RESUMEN
CD40L and TNF signal through engagement of their respective receptors, which are both members of the TNF receptor family. They use partially common signaling molecules leading, among others, to activation of the NF-κB pathway. However, whereas TNF activates the classical, CD40L has been reported to activate the alternative NF-κB pathway, leading to the anticipation that differences in the pattern of inflammatory gene expression would occur. Here, we have compared the gene expression repertoire of CD40L (CD154) and TNF stimulated HUVEC and report that unexpectedly, apart from a stronger response to TNF, no major qualitative differences could be observed. This applies for the period of up to 6 h, a time where the alternative pathway has already been activated. Analysis of the early events after receptor engagement revealed that both TNF and CD40L activate the classical NF-κB pathway, and confirm activation of the alternative by the latter. Furthermore, using genetic and pharmacological inhibition of the classical pathway we show that activation of the alternative occurs independently of the former. This reveals novel insights into NF-κB signaling by CD40L and TNF in endothelial cells.
Asunto(s)
Ligando de CD40/inmunología , Células Endoteliales/inmunología , Regulación de la Expresión Génica/inmunología , Redes y Vías Metabólicas/inmunología , FN-kappa B/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Células Cultivadas , Humanos , Transducción de Señal/inmunologíaRESUMEN
Animals self-medicate using a variety of plant and arthropod secondary metabolites by either ingesting them or anointing them to their fur or skin apparently to repel ectoparasites and treat skin diseases. In this respect, much attention has been focused on primates. Direct evidence for self-medication among the great apes has been limited to Africa. Here we document self-medication in the only Asian great ape, orang-utans (Pongo pygmaeus), and for the first time, to our knowledge, the external application of an anti-inflammatory agent in animals. The use of leaf extracts from Dracaena cantleyi by orang-utan has been observed on several occasions; rubbing a foamy mixture of saliva and leaf onto specific parts of the body. Interestingly, the local indigenous human population also use a poultice of these leaves for the relief of body pains. We present pharmacological analyses of the leaf extracts from this species, showing that they inhibit TNFα-induced inflammatory cytokine production (E-selectin, ICAM-1, VCAM-1 and IL-6). This validates the topical anti-inflammatory properties of this plant and provides a possible function for its use by orang-utans. This is the first evidence for the deliberate external application of substances with demonstrated bioactive potential for self-medication in great apes.
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Conducta Animal , Productos Biológicos , Dracaena/química , Plantas Medicinales , Pongo pygmaeus , Automedicación , Animales , Productos Biológicos/química , Productos Biológicos/farmacología , Biomarcadores , Línea Celular , Citocinas/metabolismo , Dracaena/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacologíaRESUMEN
PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.
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Antiinflamatorios/farmacología , Asteraceae/química , Lactonas/farmacología , Sesquiterpenos/farmacología , Animales , Selectina E/metabolismo , Edema/tratamiento farmacológico , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Estructura Molecular , Monocitos/efectos de los fármacos , Componentes Aéreos de las Plantas/química , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
BACKGROUND: Urokinase-type plasminogen activator (u-PA) plays a pivotal role in extracellular proteolysis and is thought to be critically involved in the modulation of angiogenesis. Interleukin (IL)-33 is a member of the IL-1 cytokine family, which is thought to act as danger signal that is released from cells after injury. IL-33 is involved in the pathogenesis of various inflammatory diseases and previously was shown to induce angiogenesis and inflammatory activation of endothelial cells. OBJECTIVE: We investigated the impact of IL-33 on u-PA in endothelial cells as a new possible function for IL-33. METHODS AND RESULTS: We could demonstrate that IL-33 upregulated u-PA mRNA expression and protein production in human coronary artery and human umbilical vein endothelial cells in a time- and concentration-dependent manner via interaction with its receptor ST2 and activation of the nuclear factor-κB pathway but independent of autocrine IL-1-induced effects. The hydroxymethylglutaryl-coenzyme A reductase inhibitor simvastatin abrogated the IL-33-induced increase in u-PA, thus providing further evidence for pleiotropic effects of statins. IL-33 induced u-PA-dependent capillary-like tube formation and vessel sprouting. In human carotid atherosclerotic plaques (n = 16), u-PA mRNA positively correlated with IL-33 mRNA expression (r = 0.780, P < 0.001). Furthermore, IL-33 and u-PA protein were detected in endothelial cells in these samples using fluorescence immunohistochemistry. CONCLUSIONS: We hypothesize that IL-33, representing a danger signal that is released after tissue damage, in addition to its role in the inflammatory activation of endothelial cells, is involved in u-PA-driven angiogenesis, a process that has been shown before to be linked to inflammation in various pathologies.
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Inductores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Interleucinas/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/enzimología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteína 1 Similar al Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Interleucinas/metabolismo , FN-kappa B/metabolismo , Placa Aterosclerótica , Interferencia de ARN , ARN Mensajero/metabolismo , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Simvastatina/farmacología , Factores de Tiempo , Transfección , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Adhesión Celular/efectos de los fármacos , Endotelio Linfático/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/química , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Esferoides Celulares/efectos de los fármacos , Sulfonas/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular , Quimiotaxis/efectos de los fármacos , Endotelio Linfático/citología , Endotelio Linfático/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-kappaB, serum response factor and C/EBP. alpha-Catulin, an alpha-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that alpha-catulin also interacts with a key component of the NF-kappaB signalling pathway, namely the IkappaB kinase (IKK)-beta. In co-immunoprecipitations, alpha-catulin can bind IKK-beta and Lbc. Ectopic expression of alpha-catulin augmented NF-kappaB activity, promoted cell migration and increased resistance to apoptosis, whereas knockdown experiments showed the opposite effects. Together, these features suggest that alpha-catulin has tumorigenic potential.
Asunto(s)
Apoptosis/genética , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , alfa Catenina/metabolismo , alfa Catenina/fisiología , Apoptosis/efectos de los fármacos , Movimiento Celular/genética , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Citoprotección/genética , Células HeLa , Humanos , Mediadores de Inflamación/metabolismo , Unión Proteica , Factor Rho/metabolismo , Factor Rho/fisiología , Transducción de Señal/fisiología , Distribución Tisular , Transfección , Factor de Necrosis Tumoral alfa/farmacología , alfa Catenina/genéticaRESUMEN
INTRODUCTION: Macrophage colony stimulating factor (M-CSF) is a key factor for monocyte and macrophage survival and proliferation. M-CSF has been implicated in cardiac healing and repair after myocardial infarction. METHODS AND RESULTS: We show by immunohistochemistry and Western blotting analysis that M-CSF protein is present in human heart tissue. Cultured human adult cardiac myocytes (HACM) and human adult cardiac fibroblasts (HACF) isolated from human myocardial tissue constitutively express M-CSF. When HACM and HACF were treated with tumor necrosis factor-alpha (TNF-alpha) M-CSF protein production and M-CSF mRNA expression, determined by ELISA or by using RT-PCR, respectively, was significantly increased. To determine a possible role of nuclear factor kappaB (NF-kappaB) and activating protein 1 (AP-1) in M-CSF regulation, blockers to both pathways and an adenovirus overexpressing a dominant negative (dn) form of IkappaB kinase 2 (IKK2) were used. Only the NF-kappaB blocker dimethylfumarate and the dn IKK2, but not januskinase inhibitor-1 (JNK-I), were able to block the TNF-alpha-induced increase in M-CSF production in these cells, suggesting that the induction of M-CSF through TNF-alpha is mainly dependent on the activation of the NF-kappaB pathway. The monocyte activation marker CD11b was significantly increased after incubating U937 cells with conditioned medium from HACM or HACF as determined by FACS analysis. CONCLUSIONS: Our in vitro data taken together with our immunohistochemistry data suggest that human cardiac cells constitutively express M-CSF. This expression of M-CSF in the human heart and its upregulation by TNF-alpha might contribute to monocyte and macrophage survival and differentiation.
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Fibroblastos/metabolismo , Factor Estimulante de Colonias de Macrófagos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , FN-kappa B/metabolismo , Fragmentos de Péptidos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Western Blotting , Antígeno CD11b/metabolismo , Separación Celular , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Dimetilfumarato , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Fumaratos/farmacología , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunohistoquímica , Factor Estimulante de Colonias de Macrófagos/genética , Monocitos/inmunología , Monocitos/metabolismo , Mutación , Miocardio/citología , Miocitos Cardíacos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Células U937 , Regulación hacia ArribaRESUMEN
We describe here the identification and initial characterization of a novel human gene termed IKIP (I kappa B kinase interacting protein) that is located on chromosome 12 in close proximity to APAF1 (apoptotic protease-activating factor-1). IKIP and APAF1 share a common 488 bp promoter from which the two genes are transcribed in opposite directions. Three IKIP transcripts are generated by differential splicing and alternative exon usage that do not show significant homology to other genes in the databases. Similar to APAF1, expression of IKIP is enhanced by X-irradiation, and both genes are dependent on p53. Moreover, IKIP promotes apoptosis when transfected into endothelial cells. We conclude that IKIP is a novel p53 target gene with proapoptotic function.
Asunto(s)
Apoptosis/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos Par 12/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos/genética , Animales , Factor Apoptótico 1 Activador de Proteasas , Secuencia de Bases/genética , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Bovinos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Línea Celular , Secuencia Conservada/genética , ADN Complementario/análisis , ADN Complementario/genética , Exones/genética , Regulación de la Expresión Génica/genética , Humanos , Quinasa I-kappa B , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Proteínas/metabolismo , Ratas , Transducción de Señal/genética , Transcripción Genética/genética , Transfección , Regulación hacia Arriba/genética , Regulación hacia Arriba/efectos de la radiación , Rayos X , XenopusRESUMEN
BACKGROUND: To investigate the contribution of inflammation to postangioplasty lumen loss, we used an adenoviral gene therapy approach to inhibit the central inflammatory mediator nuclear factor-kappaB (NF-kappaB) by overexpression of its natural inhibitor, IkappaBalpha. METHODS AND RESULTS: The adenovirus carrying human IkappaBalpha was applied immediately after balloon dilatation by a double-balloon catheter in a rabbit iliac artery restenosis model. Immunohistochemistry of IkappaBalpha revealed that mainly smooth muscle cells of the media but also cells of the adventitia were transduced and expressed the transgene IkappaB alpha for >/= 8 days. At this time point, intercellular adhesion molecule-1 (30%) and monocyte chemotactic protein-1 (50%) expression, as well as recruitment of macrophages into the wounded area (90%), were significantly reduced in IkappaB alpha-treated vessels. In addition, expression of inhibitor of apoptosis proteins was reduced and the percentage of apoptotic cells was increased compared with control-treated contralateral vessels. Animals killed 5 weeks after treatment exhibited a significantly reduced degree of lumen narrowing (P<0.02) on the side treated with adenovirus IkappaBalpha. The lumen gain of approximately 40% was due to positive remodeling. CONCLUSIONS: From these data, we conclude that balloon angioplasty-induced activation of NF-kappaB contributes to lumen loss likely via induction of an inflammatory response and a decrease in the rate of apoptosis. These data show for the first time that inflammation mediated by NF-kappaB is involved in postangioplasty lumen narrowing. Specific and more potent inhibitors of NF-kappaB might therefore be a useful therapeutic measure to improve clinical outcome after balloon dilatation.
Asunto(s)
Oclusión de Injerto Vascular/metabolismo , Oclusión de Injerto Vascular/prevención & control , Proteínas I-kappa B , FN-kappa B/metabolismo , Adenoviridae/genética , Angiografía de Substracción Digital , Angioplastia de Balón/efectos adversos , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Dieta Aterogénica , Modelos Animales de Enfermedad , Expresión Génica , Oclusión de Injerto Vascular/patología , Humanos , Arteria Ilíaca/diagnóstico por imagen , Arteria Ilíaca/metabolismo , Arteria Ilíaca/patología , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , Infiltración Neutrófila/efectos de los fármacos , Conejos , Transgenes , Grado de Desobstrucción Vascular/efectos de los fármacosRESUMEN
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.
Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-8/genética , Lipoproteínas LDL/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/efectos de los fármacos , Genes Reporteros , Células HeLa , Factor C1 de la Célula Huésped , Humanos , Interleucina-8/biosíntesis , Cinética , FN-kappa B/metabolismo , Factor 1 de Transcripción de Unión a Octámeros , Oxidación-Reducción , Éteres Fosfolípidos/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Elementos de Respuesta , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Gem belongs to the Rad/Gem/Kir (RGK) subfamily of Ras-related GTPases, which also comprises Rem, Rem2 and Ges. The RGK family members Ges and Rem have been shown to produce endothelial cell sprouting and reorganization of the actin cytoskeleton upon overexpression. Here we show that high intracellular Gem levels promote profound changes in cell morphology and we investigate how this phenotype arises dynamically. We also show that this effect requires intact microtubules and microfilaments, and that Gem is associated with both cytoskeletal components. In order to investigate the mechanisms of Gem recruitment to the cytoskeleton, we performed a yeast two-hybrid screen and identified a novel kinesin-like protein, termed KIF9, as a new Gem interacting partner. We further show that Gem and KIF9 interact by co-immunoprecipitation. Furthermore, Gem and KIF9 display identical patterns of gene expression in different tissues and developmental stages. The Gem- KIF9 interaction reported here is the first molecular link between RGK family members and the microtubule cytoskeleton.
Asunto(s)
Proteínas Inmediatas-Precoces/metabolismo , Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular , Membrana Celular/metabolismo , Tamaño de la Célula , Células Cultivadas , Chlorocebus aethiops , Citoesqueleto/metabolismo , ADN Complementario , Endotelio Vascular/citología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Cinesinas/genética , Proteínas de la Membrana/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Pruebas de Precipitina/métodos , Saccharomyces cerevisiae , Técnicas del Sistema de Dos Híbridos , Proteínas rasRESUMEN
In a variety of cell types, the transcription factor nuclear factor kappaB (NF-kappaB) functions as a mediator of stress and immune responses. In endothelial cells (ECs), it controls the expression of genes encoding, eg, cytokines, cell adhesion molecules, and procoagulatory proteins. This study investigates the effect of NF-kappaB suppression on several pathophysiologic functions of ECs, including inflammation, coagulation, and angiogenesis. A recombinant adenovirus was generated for expression of a dominant negative (dn) mutant of IkappaB kinase 2 (IKK2), a kinase that acts as an upstream activator of NF-kappaB. dnIKK2 inhibited NF-kappaB, resulting in strongly reduced nuclear translocation and DNA binding activity of the transcription factor and lack of expression of several proinflammatory markers, including E-selectin, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and interleukin-8. Concomitantly, inhibition of leukocyte binding to dnIKK2-expressing ECs could be demonstrated in a cell adhesion assay. Furthermore, expression of tissue factor as well as the ability to form capillary tubes in a matrigel assay was impaired in dnIKK2-expressing ECs. These data demonstrate that NF-kappaB is of central importance not only for the inflammatory response but also for a number of other EC functions. Therefore, this transcription factor as well as its upstream regulatory signaling molecules may represent favorable targets for therapeutic interference.
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Endotelio Vascular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/farmacología , Transfección/métodos , Adenoviridae/genética , Coagulación Sanguínea/efectos de los fármacos , Pruebas de Coagulación Sanguínea , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Citocinas/efectos de los fármacos , Endotelio Vascular/patología , Endotelio Vascular/fisiopatología , Humanos , Quinasa I-kappa B , Inflamación/metabolismo , Mutación , FN-kappa B/antagonistas & inhibidores , FN-kappa B/farmacología , FN-kappa B/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/patología , Venas Umbilicales/fisiopatologíaRESUMEN
Fumaric acid esters are thought to improve psoriasis by altering leukocyte, keratinocyte, and/or endothelial functions. To determine specificity, kinetics, and molecular mechanisms of different fumaric acid esters in their ability to inhibit endothelial cell activation, we analyzed CD62E and CD54 expression in endothelial cells in vivo and in vitro. In lesional skin of psoriatic patients, oral fumaric acid ester treatment resulted in a marked reduction of CD62E but not CD54 expression on dermal microvessels. Using human umbilical vein endothelial cells, dimethylfumarate almost completely inhibited tumor-necrosis-factor-induced CD62E, but not CD54 expression at concentrations < or = 70 microM, mimicking the situation in vivo. A 60 min dimethylfumarate preincubation was sufficient to block tumor-necrosis-factor-induced CD62E expression for up to 24 h. In contrast, equimolar concentrations of methylhydrogenfumarate, the hydrolysis product of dimethylfumarate, did not suppress tumor-necrosis-factor-induced CD62E expression. Likewise, all fumaric acid esters other than dimethylfumarate were ineffective. Using CD62E, NF-kappa B, or AP-1-responsive promoter constructs, dimethylfumarate inhibited tumor-necrosis-factor-induced activation of the CD62E and the NF-kappa B but not the AP-1 promoter construct. In summary, at a dose range < or = 70 microM, dimethylfumarate appeared to be a specific inhibitor of CD62E expression in an NF-kappa B-dependent manner.
Asunto(s)
Fármacos Dermatológicos/farmacología , Selectina E/genética , Fumaratos/farmacología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Capilares/química , Capilares/efectos de los fármacos , Capilares/fisiología , Células Cultivadas , Dimetilfumarato , Selectina E/análisis , Endotelio Vascular/química , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Molécula 1 de Adhesión Intercelular/genética , Psoriasis/tratamiento farmacológico , Psoriasis/fisiopatología , ARN Mensajero/análisis , Piel/irrigación sanguínea , Venas Umbilicales/citologíaRESUMEN
A variety of pathophysiological situations that affect cells of the vasculature, including endothelial and smooth muscle cells, leads to the expression of genes such as adhesion molecules and chemokines that are dependent on members of the nuclear factor (NF)-kappaB family of transcription factors. The corresponding gene products mediate important biological functions such as immune and inflammatory reactions, smooth muscle cell proliferation, and angiogenesis. The beneficial and usually transient NF-kappaB-dependent gene expression may be exaggerated in pathological situations and results in damage to the vessel wall and impaired vascular cell function. In this review, we will capitalize on the favorable and adverse roles of NF-kappaB in the context of vascular disease, eg, chronic and localized inflammation, arteriosclerosis, and neoangiogenesis.
Asunto(s)
Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , FN-kappa B/fisiología , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Endotelio Vascular/citología , Endotelio Vascular/patología , Regulación de la Expresión Génica , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/patología , FN-kappa B/metabolismo , Neovascularización Patológica/genética , Neovascularización Fisiológica/genéticaAsunto(s)
Activación de Complemento , Endotelio Vascular/inmunología , Regulación de la Expresión Génica/inmunología , Animales , Aorta , Antígenos CD55/genética , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor alpha-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-kappaB activity. Here we demonstrate that XIAP, a NF-kappaB-dependent member of the IAP gene family, is a strong stimulator of NF-kappaB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-kappaB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cell's response to environmental stimuli.
Asunto(s)
Apoptosis , Endotelio Vascular/metabolismo , Quinasas Quinasa Quinasa PAM , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Línea Celular , Endotelio Vascular/patología , Humanos , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.
Asunto(s)
Proteínas I-kappa B/metabolismo , Proteínas Luminiscentes/metabolismo , FN-kappa B/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Secuencia de Bases , Células CHO , Cricetinae , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes , Cinética , Microscopía Fluorescente , Unión Proteica , Espectrometría de Fluorescencia , Factor de Transcripción ReIARESUMEN
Cystic fibrosis (CF) is characterised by chronic airway inflammation. Pro-inflammatory mediators in the lung are regulated by the transcription factor nuclear factor kappa B (NFkappaB). We have assessed the effect of adenovirus and liposome-mediated overexpression of the NFkappaB inhibitor IkappaBalpha, as well as liposome-mediated transfection with oligonucleotides resembling NFkappaB consensus binding sites (decoys) in a cystic fibrosis airway epithelial cell line (CFTE). Electrophoretic mobility shift assays (EMSA) were used to assess NFkappaB activity and secretion of the pro-inflammatory cytokine interleukin-8 (IL-8) was measured by ELISA. At a MOI of 30, Ad-IkappaBalpha significantly decreased IL-8 secretion to 60% and 43% of control unstimulated and TNF-alpha stimulated cells, respectively. At this MOI, approximately 70% of cells are transduced. EMSA showed an approximately 50% decrease in NFkappaB activation. Liposome-mediated transfection of IkappaBalpha did not reduce IL-8 secretion, probably due to low transfection efficiency (approximately 5% of cells). Liposome-mediated transfection of CFTE cells with rhodamine-labeled decoy oligonucleotides indicated a transfection efficiency close to 100%. TNF-alpha stimulated IL-8 secretion was reduced by approximately 40% using this approach. EMSA confirmed a significant decrease of NFkappaB activation. Decoy oligonucleotides may be a promising approach for reduction of NFkappaB-mediated pulmonary inflammation. Gene Therapy (2000) 7, 306-313.
Asunto(s)
Fibrosis Quística/terapia , Terapia Genética/métodos , FN-kappa B/genética , Adenoviridae/genética , Células Cultivadas , Fibrosis Quística/metabolismo , Electroforesis , Vectores Genéticos/genética , Humanos , Interleucina-8/metabolismo , Liposomas/genética , Liposomas/farmacología , FN-kappa B/antagonistas & inhibidores , Oligonucleótidos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/genéticaRESUMEN
Recent progress in the identification and functional analysis of protein kinases and adapter molecules that lead to activation of NF-kappaB family transcription factors has lead to a quite detailed understanding of one of the major signalling pathways that mediate a cell's response to environmental stress in a variety of host-defense situations. NF-kappaB is recognized as a key regulatory factor mediating the coordinate expression of genes which are part of the cellular machinery that functions to protect an organism against damage posed by physical, chemical or microbial noxae. In a wide variety of patho-physiological situations such as immune and inflammatory reactions, the expression of cytokines, interleukins and adhesion molecules in cells of the immune system including T and B cells, endothelial as well as phagocytic/antigen presenting cells is to a large extent regulated by NF-kappaB. Moreover, this transcription factor appears to play a central role in the regulation of apoptosis, an important cellular program that decides upon a cell's fate not only during embryonic development but also on its way from normal to the transformed phenotype. Thus, NF-kappaB has emerged also as an attractive target for therapeutic interference in a variety of pathological situations, including chronic inflammatory and autoimmune diseases, HIV infection and cancer.
Asunto(s)
Apoptosis/genética , Transformación Celular Neoplásica/genética , FN-kappa B/genética , Animales , División Celular , Regulación Neoplásica de la Expresión Génica/genética , Transducción de Señal , Factores de TranscripciónRESUMEN
Mobilization of nuclear factor-kappaB (NF-kappaB) activates transcription of genes encoding endothelial adhesion molecules and chemokines that contribute to monocyte infiltration critical in atherogenesis. Inhibition of NF-kappaB has been achieved by pharmacological and genetic approaches; however, monocyte interactions with activated endothelium in shear flow following gene transfer of the NF-kappaB inhibitor IkappaB-alpha have not been studied. We found that overexpression of IkappaB-alpha in endothelial cells using a recombinant adenovirus prevented tumor necrosis factor-alpha (TNF-alpha)-induced degradation of IkappaB-alpha and suppressed the upregulation of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin mRNA and surface protein expression and the upregulation of transcripts for the chemokines monocyte chemoattractant protein 1 (MCP-1) and growth-related activity-alpha (GRO-alpha) by TNF-alpha. This was associated with a reduction in endothelial MCP-1 secretion and GRO-alpha immobilization. Adhesion assays under physiological shear flow conditions showed that firm arrest, spreading, and transmigration of monocytes on TNF-alpha-activated endothelium was markedly inhibited by IkappaB-alpha overexpression. Inhibition with monoclonal antibodies and peptide antagonists inferred that this was due to reduced expression of Ig integrin ligand as well as of chemokines specifically involved in these events. In contrast, rolling of monocytes was increased by IkappaB-alpha transfer and was partly mediated by P-selectin; however, it appeared to be unaffected by the inhibition of E-selectin induction. Thus, our data provide novel evidence that selective modulation of NF-kappaB by adenoviral transfer of IkappaB-alpha impairs the expression of multiple endothelial gene products required for subsequent monocyte arrest and emigration in shear flow and thus for monocyte infiltration in atherosclerotic plaques.