RESUMEN
Overexpression of vascular endothelial growth factor (VEGF) is implicated in a number of diseases. It is therefore critical that mechanisms exist to strictly regulate VEGF expression. A hypoxia-responsive (HR) region of the VEGF promoter which binds the HIF-1 transcription factor is a target for many signals that up-regulate VEGF transcription. Repressors targeting the HIF-1 transcription factor have been identified but no repressors directly binding the HR promoter region had been reported. We now report a novel mechanism of repression of the VEGF HR region involving DNA binding. We find that single strand DNA-specific cold shock domain (CSD or Y-box) proteins repress the HR region via a binding site downstream of the HIF-1 site. The repressor site is functional in unstimulated, normoxic fibroblasts and represents a novel means to prevent expression of VEGF in the absence of appropriate stimuli. We characterized complexes forming on the VEGF repressor site and identified a previously unreported nuclear CSD protein complex containing dbpA. Nuclear dbpA appears to bind as a dimer and we determined a means by which nuclear CSD proteins may enter double strand DNA to bind to their single strand sites to bring about repression of the VEGF HR region.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento Endotelial/genética , Silenciador del Gen , Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Factores de Transcripción , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Hipoxia de la Célula , Núcleo Celular/metabolismo , ADN de Cadena Simple/metabolismo , ADN Superhelicoidal/metabolismo , Dimerización , Factores de Crecimiento Endotelial/biosíntesis , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Linfocinas/biosíntesis , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factores de Transcripción NFI , Elementos Silenciadores Transcripcionales , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The hypoxia responsive region (HRR) of the VEGF promoter plays a key role in regulating VEGF expression. We found that the cold shock domain (Y-box) repressor proteins, dbpA and dbpB/YB-1, bind distinct strands of the human VEGF HRR. We find both dbpA and dbpB are phosphorylated by ERK2 and GSK3beta in vitro, and the binding of dbpB to single-strand VEGF HRR DNA is regulated by this phosphorylation. These findings suggest the ERK/MAPK and PI3K pathways may regulate VEGF expression in part through regulating the action of these repressor proteins.