Cooperative interaction of hypoxia-inducible factor-2alpha (HIF-2alpha ) and Ets-1 in the transcriptional activation of vascular endothelial growth factor receptor-2 (Flk-1).
Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. Here we show that the endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, we show here that HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element conferred strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and was most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis.
Elvert
Gerd
G
Zentrum für Molekulare Medizin der Universität zu Köln, Joseph-Stelzmann-Strasse 9, 50931 Köln, Germany.
Kappel
Andreas
A
Heidenreich
Regina
R
Englmeier
Ursula
U
Lanz
Stephan
S
Acker
Till
T
Rauter
Manuel
M
Plate
Karl
K
Sieweke
Michael
M
Breier
Georg
G
Flamme
Ingo
I
D000367:Age Factors; D020816:Amino Acid Motifs; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D015153:Blotting, Western; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009389:Neovascularization, Pathologic; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics* / Q000378:metabolism*; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D005982:Glutathione Transferase; D008156:Luciferases; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099162:Endothelial PAS Domain-Containing Protein 1; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse