RESUMEN
We recently showed that Etk/Bmx, a member of the Tec family of nonreceptor protein tyrosine kinases, promotes tight junction formation during chronic hypoxic exposure and augments normoxic VEGF expression via a feedforward mechanism. Here we further characterized Etk's role in potentiating hypoxia-induced gene expression in salivary epithelial Pa-4 cells. Using transient transfection in conditionally activated Etk (DeltaEtk:ER) cells, we demonstrated that Etk enhances hypoxia-response element-dependent reporter activation in normoxia and hypoxia. This Etk-driven reporter activation is ameliorated by treatment with wortmannin or LFM-A13. Using lentivirus-mediated gene delivery and small interfering RNA, we provided direct evidence that hypoxia leads to transient Etk and Akt activation and hypoxia-mediated Akt activation is Etk dependent. Northern blot analyses confirmed that Etk activation led to induction of steady-state mRNA levels of endogenous VEGF and plasminogen activator inhibitor (PAI)-1, a hallmark of hypoxia-mediated gene regulation. We also demonstrated that Etk utilizes a phosphatidylinositol 3-kinase/Akt pathway to promote reporter activation driven by NF-kappaB, another oxygen-sensitive transcription factor, and to augment cytokine-induced inducible nitric oxide synthase expression in endothelial cells. To establish the clinical relevance of Etk-induced, hypoxia-mediated gene regulation, we examined Etk expression in keloid, which has elevated VEGF and PAI-1. We found that Etk is overexpressed in keloid (but not normal skin) tissues. The differential steady-state Etk protein levels were further confirmed in primary fibroblast cultures derived from these tissues, suggesting an Etk role in tissue fibrosis. Our results provide further understanding of Etk function within multiple signaling cascades to govern adaptive cytoprotection against extracellular stress in different cell systems, salivary epithelial cells, brain endothelial cells, and dermal fibroblasts.
Asunto(s)
Óxido Nítrico Sintasa/biosíntesis , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Northern Blotting , Western Blotting , Línea Celular , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Hipoxia/metabolismo , Queloide/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Tirosina Quinasas/efectos de los fármacos , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Factor A de Crecimiento Endotelial Vascular/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Little is known about the molecular mechanisms underlying the release of merozoites from malaria infected erythrocytes. In this study membranous structures present in the culture medium at the time of merozoite release have been characterized. Biochemical and ultrastructural evidence indicate that membranous structures consist of the infected erythrocytes membrane, the parasitophorous vacuolar membrane and the residual body containing electron dense material. These are subcellular compartments expected in a structure that arises as a consequence of merozoite release from the infected cell. Ultrastrutural studies show that a novel structure extends from the former parasite compartment to the membrane. Since these membrane modifications are detected only after merozoites have been released from the infected erythrocyte, it is proposed that they might play a role in the release of merozoites from the host cell.