Activation of hypoxia-induced transcription in normoxia

Abstract

Hypoxia-inducible factor-1 (HIF-1), the master regulator of transcriptional responses to reduced oxygen tension (hypoxia) in mammalian cells, consists of one HIF-1α and one HIF-1β subunit. In normoxia, HIF-1α subunits are hydroxylated on specific proline residues; modifications that signal ubiquitination and degradation of HIF-1α by the proteasome. To test the effect of saturating HIF-1α degradation, we generated a construct, denoted the saturating domain (SD), based on a region surrounding proline 564 (Pro564) in HIF-1α. Expression of the SD led to accumulation of endogenous HIF-1α proteins in nuclei of normoxic cells. The induced HIF-1α was functional as it activated expression from a hypoxia-regulated reporter gene and from the endogenous vascular endothelial growth facor-a ( Vegf-a) and carbonic anhydrase 9 ( Ca9) 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 The effect of the SD was dependent on Pro564 since a mutated SD, in which Pro564 had been replaced by a glycine residue, failed to bind the von Hippel–Lindau protein (pVHL) and to stabilise HIF-1α. 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-1α degradation, either by saturation, or inhibition of prolyl hydroxylases or proteosomal degradation, leads to nuclear localisation of active HIF-1α proteins.