Hyperoxia attenuates the inhibitory effect of nitric oxide donors on HIF prolyl-4-hydroxylase-2: Implication on discriminative effect of nitric oxide on HIF prolyl-4-hydroxylase-2 and collagen prolyl-4-hydroxylase

Prolyl 4-hydroxylases (P4Hs), such as collagen prolyl-4-hydroxylases (CPHs) and hypoxia inducible factor prolyl-4-hydroxylases (HPHs), have recently been recognized as promising drug targets for the treatment of fibrotic and ischemic diseases. CPHs and HPHs catalyze identical metabolic reactions, yet lead to quite different physiological consequences, collagen synthesis and the regulation of oxygen homeostasis. Selective modulation of the two enzymes should provide a therapeutic benefit upon pharmacotherapy. In an in vitro VHL capture assay, hydroxylation of the 19mer HIF peptide (corresponding to HIF-1 alpha residues 556-574) by HPH-2 was effectively prevented by nitric oxide (NO) donors, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The NO donors also caused inhibition of HPHs and accumulation of nonhydroxylated HIF-1 alpha protein in A549 human lung adenocarcinoma cells. Hyperoxia (100% O(2)) attenuated both NO donor-induced accumulation of HIF-1 alpha and inhibition of HPH-mediated hydroxylation. In the presence of a proteasome inhibitor, MG132, the hyperoxia-mediated degradation of HIF-1 alpha was deterred and hydroxylated HIF-1 alpha was detected. SNAP, while being an effective inhibitor of proline 4-hydroxylation of HIF-1 alpha by HPH-2, did not diminish praline hydroxylation of collagen by CPHs. Our data suggest that NO inhibits HPH-2 via competing with dioxygen and that the discriminative effect of NO on CPHs and HPH-2 is attributable to the difference in the affinity of the two enzymes toward dioxygen. (C) 2011 Elsevier Inc. All rights reserved.