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

Georgia Schäfer,Thorsten Cramer,Guntram Suske,Wolfgang Kemmner,Bertram Wiedenmann,Michael Höcker

doi:10.1074/jbc.m211999200

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.

Highlights

Reactive oxygen species (ROS),1 such as superoxide O2., hydroxyl radical OH1⁄7, and H2O2 are continuously generated as products of cellular metabolism [1,2,3]
Oxidative Stress Stimulates Production and Release of VEGF-A Protein and Increases VEGF-A mRNA Levels in AGS Cells—To determine the effects of oxidative stress on VEGF-A gene expression, AGS cells were exposed to H2O2 and analyzed for VEGF-A protein and mRNA levels
We demonstrate oxidative stress-dependent stimulation of VEGF-A production and release in gastric cancer cells and provide clear evidence that oxidative stress regulates VEGF-A gene expression through transcriptional mechanisms

Summary

Introduction

Reactive oxygen species (ROS), such as superoxide O2., hydroxyl radical OH1⁄7, and H2O2 are continuously generated as products of cellular metabolism [1,2,3]. Increased ROS levels have been linked to peptic lesions of the gastric mucosa triggered by ethanol, nonsteroidal anti-inflammatory drugs, stress, ischemia-reperfusion, and Helicobacter pylori infection (10 –12). In addition to these deleterious effects, ROS are capable of influencing mucosal repair processes by stimulating epithelial proliferation, production, and release of mucosal growth factors as well as activation of proangiogenic pathways [13]. Despite the fact that these studies clearly established an important role of VEGF-A-dependent angiogenesis in mucosal regeneration, peptic ulcer healing, and gastric cancer, the pathways controlling VEGF-A gene expression in these settings have not yet been defined. Signaling pathways mediating the effects of oxidative stress on the VEGF-A gene await clarification

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