Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements.
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.
Schäfer
Georgia
G
Medizinische Klinik mit Schwerpunkt Hepatologie, Gastroenterologie, Endokrinologie und Stoffwechsel, Charité, Campus Virchow-Klinikum, 13353 Berlin, Germany.
Cramer
Thorsten
T
Suske
Guntram
G
Kemmner
Wolfgang
W
Wiedenmann
Bertram
B
Höcker
Michael
M
D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016228:Endothelial Growth Factors / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A
D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; D012333:RNA, Messenger; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D051705:Sp3 Transcription Factor