Hypoxia Up-regulates Prolyl Hydroxylase Activity

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (40), p.38183-38187
• Main Authors: D'Angelo, Gisela, Duplan, Eric, Boyer, Nicole, Vigne, Paul, Frelin, Christian
• Format: Article
• Language: English
• Subjects: Life Sciences
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302244200

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-1α (HIF-1α). As a consequence, HIF-1α 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-1α 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.