Activation of hypoxia-induced transcription in normoxia

Hypoxia-inducible factor-1 (HIF-1), the master regulator of transcriptional responses to reduced oxygen tension (hypoxia) in mammalian cells, consists of one HIF-1 alpha and one HIF-1 beta subunit. In normoxia, HIF-1 alpha subunits are hydroxylated on specific proline residues; modifications that signal ubiquitination and degradation of HIF-1 alpha by the proteasoine. To test the effect of saturating HIF-1 alpha degradation, we generated a construct, denoted the saturating domain (SD), based on a region surrounding proline 564 (Pro564) in HIF-1 alpha. Expression of the SD led to accumulation of endogenous HIF-1 alpha proteins in nuclei of nonrioxic cells. The induced HTF-1 alpha was functional as it activated expression from a hypoxia-regulated reporter gene and from the endogenous vascular endothelial growth facor-alpha (Vegf-a) and carbonic anhydrase 9 (CO) genes. The effect of the SD was dependent on Pro564 since a mutated SD, in which Pro564 had been replaced by a glycine residue, failed to bind the von Hippel-Lindau protein (pVHL) and to stabilise HIF-1 alpha. Treatment of cells with the prolylhydroxylase inhibitor dimethyloxalylglycine, or the proteasome inhibitor MG-132, mimicked the effect of the SD. In conclusion, we show that blocking HIF-1 alpha degradation, either by saturation, or inhibition of prolyl hydroxylases or proteosomal degradation, leads to nuclear localisation of active HIF-1 alpha proteins. (c) 2005 Elsevier Inc. All rights reserved.