Critical Review. Regulation of HIF by the von Hippel-Lindau Tumour Suppressor: Implications for Cellular Oxygen Sensing

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1 IUBMB Life, 52: 43 47, 2001 Copyright c 2001 IUBMB /01 $ Critical Review Regulation of HIF by the von Hippel-Lindau Tumour Suppressor: Implications for Cellular Oxygen Sensing D. R. Mole, P. H. Maxwell, C. W. Pugh, and P. J. Ratcliffe The Henry Wellcome Building of Genomic Medicine, University of Oxford, Oxford, United Kingdom Summary Hypoxia-inducible factor (HIF) is central in coordinating many of the transcriptional adaptations to hypoxia. Composed of a heterodimer of and subunits, the 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 nding has been the ability to mimic hypoxia by chelation or substitution of iron. A key insight has been the recognition that HIF- 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-. Importantly, the classical features of regulation by iron and oxygen availability are re ected in regulation of the HIF- /p VHL interaction. It has recently been shown that HIF- undergoes an iron- and oxygen-dependent modi cation before it can interact with pvhl, and that this results in hydroxylation of at least one prolyl residue (HIF-1, Pro 564). This modi- cation 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-, 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 de ned. IUBMB Life, 52: 43 47, 2001 Keywords HIF prolyl hydroxylase; hypoxia-inducible factor; oxygen; von Hippel-Lindau. Regulation of oxygen homeostasis is a fundamental cellular and systemic response that is conserved across many species. Received 21 June 2001; accepted 26 July Address correspondence to Peter J. Ratcliffe, The Henry Wellcome Building of Genomic Medicine, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, United Kingdom. Fax: peter.ratcliffe@imm.ox.ac.uk It is now recognised that all mammalian cells, and not just specialised chemoreceptors, respond to changes in oxygen level (1). Furthermore, hypoxia is critical to the pathophysiology of major human diseases such as myocardial ischaemia, stroke, and cancer (2). Therefore, pharmacological manipulation of the cellular oxygen response is attractive as a therapeutic approach. Hypoxia Inducible Factor: A Key Effector of the Hypoxic Response Studies of the transcriptional response to hypoxia have de- ned hypoxia-inducible factor (HIF) as a central effector mechanism coordinating cellular responses to oxygen tension. HIF is a heterodimeric transcription factor formed by association of a constitutively expressed beta subunit and an alpha subunit that is rapidly degraded in the presence of oxygen (3 5). At least two isoforms of the regulatory alpha subunit (HIF-1 and HIF-2 ) have been de ned that operate in a similar manner (6). In hypoxia these alpha subunits are stabilised leading to accumulation, and formation of the HIF complex. The complex recruits coactivators such as p300/cbp (7 ), and binds to hypoxia response elements (HRE) within the cis-acting regulatory elements of HIF target genes thereby directing transcription. Notable among these target genes are erythropoietin; genes controlling angiogenesis and vascular tone such as vascular endothelial growth factor (VEGF) (8) and nitric oxide synthetase (NOS); genes controlling cell proliferation; and genes co-ordinating energy metabolism (Fig. 1) (9). HIF- and HIF- both contain basic helix-loop-helix domains (bhlh) and PAS domains that function in dimerisation, binding of the complex to DNA (10), and cooperative interactions with other transcriptional factors. Studies of fusion proteins containing different regions of the HIF- subunits have demonstrated that each isoform contains a number of independently acting regulatory regions (11 15 ). Of particular importance was the de nition of a transferable oxygen-dependent degradation domain (ODDD), extending over approximately 200 residues 43

2 44 MOLE ET AL. Figure 1. Genes regulating the physiological response to oxygen are under the transcriptional control of hypoxia-inducible factor (HIF). These include genes responsible for angiogenesis, vasomotor contorl, cell proliferation and viability, energy metabolism, iron metabolism, and erythropoiesis. within the central region of HIF-1 and HIF-2. Metabolic labelling, transfection, and inhibitor studies indicated that this domain mediates destruction of HIF- subunits by the proteasomal pathway (5, 12). Furthermore, because the domain confers the property of regulation by oxygen, these studies also indicated that the ODDD sequences must in some way interact with the oxygen sensing/transduction pathway. Models of Oxygen Sensing Many models have been proposed for the oxygen-sensing process. Important in shaping these ideas were the observations that iron chelation or exposure to transition metals such as cobalt, manganese, and nickel mimic the hypoxia response, stabilising HIF- and up-regulating the expression of HIF target genes (16, 17 ). Moreover, the ef cacy of cobalt was noted to be inversely related to the availability of iron, suggesting that metal might be competing at an oxygen-sensing iron centre. Two broad categories of process have been proposed. In the rst type of model, a speci c oxygen-sensing protein has been postulated. Several examples of such sensing proteins have been de ned in prokaryotic systems and include the dioxygen-liganding haemoprotein xl in Rhizobia (18), and oxygen radical sensitive iron sulphur clusters such as sox r/s in E. coli (19). In such models, it has been proposed that cobalt might substitute for iron and inhibit activation by oxygen. One potential problem with such models is that many iron centres (for instance haem iron) do not exchange in this way, so that it was necessary to propose that the sensing molecule was itself turning over rapidly to allow incorporation of cobalt into the nascent protein. The second type of model followed from the recognition that exposure of cells to hydrogen peroxide inhibits activation of the HIF system by hypoxia (20), and proposed that the ironand oxygen-sensitive degradation of HIF derived from Fenton chemistry. In the majority of such models, it has been proposed that increases in the generation of reactive oxygen species (ROS) at higher dioxygen concentrations were responsible for activating pathways that promote HIF- degradation. Several sources of such ROS have been proposed, including different isoforms of NADPH oxidase (21) and speci c cytochrome/cytochrome reductase systems (22). In contrast, in another model it has been proposed (23) that an increase in mitochondrial ROS production in hypoxia provides a signal that stabilises HIF-. In considering the problem of cellular oxygen sensing, postulated ROS signalling mechanisms have the potential to provide responses over a broad range of cellular oxygen levels. However there are dif culties in understanding how precise physiological responses to dioxygen availability (for instance feedback control of haematocrit by erythropoietin) could be regulated by such broadly reactive species. To address this, two proposals have been put forward. First, it has been suggested that a speci c sensing/signalling molecule might contain speci c sensitive residues, such as the highly reactive cysteine residues in the bacterial ROS sensor oxyr/s (24). Second, it has been proposed that speci c local interactions of iron with metal-binding sites in proteins might generate localised ROS by Fenton chemistry, allowing speci c modi cation of adjacent amino acids. Although pharmacological approaches have been used to gain indirect evidence for or against these models, none of these studies has been conclusive and many groups have sought to move proximally from an understanding of the regulation of HIF to the sensing/transduction process by molecular biochemical and genetic approaches. HIF- is Targeted for Proteasomal Degradation by pvhl An important genetic insight into the regulation of HIF has come from observations on the von Hippel-Lindau (VHL) syndrome. Enhanced glucose metabolism and angiogenesis are classical features of cancer, involving up-regulation of HIF target genes. In addition to stimulation by the hypoxic microenvironment, genetic alterations contribute to these effects. VHL disease provides a striking example. Affected individuals bear a germ-line mutation in the VHL tumour suppressor gene, and tumour formation is associated with somatic loss or inactivation of the remaining wild-type allele. Cardinal features are tumours rich in blood vessels, such as haemangioblastomas, affecting the CNS and retina, and renal clear cell carcinomas. In addition, there is a predisposition to phaeochromocytoma and other rarer tumours. Many of these tumours overexpress HIF target genes, such as VEGF, and more rarely erythropoietin, providing early evidence of a connection with this system (25, 26). The VHL gene encodes a 213-amino acid protein (pvhl). Although the primary sequence did not immediately suggest a function, protein-association experiments de ned a series of pvhl-interacting molecules, including elongins B and C and CUL2 (27, 28), a member of the Cullin family. This suggested homology to yeast ubiquitin ligase complexes known as SCF

3 REGULATION OF HIF 45 Figure 2. In hypoxic conditions HIF- subunits are stable, leading to accumulation and heterodimerisation with HIF-. HIF then binds to cis-acting hypoxia response elements (HRE) of HIF target genes, as part of a larger complex, initiating transcription. In normoxia HIF- is recognised by the -domain of pvhl, which as part of a E3 ubiquitin ligase complex, targets it for proteasomal degradation by covalent attachment of a polyubiquitin chain, leading to its rapid disappearance. interaction, suggesting that the oxygen sensing mechanism might impinge directly on this protein interactio. Surprisingly, however, the rst studies indicated that the HIF- /p VHL complex could be retrieved intact from hypoxic cells (29). Given the rapidity of pvhl-dependent proteolysis of HIF- in oxygenated cells, it seemed possible that the apparent association in hypoxia could have arisen in vitro by re-oxygenation of cells during lysis. This was clearly demonstrated by repeating the pvhl co-immunoprecipitation experiments using extracts harvested in an enclosed hypoxic workstation with de-oxygenated buffers (34). Maintenance of the hypoxic setting dramatically reduced the proportion of HIF- associated with pvhl, compared to harvesting in an oxygenated environment. Thus, the classical features of regulation by iron and oxygen availability are re ected in regulation of the HIF- /pvhl interaction. Furthermore, the same features were demonstrated in in vitro interactions between recombinant proteins produced in reticulocyte lysates, and were also observed in interactions with the minimal pvhl-binding domain of HIF-1 outlined previously (34). HIF- is Targeted for pvhl Binding by Oxygen-Regulated Prolyl Hydroxylation The development of an in vitro system for studying interactions between HIF- and pvhl provided a means for detailed biochemical analysis of this process (34, 35) (Fig. 3). These studies showed that whereas HIF- produced in reticulocyte complexes (Skp1/Cdc53/F-box protein), with CUL2 and elongin C resembling yeast Cdc53 and Skp1, respectively. A critical link was established between pvhl and HIF- regulation through the study of renal carcinoma cell lines de - cient in pvhl (29). In a series of such cell lines, HIF- subunits were found to be constitutively stabilized and HIF was activated, leading to increased trascription of reporter genes linked to hypoxia response elements (HREs) and dysregulation of a wide range of hypoxically regulable native genes. Re-expression of wild-type pvhl in stable transfectants restored the normal pattern of oxygen dependent HIF- instability. Following culture of these stable transfectants, in the presence of proteasomal inhibitors, pvhl and HIF- subunits co-immunoprecipitated, demonstrating a physical interaction between pvhl and HIF-. Further experiments con rmed the function of pvhl as part of a ubiquitin ligase complex that targeted HIF- subunits by interaction between the -domain of pvhl and speci c sequences within the HIF- ODDD (30 33) (Fig. 2). Precise domain analysis de ned HIF- residues as a targeting motif for pvhl (34). Regulated Destruction of HIF- How does targeting by pvhl binding lead to oxygen dependent regulation of HIF-? Interestingly treatment of cells with cobaltous ions or iron chelators prevents the HIF- /p VHL Figure 3. (a) In vitro assay for studying interactions between HIF- and pvhl. HIF- incapable of binding pvhl was produced by preparation in the presence of DFO or from nonmammalian sources and puri ed on beads. This substrate was then incubated with a variety of cell lysates, in the presence or absence of iron and/or oxygen, and subsequently tested for its ability to bind radiolabelled pvhl. (b) HIF- incubated with extract and iron in the presence of oxygen is rapidly converted to its pvhl-binding form, whereas in hypoxia this conversion is dramatically reduced.

4 46 MOLE ET AL. lysate could interact with pvhl recombinant HIF- produced in other bacterial or insect cell expression systems could not interact and required modi cation with a mammalian cell extract to promote the ability to capture pvhl. Further experiments showed that the HIF-modifying activity was dependent on the provision of oxygen and iron, was heat labile and temperature dependent, and was not present in an ultra ltrate of cell extract. Mutational and mass spectrometric analysis of the minimal pvhl-binding domain implicated an oxidation of HIF- 1 proline residue 564 as the critical modi cation, and represents a novel targeting modi cation for proteasomal degradation. This modi cation was demonstrated further using the ability of synthesised HIF-1 peptides, containing a trans-4- hydroxy-s-proline at residue 564, to bind pvhl. Whereas wildtype peptides required pretreatment with cell extract to promote pvhl capture, hydroxyproline-substitute d versions could capture pvhl ef ciently without prior exposure to cell extracts (34). These results demonstrated that the enzymatic activity promoting interaction of HIF-1 with pvhl is a prolyl-4- hydroxylase (Fig. 4). Further experiments indicated that HIF- is not a substrate for the known collagen-modifying prolyl hydroxylases, indicating the operation of one or more novel prolyl hydroxylases, which we have termed HIF- prolyl hydroxylase (HIF-PH). To date, all previously described prolyl-4-hydroxylase s are members of the super-family of 2-oxoglutarate-dependent dioxygenases (36). Consistent with this, 2-oxoglutarate analogues competitively inhibit HIF-PH activity in a stereospeci c manner, providing a potential for the development of HIF-activating therapies (34). Implications for Cellular Oxygen Sensing To what extent do these ndings explain the inducible properties of the HIF system? The requirement for dioxygen as cosubstrate provides a direct link to the availability of molecular oxygen and thus the potential for HIF-PH(s) to function directly as a cellular oxygen sensor. Moreover the properties of the catalytic iron centre readily explain the actions of cobaltous ions and iron chelators on HIF. Structural studies of dioxygenases have identi ed a labile nonheme iron centre coordinated by a 2-histidine-1-carboxylate (HXD/E...H) motif (37 ). Interestingly, the iron(ii) can be readily removed by chelating agents and enzyme activity is inhibited by substitution of iron(ii) with cobalt(ii) or nickel(ii) (38). Studies of the catalytic mechanism of other members of this class suggest that substrate binding occurs in the order iron, 2-oxoglutarate, peptide (37 ). Activation of dioxygen by iron most likely occurs though the generation of a reactive ferryl intermediate at the iron centre, with one oxygen atom being incorporated into the prolyl residue and the other yielding succinate via the decarboxylation of 2-oxoglutarate. Whereas the requirement for dioxygen strongly suggests an oxygen-sensing function for HIF-PH, it remains unclear as to what extent and how the properties of such enzymes can account for the full range of physiological regulation displayed by the HIF system. Following isolation of the HIF-PH(s), it will be of interest to determine whether agents that have been shown to modulate the HIF system such as ROS, nitric oxide, and carbon monoxide interact at the catalytic centre, or whether the HIF- PH enzymes are responsive to other signalling pathways via allosteric mechanisms or whether they are themselves subject to transcriptional regulation of expression. Figure 4. Recognition of HIF-1 by pvhl is regulated, at least in part, through an oxygen-, iron-, and 2-oxoglutaratedependent enzymatic hydroxylation of its proline 564 residue, by an enzyme termed HIF prolyl hydroxylase (HIF-PH). The key position of this hydroxylation in the degradation pathway of HIF-, together with its requirement for molecular dioxygen as a cosubstrate, provides the potential for HIF-PH to function directly as a cellular oxygen sensor. REFERENCES 1. Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (1993) Inducible operation of the erythropoietin 3 0 enhancer in multiple cell lines: evidence for a widespread oxygen sensing mechanism. Proc. Natl. Acad. Sci. USA 90, Semenza, G. (2000) HIF-1 and human disease: one highly involved factor. Genes Devel. 14, Wang, G. L., Jiang, B.-H., Rue, E. A., and Semenza, G. L. (1995) Hypoxia-inducibl e factor 1 is a basic-helix-loop-helix-pa S heterodimer regulated by cellular O 2 tension. Proc. Natl. Acad. Sci. USA 92, Huang, L. 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