HIF-1 (2003)

Requirement of Cells for Oxygen All metazoan organisms are committed to respiration, using oxygen as the terminal electron acceptor in a reaction that is carried out by cytochrome c oxidase. The consequence is that cells need a continuous supply of oxygen. In higher organisms such as humans, an extensive infrastructure is required to achieve this. It includes the airways, lungs, heart, and vascular network, but the “organ” most clearly devoted to this task is the erythron—consisting of 2 × 1013 cells, occupying 2 L, and carrying 99% of the oxygen in the blood. The size of this circulating organ is regulated through the action of erythropoietin (EPO), which acts as a survival factor for red blood cell precursors. A fundamental challenge underlying much of the physiology and anatomy of higher organisms is avoiding local oxygen starvation. The efficiency with which we meet this challenge means that it easy to take it for granted. Adding to the precision that is required, excess oxygen is potentially dangerous, with the ability to generate reactive species that may damage proteins and nucleic acid. Hypoxia-inducible factor-1 (HIF-1) is a transcription complex that was isolated as a factor controlling EPO gene expression (1) and is now recognized to be centrally involved in many aspects of meeting these challenges. The HIF-1 system operates in all mammalian cell types examined to date but has specific relevance to the kidney in several ways. First, HIF-1 underlies regulation of EPO, and the kidney is the dominant site of production. Intriguing is that compromised EPO production is a striking feature of many kidney diseases; understanding why this is so could provide insight into the events that lead to continuing renal damage and clues as to how it could be prevented. Second, parts of the kidney experience low oxygen tensions even under normal circumstances (2), and the kidney is very sensitive to hypoperfusion injury (3). Third, constitutive activation of HIF-1 occurs in the great majority of clear cell renal cell carcinomas (CCRCC), which is the most common form of cancer affecting the kidney (4). This is because the von Hippel-Lindau (VHL) tumor suppressor gene has a critical role in the regulation of HIF-1 (5). Furthermore, inheriting a defective VHL gene results in a very high risk of CCRCC and does not predispose to cancer in other organs (6). This suggests that HIF-1 may have a very specific role in controlling the behavior of kidney epithelial cells. This review summarizes how the HIF-1 system works and what it does, with some emphasis on two areas that have been important in understanding how HIF-1 is regulated and are relevant to renal physiology and pathology. These are the control of EPO production and the effect of activation of HIF-1 in renal cells when the VHL tumor suppressor gene is inactivated. HIF-1 Oxygen Response System An HIF-1 complex contains an α and a β subunit. Both subunits are members of multiprotein families and belong to the extended family of basic helix-loop-helix PAS domain transcription factors (1). The β subunit is constitutively expressed, and HIF-1β is also known as the aryl hydrocarbon receptor nuclear translocator. This protein has an important role in responses to xenobiotics, where it forms a heterodimeric transcription complex with the aryl hydrocarbon receptor. The alternate α subunits of the HIF-1 complex, HIF-1α and HIF-2α, are regulated by oxygen and are unique to the oxygen response pathway. How HIF-1 Responds to Oxygen At the heart of the HIF-1 system, molecular oxygen reacts with HIF-α proteins in a way that switches their ability to interact with other proteins (Figure 1). The HIF-α chain is regulated by two enzymatic reactions in which molecular oxygen reacts with specific amino acid residues (7–9). The addition of a single 16-Da oxygen atom to a prolyl residue in the central region of these approximately 120 kD proteins leads to capture by the VHL tumor suppressor protein (7,8). In a second reaction, addition of a single oxygen atom to an asparaginyl residue near the C terminus of HIF-α chains prevents HIF-1 from interacting with the co-activator required to switch on target genes (9). Figure 1. : Regulation of hypoxia-inducible factor (HIF). At low levels of oxygen, HIF-α forms an active HIF-1 complex with a β subunit and recruits P300 (left). As the oxygen concentration increases, the rate of the reactions in the right panel increases. This inactivates HIF-α by hydroxylation of a prolyl residue in the oxygen-dependent destruction domain (Pro402 or 564 of HIF-1α) and an asparaginyl residue in the C terminal transcativation domain (Asn803 of HIF-1α). The prolyl modification is carried out by prolyl hydroxylase domain enzymes and enables capture by the von Hippel-Lindau (VHL) gene. The asparaginyl modification prevents the interaction with P300.Both of these reactions are catalyzed by 2-oxoglutarate–dependent dioxygenases, which belong to an extended family of enzymes with diverse biologic roles (10,11). The extended family includes collagen prolyl 4 hydroxylase (necessary for collagen chain assembly), Alk B (involved in DNA repair), and Phytanoyl Co-A 2-hydroxylase (defects in which underlie many cases of Refsum’s disease) (10–13). The structures of several enzymes in the family (including the HIF asparaginyl hydroxylase FIH-1) have now been solved, and the enzymes are based on a β barrel jelly roll motif, coordinating a non-heme ferrous iron atom at the catalytic center (14–16). The iron is coordinated at three positions, by two histidines and a carboxylate, leaving the other three positions available to interact with oxygen, 2-oxoglutarate, and the prime substrate during the reaction process. In the HIF hydroxylase reactions, one atom of molecular oxygen is used to convert 2-oxoglutarate to succinate and carbon dioxide. The other atom from the oxygen is added to the HIF substrate. Several characteristics of EPO regulation can be explained by the properties of the enzymes. First, EPO production can be provoked by exposure to cobalt. In fact, accidental or experimental exposure of humans to cobalt increases red blood cell production sufficiently to cause erythrocytosis, and it seems that heavy metal exposure contributes to chronic mountain sickness in some areas (17,18). The explanation is that when other atoms are substituted for ferrous iron in the catalytic center, the enzymes can no longer oxidize HIF, which remains able to activate target genes despite the presence of oxygen. Second, depleting intracellular iron can also inactivate the enzymes, explaining why iron chelators can mimic the effect of hypoxia in cultured cells and why systemic administration of desferrioxamine in humans and mice can increase EPO production (19,20). HIF transactivation is regulated by hydroxylation of an asparaginyl residue (9). In the absence of this modification, HIF-α interacts with the CH1 pocket of CBP/P300, which results in transactivation of target genes (21,22). However, in the presence of oxygen, the enzyme FIH-1 (for factor-inhibiting HIF) adds an oxygen to the β carbon of Asn803 of HIF-1α, or the equivalent residue in HIF-2α (23–25). This modification is highly unfavorable to the interaction with CBP/P300 and thus prevents HIF-α from recruiting the basal transcription machinery when cells are oxygenated. HIF destruction is regulated by conversion of a prolyl residue in the middle portion of the molecule. The system is most clearly understood in the nematode Caenorhabditis elegans, where it is less complicated than in mammals and more genetic information is available. C. elegans has a single isoform of each of HIF-α, the hydroxylase enzyme egl9, and the E3 ligase recognition component vhl. In the presence of oxygen, egl9 converts Pro621 of hif1 to 4-hydroxyproline (26). The modified hif1 is then captured by vhl, which leads to ubiquitylation and destruction. In low oxygen, hif1 is not oxidized (because egl9 requires molecular oxygen as a co-substrate) and is stabilized. Worms that carry mutant alleles for vhl or egl9 show constitutive activation of hif1. Complexity of HIF-α Prolyl Hydroxylation in Mammals In mammals, the HIF system is more complicated than in the worm. This is not entirely surprising given that the worm has no specialized oxygen distribution systems. Thus, in mammalian cells, there are at least two different oxygen-responsive HIF-α chains, termed HIF-1α and HIF-2α, both of which are subject to VHL-dependent destruction by the proteasome (5). Homozygous genetic of HIF-1α or HIF-2α in the is that in these proteins are not It is to what HIF-1α and HIF-2α may be for of HIF-1 genes when the proteins are at normal levels and the target genes are in their normal HIF-2α was to be it is by a very of cell types cells in the kidney HIF-2α, than HIF-1α, in response to hypoxia suggests that HIF-2α may be involved in control of EPO production This is by the that HIF-2α expression EPO production in hypoxia has also been to be a target for prolyl hydroxylation and ubiquitylation An of this which the transactivation can as a dominant of the HIF response and seems to be important in the response in such as the In mammalian HIF-1α and HIF-2α, two different prolyl residues can be subject to oxygen-dependent hydroxylation by VHL capture of site HIF-α chains form but of both increases the different prolyl hydroxylase genes on the of to egl9 and other have been termed and (for prolyl hydroxylase (26). are to (for HIF prolyl and to (for of of the three enzymes is of the target prolyl residues in mammalian HIF-α chains in in and of each enzyme has been to As the of these three enzymes to the regulation of HIF-1 is not but in a of the prolyl hydroxylase from isolated An HIF prolyl hydroxylase gene has been termed The of the three different prolyl hydroxylase enzymes is the hydroxylation reaction is not at it is that the of hydroxylase enzyme the HIF response to of the prolyl hydroxylase genes can be in cells, which is to provide an of on the HIF-1 response Furthermore, it is clear that the expression of the three genes is from one cell to and that expression is by many In addition to the hydroxylation HIF is in other ways. HIF is and was to be and these to HIF there is for in the way that HIF operates in mammalian cells, which is important in the basic oxygen response system to different of the VHL in HIF Regulation The VHL tumor suppressor protein is critical for the normal regulation of HIF-1 because it is required to HIF-α subunits in the presence of oxygen. In this VHL acts as the recognition component of a E3 ligase complex that includes several other and The that VHL is required for HIF regulation in renal cell (5). it is now clear that VHL is for regulated HIF destruction in many other cell types in metazoan because VHL in C elegans, cells, cells, cells, and results in constitutive HIF activation in each hydroxylation of or HIF-1α is captured by the VHL tumor suppressor protein A of the complex of the HIF target with B and C that the into a with hydroxylation the of two HIF-α chains are and by the In cells that HIF-α chains are constitutively and the HIF-1 system is even when oxygen is (5). 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