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) 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 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 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 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 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 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.
Liu
Qing
Q
Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D-37073 Göttingen, Germany.
Berchner-Pfannschmidt
Utta
U
Möller
Ulrike
U
Brecht
Martina
M
Wotzlaw
Christoph
C
Acker
Helmut
H
Jungermann
Kurt
K
Kietzmann
Thomas
T
D002467:Cell Nucleus / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D020112:Rhodamine 123 / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012235:Rhodamines; D014157:Transcription Factors; C058319:dihydrorhodamine 123; D020112:Rhodamine 123