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.