Reactive oxygen species: important regulators of the oxygen sensing pathway

Various studies have demonstrated the influence of reactive oxygen species (ROS) in oxygen signaling. However, reports on the effect of ROS on HIF function are controversial as different, even contrasting outcomes on HIF activity have been observed depending on whether ROS are part of a normoxic, hypoxic or growth factor signalling cascade response. Thus, a HIF upregulation upon ROS depletion under normoxia and a HIF downregulation upon ROS increase under hypoxia have been documented. These observations are in line with the concept that declining O₂ concentrations trigger the hypoxia response as a result of decreased ROS intermediate production. In contrast, other groups reported that exposure of cells to ROS under normoxia (within a signalling cascade response) or ROS production by mitochondria under hypoxia triggered HIF-1 stabilization. It was proposed that ROS mediated oxidation of ferrous iron (Fe II) depletes PHD of a crucial co-factor resulting in reduced PHD activity. We were interested to further analyze how ROS signalling interconnects with the oxygen sensing pathway.

In line with the first model, H₂O₂ treatment of a panel of glioblastoma cell lines suppressed hypoxia-induced HIF-1£\, HIF-2£\ and HIF-target gene expression in a dose- and time-dependent fashion. In contrast, ROS depletion by antioxidant treatment upregulated HIF-1£\/2£\ expression under normoxia and after reoxygenation. ROS regulation of HIF-£\ was dependent on PHD function as VHL-deficiency or PHD inhibition by the 2-oxoglutarate analogue DMOG abolished ROS dependent downregulation of HIF-£\. Likewise, HIF-1/2£\ constructs with mutated prolyl residues failed to respond to ROS treatment. Previously, ascorbate has been shown to regulate PHD activity in a similar fashion by increasing the availability of Fe II. Indeed, while ascorbate and ROS decreased HIF-upregulation by Fe III iron chelator, Fe II iron chelator mediated HIF induction remained unaffected, suggesting that ascorbate and ROS use analogous mechanisms to change the redox state of iron.

The low O₂ affinity of PHD renders this enzyme highly sensitive to alterations in co-factor concentration such as ferrous iron. Our results indicate that ROS regulate HIF-£\ levels by augmenting PHD activity and imply that, paradoxically similar to the antioxidant ascorbate, ROS interface with the PHD/HIF system via changing the redox state of iron. ROS may thus crucially help to fine-tune the oxygen sensing response.