RESUMEN
OBJECTIVE: Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown. METHODS AND RESULTS: We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription-polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5' untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis. CONCLUSIONS: We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.
Asunto(s)
Factor Inductor de la Apoptosis/biosíntesis , Células Endoteliales/virología , Poliovirus/patogenicidad , Proteínas Proto-Oncogénicas c-jun/biosíntesis , ARN Mensajero/metabolismo , Ribosomas/virología , Regiones no Traducidas 5' , Factor Inductor de la Apoptosis/genética , Línea Celular , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Genes Reporteros , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-jun/genética , Caperuzas de ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/metabolismo , TransfecciónRESUMEN
Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs). NETs are lattices of extracellular DNA, chromatin, and antibacterial proteins that mediate extracellular killing of microorganisms and are thought to form via a unique death pathway signaled by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated reactive oxygen species (ROS). We found that neutrophils from term and preterm infants fail to form NETs when activated by inflammatory agonists-in contrast to leukocytes from healthy adults. The deficiency in NET formation is paralleled by a previously unrecognized deficit in extracellular bacterial killing. Generation of ROSs did not complement the defect in NET formation by neonatal neutrophils, as it did in adult cells with inactivated NADPH oxidase, demonstrating that ROSs are necessary but not sufficient signaling intermediaries and identifying a deficiency in linked or downstream pathways in neonatal leukocytes. Impaired NET formation may be a critical facet of a common developmental immunodeficiency that predisposes newborn infants to infection.
Asunto(s)
Actividad Bactericida de la Sangre , Recién Nacido/inmunología , Recien Nacido Prematuro/inmunología , Sustancias Macromoleculares/inmunología , Neutrófilos/patología , Adulto , Envejecimiento/inmunología , Cromatina/fisiología , ADN/fisiología , Susceptibilidad a Enfermedades , Espacio Extracelular , Sangre Fetal/citología , Sangre Fetal/inmunología , Humanos , Infecciones/inmunología , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Factor de Activación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Glicoproteínas de Membrana Plaquetaria/genética , ARN Mensajero/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Estallido Respiratorio , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/genéticaRESUMEN
Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-kappaB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1beta, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3'-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1beta treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.