Inhibition of mitochondrial respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF) intact

Abstract

The transcription factor hypoxia-inducible factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published regarding the role of the mitochondrial electron transport chain (ETC) in the regulation of HIF-1␣. We assessed cellular hypoxia by pimonidazole staining and blotting of the O 2 -labile HIF-1 ␣-subunit in human osteosarcoma cell cultures (U2OS and 143B). In conventional, gas-impermeable cell culture dishes, ETC inhibitors had no effect on pimonidazole staining or HIF-1␣ abundance in a 20% O 2 atmosphere; both parameters were undetectable. Pimonidazole staining and HIF activity were substantial in 0.1% O 2 irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O 2 ) pimonidazole staining and HIF-␣ expression were detectable but strongly reduced after ETC inhibition in conventional cell cultures. All effects of ETC inhibition on HIF-1␣ regulation were eliminated in gas-permeable IntroductionAdaptation to low oxygen concentration leads to a series of responses through the transactivation of more than 70 target genes 1 mediated by the hypoxia-inducible factor-1 (HIF-1). The hematopoietic growth factor erythropoietin (EPO) and the angiogenic peptide vascular endothelial growth factor (VEGF) are the most prominent examples of HIF-1 target genes. Actually, the HIF binding site embedded in the 3Ј enhancer of the EPO gene allowed purification and characterization of HIF-1. 2 HIF is also known to be involved in angiogenesis, tumor growth, and apoptosis, 3,4 which makes it a challenging target for therapeutic manipulation. HIF-1 is a heterodimeric transcription factor composed of 2 subunits. 2 Each HIF-1 subunit contains an N-terminal basic helix-loop-helix domain responsible for dimerization and DNA binding followed by a PAS domain (Per, aryl hydrocarbon nuclear translocator [ARNT], and Sim were the first members of this protein family) that is involved in protein interactions. 5,6 Both subunits are constitutively expressed. While the ␤-subunit is present in the nucleus at detectable levels under normoxia and hypoxia, HIF-1␣ levels are affected by changes in the cellular oxygen partial pressure (pO 2 ). 7,8 In hypoxia, HIF-1␣ accumulates and is translocated to the nucleus where it dimerizes with HIF-1␤, forming an active DNA-binding complex (reviewed by Semenza 1 ). In contrast, atmospheric pO 2 leads to a rapid destruction by the ubiquitin-proteasome system. 9,10 Initially, HIF-1␣ is hydroxylated at Pro564 and Pro402 by 2-oxoglutarate-dependent dioxygenases, 11,12 which have been termed "prolyl hydroxylase domain," containing protein 1, 2, and 3 (PHD-1, 2, 3) 13 or HIF-1␣ prolyl hydroxylase 3, 2, and 1, 14 which prevent the stabilization. Furthermore, HIF-1␣ is hydroxylated at Asn803 by an enzyme first identified as "factor inhibiting HIF-1" (FIH-1), 15 which abrogates the transactivation by preventing the binding of transcriptional coactivators such as cyclic adenosine monophosphate response...

Reactive oxygen species and cellular oxygen sensing

“…In direct contrast to previous findings, some groups found that ρ° cell lines lacking functional mitochondria still stabilize HIFα under hypoxia [76,77]. Other studies suggested that cells with impaired mitochondrial function fail to stabilize HIFα under hypoxia, not because of defective ROS-generation, but because of intracellular O 2 re-distribution which leads to increased availability of O 2 as a substrate for the hydroxylation reaction [72,78].…”

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

“…This contrasts findings in mammalian cellular systems (e.g. human hepatoma cell lines) where the HIF-1a protein is detectable only under severe oxygen deprivation (1-0.1% oxygen) because PHD activity is compromised due to the lack of oxygen (Huang et al 1996;Doege et al 2005). Actually, the half-life of HIF-1a at atmospheric pO 2 in these cellular systems has been estimated to <5 min whereas its hypoxic stabilization happens instantaneously (Huang and Bunn 2003).…”

Mitochondria: Oxygen sinks rather than sensors?

“…However, Metzen and co-workers recently reported that under mild hypoxic conditions (3% O 2 ) chemical inhibition of mitochondria impaired hypoxic HIF-1α stabilization in normal cell culture dishes but not in oxygen-permeable dishes. Importantly, no difference was seen in severe (0.1% O2) hypoxia [45]. These data suggest that enough oxygen could be redirected from mitochondria (when the sink is blocked) towards the PHD oxygen sensors in mild hypoxia when the dishes (as usual) are gas-impermeable.…”