Activation of C-transactivation domain is essential for optimal HIF-1α-mediated transcriptional and angiogenic effects

Reshef Tal, Aviv Shaish, Livnat Bangio, Michael Peled, Eyal Breitbart, Dror Harats*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

HIF-1 is a transcription factor that regulates genes involved in oxygen homeostasis. In normoxia, degradation of the HIF-1α subunit is enabled by two prolyl hydroxylations at residues P402 and P564, while inactivation occurs through asparaginyl hydroxylation at residue N803 within its C-transactivation domain (C-TAD). For therapeutic angiogenesis purposes, HIF-1α stabilization was previously achieved by either deleting its oxygen-dependent degradation domains, or introducing two proline point mutations at residues P402 and P564. We assessed the hypothesis that constitutive activation of HIF-1α in addition to its stabilization would result in greater HIF-1α transcriptional activity and angiogenic effects than mere stabilization of the molecule. For this, we constructed a Triple mutant HIF-1α (TM), bearing mutations P402A and P564G N803A. Transient co-transfections with hypoxia-responsive element-luciferase construct revealed 2 to 2.5-fold increase in transcriptional activity of TM compared with P402A P564G double mutant and wild-type HIF-1α. In-vitro angiogenesis assay using transfected human umbilical vein endothelial cells (HUVEC) showed that TM stimulated tube formation to a greater extent than both P402A P564G mutant and wild-type HIF-1α. Accordingly, ELISA revealed that VEGF levels within the transfected HUVEC were about 10-fold greater with the TM. Conclusions: Constitutive activation of the HIF-1α C-TAD, and not merely stabilization of the HIF-1α molecule, is essential for optimal HIF-mediated transcriptional and angiogenic effects. This finding could have important implications for therapeutic angiogenesis using HIF-1α.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalMicrovascular Research
Volume76
Issue number1
DOIs
StatePublished - May 2008

Keywords

  • Angiogenesis
  • Endothelium
  • Hypoxia
  • Hypoxia-inducible factor 1

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