Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression.
Endothelin-1 (ET-1) is a 21-amino-acid potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells. Expression of the ET-1 gene is subject to complex regulation by numerous factors, among which transforming growth factor-beta (TGF-beta) is one of the most important. It has been widely documented that TGF-beta increases ET-1 mRNA and peptide levels. We have explored the mechanism by which TGF-beta upregulates ET-1 expression in endothelial cells. Transcriptional activation of the ET-1 promoter accounted for the TGF-beta-induced increase in ET-1 mRNA levels. We have identified within the ET-1 promoter two DNA elements indispensable for TGF-beta-mediated induction of ET-1: an activator protein-1 (AP-1) site at -108/-102, known to be important for constitutive and induced expression, and a novel regulatory sequence located at -193/-171, which constitutes a specific binding site for Smad transcription factors. Mutation of both elements abolished TGF-beta responsiveness. Binding of Smad3/Smad4 and c-Jun to their corresponding DNA elements was evidenced by electrophoretic mobility shift assays. Furthermore, the coactivator CREB-binding protein (CBP)/p300 was found to play an essential role in the induction of the gene. The simultaneous requirement for two distinct and independent DNA elements suggests that Smads and activator protein-1 functionally cooperate through CBP/p300 to mediate TGF-beta-induced transcriptional activation of the ET-1 gene.
Rodríguez-Pascual
Fernando
F
Departamento de Estructura y Función de Proteínas, Centro de Investigaciones Biológicas, C.S.I.C., Instituto Reina Sofía de Investigaciones Nefrológicas, Velázquez 144, E-28006 Madrid, Spain. frodriguez@cib.csic.es
Redondo-Horcajo
Mariano
M
Lamas
Santiago
S
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D019332:Endothelin-1 / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D019332:Endothelin-1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D008156:Luciferases