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1.
Circ Res ; 102(8): 888-95, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18309103

RESUMO

Inflammatory activation of the endothelium by Chlamydophila pneumoniae infection has been implicated in the development of chronic vascular lesions and coronary heart disease by seroepidemiological and animal studies. We tested the hypothesis that C. pneumoniae induced inflammatory gene expression is regulated by Rho-GTPase-related histone modifications. C. pneumoniae infection induced the liberation of proinflammatory interleukin-6, interleukin-8, granulocyte colony-stimulating factor, macrophage inflammatory protein-1beta, granulocyte/macrophage colony-stimulating factor, and interferon-gamma by human endothelial cells. Cytokine secretion was reduced by simvastatin and the specific Rac1 inhibitor NSC23766 but was synergistically enhanced by inhibitors of histone deacetylases trichostatin A and suberoylanilide hydroxamic acid. Infection of endothelial cells with viable C. pneumoniae, but not exposure to heat-inactivated C. pneumoniae or infection with C. trachomatis, induced acetylation of histone H4 and phosphorylation and acetylation of histone H3. Pretreatment of C. pneumoniae-infected cells with simvastatin or NSC23766 reduced global histone modifications as well as specific modifications at the il8 gene promoter, as shown by chromatin immunoprecipitation. Reduced recruitment of nuclear factor kappaB p65/RelA as well as of RNA polymerase II was observed in statin-treated cells. Taken together, Rac1-mediated histone modifications seem to play an important role in C. pneumoniae-induced cytokine production by human endothelial cells.


Assuntos
Chlamydophila pneumoniae/fisiologia , Histonas/metabolismo , Sinvastatina/farmacologia , Células Cultivadas , Chlamydophila pneumoniae/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/metabolismo , Células Endoteliais/microbiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
J Virol ; 81(7): 3058-67, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17229704

RESUMO

Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3beta and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.


Assuntos
Apoptose , Vírus da Influenza A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Cães , Ativação Enzimática , Regulação Viral da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A Subtipo H7N7 , Vírus da Influenza A/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação/genética , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas não Estruturais Virais/genética
3.
Biochem Pharmacol ; 72(11): 1367-74, 2006 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-16884694

RESUMO

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. Small GTPases of the Rho family act as molecular switches in signal transduction. We tested the hypothesis that Rho GTPases contribute to the regulation of cytokine expression following L. monocytogenes infection. L. monocytogenes induced release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by endothelial cells and activated RhoA and Rac1. Inhibition of Rac1 by inhibitor Nsc23766 reduced cytokine expression, and slightly yet significantly the uptake of bacteria. Blocking of Rho proteins by Clostridium difficile toxin B-10463 (TcdB) reduced Listeria-dependent cytokine expression, whereas activating Rho proteins by Escherichia coli CNF1 increased it. We analyzed regulation of IL-8 expression in more detail: Listeria-induced IL-8 release was reduced by inhibition of RhoA, Rac1 and Cdc42 (TcdB) or Rac1 while blocking of RhoA/B/C by Clostridium limosum C3 fusion toxin (C3FT) or Rho kinase by Y27632 reduced cytokine expression only slightly. Activation of RhoA, Rac1 and Cdc42 (CNF1), but not of RhoA alone (CNF(Y)), enhanced Listeria-dependent IL-8 release significantly. Furthermore, inhibition of RhoA, Rac1 and Cdc42 (TcdB) and Rac1 (Nsc23766), but not of RhoA (C3FT) reduced Listeria-related recruitment of NF-kappaB/p65 and RNA polymerase II to the il8 promoter, as well as acetylation of histone H4 and Ser10/Lys14-phosphorylation/acetylation of histone H3 at the il8 gene promoter in HUVEC. In conclusion, Rac1 contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.


Assuntos
Endotélio Vascular/enzimologia , Listeria monocytogenes/patogenicidade , Transdução de Sinais , Veias Umbilicais/enzimologia , Proteínas rac1 de Ligação ao GTP/biossíntese , Aminoquinolinas/farmacologia , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Células Cultivadas , Citocinas/biossíntese , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/microbiologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/farmacologia , Humanos , Pirimidinas/farmacologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/microbiologia
4.
Am J Physiol Lung Cell Mol Physiol ; 290(5): L818-26, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16399788

RESUMO

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease (COPD) and may also contribute to the pathogenesis of COPD. Little is known about M. catarrhalis-bronchial epithelium interaction. We investigated activation of M. catarrhalis infected bronchial epithelial cells and characterized the signal transduction pathways. Moreover, we tested the hypothesis that the M. catarrhalis-induced cytokine expression is regulated by acetylation of histone residues and controlled by histone deacetylase activity (HDAC). We demonstrated that M. catarrhalis induced a strong time- and dose-dependent inflammatory response in the bronchial epithelial cell line (BEAS-2B), characterized by the release of IL-8 and GM-CSF. For this cytokine liberation activation of the ERK and p38 mitogen-activated protein (MAP) kinases and transcription factor NF-kappaB was required. Furthermore, M. catarrhalis-infected bronchial epithelial cells showed an enhanced acetylation of histone H3 and H4 globally and at the promoter of the il8 gene. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A augmented the M. catarrhalis-induced IL-8 response. After exposure to M. catarrhalis, we found a decrease in global histone deacetylase expression and activity. Our findings suggest that M. catarrhalis-induced activation of il8 gene transcription was caused by interference with epigenetic mechanisms regulating il8 gene accessibility. Our findings provide insight into important molecular and cellular mechanisms of M. catarrhalis-induced activation of human bronchial epithelium.


Assuntos
Brônquios/microbiologia , Brônquios/fisiopatologia , Histona Desacetilases/metabolismo , Inflamação/microbiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Moraxella catarrhalis/fisiologia , NF-kappa B/metabolismo , Mucosa Respiratória/fisiopatologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Inibidores de Histona Desacetilases , Humanos , Interleucina-8/fisiologia
5.
Am J Physiol Lung Cell Mol Physiol ; 290(6): L1131-8, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16414978

RESUMO

Streptococcus pneumoniae is a major cause of community-acquired pneumonia and death from infectious diseases in industrialized countries. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX)-derived prostaglandins, such as PGE(2), are considered to be important regulators of lung function. Herein, we tested the hypothesis that pneumococci induced COX-2-dependent PGE(2) production in pulmonary epithelial cells. Pneumococci-infected human pulmonary epithelial BEAS-2B cells released PGE(2). Expression of COX-2 but not COX-1 was dose and time dependently increased in S. pneumoniae-infected BEAS-2B cells as well as in lungs of mice with pneumococcal pneumonia. S. pneumoniae induced degradation of IkappaBalpha and DNA binding of NF-kappaB. A specific peptide inhibitor of the IkappaBalpha kinase complex blocked pneumococci-induced PGE(2) release and COX-2 expression. In addition, we noted activation of p38 MAPK and JNK in pneumococci-infected BEAS-2B cells. PGE(2) release and COX-2 expression were reduced by p38 MAPK inhibitor SB-202190 but not by JNK inhibitor SP-600125. We analyzed interaction of kinase pathways and NF-kappaB activation: dominant-negative mutants of p38 MAPK isoforms alpha, beta(2), gamma, and delta blocked S. pneumoniae-induced NF-kappaB activation. In addition, recruitment of NF-kappaB subunit p65/RelA and RNA polymerase II to the cox2 promoter depended on p38 MAPK but not on JNK activity. In summary, p38 MAPK- and NF-kappaB-controlled COX-2 expression and subsequent PGE(2) release by lung epithelial cells may contribute significantly to the host response in pneumococcal pneumonia.


Assuntos
Ciclo-Oxigenase 2/genética , Pulmão/citologia , NF-kappa B/metabolismo , Mucosa Respiratória/enzimologia , Streptococcus pneumoniae/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Sequência de Bases , Linhagem Celular , Primers do DNA , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Plasmídeos , Infecções Pneumocócicas/fisiopatologia , Piridinas/farmacologia , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genética
6.
J Immunol ; 176(1): 484-90, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16365441

RESUMO

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-kappaB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-kappaB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Interleucina-8/metabolismo , Listeria monocytogenes/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Cultivadas , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Interleucina-8/imunologia , Microscopia Confocal , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1 , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
7.
J Immunol ; 175(5): 2843-50, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16116170

RESUMO

Epigenetic histone modifications contribute to the regulation of eukaryotic gene transcription. The role of epigenetic regulation in immunity to intracellular pathogens is poorly understood. We tested the hypothesis that epigenetic histone modifications influence cytokine expression by intracellular bacteria. Intracellular Listeria monocytogenes, but not noninvasive Listeria innocua, induced release of distinct CC and CXC chemokines, as well as Th1 and Th2 cytokines and growth factors by endothelial cells. Cytokine expression was in part dependent on p38 MAPK and MEK1. We analyzed global histone modification and modifications in detail at the gene promoter of IL-8, which depended on both kinase pathways, and of IFN-gamma, which was not blocked by kinase inhibition. Intracellular Listeria induced time-dependent acetylation (lysine 8) of histone H4 and phosphorylation/acetylation (serine 10/lysine 14) of histone H3 globally and at the il8 promoter in HUVEC, as well as recruitment of the histone acetylase CREB-binding protein. Inhibitors of p38 MAPK and MEK1 reduced lysine 8 acetylation of histone H4 and serine 10/lysine 14 phosphorylation/acetylation of histone H3 in Listeria-infected endothelial cells and disappearance of histone deacetylase 1 at the il8 promoter in HUVEC. In contrast, IFN-gamma gene transcription was activated by Listeria monocytogenes independent of p38 MAPK and MEK1, and histone phosphorylation/acetylation remained unchanged in infected cells at the IFN-gamma promoter. Specific inhibition of histone deacetylases by trichostatin A increased Listeria-induced expression of IL-8, but not of IFN-gamma, underlining the specific physiological impact of histone acetylation. In conclusion, MAPK-dependent epigenetic modifications differentially contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.


Assuntos
Citocinas/biossíntese , Células Endoteliais/imunologia , Epigênese Genética , Histonas/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Acetilação , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Interferon gama/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Sistema de Sinalização das MAP Quinases , Regiões Promotoras Genéticas , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
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