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Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1-and Sp3-dependent activation of two proximal GC-rich promoter elements

Item Type:Article
Title:Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1-and Sp3-dependent activation of two proximal GC-rich promoter elements
Creators Name:Schaefer, G. and Cramer, T. and Suske, G. and Kemmner, W. and Wiedenmann, B. and Hoecker, M.
Abstract:Enhanced VEGF-A (vascular endothelial growth factor A) gene expression is associated with increased tumor growth and metastatic spread of solid malignancies including gastric cancer. Oxidative stress has been linked to tumor-associated neoangiogenesis; underlying mechanisms, however, remained poorly understood. Therefore, we studied the effect of oxidative stress on VEGF-A gene expression in gastric cancer cells. Oxidative stress generated by H(2)O(2) application potently stimulated VEGF-A protein and mRNA levels as determined by enzyme-linked immunosorbent assay and real-time PCR techniques, respectively, and elevated the activity of a transfected (-2018) VEGF-A promoter reporter gene construct in a time- and dose-dependent manner (4-8-fold). These effects were abolished by the antioxidant N-acetylcysteine, demonstrating specificity of oxidative stress responses. Functional 5' deletion analysis mapped the oxidative stress response element of the human VEGF-A promoter to the sequence -88/-50, and a single copy of this element was sufficient to confer basal promoter activity as well as oxidative stress responsiveness to a heterologous promoter system. Combination of EMSA studies, Sp1/Sp3 overexpression experiments in Drosophila SL-2 cells, and systematic promoter mutagenesis identified enhanced Sp1 and Sp3 binding to two GC-boxes at -73/-66 and -58/-52 as the core mechanism of oxidative stress-triggered VEGF-A transactivation. Additionally, in Gal4-Sp1/-Sp3-Gal4-luciferase assays, oxidative stress increased Sp1 but not Sp3 transactivating capacity, indicating additional mechanism(s) of VEGF-A gene regulation. Signaling studies identified a cascade comprising Ras --> Raf --> MEK1 --> ERK1/2 as the main pathway mediating oxidative stress-stimulated VEGF-A transcription. This study for the first time delineates the mechanisms underlying regulation of VEGF-A gene transcription by oxidative stress and thereby further elucidates potential pathways underlying redox control of neoangiogenesis.
Keywords:Cultured Tumor Cells, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Endothelial Growth Factors, Enzyme-Linked Immunosorbent Assay, Genetic Promoter Regions, Genetic Transcription, Messenger RNA, Oxidative Stress, Protein Binding, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors, Vascular Endothelial Growth Factor A
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:278
Number:10
Page Range:8190-8198
Date:7 March 2003
Official Publication:https://doi.org/10.1074/jbc.M211999200
PubMed:View item in PubMed

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