RESUMEN
The inflammatory response is a self-limiting process which involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Galectin-1 (Gal-1), an endogenous lectin found in peripheral lymphoid organs and inflammatory sites, elicits a broad spectrum of biological functions predominantly by acting as a potent anti-inflammatory factor and as a suppressive agent for T-cell responses. However, the molecular pathways underlying Gal-1 expression and function remain poorly understood. Here we identified a regulatory loop linking Gal-1 expression and function to NF-κB activation. NF-κB-activating stimuli increased Gal-1 expression on T cells, an effect which could be selectively prevented by inhibitors of NF-κB signaling. Accordingly, transient transfection of the p65 subunit of NF-κB was sufficient to induce high Gal-1 expression. Using in silico studies and chromatin immunoprecipitation analysis we have identified a functional NF-κB binding site within the first intron of the LGALS1 gene. In addition, our results show that exogenous Gal-1 can attenuate NF-κB activation, as shown by inhibition of IκB-α degradation induced by pro-inflammatory stimuli, higher cytoplasmic retention of p65, lower NF-κB DNA binding activity and impaired transcriptional activation of target genes. The present study suggest a novel regulatory loop by which NF-κB induces expression of Gal-1, which in turn may lead to negative control of NF-κB signaling.
Asunto(s)
Galectina 1/biosíntesis , Regulación de la Expresión Génica/inmunología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Sitios de Unión , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Retroalimentación Fisiológica/fisiología , Galectina 1/genética , Galectina 1/inmunología , Expresión Génica , Humanos , Microscopía Confocal , FN-kappa B/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismo , TransfecciónRESUMEN
We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50% NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.
Asunto(s)
FN-kappa B/metabolismo , Factores de Transcripción/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Activación Enzimática , Humanos , Coactivador 2 del Receptor Nuclear , Fosforilación , Activación Transcripcional , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
We have previously shown that nuclear receptor coactivator overexpression significantly enhanced NF-kappaB activity in a dose response manner. We studied the mechanism by which TIF2 regulates NF-kappaB activity. We determined that: 1) the p38 specific inhibitor reduces 50
NF-kappaB transcriptional activity, even in cells that overexpress distinct TIF2 deletions; 2) there is a physical interaction between TIF2 and p38 and RelA determined through in vitro translated protein binding assays; 3) TIF2 is a p38 substrate; 4) there is a physical interaction between TIF2 and IKK in TNF-alpha 20 ng/ml stimulated or not HEK 293 cell protein extract, and IkappaB only in basal conditions, determined by binding pull down assays. This NF-kappaB complex regulates its activity and targets gene expression in a determined physiologic context depending on the coactivator complex content.