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

Abstract

Angiopoietins are ligands of the endothelial cell tyrosine kinase receptor Tie2. Angiopoietin-1 (Ang-1) 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-1 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-1alpha 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-1 and particularly GATA-2 as central factors in endothelial specific Ang-2 expression.

Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature

“…7,8 Multiple lines of evidence have implicated GATA2 in vascular development. GATA2 has been demonstrated to bind and regulate the promoter activity of endothelial genes, including PECAM1, 10 Kdr (encoding VEGFR-2), 11 ANGPT2, 12 and EMCN, 13 and GFP knocked into the Gata2 locus is expressed in endothelial cells of the heart, blood vessels, and lymphatic vessels during mouse embryogenesis. 14 A definitive role for GATA2 during vascular development has, however, remained enigmatic, as vascular defects are not obvious in Gata2 Ϫ/Ϫ mice before their death at approximately embryonic day 10 (E10), 1 and very few studies have documented GATA2 protein expression in the vasculature in vivo.…”

HIF1α and physiological responses to hypoxia are correlated in mice but not in rats

“…We also observed an increased expression of Angpt2 in the brainstem of mice exposed to 12% O 2 for 6 h. Angpt2 is a proangiogenic factor, whose expression in the brain is enhanced by HIF-1α in hypoxia (Simon et al, 2008), and it contributes to vascular remodeling (Benderro and LaManna, 2014).…”

Ets-1 Is Required for the Activation of VEGFR3 during Latent Kaposi's Sarcoma-Associated Herpesvirus Infection of Endothelial Cells

“…Ets-1 cooperatively interacts physically and functionally with a number of proteins and transcription factors, including AP-1, NF-B, HIF 2␣, and STAT5 (39)(40)(41)(42)(43). Known gene targets of Ets-1 include early growth response 1 (Egr1), Tie1 and -2, MMPs, p53, osteopontin, and VEGFR1 genes (44)(45)(46)(47)(48)(49)(50). Ets-1 is upregulated by another human gammaherpesvirus, Epstein-Barr virus (EBV), mediated by the viral latent membrane protein 1, resulting in the upregulation of c-Met and proliferation of virus-infected epithelial cells (51).…”