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1.
Cell Microbiol ; 13(12): 2007-21, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21899702

RESUMO

In human disease induced by Salmonella enterica serovar Typhimurium (S. Typhimurium), transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously shown that during S. Typhimurium infection the multidrug resistance-associated protein 2 (MRP2) is highly expressed at the apical surface of the intestinal epithelia, and that it functions as an efflux pump for the potent neutrophil chemoattractant hepoxilin A(3) . However, the molecular mechanisms regulating its apical localization during active states of inflammation remain unknown. Thus, our objective was to determine the mechanistic basis for the translocation of MRP2 to the apical surface of intestinal epithelial cells during S. Typhimurium infection. We show that suppression of ezrin, through either RNAi or truncation of the C-terminus, results not only in a decrease in S. Typhimurium-induced neutrophil transmigration but also significantly attenuates the apical membrane expression of MRP2 during Salmonella infection. In addition, we determined that S. Typhimurium induces the activation of ezrin via a PKC-α-dependent pathway and that ezrin activation is coupled to apical localization of MRP2. Based on these results we propose that activation of ezrin is required for the apical localization of MRP2 during S. Typhimurium infection.


Assuntos
Proteínas de Bactérias/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neutrófilos/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas de Bactérias/genética , Benzofenantridinas/farmacologia , Western Blotting , Carbazóis/farmacologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Proteínas dos Microfilamentos/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosforilação , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transfecção
2.
J Immunol ; 181(11): 8044-52, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017997

RESUMO

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A(3), an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A(3) is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A(3) is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A(3) synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A(3) synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.


Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Enteropatias/imunologia , Mucosa Intestinal/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Ácido 8,11,14-Eicosatrienoico/imunologia , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Araquidonato 12-Lipoxigenase/imunologia , Araquidonato 12-Lipoxigenase/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Enteropatias/metabolismo , Enteropatias/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Neutrófilos/metabolismo , Neutrófilos/patologia
3.
Infect Immun ; 76(8): 3614-27, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18505810

RESUMO

Salmonella spp. and Shigella spp. are responsible for millions of cases of enteric disease each year worldwide. While these pathogens have evolved distinct strategies for interacting with the human intestinal epithelium, they both induce significant proinflammatory responses that result in massive transepithelial migration of neutrophils across the intestinal mucosa. It has previously been shown with Salmonella enterica serotype Typhimurium that the process of neutrophil transmigration is mediated in part by the secretion of hepoxilin A(3) (HXA(3); 8-hydroxy-11,12-epoxy-eicosatetraenoic acid), a potent neutrophil chemoattractant, from the apical surface of infected model intestinal epithelium. This study confirms that HXA(3) is also secreted in response to infection by Shigella flexneri, that it is produced by a pathway involving 12/15-lipoxygenase (12/15-LOX), and that S. enterica serovar Typhimurium and S. flexneri share certain elements in the mechanism(s) that underlies the otherwise separate signal transduction pathways that are engaged to induce polymorphonuclear leukocyte (PMN) transepithelial migration (protein kinase C and extracellular signal-regulated kinases 1 and 2, respectively). PMN transepithelial migration in response to infection with S. flexneri was dependent on 12/15-LOX activity, the enzyme responsible for the initial metabolism of arachidonic acid to HXA(3). Probing further into this pathway, we also found that S. enterica serovar Typhimurium and S. flexneri activate different subtypes of phospholipase A(2), a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. Thus, although S. enterica serovar Typhimurium and S. flexneri utilize different mechanisms for triggering the induction of PMN transepithelial migration, we found that their reliance on 12/15-LOX is conserved, suggesting that enteric pathogens may ultimately stimulate similar pathways for the synthesis and release of HXA(3).


Assuntos
Movimento Celular/imunologia , Neutrófilos/imunologia , Fosfolipases A2/química , Fosfolipases A2/imunologia , Salmonella typhimurium/enzimologia , Salmonella typhimurium/imunologia , Shigella flexneri/enzimologia , Shigella flexneri/imunologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Linhagem Celular , Ensaios de Migração de Leucócitos , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Isoenzimas
4.
Am J Physiol Gastrointest Liver Physiol ; 294(6): G1392-400, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18403618

RESUMO

Studies over the last decade have shown that Salmonella enterica serovar Typhimurium (S. typhimurium) is able to preferentially locate to sites of tumor growth and modulate (shrink) the growth of many cancers. Given this unique association between S. typhimurium and cancer cells, the objective of this study was to investigate the capacity of this microorganism to modulate the plasma membrane multidrug resistance (MDR) protein P-glycoprotein (P-gp), an ATP-binding cassette transporter responsible for effluxing many cancer drugs. Using an in vitro model of S. typhimurium infection of polarized human cancer intestinal cell lines, we have found that this enteric pathogen functionally downregulates the efflux capabilities of P-gp. Specifically, we show that S. typhimurium infection of human intestinal cancer cells results in the enhanced intracellular accumulation of a number of P-gp substrates that corresponds to the posttranscriptional downregulation of P-gp expression. Furthermore, cells expressing small interfering RNAs against MDR1, the gene encoding P-gp, were significantly more susceptible to the cytotoxic effects of bacterial infection. This result is consistent with our observation that S. typhimurium was significantly less able to invade cells overexpressing MDR1. Taken together, these results reveal a novel role for P-gp in the maintenance of homeostasis in the gastrointestinal tract in regard to bacterial infection. Thus the regulation of P-gp by S. typhimurium has important implications not only for the development of new cancer therapeutics aimed at reversing drug resistance but also in the understanding of how microbes have evolved diverse strategies to interact with their host.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/microbiologia , Salmonella enterica/fisiologia , Linhagem Celular , Humanos
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