Abstract
Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS.
Highlights
Hypoxia, defined as a decrease in oxygen levels, is a common feature of physiological and pathological processes, including embryonic development, adaptation to high altitudes, wound healing, inflammation, ischemic diseases and solid cancer growth
To monitor the hypoxic conditions we measured the mRNA levels of vascular endothelial growth factor (VEGF) and adrenomedullin (ADM), as both genes are well known to be induced under hypoxic conditions [19,20,21]
Since we did not observe a significant increase in reactive oxygen species (ROS) levels in thioredoxin reductase 1 (TR1) over-expressing cells under hypoxic conditions compared to normoxic conditions, we examined how TR1 overexpression affected hypoxia inducible factors (HIFs) stabilization or activity
Summary
Hypoxia, defined as a decrease in oxygen levels, is a common feature of physiological and pathological processes, including embryonic development, adaptation to high altitudes, wound healing, inflammation, ischemic diseases and solid cancer growth. HIF is a heterodimeric transcription factor composed of an a subunit, which under normoxic conditions is degraded by the proteasome, and a b subunit, whose protein levels are not affected by hypoxia. The hydroxyprolines are recognized by the Von Hippel Lindau protein (VHL) which promotes the ubiquitination of HIF-a thereby targeting it for proteasome degradation. The enzymatic activity of the prolyl hydroxylases is impaired and, HIF-a is no longer hydroxylated and its interaction and ubiquitination by VHL no longer occurs. This leads to its accumulation, translocation to the nucleus, dimerization with the b subunit and regulation of its target genes
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