Oxygen sensing with Peter Ratcliffe

Two Wellcome Trust-funded researchers were honoured in this year’s Canada Gairdner Awards – we catch up with one of the winners and learn that oxygen sensing amongst cells is more prevalent than previously thought

Professor Peter Ratcliffe is head of the Nuffield Department of Clinical Medicine at the University of Oxford and has recently been honoured in the 2010 Canada Gairdner Awards – Canada’s only international science awards. Created in 1959, the award was set up to recognise and reward the achievements of medical researchers whose work contributes significantly to improving the quality of life.

Ratcliffe receives his award for identifying how cells in the body monitor and respond to oxygen levels. Low oxygen levels – or hypoxia – is an important component of many human disease including cancer, heart disease, stroke, vascular disease and anaemia.

Ratcliffe’s research paves the way to therapies that manipulate responses to hypoxia, for instance to improve the supply of oxygen to tissues in those with diseases of the heart and circulation.

You’ve just been honoured in the 2010 Canada Gairdner Awards – how does it feel?

In research there is immense satisfaction in simply gaining an understanding of the problems you’ve been grappling with – most of us are addicted to it. This is true whether or not other people think the work is important – but if they do recognise its importance (and the Gairdner Award must be evidence of that) then this is doubly satisfying.

Could you tell us about your award-winning work?

The work has defined mechanisms by which cells sense and respond to changes in oxygen levels. In humans (and all other large organisms) oxygen must be delivered to all cells in the body in precisely the right amounts. Too little will impair metabolism, but too much causes toxicity, so there is a need for precise control. Many human diseases cause mis-regulation of this oxygen balance. For instance, heart, lung, vascular and anaemic diseases cause low oxygen delivery, whereas cancers need oxygen to grow and if they grow rapidly this causes hypoxia. We made two main discoveries.

The first was of the widespread existence of this type of oxygen sensing mechanism in the cells of higher organisms – i.e. that essentially all animal cells possess a similar mechanism for sensing oxygen levels. Previously it was believed that ‘oxygen sensing’ was a property of only a few special cells that operated a specific and limited set of responses to hypoxia. For instance the hormone erythropoietin (Epo) is produced by the kidney when blood oxygen levels are low and stimulates the production of more (oxygen carrying) red blood cells. Epo is made in particular cells in the kidney and it was thought that these cells had special ‘oxygen sensing’ properties. A major goal of physiologists in this area, for much of the latter part of the twentieth century, was to understand this special property. Surprisingly, we showed that the oxygen sensing property was present in all animals cells even those from primitive invertebrate species that don’t make Epo and don’t even have red blood cells. The implication was the oxygen sensing system must be doing many other things. This changed the way scientists thought about it. It is now recognised that this oxygen sensing pathway (mediated by hypoxia inducible factor, HIF, which was discovered and characterised by Gregg Semenza, one of the other Gairdner Awardees) regulates hundreds of genes and does many other things (e.g. controlling blood vessel growth, controlling cell metabolism, determining cell migration, controlling cell survival).

The second was the nature of the oxygen sensing and signalling pathway. The essential oxygen sensitive signal is generated by a set of non-haem Fe(II) and 2-oxoglutarate dependent dioxygenases that catalyse the post-translational hydroxylation of specific amino acid residues in the transcriptional factor HIF. Hydroxylation of two prolyl residues in HIF-alpha subunits targets the protein for destruction by the von Hippel-Lindau (VHL) ubiquitin ligase. This work involved determining the regions of the HIF molecule that were critical for oxygen sensitive behaviour, defining the interaction with the VHL protein, working out that the critical modification that promotes the interaction of HIF and VHL is prolyl hydroxylation of HIF and (with Christopher Schofield) identifying the hydroxylase enzymes that catalyse the process. Because these enzymes absolutely require molecular oxygen the process is suppressed in hypoxia allowing HIF-alpha to escape proteolytic destruction and build up an active complex.

My laboratory and that of William Kaelin (also a Gairdner awardee) worked independently on this pathway, but made many of these discoveries almost simultaneously. In the later stages of the work I was also very fortunate in teaming up with Christopher Schofield in Oxford, a leader in the 2-G oxygenase field with whom we continue to work closely.

From the medical perspective there is hope that by manipulating these pathways (e.g. by using drugs to block the hydroxylase enzymes and upregulate HIF) it would be possible to enhance the body’s natural defences against low oxygen (e.g. to treat heart and vascular diseases). In cancer it might be desirable to switch the system in the other direction i.e. to increase hydroxylation or down-regulate the HIF pathway. This might reduce a cancer’s ability to make new blood vessels and hence limit its ability to grow.

How did you get into this area of research?

I trained as a clinical nephrologist and was originally interested in understanding why the kidney is susceptible to ischaemic injury in shock. This led to an interest in understanding the renal circulation, its effects on oxygen levels in the kidney tissue, and how this controlled Epo production. The overwhelming prejudice at the time was that Epo was regulated by a highly specialised ‘oxygen sensor’ within the kidney; understanding that mechanism was my goal. We were surprised to find Epo-producing cells in the kidney were rather unremarkable fibroblasts. We therefore began looking for the Epo ‘oxygen sensing’ system in a wide range of non-Epo producing cells (by transfecting them with Epo control sequences) and were even more surprised to find that the sensing system was present in essentially all cells that we examined. That took us away from the kidney, but into what turned out to be a truly exciting field of research.

What’s the next stage for your research?

This is an unprecedented type of signalling process – so a major thrust in the laboratory is finding out whether it regulates other biological pathways i.e. whether the HIF hydroxylase enzymes have other (non-HIF) substrates.  At least for one enzyme, we now have good evidence that this is the case. HIF is also regulated by asparaginyl hydroxylation, which modulates co-activator recruitment to the complex. The HIF asparaginyl hydroxylase has a wide range of targets amongst ankyrin repeat domain (ARD) containing proteins. We think that at least some of these targets might regulate other oxygen sensitive pathways, but so far their function(s) are unclear.

The second major question is whether the work will led to new treatments for disease. The HIF hydroxylases are enzymes with substrate and co-substrate binding pockets. As such they are classic drug targets. We are particularly interested in whether inhibition might be used to augment protective responses to hypoxia and to improve the outcome in ischaemic/hypoxic or anaemic diseases. The problem is that activation of HIF entrains many different biological responses. So the challenge is to develop something

[caption id="attachment_23445" align="alignright" width="150" caption="Peter Ratcliffe"][/caption]

that is sufficiently specific for a given clinical situation i.e. it promotes effective angiogenesis or tissue protection, but not erythropoiesis, or vice-versa.

Will this award change anything in your working life?

I guess I’ll need to wait and see – perhaps it’s like scoring goal a football game (which I never could). The game is the same but one carries on playing hard or harder perhaps.