HIF-2alpha specifically activates the VE-cadherin promoter independently of hypoxia and in synergy with Ets-1 through two essential ETS-binding sites.

The mechanisms that are responsible for the restricted pattern of expression of the VE-cadherin gene in endothelial cells are not clearly understood. Regulation of expression is under the control of an approximately 140 bp proximal promoter that provides basal, non-endothelial specific expression. A larger region contained within the 2.5 kb genomic DNA sequence located ahead of the transcription start is involved in the specific expression of the gene in endothelial cells. We show here that the VE-cadherin promoter contains several putative hypoxia response elements (HRE) which are able to bind endothelial nuclear factors under normoxia. The VE-cadherin gene is not responsive to hypoxia but hypoxia-inducible factor (HIF)-2alpha specifically activates the promoter while HIF-1alpha does not. The HRE, that are involved in this activity have been identified. Further, we show that HIF-2alpha cooperates with the Ets-1 transcription factor for activation of the VE-cadherin promoter and that this synergy is dependent on the binding of Ets-1 to DNA. This cooperative action of HIF-2alpha with Ets-1 most probably participates to the transcriptional regulation of expression of the gene in endothelial cells. This mechanism may also be involved in the expression of the VE-cadherin gene by tumor cells in the process of vascular mimicry.

The Ets family contains transcriptional activators and repressors involved in angiogenesis.

The Ets family contains a growing number of transcriptional activators and inhibitors, which activity is regulated by phosphorylation and protein-protein interactions. Among these factors, Ets1, Erg1 and Fli1 are expressed in endothelial cells during angiogenesis in normal and pathological development. The expression of these transcription factors is regulated by angiogenic factors in cultured endothelial cells, as well as by various stresses occurring during angiogenesis. Transfection experiments and transgenic mice analysis revealed that Ets family members are involved in the transcriptional regulation of endothelial specific genes such as those encoding Tie1 and -2, VEGFR1 and -2 and VE-Cadherin. In vitro studies plead for a role of Ets family members in endothelial cell adhesion, spreading and motility. Gene inactivation experiments show that Ets1 is dispensable for embryonic development. The phenotype of knocked-out embryos indicates that Tel is required for maintenance of the developing vascular network in the yolk sac. Altogether, we suggest that Ets family members act both positively and negatively during the different steps of the angiogenic process. The regulation of the initiation of gene transcription arises from the combined activity of different transcriptional regulators. Therefore very few transcription factors are specific for a physiological process, or a given cell type. The transcriptional network that regulates blood vessel formation involves transcription factors which are expressed in a variety of situations. The Lung Kruppel Like Factor (LKLF) which is required for blood vessel stabilisation during murine development is also expressed in the primitive vertebrae and in the lung of the adult (C.T. Kuo, M.L. Veselits, K.P. Barton, M.M. Lu, C. Clendenin, J.M. Leiden, The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilisation during murine embryogenesis, Genes Dev. 11 (22) (1997) 2996-3006). Scl/Tal1 which is essential for angiogenic remodelling of the yolk sac capillary network (J.E. Visvader, Y. Fujiwara, S.H. Orkin, Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development, Genes Dev. 12 (4) (1998) 473-479), is involved in blood cell development and is also expressed in the developing brain. The EPAS transcription factor which was thought to be endothelial cell specific in the mouse embryo (H. Tian, S.L. McKnight, D.W. Russell, Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells, Genes Dev. 11 (1) (1997) 72-82) is also expressed in the liver, kidney and cells of the sympathetic nervous system of the chick embryo (J. Favier, H. Kempf, P. Corvol, J.M. Gasc, Cloning and expression pattern of EPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase, FEBS Lett. 462 (1-2) (1999) 19-24). Ets1, which expression was originally detected in lymphoid cells of adult tissues, has been the first transcription factor to be identified in endothelial cells during angiogenesis in the embryo (B. Vandenbunder, L. Pardanaud, T. Jaffredo, M.A. Mirabel, D. Stehelin, Complementary patterns of expression of c-etsl, c-myb and c-myc in the blood-forming system of the chick embryo, Development 107 (1989) 265-274 [5]) and in tumours (N. Wernert, M.B. Raes, P. Lassalle, M.P. Dehouck, B. Gosselin, B. Vandenbunder, D. Stehelin, The c-ets 1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularisation and other forms of angiogenesis in man, Am. J. Path. 140 (1992) 119-127 [6]). Since then, the Ets family has extended and this review will emphasise the relationships between these factors and angiogenesis.

Expression and purification of recombinant mouse Ets-1 transcription factor.

Ets-1 is a transcription factor which belongs to the ETS family. Its mRNA is expressed in the embryo during normal development and also in tumors. In order to sort out functional Ets-1-binding sites among those present in gene promoters, we constructed an expression vector and designed a purification protocol for the production of the 440-amino-acid form of mouse Ets-1, based on heparin-Sepharose affinity and anion-exchange chromatographies. This protocol allows the purification of large amounts of pure recombinant protein as assessed by SDS-PAGE, C18 reverse-phase HPLC, amino-terminal sequencing, and mass spectrometry. The purified protein is recognized by specific anti-Ets-1 antibodies and binds to DNA ETS-binding sites.

[Role of the ETS transcription factors in the control of endothelial-specific gene expression and in angiogenesis]. / Rôle des facteurs de transcription ETS dans le contrôle de la spécificité d'expression endothéliale et l'angiogenèse.

The transcription factors of the ETS family are involved in the control of the endothelial-specific expression of genes that are important for the formation of new blood vessels. The analysis of the expression pattern of ets1, the gene inactivation of tel and fli1, the in vitro analysis of potential target genes of ETS factors in endothelial cells, the in vivo studies of the promoter regions of endothelial-specific genes all demonstrate a role for ETS factors in this specificity. However, the precise role of individual ETS factors in the endothelial identity and in angiogenesis in general remains difficult to understand in vivo.