Hypoxia plays a central role in many common diseases including cardiovascular and cerebrovascular disease and cancer. The regulation of gene expression in response to variation in oxygen is essential in physiological control of vascular supply and metabolism and its pathological disturbance. This regulation of gene expression is mediated largely via the transcription factor HIF-1. It controls the expression of a wide variety of genes involved in erythropoiesis, angiogenesis, vascular tone and metabolism. The nature of the oxygen sensor which underlies regulation of HIF-1 has recently received further definition.

The regulation of HIF-1 by oxygen occurs principally through oxygen regulated degradation via the ubiquitin-proteasome system. In the presence of oxygen there is rapid degradation but in hypoxia HIF-1 is stabilised. This degradation requires recognition of HIF-1 by the von Hippel Lindau gene product VHL which then facilitates subsequent ubiquitylation and subsequent proteasomal degradation. The mechanism by which oxygen renders the HIF molecule available for VHL binding and subsequent destruction has recently been established. Two critical proline residues within the HIF-1 molecule are necessary for the binding of VHL. In the presence of oxygen these residues are hydroxylated to hydroxyproline. This process is enzymatic and requires the presence of ferrous ions and 2-oxoglutarate. The other well established role for prolyl hydroxylation is in the synthesis of collagen. The sequence of the collagen prolyl hydroxylase was used in database searches to identify homologous gene products which might function as HIF prolyl hydroxylases. Three highly homologous human proteins were identified which were able to convert HIF-1 to a VHL binding form by prolyl hydroxylation.

We undertook studies to examine whether these enzymes showed oxygen dependence of activity. The conversion of HIF-1 to a VHL binding form by these proteins (named PHD1, 2 and 3) was oxygen sensitive and also showed characteristic inhibition by desferrioxamine and cobalt. The ability of these enzymes to regulate HIF in vivo has been confirmed by creating cells with inducible expression of each enzyme and utilising RNAi techniques. Recently a further oxygen-dependent modification of HIF has been defined – the hydroxylation of a specific asparaginyl residue which regulates the transcriptional activity of HIF and its interaction with p300. FIH has been identified as the 2-oxoglutarate-dependent oxygenase which mediates this hydroxylation and its structure has been determined.