RESUMEN
The epidermal growth factor-like protein DLK2, highly homologous to DLK1, has been identified as a modulator of adipogenesis in vitro. Knocking down Dlk2 expression prevents adipogenesis of 3T3-L1 cells but enhances that of the mesenchymal cell line C3H10T1/2. The expression of Dlk2 shows two peaks along this differentiation process: the first one, in response to 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (Dex), and the second, shortly after exposure to insulin. Nothing is known about the transcriptional regulation of Dlk2 during adipogenesis. Here, we report that, during early adipogenesis of 3T3-L1 cells, Dlk2 expression is controlled independently by IBMX and Dex. We also show that KLF4, a transcription factor critical for the control of early adipogenesis, binds directly to the Dlk2 promoter and increases Dlk2 expression in response to IBMX. Overexpression of KLF4 leads to an increase in DLK2 expression levels, whereas KLF4 knockdown downregulates the transcriptional activity of the Dlk2 promoter. Finally, we demonstrate that KLF4 regulates the basal expression of Dlk2 in C3H10T1/2 cells, and it is required for the adipogenic differentiation of those cells. These results indicate that KLF4 mediates the transcriptional regulation of Dlk2 in response to IBMX during the early stages of adipogenesis.
Asunto(s)
Adipogénesis/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Factores de Transcripción de Tipo Kruppel/genética , Transcripción Genética , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3-L1 , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Inhibidores de Fosfodiesterasa/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: DLK2 is an EGF-like membrane protein, closely related to DLK1, which is involved in adipogenesis. Both proteins interact with the NOTCH1 receptor and are able to modulate its activation. The expression of the gene Dlk2 is coordinated with that of Dlk1 in several tissues and cell lines. Unlike Dlk1, the mouse Dlk2 gene and its locus at chromosome 17 are not fully characterized. RESULTS: The goal of this work was the characterization of Dlk2 mRNA, as well as the analysis of the mechanisms that control its basal transcription. First, we analyzed the Dlk2 transcripts expressed by several mouse cells lines and tissues, and mapped the transcription start site by 5' Rapid Amplification of cDNA Ends. In silico analysis revealed that Dlk2 possesses a TATA-less promoter containing minimal promoter elements associated with a CpG island, and sequences for Inr and DPE elements. Besides, it possesses six GC-boxes, considered as consensus sites for the transcription factor Sp1. Indeed, we report that Sp1 directly binds to the Dlk2 promoter, activates its transcription, and regulates its level of expression. CONCLUSIONS: Our results provide the first characterization of Dlk2 transcripts, map the location of the Dlk2 core promoter, and show the role of Sp1 as a key regulator of Dlk2 transcription, providing new insights into the molecular mechanisms that contribute to the expression of the Dlk2 gene.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/genética , Elementos de Respuesta , Factor de Transcripción Sp1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Islas de CpG , Regulación de la Expresión Génica , Orden Génico , Silenciador del Gen , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Motivos de Nucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/química , ARN Interferente Pequeño , Factor de Transcripción Sp1/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética , Activación TranscripcionalRESUMEN
The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.
Asunto(s)
Fibroblastos/virología , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Muromegalovirus/metabolismo , Proteínas Virales/metabolismo , Animales , Genoma Viral , Infecciones por Herpesviridae/virología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Mutación , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Tiempo , Proteínas Virales/genéticaRESUMEN
Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-kappaB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-alpha and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-kappaB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-kappaB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IkappaB kinase alpha/beta complex and the expression of some NF-kappaB family members. Treatment of macrophages with the gamma-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-kappaB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-alpha and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-kappaB signaling.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , FN-kappa B/inmunología , Receptor Notch1/inmunología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Masculino , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , FN-kappa B/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
BACKGROUND: The Dlk1 gene encodes for dlk1, a transmembrane protein belonging to the EGF-like repeat-containing family. Dlk1 has been shown to act as a regulator of adipogenesis. Fc-dlk1 transgenic mice show a decrease in adipose tissue and glucose tolerance, hypertriglyceridaemia and lower insulin sensitivity. Dlk1-deficient mice show growth retardation, increased serum lipid metabolites and develop obesity. These data advocate for a role of dlk1 in the maintenance of lipid homeostasis, and suggest that dlk1 levels may influence the development of cardiovascular disease. AIM AND METHODS: In this study, we analysed whether dlk1 serum levels could be indicative of the different hormonal or metabolic status shown by two Spanish children populations (6-8 years-old), Orense and Murcia. We determined dlk1 serum levels by ELISA assay, using an antibody raised against the recombinant protein, and performed a correlation analysis against measurements of several hormonal and biochemical parameters in samples from 494 subjects. RESULTS: We found a statistically significant positive correlation between serum levels of dlk1 and those of glucose (P < 0.05), total cholesterol (P < 0.01) and high-density lipoprotein-cholesterol (HDL-C) (P < 0.01) in children from Murcia, but not from Orense's population, where dehydroepiandrosterone-sulphate (DHEA-S) levels were significantly higher (P < 0.01) and dlk1 correlated positively with insulin (P < 0.01), homeostasis model assessment (HOMA) (P < 0.01) and free fatty acids (FFA) (P < 0.05). CONCLUSIONS: dlk1 serum levels appear related to the anabolic status of the children in association with changes in the levels of DHEA-S, which have been associated with hyperinsulinaemia and diabetes. Monitoring dlk1 levels may be important to evaluate the metabolic and hormonal stage of child development.
Asunto(s)
Metabolismo de los Hidratos de Carbono/fisiología , Desarrollo Infantil/fisiología , Hormonas/sangre , Péptidos y Proteínas de Señalización Intercelular/sangre , Metabolismo de los Lípidos/fisiología , Proteínas de la Membrana/sangre , Células 3T3-L1 , Animales , Células 3T3 BALB , Biomarcadores/sangre , Biomarcadores/metabolismo , Proteínas de Unión al Calcio , Niño , Indicadores de Salud , Hormonas/análisis , Hormonas/metabolismo , Humanos , Ratones , Pichia , EspañaRESUMEN
Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-gamma, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-gamma, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.
Asunto(s)
Presentación de Antígeno/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular , ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Inflamación/inmunología , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/biosíntesis , Proteína Jagged-1 , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , Receptor Notch1/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteínas Serrate-Jagged , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The murine gammaherpesvirus-68 genome encodes 73 protein-coding open reading frames with extensive similarities to human gamma(2) herpesviruses, as well as unique genes and cellular homologues. We performed transcriptome analysis of stage-specific viral RNA during permissive infection using an oligonucleotide-based microarray. Using this approach, M4, K3, ORF38, ORF50, ORF57 and ORF73 were designated as immediate-early genes based on cycloheximide treatment. The microarray analysis also identified 10 transcripts with early expression kinetics, 32 transcripts with early-late expression kinetics and 29 transcripts with late expression kinetics. The latter group consisted mainly of structural proteins, and showed high expression levels relative to other viral transcripts. Moreover, we detected all eight tRNA-like transcripts in the presence of cycloheximide and phosphonoacetic acid. Lytic infection with MHV-68 also resulted in a significant reduction in the expression of cellular transcripts included in the DNA chip. This global approach to viral transcript analysis offers a powerful system for examining molecular transitions between lytic and latent virus infections associated with disease pathogenesis.