The Angiopoietin-2 Gene of Endothelial Cells is Up-Regulated in Hypoxia by a HIF Binding Site Located in Its First Intron and by the Central Factors GATA-2 and Ets-1

Angiopoietins are ligands of the endothelial cell tyrosine kinase receptor Tie2. Angiopoietin-I (Ang-I) is widely expressed in human normal adult tissues and promotes blood vessel maturation and stabilization by inducing Tie2 receptor phosphorylation. In contrast, the antagonistic ligand Angiopoietin-2 (Ang-2) is up-regulated by hypoxia, expressed only at sites of vascular remodeling and plays a crucial role in destabilizing vessels for normal or pathological angiogenesis. Ang-2 expression is tightly regulated at transcriptional and post-transcriptional levels. To characterize the regulatory sequences of the human Ang-2 gene we cloned a fragment of around 8.5 kb upstream of the Ang-2 coding sequence and analyzed the luciferase reporter activity of constructs of various lengths in endothelial and non-endothelial cells. We isolated a minimal promoter sequence sufficient to promote significant Ang-2 non-cell type specific transcription. Moreover, we identified sequences conferring endothelial specificity. Indeed, sequence analysis of the fragment revealed the presence of several potential binding sites for specific endothelial regulatory factors like GATA or Ets. Using GATA-2 and Ets-I co-transfection and overexpression assay, we showed that these two factors are able to induce Ang-2 promoter activation. We also show that hypoxic regulation of Ang-2 is HIF-dependent and demonstrate that HIF-I alpha binds in human microvascular endothelial cells (HMVEC) to an evolutionary conserved Hypoxia-Responsive Element (HRE) located in the first intron of the Ang-2 gene. In conclusion, our study provides new elements in favor of HIF involvement in Ang-2 hypoxic regulation and identifies Ets-I and particularly GATA-2 as central factors in endothelial specific Ang-2 expression.