Activation of C-transactivation domain is essential for optimal HIF-1 alpha-mediated transcriptional and angiogenic effects.
HIF-1 is a transcription factor that regulates genes involved in oxygen homeostasis. In normoxia, degradation of the HIF-1 alpha 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-1 alpha 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-1 alpha in addition to its stabilization would result in greater HIF-1 alpha transcriptional activity and angiogenic effects than mere stabilization of the molecule. For this, we constructed a Triple mutant HIF-1 alpha (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-1 alpha. 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-1 alpha. Accordingly, ELISA revealed that VEGF levels within the transfected HUVEC were about 10-fold greater with the TM.


CONCLUSIONS
Constitutive activation of the HIF-1 alpha C-TAD, and not merely stabilization of the HIF-1 alpha molecule, is essential for optimal HIF-mediated transcriptional and angiogenic effects. This finding could have important implications for therapeutic angiogenesis using HIF-1 alpha.
Tal
Reshef
R
Vascular Biogenics Ltd., Or-Yehuda, Israel.
Shaish
Aviv
A
Bangio
Livnat
L
Peled
Michael
M
Breitbart
Eyal
E
Harats
Dror
D
D019943:Amino Acid Substitution; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D016373:DNA, Antisense / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D006899:Mixed Function Oxygenases; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000502:physiology*; D020574:Proto-Oncogene Proteins c-sis / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics
D000951:Antigens, Neoplasm; D016373:DNA, Antisense; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020574:Proto-Oncogene Proteins c-sis; D012097:Repressor Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases