A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression

It has been proposed that hydroxyl radicals ((OH)-O-.) generated in a perinuclear iron-dependent Fenton reaction are involved in O-2-dependent gene expression. Thus, it was the aim of this study to localize the cellular compartment in which the Fenton reaction takes place and to determine whether scavenging of (OH)-O-. can modulate hypoxia-inducible factor 1 (HIF-1)-dependent gene expression. The Fenton reaction was localized by using the nonfluorescent dihydrorhodamine (DHR) 123 that is irreversibly oxidized to fluorescent rhodamine 123 while scavenging (OH)-O-. together with gene constructs allowing fluorescent labeling of mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, or lysosomes. A 3D two-photon confocal laser scanning microscopy showed (OH)-O-. generation in distinct hot spots of perinuclear ER pockets. This ER-based Fenton reaction was strictly pO(2)-dependent. Further colocalization experiments showed that the O-2-sensitive transcription factor HIF-1alpha was present at the ER under normoxia, whereas HIF-1alpha was present only in the nucleus under hypoxia. inhibition of the Fenton reaction by the (OH)-O-. scavenger DHR attenuated HIF-prolyl hydroxylase activity and interaction with von Hippel-Lindau protein, leading to enhanced HIF-1alpha levels, HIF-1alpha transactivation, and activated expression of the HIF-1 target genes plasminogen activator inhibitor 1 and heme oxygenase 1. Further, (OH)-O-. scavenging appeared to enhance redox factor 1 (Ref-1) binding and, thus, recruitment of p300 to the transactivation domain C because mutation of the Ref-1 binding site cysteine 800 abolished DHR-induced transactivation. Thus, the localized Fenton reaction appears to impact the expression of hypoxia-regulated genes by means of HIF-1alpha stabilization and coactivator recruitment.