Oxidative Stress Regulates Vascular Endothelial Growth Factor-A Gene Transcription through Sp1- and Sp3-dependent Activation of Two Proximal GC-rich Promoter Elements

• Published in: The Journal of biological chemistry 2003-03, Vol.278 (10), p.8190-8198
• Main Authors: Schaefer, G, Cramer, T, Suske, G, Kemmner, W, Wiedenmann, B, Hoecker, M
• Format: Article
• Language: English
• Subjects: DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Endothelial Growth Factors - genetics; Enzyme-Linked Immunosorbent Assay; Humans; Oxidative Stress; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Sp1 Transcription Factor - metabolism; Sp3 Transcription Factor; Transcription Factors - metabolism; Transcription, Genetic; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M211999200

Enhanced VEGF-A ( v ascular e ndothelial g rowth f actor 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.