A significant drawback to living in air is that there is always a risk of death from metabolic failure if oxygen levels fall too low (hypoxia), and from oxidative damage if O2 gets too high (hyperoxia). Much recent attention has focused on the cellular mechanisms that protect us from oxygen damage, as well as the sensors that trigger the responses, but a recent study by Savita Khanna and colleagues in Free Radical Biology and Medicineapproaches the question from a different angle: how does a cell decide what are normal and abnormal oxygen levels and can it reset the `normal' level?

All aerobic organisms have developed elaborate adaptive mechanisms to maintain oxygen homeostasis, but in order to control oxygen levels the organisms must first detect it before initiating a signalling cascade to induce the appropriate survival responses. One such survival response is an increase in HIF (hypoxia inducible factor). HIF regulates more than 60 putative target genes involved in hypoxia tolerance via a heat shock response element (HRE). During normoxic conditions, HIF is continuously degraded by an oxygen-dependent process. The enzyme proline hydoxylase (PHD)initiates the degradation by hydroxylation of the transcription factor's proline residues. When oxygen levels decline, HIF is no longer degraded so the protein levels rise, ready to upregulate genes involved in protecting the organism during hypoxia. As tissue oxygen levels vary widely within an organism, from organ to organ and cell to cell and even within a cell, oxygen sensing must be a flexible and highly adaptive system. However, instead of focusing on the O2 sensor itself, Khanna and colleagues decided to investigate how a cell decides what its `normal' oxygen pressure(PO2) is by testing the hypothesis that cells are capable of resetting their normoxic set point by changing expression levels of proline hydoxylase.

The team used a murine neuronal cell line (HT22 cells), which they transfected with a luciferase reporter gene; this expressed light-emitting luciferase when HIF bound the heat shock response element. Using this HIF indicator, Khanna and colleagues tested cellular responses to hypoxia; hypoxia should increase HIF levels and induce clearly visible luciferase expression. Cells exposed to 5% O2 (instead of the 20% O2 cell cultures are generally grown in) showed significantly higher HRE-driven luciferase expression, with further luciferase activity induced when O2 levels were decreased to 0.5%. However, cells maintained at 5%O2 for 4 weeks no longer increased HIF activity in response to that PO2, and when the oxygen levels was decreased further to 0.5% O2 the resulting induction of HIF activity was significantly less than in cells adjusted to a norm of 20% O2. This suggested that the biological response to a given PO2 is not so much dependent on the PO2 itself but on conditioning of the cells.

To further test the hypothesis that the normoxic set point in cells is adjustable, additional HT22 cells were grown in hyperoxic conditions at 30%O2. Acute exposure of cells maintained in 30% to `normal' oxygen levels of 20% resulted in increased HIF activity; the normoxic set point can thus clearly be adjusted both upwards and downwards.

The team then went on to investigate proline hydroxylase mRNA expression and found that cells maintained at low O2 increased proline hydroxylase expression compared to the 20% O2 cells, while decreasing proline hydroxylase expression in high O2. They also looked at proline hydroxylase levels in mice maintained in hypoxia (10%O2), which reduced brain PO2, and found that the animals had significantly increased proline hydroxylase expression. Taken together the data indicate that changes in proline hydroxylase expression may be effective in resetting the normoxic set point of cells, and that `normal' PO2 is clearly adjustable over a wide range of oxygen levels even within a single cell type.

Khanna, S., Roy, S., Maurer, M., Ratan, R. R. and Sen, C. K.(
). Oxygen sensitive reset of hypoxia-inducible factor transactivation response: Prolyl hydroxylases tune the biological normoxic set point.
Free Rad. Biol. Med.