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Hypoxia-inducible factor (HIF) is central in coordinating many of the transcriptional adaptations to hypoxia. Composed of a heterodimer of alpha and beta subunits, the alpha subunit is rapidly degraded in normoxia, leading to inactivation of the hypoxic response. Many models for a molecular oxygen sensor regulating this system have been proposed, but an important finding has been the ability to mimic hypoxia by chelation or substitution of iron. A key insight has been the recognition that HIF-alpha is targeted for degradation by the ubiquitin-proteasome pathway through binding to the von Hippel-Lindau tumour suppressor protein (pVHL), which forms the recognition component of an E3 ubiquitin ligase complex leading to ubiquitylation of HIF-alpha. Importantly, the classical features of regulation by iron and oxygen availability are reflected in regulation of the HIF-alpha/pVHL interaction. It has recently been shown that HIF-alpha undergoes an iron- and oxygen-dependent modification before it can interact with pVHL, and that this results in hydroxylation of at least one prolyl residue (HIF-1alpha, Pro 564). This modification is catalysed by an enzyme termed HIF-prolyl hydroxylase (HIF-PH), and compatible with all previously described prolyl-4-hydroxylases HIF-PH also requires 2-oxoglutarate as a cosubstrate. The key position of this hydroxylation in the degradation pathway of HIF-alpha, together with its requirement for molecular dioxygen as a co-substrate, provides the potential for HIF-PH to function directly as a cellular oxygen sensor. However, the ability of these enzyme(s) to account for the full range of physiological regulation displayed by the HIF system remains to be defined.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Matching oxygen consumption and supply represents a fundamental challenge to multicellular organisms. HIF-1 is a transcription complex which is emerging as a key mediator of oxygen homeostasis. HIF-1 controls the expression of many genes, including erythropoietin, angiogenic growth factors, glucose transporters and glycolytic enzymes. The HIF-1 complex, which contains an alpha and beta subunit (both basic helix-loop-helix proteins of the PAS family) is formed in hypoxia and modulates gene expression through hypoxia response elements. Regulation involves ubiquitin-mediated oxygen-dependent destruction of the alpha subunit. Oxygen-regulated destruction of HIF-alpha requires the von Hippel Lindau tumour suppressor protein (pVHL). pVHL acts as the recognition component of a ubiquitin E3 ligase complex which binds HIF-alpha. Loss of pVHL function, which results in constitutive activation of the hypoxic response, is important in the development of clear cell renal cancer, where both copies of the gene are usually inactivated. The importance of the VHL-HIF system in multicellular organisms is supported by conservation in the nematode C. elegans. Understanding the events resulting in HIF activation should provide novel therapeutic targets. This would be useful in preventing angiogenesis in cancers and promoting adaptive changes in hypoxic/ischaemic tissue.

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing.

A great many aspects of the anatomy and physiology of large animals are constrained by the need to match oxygen supply to cellular metabolism and appear likely to involve the regulation of gene expression by oxygen. Some insight into possible underlying mechanisms has been provided by studies of erythropoietin, a haemopoietic growth factor which stimulates red cell production in response to hypoxia. Studies of hypoxia-inducible cis-acting sequences from the erythropoietin gene have led to the recognition of a widespread transcriptional response to hypoxia based on the activation of a DNA-binding complex termed hypoxia-inducible factor-1 (HIF-1). Perturbation of the transcriptional response by particular transition metal ions, iron chelators and certain redox-active agents have suggested a specific oxygen sensing mechanism, perhaps involving a haem protein in a flavoprotein/cytochrome system. In addition to erythropoietin, HIF-1-responsive genes include examples with functions in cellular energy metabolism, iron metabolism, catecholamine metabolism, vasomotor control and angiogenesis, suggesting an important role in the coordination of oxygen supply and cellular metabolism. In support of this, we have demonstrated an important role for HIF-1 in tumour angiogenesis. HIF-1 itself consists of a heterodimer of two basic-helix-loop-helix proteins of the PAS family, termed HIF-1alpha and HIF-1beta, although other closely related members of this family may also contribute to the response to hypoxia. We have fused domains of HIF-1 genes to heterologous transcription factors to assay for regulatory function. These experiments have defined several domains in HIF-1alpha which can independently confer the hypoxia-inducible property, and they suggest a mechanism of HIF-1 activation in which post-translational activation/derepression of HIF-1alpha is amplified by changes in HIF-1alpha abundance most probably arising from suppression of proteolytic breakdown. Pursuit of the mechanism(s) underlying these processes should ultimately lead to better definition of the oxygen-sensing process.

Adaptive responses to hypoxia occur in many biological systems. A well-characterized example is the hypoxic induction of the synthesis of erythropoietin, a hormone which regulates erythropoiesis and hence blood oxygen content. The restricted expression of the erythropoietin gene in subsets of cells within kidney and liver has suggested that this specific oxygen-sensing mechanism is restricted to specialized cells in those organs. Using transient transfection of reporter genes coupled to a transcriptional enhancer lying 3' to the erythropoietin gene, we show that an oxygen-sensing system similar, or identical, to that controlling erythropoietin expression is wide-spread in mammalian cells. The extensive distribution of this sensing mechanism contrasts with the restricted expression of erythropoietin, suggesting that it mediates other adaptive responses to hypoxia.