RESUMEN
RATIONALE: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases. OBJECTIVES: To define changes in gene expression profiles during in vivo rhinovirus infections. METHODS: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections. MEASUREMENTS AND MAIN RESULTS: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells. CONCLUSIONS: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/genética , Infecciones por Picornaviridae/genética , Rhinovirus/genética , Adolescente , Técnicas de Cultivo de Célula , Quimiocinas/genética , Femenino , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Masculino , Mucosa Nasal/virología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba/genética , Adulto JovenRESUMEN
This study tested the hypothesis that nitric oxide (NO) inhibits the rate-limiting catalytic step in steroidogenesis, cytochrome P450 cholesterol side-chain cleaving enzyme (CYP11A1), independent of soluble guanylyl cyclase (GC-S) stimulation. To assess CYP11A1 activity, pregnenolone levels were quantified in murine adrenocortical Y1 cells in the presence of the 3beta-hydroxy-Delta(5)-steroid dehydrogenase inhibitor, 2alpha-cyano-17beta-hydroxy-4,4',17alpha-trimethylandrost-5-ene-3-one. The NO donor, (Z)-1-[2-(2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate(deta nonoate), inhibited vasoactive intestinal peptide-, forskolin- and 22alpha-hydroxycholesterol (22HC)-facilitated pregnenolonogenesis in the absence of GC-S activation and in the presence of a GC-S inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). CYP11A1 was also heterologously expressed in monkey COS7 cells. Deta nonoate inhibited 22HC-facilitated activity of the over-expressed enzyme in the absence of GC-S activation and in the presence of ODQ. The NO-independent, GC-S agonist, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole did not inhibit steroidogenesis. The IC(50) for effects of free NO on CYP11A1 was potent and in the 0.4-2 microM range. These results support the hypothesis that NO inhibits the rate-limiting enzyme in steroidogenesis independent of GC-S activation.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/antagonistas & inhibidores , Óxido Nítrico/fisiología , Compuestos Nitrosos , Esteroides/biosíntesis , Animales , Células COS , Catálisis/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , GMP Cíclico/análisis , Guanilato Ciclasa/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Humanos , Hidrazinas/farmacología , Ratones , Proteínas Mitocondriales , Donantes de Óxido Nítrico/farmacología , Células PC12 , Pregnenolona/biosíntesis , Ratas , TransfecciónRESUMEN
The cannabinoid CB(1) but not the CB(2) receptor was demonstrated to couple via G(alpha16) to activate phospholipase C after co-expression in COS7 cells. Chimeric CB(1)/CB(2) receptors were used as a model to study receptor-G(alpha16) interaction. Sequences of the second and third intracellular loops and the carboxy-terminus were substituted from the CB(1) into the CB(2) receptor. Only the triple mutant with all three regions replaced activated phospholipase C to a similar extent as the CB(1) receptor, suggesting that all three intracellular regions are required for interacting with G(alpha16). Several sub-domains within the third intracellular loop were identified for receptor-G(alpha16) interaction.
