Introduction ATF2 also known as cyclic AMP response element binding protein 2 (CREB2) is a member of the leucine zipper (bZIP) transcription factor family that binds to specific DNA sequences and regulates the transcriptional activation of target genes. ATF2 regulation and functions have been studied in a number of developmental and pathological conditions. Studies have shown that activation of ATF2 by VEGF mediates angiogenic processes such as endothelial cell migration and tubular morphogenesis but the molecular role of ATF2 in these processes is largely unknown. To shed some light on this matter we are identifying ATF2-target genes induced by stimulation of endothelial cells with pro-angiogenic stimuli.
Methods HUVEC were stimulated with various pro-angiogenic stimuli at different times, and the activation (phosphorylation) status of ATF2 was determined by Western Blot.
ATF2 functionality in endothelial cells was suppressed by infecting HUVEC with an adenovirus encoding a phosphorylation-mutant, dominant-negative version of ATF2 (Ad-ATF2AA) where phosphorylation residues Thr69 and Thr71 have been mutated to Ala. HUVEC infection with an adenovirus encoding GFP was used as a control. PCR-based screening of specific gene arrays was used to identify the effect of ATF2 loss-of-function in the transcriptional expression of genes related to the NOTCH signalling pathway. The screening results were validated using TaqMan-based qPCR.
Results Treatment of HUVEC with the pro-angiogenic stimuli VEGF, bFGF, HGF, or HB-EGF induced in transient phosphorylation (activation) of ATF2 at Thr69 and Thr71. Suppression of ATF2 function by overexpression of ATF2AA in endothelial cells strongly attenuated VEGF- and bFGF-induced tubular morphogenesis. Loss of ATF2 function resulted in a significant increase in the VEGF- and bFGF-induced upregulation of Notch ligands DLL1 and DLL4 suggesting ATF2 involvement in the upregulation of these proteins.
Conclusion Our study demonstrates that growth factor-induced phosphorylation of ATF2 in residues 69 and 71 is essential for proper angiogenesis. Moreover, our results suggest that ATF2 plays a fundamental role in the regulation of Notch signalling-induced angiogenesis in endothelial cells.
Conflict of interest None
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