RESUMEN
Commercially available and widely used cat expression vectors were found to contain a forskolin (Fs)-inducible element capable of co-operation with NF-kappa B-sites in test promoters. An alternative NF-kappa B-dependent reporter system is presented that allows investigation of the effects of Fs and other agents that augment intracellular cyclic AMP.
Asunto(s)
Moléculas de Adhesión Celular/genética , Cloranfenicol O-Acetiltransferasa/biosíntesis , AMP Cíclico/metabolismo , Endotelio/metabolismo , Elementos de Facilitación Genéticos , Vectores Genéticos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Sitios de Unión , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Selectina E , Inducción Enzimática , Humanos , Venas UmbilicalesRESUMEN
Cytokines induce the expression of E-selectin, VCAM-1, and ICAM-1 on human umbilical vein endothelial cells (HUVECs). We show that expression of these surface proteins is differently affected by cAMP. Increased cAMP levels decrease E-selectin and VCAM-1 but increase ICAM-1 expression. We demonstrate by mRNA half-life analysis and nuclear run-on assays that the cAMP repression of E-selectin occurs at the transcription level. This effect is abolished by protein kinase A inhibition, suggesting that repression is mediated by protein kinase A-driven phosphorylation. We found that a minimal E-selectin promoter sequence necessary to confer cytokine inducibility is also sufficient to mimic the cAMP effect in transfected HUVECs. Previously we characterized two regions (NF-kappa B and NF-ELAM1) of the minimal promoter that bind transcription factors necessary for E-selectin induction, Increased cAMP did not alter the binding of the complexes formed on either the NF-kappa B or NF-ELAM1 site. In contrast, in interleukin-1-treated HUVECs transactivity due to an NF-kappa B site is reduced by elevated cAMP. Increased cAMP in HUVECs appears to induce a protein kinase activity that reduces the cytokine signal for E-selectin and VCAM-1 expression. The reduction in signal may occur through an inhibitory phosphorylation of one or more of the factors responsible for regulating E-selectin expression.
Asunto(s)
Moléculas de Adhesión Celular/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transcripción Genética , Secuencia de Bases , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , ADN/metabolismo , Selectina E , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos , Fosforilación , Regiones Promotoras Genéticas , Acetato de Tetradecanoilforbol/farmacología , Molécula 1 de Adhesión Celular VascularRESUMEN
A regulatable binary expression system for eukaryotes was recently developed based on the tetracycline repressor and its operator. Here we show that this system can be successfully applied to express antisense RNA and completely inhibit gene expression in a tetracycline-repressible fashion.
Asunto(s)
Expresión Génica/genética , Proteínas Represoras/metabolismo , ADN Complementario , Células HeLa , Humanos , ARN sin Sentido/genética , Receptores de Interleucina/genética , Receptores de Interleucina-8A , Tetraciclina , Factores de Transcripción/metabolismoRESUMEN
We have isolated a cDNA encoding a variant of the transcription factor ATF-a (called ATF-a0) by screening a HeLa cDNA expression library with a regulatory element of the E-selectin promoter, NF-ELAM1/delta A. Relative to full-length ATF-a, the ATF-a0 cDNA contains a large in-frame deletion of 525 base pairs that removes the P/S/T-rich putative transactivation domain. Using reverse-transcription-polymerase chain reaction and Northern blot hybridization to characterize ATF-a0 expression, we found that putative mRNAs for ATF-a0 and ATF-a are present at varying ratios in different tissues. Full-length ATF-a is a transcriptional activator for the NF-ELAM1/delta A site of the E-selectin promoter. In contrast, we show ATF-a0 has no measurable transactivating function on this element. Moreover, we demonstrate that co-expressed ATP-a0 and ATF-a preferentially heterodimerize. In the heterodimer ATF-a0 is a dominant inhibitor that completely blocks the transactivating activity of ATF-a. Both forms of ATF-a bind the p50 subunit of NF-kappa B as shown by affinity chromatography. ATF-a0 appears to be a splice variant similar to the one found for ATF-2, its closest homologue in structure and function. Taken together, our results suggest that ATF-a0 is an important member of the ATF family with a negative regulatory role in transactivation.
Asunto(s)
Proteínas Sanguíneas/genética , Regulación de la Expresión Génica , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción Activadores , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Genes Dominantes , Células HeLa , Humanos , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/química , Unión Proteica , ARN Mensajero/genética , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.
Asunto(s)
Moléculas de Adhesión Celular/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Secuencia de Bases , ADN , Selectina E , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , FN-kappa B/química , Unión Proteica , Células Tumorales CultivadasRESUMEN
E-selectin is an adhesion molecule transiently and specifically expressed on endothelial cells upon stimulation with cytokines. We wished to determine whether methylation could play a role in cell-type specific expression of this gene. We found that the E-selectin promoter in cultured endothelial cells is under-methylated in comparison with non-expressing HeLa cells. Plasmid constructs carrying a reporter driven by the E-selectin promoter and methylated in vitro are no longer transcribed in either an in vitro transcription system or in transiently transfected cells. We identified the NF-kappa B site in the promoter as the likely target for this methylation-mediated repression by testing a minimal promoter carrying only this and an associated element. We conclude that methylation is likely to play a role in blocking E-selectin expression in non-endothelial cells.
Asunto(s)
Moléculas de Adhesión Celular/genética , ADN/metabolismo , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/genética , FN-kappa B/antagonistas & inhibidores , Regiones Promotoras Genéticas , Secuencia de Bases , Células Cultivadas , Cromatografía de Afinidad , ADN/aislamiento & purificación , Selectina E , Células HeLa , Humanos , Metilación , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa , Transcripción Genética , Activación Transcripcional , Transfección , Venas UmbilicalesRESUMEN
ELAM1 (endothelial leukocyte adhesion molecule 1, also known as E-selectin) is a highly tissue-specific adhesion molecule that is transiently and exclusively expressed on cytokine-induced endothelial cells. We have identified two proximal ELAM1 promoter elements and their DNA-binding factors that are, in addition to NF-kappa B, essential for ELAM1 transcription. Mutation of either element in promoter constructs carrying the first 383 nucleotides of the ELAM1 promoter markedly diminshed the expression of a fused chloramphenicol acetyltransferase reporter gene. Although multimers of either element failed to display enhancer activity on its own, fusion of the most upstream of these to the NF-kappa B element had a strong stimulatory effect. This site, ACATCAT, is recognized by a factor we have called NF-ELAM1. The site corresponds to NF-ELAM1's preferential binding sequence (A/T)CA(G/T)CA(G/T) as determined in a target definition assay. This element is identical to the T-cell delta A enhancer found in the T-cell receptor-alpha, -beta, and CD3 delta genes. Our results suggest that the delta A/NF-ELAM1 element can function as a modulator of NF-kappa B in endothelial cells both as well as a T-cell enhancer.
Asunto(s)
Moléculas de Adhesión Celular/genética , Citocinas/farmacología , Endotelio Vascular/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , FN-kappa B/fisiología , Secuencia de Bases , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Selectina E , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Regiones Promotoras Genéticas , ARN Mensajero/genética , Linfocitos T/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
The aim of the present work was to study the effect of hypothyroidism on the expression of the beta-adrenergic receptor (beta-AR) in interscapular brown adipose tissue and heart. The total density of plasma membrane beta-AR per tissue is decreased by 44% in hypothyroid rat interscapular brown adipose tissue and by 55% in hypothyroid rat heart compared with euthyroid controls. The effects of hypothyroidism on the density of both beta 1- and beta 2-AR subtypes were also determined in competition displacement experiments. The densities of beta 1- and beta 2-AR per tissue are decreased by 50% and 48% respectively in interscapular brown adipose tissue and by 52% and 54% in the heart. Northern blot analysis of poly(A)+ RNA from hypothyroid rat interscapular brown adipose tissue demonstrated that the levels of beta 1- and beta 2-AR mRNA per tissue are decreased by 73% and 58% respectively, whereas in hypothyroid heart, only the beta 1-AR mRNA is decreased, by 43%. The effect of hypothyroidism on the beta 1-AR mRNA is significantly more marked in the interscapular brown adipose tissue than in the heart. These results indicate that beta-AR mRNA levels are differentially regulated in rat interscapular brown adipose tissue and heart, and suggest that the decrease in beta-AR number in interscapular brown adipose tissue and heart of hypothyroid animals may in part be explained by a decreased steady-state level of beta-AR mRNA.