in this lineage of differentiation and is not necessary for the expression of the differentiated phenotype.
Asunto(s)
Células HL-60/citología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Monocitos/metabolismo , Factor de Transcripción AP-1/metabolismo , Antígenos de Diferenciación Mielomonocítica/efectos de los fármacos , Antígenos de Superficie/efectos de los fármacos , Calcitriol/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Granulocitos/citología , Granulocitos/metabolismo , Células HL-60/metabolismo , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Macrófagos/citología , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Piridinas/metabolismo , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/agonistas , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Vascular endothelial growth factor (VEGF) is a potent mitogen in endothelial cells, but little is known about its activity in other cell types. To clarify the role of VEGF in human dental pulp cells and pulp tissue, we investigated the effects of VEGF on the chemotaxis, proliferation, and differentiation of human dental pulp cells. VEGF induced a strong chemotactic response in human dental pulp cells in a dose-dependent manner. VEGF also marginally enhanced the proliferation of human dental pulp cells and induced an increase in alkaline phosphatase in human dental pulp cells. However, these effects of VEGF were not observed in reference to human skin fibroblasts. Analyses by the reverse-transcription/polymerase-chain-reaction method and flow cytometry showed that the mRNAs of two VEGF receptors, fins-like tyrosine kinase and kinase insert domain-containing receptor, were expressed in human dental pulp cells, whereas only fms-like tyrosine kinase mRNA was expressed in human skin fibroblasts. VEGF induced the activation of activator protein 1 (AP-1) and c-fos mRNA expression in human dental pulp cells. The AP-1 inhibitor curcumin strongly inhibited VEGF-induced alkaline phosphatase production in human dental pulp cells. In addition, VEGF antisense oligonucleotide suppressed the production of VEGF and alkaline phosphatase in human dental pulp cells. These results suggest that VEGF produced by human dental pulp cells acts directly upon human dental pulp cells in an autocrine manner, and may promote the chemotaxis, proliferation, and/or differentiation of human dental pulp cells via the utilization of kinase insert domain-containing receptor and in part through AP-1 by increasing c-fos.