Hypoxia up-regulates prolyl hydroxylase activity: a feedback mechanism that limits HIF-1 responses during reoxygenation.
The mechanism by which hypoxia induces gene transcription is now well established. Hypoxia reduces activity of prolyl hydroxylases (PHD) that hydroxylate specific proline residues in the oxygen-dependent degradation domain (ODD) of hypoxia-inducible factor-1alpha (HIF-1alpha). As a consequence, HIF-1alpha accumulates and promotes hypoxic tolerance by activating gene transcription. This paper identifies the three forms of PHDs in rats and shows that a period of hypoxia selectively increases expression of PHD-2 mRNAs levels. We developed assays for PHD activity that used (i) the peptide-specific conversion of labeled 2-oxoglutarate into succinate and (ii) the binding of the von Hippel-Lindau protein to a glutathione S-transferase-ODD fusion protein. The two assays indicated a low enzymatic activity in normoxic and hypoxic cells and a rapid increase during reoxygenation. We also developed hydroxyproline-specific antibodies that recognized hydroxylated forms of a fusion protein (ODD-green fluorescent protein) that combined the ODD domain of HIF-1alpha and the green fluorescent protein. Using this antibody, we demonstrated that reoxygenation induced a rapid hydroxylation of Pro-564, which was followed by a massive degradation of the proteins. The results suggest that a hypoxic upregulation of PHD (presumably PHD-2) acts as a feedback mechanism to stop hypoxic responses in reoxygenated cells. We propose that proline hydroxylation might play a role in hypoxic preconditioning.
D'Angelo
Gisela
G
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia-Antipolis, 06560 Valbonne, France. dangelo@ipmc.cnrs.fr
Duplan
Eric
E
Boyer
Nicole
N
Vigne
Paul
P
Frelin
Christian
C
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D025461:Feedback, Physiological; D005982:Glutathione Transferase / Q000378:metabolism; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D015151:Immunoblotting; D007656:Ketoglutaric Acids / Q000737:chemistry; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D019802:Succinic Acid / Q000737:chemistry; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D015854:Up-Regulation; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D009687:Nuclear Proteins; D010455:Peptides; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D011392:Proline; D019802:Succinic Acid; D011393:Procollagen-Proline Dioxygenase; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D005982:Glutathione Transferase; D008025:Ligases; D010100:Oxygen