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1.
Biochem Biophys Res Commun ; 495(1): 1389-1394, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29183724

RESUMO

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.


Assuntos
Ligante de CD40/imunologia , Células Endoteliais/imunologia , Regulação da Expressão Gênica/imunologia , Redes e Vias Metabólicas/imunologia , NF-kappa B/imunologia , Fator de Necrose Tumoral alfa/imunologia , Células Cultivadas , Humanos , Transdução de Sinais/imunologia
2.
Sci Rep ; 7(1): 16653, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29192145

RESUMO

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.


Assuntos
Comportamento Animal , Produtos Biológicos , Dracaena/química , Plantas Medicinais , Pongo pygmaeus , Automedicação , Animais , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Biomarcadores , Linhagem Celular , Citocinas/metabolismo , Dracaena/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia
3.
Phytomedicine ; 21(12): 1695-701, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25442279

RESUMO

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.


Assuntos
Anti-Inflamatórios/farmacologia , Asteraceae/química , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Animais , Selectina E/metabolismo , Edema/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Estrutura Molecular , Monócitos/efeitos dos fármacos , Componentes Aéreos da Planta/química , Ratos Sprague-Dawley , Relação Estrutura-Atividade
4.
J Thromb Haemost ; 12(6): 948-57, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24702774

RESUMO

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.


Assuntos
Indutores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Interleucinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Interleucinas/metabolismo , NF-kappa B/metabolismo , Placa Aterosclerótica , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Fatores de Tempo , Transfecção , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/genética
5.
Br J Cancer ; 108(3): 564-9, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23093227

RESUMO

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.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/química , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Sulfonas/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
6.
Oncogene ; 27(15): 2159-69, 2008 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-17952117

RESUMO

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.


Assuntos
Apoptose/genética , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , alfa Catenina/metabolismo , alfa Catenina/fisiologia , Apoptose/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Citoproteção/genética , Células HeLa , Humanos , Mediadores da Inflamação/metabolismo , Ligação Proteica , Fator Rho/metabolismo , Fator Rho/fisiologia , Transdução de Sinais/fisiologia , Distribuição Tecidual , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , alfa Catenina/genética
7.
J Thromb Haemost ; 5(12): 2520-8, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17922812

RESUMO

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.


Assuntos
Fibroblastos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Fragmentos de Peptídeos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Antígeno CD11b/metabolismo , Separação Celular , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Fumarato de Dimetilo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Fumaratos/farmacologia , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Imuno-Histoquímica , Fator Estimulador de Colônias de Macrófagos/genética , Monócitos/imunologia , Monócitos/metabolismo , Mutação , Miocárdio/citologia , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Células U937 , Regulação para Cima
8.
Cell Death Differ ; 11(12): 1317-25, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15389287

RESUMO

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.


Assuntos
Apoptose/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos Par 12/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteína Supressora de Tumor p53/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos/genética , Animais , Fator Apoptótico 1 Ativador de Proteases , Sequência de Bases/genética , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Bovinos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Sequência Conservada/genética , DNA Complementar/análise , DNA Complementar/genética , Éxons/genética , Regulação da Expressão Gênica/genética , Humanos , Quinase I-kappa B , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Proteínas/metabolismo , Ratos , Transdução de Sinais/genética , Transcrição Gênica/genética , Transfecção , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação , Raios X , Xenopus
9.
Circulation ; 105(5): 633-8, 2002 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-11827931

RESUMO

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.


Assuntos
Oclusão de Enxerto Vascular/metabolismo , Oclusão de Enxerto Vascular/prevenção & controle , Proteínas I-kappa B , NF-kappa B/metabolismo , Adenoviridae/genética , Angiografia Digital , Angioplastia com Balão/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/farmacologia , Dieta Aterogênica , Modelos Animais de Doenças , Expressão Gênica , Oclusão de Enxerto Vascular/patologia , Humanos , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/metabolismo , Artéria Ilíaca/patologia , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Infiltração de Neutrófilos/efeitos dos fármacos , Coelhos , Transgenes , Grau de Desobstrução Vascular/efeitos dos fármacos
10.
Arterioscler Thromb Vasc Biol ; 21(10): 1585-91, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11597930

RESUMO

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.


Assuntos
Endotélio Vascular/metabolismo , Interleucina-8/genética , Lipoproteínas LDL/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/efeitos dos fármacos , Genes Reporter , Células HeLa , Fator C1 de Célula Hospedeira , Humanos , Interleucina-8/biossíntese , Cinética , NF-kappa B/metabolismo , Fator 1 de Transcrição de Octâmero , Oxirredução , Éteres Fosfolipídicos/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Elementos de Resposta , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
11.
EMBO J ; 20(15): 4076-87, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11483511

RESUMO

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.


Assuntos
Proteínas Imediatamente Precoces/metabolismo , Cinesinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Tamanho Celular , Células Cultivadas , Chlorocebus aethiops , Citoesqueleto/metabolismo , DNA Complementar , Endotélio Vascular/citologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Cinesinas/genética , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Testes de Precipitina/métodos , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-Híbrido , Proteínas ras
12.
Blood ; 97(6): 1611-7, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11238099

RESUMO

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.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/farmacologia , Transfecção/métodos , Adenoviridae/genética , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Citocinas/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Humanos , Quinase I-kappa B , Inflamação/metabolismo , Mutação , NF-kappa B/antagonistas & inibidores , NF-kappa B/farmacologia , NF-kappa B/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/patologia , Veias Umbilicais/fisiopatologia
13.
J Invest Dermatol ; 117(6): 1363-8, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11886496

RESUMO

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.


Assuntos
Fármacos Dermatológicos/farmacologia , Selectina E/genética , Fumaratos/farmacologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Capilares/química , Capilares/efeitos dos fármacos , Capilares/fisiologia , Células Cultivadas , Fumarato de Dimetilo , Selectina E/análise , Endotélio Vascular/química , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/genética , Psoríase/tratamento farmacológico , Psoríase/fisiopatologia , RNA Mensageiro/análise , Pele/irrigação sanguínea , Veias Umbilicais/citologia
14.
Arterioscler Thromb Vasc Biol ; 20(11): E83-8, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11073859

RESUMO

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.


Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , NF-kappa B/fisiologia , Animais , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Regulação da Expressão Gênica , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , NF-kappa B/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética
16.
J Biol Chem ; 275(29): 22064-8, 2000 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-10807933

RESUMO

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.


Assuntos
Apoptose , Endotélio Vascular/metabolismo , MAP Quinase Quinase Quinases , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Linhagem Celular , Endotélio Vascular/patologia , Humanos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X
17.
J Biol Chem ; 275(22): 17035-42, 2000 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-10747893

RESUMO

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.


Assuntos
Proteínas I-kappa B/metabolismo , Proteínas Luminescentes/metabolismo , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Cinética , Microscopia de Fluorescência , Ligação Proteica , Espectrometria de Fluorescência , Fator de Transcrição RelA
18.
Gene Ther ; 7(4): 306-13, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10694811

RESUMO

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.


Assuntos
Fibrose Cística/terapia , Terapia Genética/métodos , NF-kappa B/genética , Adenoviridae/genética , Células Cultivadas , Fibrose Cística/metabolismo , Eletroforese , Vetores Genéticos/genética , Humanos , Interleucina-8/metabolismo , Lipossomos/genética , Lipossomos/farmacologia , NF-kappa B/antagonistas & inibidores , Oligonucleotídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/genética
19.
Mutat Res ; 437(3): 231-43, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10592330

RESUMO

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.


Assuntos
Apoptose/genética , Transformação Celular Neoplásica/genética , NF-kappa B/genética , Animais , Divisão Celular , Regulação Neoplásica da Expressão Gênica/genética , Transdução de Sinais , Fatores de Transcrição
20.
Blood ; 93(11): 3685-93, 1999 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10339475

RESUMO

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.


Assuntos
Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Endotélio Vascular/patologia , Endotélio Vascular/fisiologia , Proteínas I-kappa B , Monócitos/patologia , Monócitos/fisiologia , NF-kappa B/genética , Adenoviridae , Adesão Celular/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Inibidor de NF-kappaB alfa , Estresse Mecânico , Fator de Necrose Tumoral alfa/farmacologia
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