Effect of ATF2 transcription factor on DLL4 gene expression in angiogenesis

Abstract

INTRODUCTION: ATF2 belongs to the AP1 transcription factor family that homodimerize or heterodimerize with other members of the bZIP family and regulates the transcriptional activation of target genes. Previous studies have shown that ATF2 mediates VEGF-induced angiogenic processes but the molecular mechanisms implicating ATF2 as a regulator of angiogenesis and its effect on other angiogenic related genes are largely unknown. METHODS: The sequences of the enhancers and the promoter of the DLL4, which is an angiogenic-related gene, were obtained from the ensembl website and using the ConTraV3 R software, the putative binding sites of ATF2 on the regulatory regions of DLL4 were identified. Among the four enhancers and the promoter regions identified, it was attempted to clone one enhancer sequence in a luciferase-based reporter plasmid. ATF2 functionality was suppressed by infecting HUVEC with an adenovirus expressing a phosphorylation-mutant, dominant-negative version of ATF2 (Ad-ATF2AA). HUVEC infection with an adenovirus encoding GFP (Ad-GFP) was used as a control. Alternatively, ATF2 expression in HUVEC was suppressed by siRNA-mediated knockdown. qPCR was performed to determine the effect of ATF2 functional suppression on the expression of DLL4-target genes and other genes related to angiogenesis. A colony of ATF2flox/flox mice was established by crossing ATF2flox/flox breeders with the intention of a future development of an endothelial-specific ATF2 knockout mice for future in vivo studies. RESULTS: In silico analysis revealed that ATF2 has potential binding sites on the regulatory regions of the DLL4 locus suggesting its involvement in the regulation of DLL4. HUVEC deficient in ATF2, achieved by overexpression of a mutant protein or knockdown of ATF2, showed a significant increase in the expression of the Notch ligand DLL4 in basal and VEGF-stimulated conditions. The gene expression of angiogenic related genes HEY1 and NRARP were also altered, suggesting ATF2 involvement in the regulation of these proteins. CONCLUSION: This study shows that activation of ATF2 is essential for the negative regulation of DLL4, HEY1 and NRARP. Interestingly, activation of these Notch-related genes has been reported to have an inhibitory effect on angiogenesis. These results indicate that the negative effect of ATF2 suppression observed in angiogenesis might implicate upregulation of DLL4, HEY1 and NRARP.