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PART I

The Kidney as an Endocrine Organ

Section I. Erythropoietin

CHAPTER 1

Erythropoietin: An Historical Overviewof Physiology, Molecular Biologyand Gene Regulation

DAVID R. MOLE AND PETER J. RATCLIFFEHenry Wellcome Building for Molecular Physiology, University of Oxford, Headington Campus, Roosevelt Drive, Oxford, UK

Contents

I. Introduction 3II. Hormonal regulation of erythropoiesis 4III. Identification of the site of erythropoietin

production 5IV. Assays of erythropoietin 5V. Isolation and characterization of erythropoietin 5VI. Erythropoietin effector mechanisms 6VII. Regulation of erythropoiesis by hypoxia 6VIII. Regulatory elements of erythropoietin

(EPO) gene 7IX. Erythropoietin – the paradigm for gene

regulation by hypoxia 8X. Hypoxia inducible-factor (HIF) 10XI. The elusive nature of the oxygen sensor 10XII. Degradation of HIF by the ubiquitin-proteosomal

pathway 12XIII. Disruption of the oxygen-sensing pathway

in cancer 14XIV. Disruption of the oxygen-sensing pathway in

hereditary polycythemia 15XV. Pharmacological manipulation of HIF 16XVI. Summary 16

References 16

I. INTRODUCTION

Although generally ascribed to the 19th century physicians,Richard Bright, Robert Christison and Pierre Rayer, the linkbetween kidney disease and anemia was first described18 centuries earlier by the Greek physician Aretaeus theCappadocian who noted: ‘In all the species there are presentpaleness, difficulty of breathing, occasional cough; they aretorpid, with much languor’. In recent years, studies into theregulation of red blood cell production by the renal hormone

erythropoietin have not only confirmed this link, but havealso provided effective therapeutic strategies for renal ane-mia and fundamental molecular insights into mechanisms ofoxygen sensing and signaling that underlie oxygen homeo-stasis throughout the animal kingdom (see Table 1.1 forsummary).

However, following Aretaeus’s description, many yearswere to pass before key discoveries in the 17th and 18thcenturies defined the importance of oxygen and oxygentransport systems to living organisms, and provided thefundamental platform necessary for modern understanding.Indeed, the description of the blood circulation by WilliamHarvey (1578–1657) in De Motu Cordis et Sanguinis inAnimalibus in 1628 left open the question of its purpose.Richard Lower (1631–1691), working in Oxford with RobertHooke (1635–1702), noted that whereas the blood leavingthe heart for the lungs was blue, that returning from the lungsto the heart was red. Lower mixed blood with air in a glassvessel and noted the same color change, concluding that:‘Nitrous spirit of the air, vital to life is mixed with the bloodduring transit through the lungs’. Furthermore, by means ofthe vacuum pump specially contrived by himself and RobertHooke, Robert Boyle (1627–1691) was able to obtain ‘air’from blood in 1670.

For centuries, it had been recognized that there was someactive part in the air. The Chinese had called it ‘yin’. TheItalian polymath, Leonardo da Vinci (1452–1519), had stat-ed that the air was not completely consumed in respiration orcombustion and had claimed that there were two gases in theair. Robert Boyle had shown that a component of air wasdepleted by living animals. However, the nature of this ‘spiritof the air’ was to remain elusive for another 100 years, in partdelayed by the erroneous phlogiston theory of combustion.By heating mercuric oxide to release a gas that supportedcombustion and respiration, Priestley and Scheele identifiedthe essential ‘dephlogistated air’ or ‘fire air’, although bypublishing first, in 1774, Priestley is commonly credited with

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the discovery. However, it was Lavoisier who overturned thephlogiston theory and, in 1777, coined the term oxygen,correctly describing the chemistry of combustion and con-cluding that biological energy metabolism was essentiallythe same process. Perhaps because of Lavoisier’s untimelyend, under the guillotine during the French Revolution, theterm did not come into general use until it was popularized inthe book ‘The Botanic Garden’ by Erasmus Darwin, grand-father of Charles Darwin, ‘The enamour’d oxygene. Thecommon air of the atmosphere appears by the analysis ofDr Priestley and other philosophers to consist of . . . aboutone-fourth of pure vital air fit for the support of animal lifeand of combustion called oxygene’.

The first consistent measurements of oxygen in bloodwere performed by Gustav Magnus in 1837. In showing thatthere was more oxygen in arterial than venous blood, heconfirmed the role of the blood circulation in deliveringoxygen to the tissues. He also showed that blood containedmore oxygen than could be accounted for by simple solubil-ity and that the uptake of oxygen by the blood could beblocked by carbon monoxide suggesting a specific carriermechanism. It was Hoppe-Seyer, in 1862, and Stokes, in1864, who demonstrated the reversible binding of oxygento the pigmented hemoglobin in the red cells that accountedfor the color change and facilitated the transport of oxygenby the blood. By independently demonstrating the produc-tion of red cells in the bone marrow, in a process initiallytermed hematopoiesis, or latterly and more specificallyerythropoiesis, Ernst Neumann and Giulio Bizzozero, in

the 1870s, set the scene for subsequent studies into the reg-ulation of this process.

II. HORMONAL REGULATIONOF ERYTHROPOIESIS

It was only a few years after the concept of hormones wasfirst suggested by Henri Brown-Sequard in 1889, that theidea of hormonal regulation of erythropoiesis was first for-mulated by Carnot and Deflandre in 1906 (Carnot andDeflandre, 1906). Their experiments involved injecting se-rum from rabbits, rendered anemic by venesection, into nor-mal rabbits leading to an increased concentration of redblood cells in the recipients within 1–2 days. They concludedthat the transferred serum contained a hematopoietic factorthat they termed ‘haematopoı̈etine’. Subsequently, haemato-poı̈etine would be substituted by the more specific term‘erythropoietin’. However, initially, the existence of haema-topoı̈etine was doubted for many years because, with a fewexceptions, most investigators failed to reproduce the resultsof Carnot and Deflandre. Interest in the possibility of ahumoral factor controlling erythropoiesis was rekindled fol-lowing observations in parabiotic animal pairs in 1950. KurtReissmann and Gerhard Ruhenstroth-Bauer observed thatinduction of anemia or hypoxemia in one of the parabioticanimals would induce erythrocytic hyperplasia and reticulo-cytosis in the partner. Allan Erslev (1919–2003) is generallycredited with providing definitive proof of the existence of

TABLE 1.1 Erythropoietin timeline

Date Event

1st century AD Aretaeus described anemia in chronic kidney disease1590 First description of the effects of altitude on the human body by Father Joseph De Acosta1628 Discovery of the circulation of the blood by William Harvey1774 Discovery of oxygen by Priestley and Scheele1837 First measurement of blood oxygenation1862/4 Description of the oxygen transport function of hemoglobin by Hoppe-Sayer/Stokes1863/1878 Description of the effects of altitude on blood concentration by Jourdanet/Bert1906 Hormonal regulation of erythropoiesis first postulated by Carnot and Deflandre1953 Definitive proof of the existence of erythropoietin provided by Erslev1956 First bioassay of erythropoietin activity1974 Direct proof that erythropoietin is produced in the adult kidney demonstrated by Erslev1977 Purification, sequencing and cloning of erythropoietin1979 Radioimmunoassay for erythropoietin developed1986 First clinical use of recombinant erythropoietin1989 Molecular identification and cloning of erythropoietin receptor1991 Identification of the hypoxia response element in the 30 erythropoietin enhancer1992 Identification of hypoxia-inducible factor (HIF)1993 Demonstration of the universal nature of the oxygen-sensing mechanism1995 Biochemical purification, molecular identification and cloning of cDNA encoding HIF1999 Demonstration of von Hippel-Lindau (VHL) dependent proteasomal degradation of HIF-a subunits2001 Oxygen-sensing process defined as oxygen-dependent prolyl hydroxylation by non-heme (FeII)-dependent dioxygenases

4 PART I � SECTION I � Erythropoietin

erythropoietin in 1953, by transfusing large quantities ofplasma from anemic rabbits. Plasma from anemic animals(but not control animals) generated a significant reticulocy-tosis in the recipient animal which, after repeated dosing,resulted in a rise in the hematocrit (Erslev, 1953). Withremarkable foresight, he also postulated: ‘Conceivablyisolation and purification of this factor would provide anagent useful in the treatment of conditions associated witherythropoietic depression, such as chronic infection andchronic renal disease’.

III. IDENTIFICATION OF THE SITE OFERYTHROPOIETIN PRODUCTION

The clinical observation that patients suffering fromhypoxemia to the lower portion of the body due to a patentductus arteriosus showed generalized erythroid hyperplasia,suggested a link between the lower part of the body and thestimulation of erythropoiesis. This was consistent with theobservation that patients suffering from significant renalimpairment were frequently anemic. The important role ofthe kidney in erythropoietin production became apparentwhen Leon Jacobson (1911–1992) and Eugene Goldwassershowed that nephrectomized rats failed to respond tovenesection or cobalt chloride with the normal increase inerythropoietin activity, while the response was intact in ratssubjected to hypophysectomy, thyroidectomy, splenectomy,adrenalectomy and gonadectomy (Jacobson et al., 1957).Nevertheless, the failure of attempts to extract erythropoietinfrom the kidney led to doubt that the kidney was the directsource of erythropoietin. Instead, an alternative hypothesiswas advanced in which the kidney secreted an enzyme(erythrogenin) that cleaved erythropoietin from a plasmaprotein. However, erythropoietin could not be reliably gen-erated by the addition of kidney extract to normal plasma.The erythrogenin concept was finally disproved by Erslev, aslate as 1974, by the demonstration of erythropoietin activityin isolated serum-free perfused kidneys from hypoxicrabbits (Erslev, 1974). Further confirmation that the kidneyproduces erythropoietin directly came with the isolation oferythropoietin mRNA from the kidneys of hypoxic rodents(Beru et al., 1986). While mRNA studies confirmed theresults of organ ablation studies in showing that the mainsites of erythropoietin synthesis were the kidney in the adultand the liver in fetal and neonatal life, the spleen, lung, bonemarrow, brain and testes were all shown to express smallamounts of erythropoietin mRNA. The translational efficien-cy and potential function of erythropoietin produced in thesesites is not known. For instance, it is unlikely that erythro-poietin produced in the brain enters the systemic circulationbecause of the blood–brain barrier. The expression of eryth-ropoietin receptor (EpoR) in the brain and the ability oferythropoietin to protect the brain from ischemic insulthas led to the assumption that erythropoietin may act as a

paracrine neuroprotective factor, though the physiologicalfunction of such an action is unclear (Sakanaka et al., 1998).

Within the kidneys of hypoxic rats, erythropoietin activ-ity was mainly found in the cortex and not in the medulla.Again, this result was borne out by later mRNA studies,which demonstrated erythropoietin expression in the inter-stitium of the renal cortex and showed co-localization withfibroblast markers implicating this cell lineage in renalerythropoietin production (Maxwell et al., 1993a).

IV. ASSAYS OF ERYTHROPOIETIN

Early erythropoietin research was hampered by the lowconcentration of the hormone in the fluids and tissues tobe studied, particularly in the basal state, which made itsdetection and quantitation unreliable. The first assays oferythropoietin activity utilized the rate of incorporation ofradioactive iron-59 into hemoglobin as a measure of eryth-ropoiesis in starved rats that had been injected with thematerial under test (Jacobson et al., 1956). These assays wererendered more sensitive by using ‘ex-hypoxic’ polycythemicmice to reduce the rate of background erythropoiesis.Initial standardization employed the ‘cobalt unit’ in whichone unit produced the same erythrogenic response in the testanimals as 5 micromol cobalt chloride. Later reference stan-dards included preparations of sheep plasma, human urinaryerythropoietin and, in 1992, a fully glycosylated purifiedrecombinant human erythropoietin.

In vivo bioassays were costly, time-consuming andlacked precision and sensitivity. Several more sensitivebioassay methods using cell culture were described. Thesemethods were generally applicable to purified erythropoietinsamples, but were often affected by non-specific inhibitorspresent in crude samples. Such methods were eventuallyreplaced by radioimmunoassay in the late 1970s andearly 1980s. Today, there are many commercially availableenzyme-linked immunoassay kits available for the determi-nation of erythropoietin levels.

V. ISOLATION AND CHARACTERIZATIONOF ERYTHROPOIETIN

Armed with the early bioassays of erythropoietic activity,researchers next turned their attention to the biochemicalpurification of erythropoietin. Early attempts at partial puri-fication of erythropoietin from anemic rabbit serum provedremarkably informative. The erythropoietic activity wasfound to have an electrophoretic mobility similar to alpha-2globulin, to be heat stable and to stain for carbohydrate.Erythropoietin was therefore deduced to be a glycoprotein.These studies also showed that erythropoietin containedhexose, hexosamine and sialic acid and that erythropoieticactivity was lost upon removal of neuraminic acid.

Chapter 1 � Erythropoietin: An Historical Overview of Physiology, Molecular Biology and Gene Regulation 5

In a mammoth effort, first ovine erythropoietin waspurified over a million-fold from anemic-sheep plasma andthen human erythropoietin was purified from 2550 liters ofurine from patients with aplastic anemia (Miyake et al.,1977). The purified human erythropoietin was subjected totryptic digestion and the resulting fragments separated andsequenced. The partial amino acid sequences obtained en-abled DNA probes to be made, which were then used toprobe both genomic and cDNA libraries to identify andsubsequently clone the erythropoietin gene (Jacobs et al.,1985; Lin et al., 1985). Expression of the erythropoietincDNA in Chinese hamster ovary cells resulted in productionof biologically active erythropoietin (Lin et al., 1985).

The human erythropoietin gene is a single copy genecontaining five exons and is located on the long arm ofchromosome 7 (7q11–q22). It encodes a 193-amino acidprohormone, from which a 27-residue leader sequence, aswell as the carboxy-terminal arginine are cleaved prior tosecretion. The resulting 165 amino acid, mature humanerythropoietin is an acidic glycoprotein with a molecularmass of 30.4 kDa that contains two bisulfide bridges. Circu-lating erythropoietin has several glycosylation isoforms with40% of the molecule consisting of carbohydrate comprisingthree tetra-antennary N-linked (Asn24, Asn38 and Asn83) andone small O-linked (Ser126) glycans. The N-linked glycansare essential for the biological activity of erythropoietin andcontain terminal sialic acid residues that protect the wholemolecule from removal by galactose receptors expressed onhepatocytes. The addition of further N-linked glycans torecombinant erythropoietin by site-directed mutagenesishas been used to prolong in vivo activity of the molecule(Egrie and Browne, 2001).

The cloning of the human erythropoietin gene and theproduction of recombinant human erythropoietin led veryquickly to its clinical application in the treatment of theanemia of chronic kidney disease (Winearls et al., 1986;Eschbach et al., 1987), thereby fulfilling Erslev’s earlierprediction.

VI. ERYTHROPOIETIN EFFECTORMECHANISMS

It is now nearly 50 years since self-replicating hematopoieticstem cells were first demonstrated in the bonemarrow (Till etal., 1961). Derived from these pluripotent stem cells, theerythroid lineage comprises ‘the burst-forming uniterythroid’ (BFU-E) and the more differentiated ‘colony-forming unit erythroid’ (CFU-E). Each BFU-E can generate50–200 erythroblasts when exposed to high concentrationsof erythropoietin. The CFU-Es are more sensitive to theeffects of erythropoietin with the number of colonies oferythroblasts derived from each increasing in response tomore modest erythropoietin concentrations. In 1992, Kouryand Bondurant demonstrated that erythropoietin promotes

red cell formation by preventing apoptosis in these celllineages (Koury and Bondurant, 1992).

Although evidence for a cell-surface erythropoietin re-ceptor was first provided in 1974, it was not until 15 yearslater that the murine erythropoietin receptor was first clonedand characterized as belonging to the cytokine class I recep-tor family (D’Andrea et al., 1989). The receptor exists as ahomodimer and crystallization studies reveal a conforma-tional change on binding of erythropoietin that leads toactivation of Janus kinase 2 (JAK2), which interacts withthe cytoplasmic region of the receptor. The affinity of eryth-ropoietin analogs for the receptor decreases with increasingglycosylation (see Chapter 4).

VII. REGULATION OF ERYTHROPOIESISBY HYPOXIA

The first description of the effects of altitude on the humanbody is generally attributed to Father Joseph De Acosta(1539–1600), a Jesuit priest who made observations duringhighland expeditions following the Spanish conquest ofSouth America. However, techniques for measuring hemo-globin concentration and red cell counts were not availableuntil the mid-19th century, so it was not until this time thatthe effects of altitude on the blood were first described.During the attempted French colonization of Mexico, DenisJourdanet (1815–1892) observed the blood of patients ataltitude to be thick, dark and to flow slowly and, on measur-ing the number of red blood corpuscles, found them to beraised despite the patients having the symptoms of anemia(Jourdanet, 1863). His prot�eg�e, Paul Bert (1833–1886),Professor of Physiology at the Sorbonne in Paris, and laterto become governor-general of French Indo-China, noted thesame phenomenon in animals living at altitude (Bert, 1878).Both workers felt that these changes were acquired overgenerations. However, on traveling from Bordeaux to Mor-ococha, in Peru, at 4500 m above sea level, Viault noted anincrease in erythrocytes within 23 days. Following this, itwas Mabel Fitzgerald (1872–1973) (a colleague of J.S. Hal-dane on the 1911 expedition to Pike’s Peak, Colorado tostudy breathing responses at altitude) who first clearly de-scribed the great sensitivity of this response, illustrating thatrelatively minor reductions in barometric pressure at modestaltitude were associated with a discernable elevation ofhematocrit (FitzGerald, 1913).

These results suggested a link between physiologicalhypoxia and erythropoiesis. However, there is also a linkbetween pathological hypoxia and polycythemia. For exam-ple, in conditions such as chronic lung disease or cyanoticheart disease, which result in systemic hypoxemia, thehematocrit is frequently raised.

Later recognition of the role of erythropoietin inregulating erythropoiesis (see above) was rapidly followedby studies of the effect of hypoxia on erythropoietin itself,


confirming the great sensitivity of erythropoietin productionto hypoxia. For instance, in the 1960s, Faura and colleaguesdemonstrated increased erythropoietin activity in responseto hypoxia in lowlanders taken to Morococha in Peru, whileprevious animal work had shown a link between hypoxiaand erythropoietic activity. Abbrecht and Littell showedthe rapid and transient nature of the erythropoietin responseduring a high altitude expedition to Colorado (4360 m) witherythropoietin peaking at 1–3 days after arrival at the newaltitude and falling close to baseline by day 10 (Abbrecht andLittell, 1972).

Given that the teleological purpose of erythropoietin is tomaintain the blood hemoglobin concentration in the normalrange and that the main function of hemoglobin is to carryand deliver oxygen from the lungs to the respiring tissues, itis appropriate that the sensed parameter in the feedback loopis tissue oxygenation. However, given that the kidney has nodirect role in erythropoiesis or oxygen transport, it is notimmediately apparent why the kidney is so well suited tosensing oxygen.

A possible explanation for this paradox lies in the unusu-al anatomical and physiological characteristics of the highlyspecialized blood supply to the kidney. In order to maintainthe osmotic gradient generated by the loop of Henle, thearterial and venous blood vessels supplying the renal tissuesrun countercurrent and in close contact. This leads to shuntdiffusion of oxygen between the arterial and venous circu-lation and the generation of an oxygen gradient throughoutthe renal parenchyma (Bauer and Kurtz, 1989). Consequent-ly, oxygen tensions fall with increasing distance from therenal surface reaching levels below 10 mmHg within themedulla. Furthermore, the glomerulus and tubule are sup-plied by the same network of vessels and, because the renaloxygen demand, arising from the work of tubular reabsorp-tion, varies in proportion with the glomerular filtration rate,the oxygen gradient is little affected by the rate of bloodflow. Therefore, as the oxygen concentration of the blood(as determined by hemoglobin concentration) falls, so thehypoxic regions of the kidney propagate outwards andincreasing numbers of peritubular fibroblasts are recruitedto produce erythropoietin in a ‘march’ effect (Koury et al.,1989). The induction of erythropoietin in this manner showsa strikingly high gain response, with even modest changes inhemoglobin leading to large changes in erythropoietin overseveral orders of magnitude.

VIII. REGULATORY ELEMENTS OFERYTHROPOIETIN (EPO) GENE

The cloning of the erythropoietin gene provided the oppor-tunity to study the regulatory pathway by examining theeffects of non-coding ‘cis-acting’ sequences on expression.Transgenic studies in mice demonstrated a role for long-range sequences, lying both 50 and 30 to the gene, in directing

erythropoietin expression to the kidney and liver, respective-ly. The mechanisms by which these enhancer sequencesinteract with local sequences at the erythropoietin promoterto direct tissue-specific expression are still unclear. At thepromoter itself, the 50 flanking sequence contains a GATAbinding site and it has been proposed that the fall in GATA-4in hepatocytes during the transition from fetal to adult lifemay underlie the switch in erythropoietin production afterbirth. Nevertheless, transgenic studies demonstrate that dis-tant 30 sequences are also needed for proper suppression ofhepatic erythropoietin expression in adult mice (Figure 1.1).In contrast, GATA-2 appears to inhibit erythropoietin geneexpression. In addition, nuclear factor kB (NF-kB) alsobinds to the erythropoietin 50 promoter and it has been pro-posed that enhanced NF-kB and GATA-2 activity might beresponsible for the suppression of erythropoietin productionseen during systemic inflammation (Ebert and Bunn, 1999).

An important finding in the transgenic mouse studies wasthat local sequences, including a hypersensitive site lying 30

to the erythropoietin gene, were sufficient to direct oxygen-sensitive expression, at least in the liver. Further analysis ofthese sequences was facilitated by the use of erythropoietinproducing hepatoma cell lines that demonstrated the samedynamic hypoxic response as the whole organism, and couldbe studied in tissue culture. Using these cell lines, reporterassays, in which 30 erythropoietin flanking sequences werefused to DNA encoding a heterologous protein, defined atranscriptional enhancer, lying 30, responsible for hypoxicregulation of erythropoietin expression. (For review, seeStockmann and Fandrey, 2006.)

These studies defined a specific point of interaction be-tween the oxygen-sensitive signaling system (at least asmanifest in the erythropoietin-producing hepatoma cells)and the erythropoietin gene locus. Unexpectedly, however,transfection studies of the 30 erythropoietin enhancer alsodemonstrated oxygen-regulated activity after introductioninto a wide variety of cells, regardless of whether the cellsproduced erythropoietin, or were derived from an erythro-poietin-producing organ. Thus, it became clear that the high-ly specific and sensitive response to hypoxia that wasmanifest in the erythropoietin-producing tissues was, in fact,a general property of mammalian cells, irrespective ofwhether they produced erythropoietin and therefore pre-dicted to have many other functions (Maxwell et al., 1993b).

Studies of proteins binding to erythropoietin 30 sequencesrevealed a trans-acting nuclear factor, termed hypoxia-in-ducible factor-1 (HIF-1), that was induced by hypoxia in aprocess requiring uninterrupted protein synthesis and whichbound to the erythropoietin gene enhancer at a site criticallyrequired for oxygen-dependent transcriptional activation(Semenza and Wang, 1992).

Taken together, these findings prompted a search forgenes other than erythropoietin that contained similar cis-acting sequences (termed hypoxia response elements) andwhich responded to HIF-1.


IX. ERYTHROPOIETIN – THE PARADIGMFOR GENE REGULATION BY HYPOXIA

Unexpectedly, the first such genes to be identified were thoseencoding the enzymes phosphoglycerate kinase and lactatedehydrogenase, genes which, though modestly inducible byhypoxia, show a much lower amplitude of induction thanerythropoietin. It is now clear that HIF target genes areinvolved in a wide range of cellular and systemic responsesto hypoxia whose dynamics differ quite markedly from thoseof erythropoietin production (Wenger, 2002) (Figure 1.2).

HIF targets included glucose transporters and keyenzymes in the glycolytic pathway such as GLUT1 (glucoseuptake), 6-phosphofructo-1-kinase L, enolase, pyruvate ki-nase, in addition to lactate dehydrogenase A. Hence, duringconditions of oxygen deficiency, under which oxidativephosphorylation cannot proceed, HIF coordinately upregu-lates the less efficient glycolytic pathway and facilitatesconversion of the resultant pyruvate to lactate for export tothe liver. In addition, in hypoxia, HIF also upregulates pyru-vate dehydrogenase kinase (PDK), which phosphorylatesand inactivates the pyruvate dehydrogenase enzyme com-plex that converts pyruvate to acetyl-coenzyme A, therebyinhibiting pyruvate entry into the tricarboxylic acid (TCA)

cycle and hence directly suppressing oxidative metabolism(Brahimi-Horn and Pouyssegur, 2007).

Also important in limiting oxygen demand are cell-baseddecisions involving cell proliferation and apoptosis throughthe expression of genes encoding apoptotic regulators suchas B-cell lymphoma-2 family members (Bcl-2) and thecell cycle regulators p21 and p27, which are themselvesregulated either directly or indirectly by the HIF transcrip-tional cascade.

Increased oxygen delivery and erythropoiesis requiresnot only stimulation of the bone marrow by erythropoietin,but also coordinated iron provision. HIF contributes to this,through processes that include enhanced gastrointestinaluptake through downregulation of the iron-regulatoryhormone, hepcidin, in addition to upregulation of transferrin,the iron carrier protein, and transferrin receptor mediatingcellular iron uptake.

During localized hypoxia, vasomotor tone is subject tocontrol by HIF-mediated transcriptional regulation of factorssuch as endothelin 1, inducible nitric-oxide synthase (iNOS),endothelial nitric-oxide synthase (eNOS), and heme oxyge-nase-1 (HO-1), endothelin-1 and atrial natriuretic peptide(ANP), though it remains unclear how these responses arecoordinated in the overall regulation of vascular tone.

HIF-1α

HIF-1β

HNF4HNF4

HIF

HA

FG

ATA

P300/CB

P

RN

A p

ol II

promoter

TATA

transcription

HAF

GATA2

GATA4

KIE NRE LIE NRLE

Distant regulatoryelements

HR

ED

R-2

enha

ncer

exon 1 exon 2 exon 3 exon 4 exon 5

erythropoietin mRNAcoding region

5’ 3’

−

+

FIGURE 1.1 Regulatory elements of theerythropoietin gene. This schematic repre-sentation of the erythropoietin gene showsthe promoter region, the five exons andthe 30 erythropoietin enhancer. Additionalcis-acting regulatory are required for tissue-specific and developmental regulation.The kidney inducible element (KIE) confersexpression in interstitial peritubular cells.The negative regulatory element (NRE) sup-presses transcription. Two cis-acting regula-tory elements 30 to the erythropoietin codingsequence, the liver-inducible element (LIE)and the negative regulatory liver element(NRLE) promote and enhance gene expres-sion in the liver. At the promoter, 50

sequences contain a ‘GATA’ motif thatregulates opposing effects of diverse GATAfactors. While the erythropoietin promoter isa weak promoter, it does contribute to hyp-oxic regulation through a hypoxia-associatedfactor (HAF) binding site. However, the 30

enhancer containing a hypoxia-responsiveelement (HRE) that binds hypoxia-induciblefactor (HIF) is responsible for the majority ofthe fold-induction of the erythropoietin genein response to hypoxia. In addition, the directrepeat of two steroid hormone receptorhalf-sites (DR2) confers regulation throughbinding of hepatic nuclear factor 4 (HNF-4).


In the adult, the vasculature is usually quiescent, withendothelial cells being among the longest-lived cells outsidethe nervous system. However, during the physiologicalprocesses of growth and development, wound healing andproliferation, as well as pathological conditions arisingin neoplasia, ischemia and inflammation, localized oxygendemand may exceed supply, leading to new blood vesselgrowth (angiogenesis) directed at restoring this balance.Both vascular endothelial growth factor (VEGF) and itsreceptor Flt-1 are transcriptionally activated by HIF andthis alone is capable of initiating angiogenesis in quiescentvessels. However, for an efficient vasculature to be formed, amore coordinated response is necessary, involving othergrowth factors such as angiopoietin and its receptor Tie-2,fibroblast growth factor (FGF) and platelet derived growth

factor (PDGF), in addition to the balanced control of matrixmetalloproteinases and tissue inhibitors of matrix metallo-proteinases (TIMPs). Again, all these processes appear to bedirectly or indirectly responsive to HIF (Pugh and Ratcliffe,2003). Though the detail of how responses are coordinated isunclear, activation of HIF does appear to be sufficient togenerate an effective angiogenic response that can enhanceor restore oxygen delivery.

Inflammatory tissues that are injured by infectious pro-cesses, trauma or other causes are characterized by lowglucose levels, high lactate concentration with resultantlow pH and, frequently, extreme degrees of hypoxia. Thisresults from a combination of changes in metabolic activity,an increased diffusion distance, disruption of blood flowthrough phagocytic plugging and damage to capillaries or,

FIGURE 1.2 Transcriptonal targets ofHIF. A representation of the increasingnumber of genes regulated by the HIFoxygen-sensing transcriptional pathway isillustrated. Broadly, these act to increaseoxygen delivery systemically by promotingerythropoiesis and iron delivery and at thetissue level by promoting angiogenesis andcontrolling vascular tone, in addition to re-ducing oxygen consumption by inhibitingTCA (tricarboxylic acid) cycle metabo-lism, promoting anaerobic glycolysis andmodulating cell proliferation and apopto-sis. Glyceraldehyde-3-phosphate dehydro-genase (G3PHD), B-cell CLL/lymphoma 2(Bcl-2), vascular endothelial growth factor(VEGF), fms-related tyrosine kinase 1/VEGF receptor (Flt-1), epidermal growthfactor (EGF), plasminogen activator inhib-itor-1 (PAI-1), endothelium-specific recep-tor tyrosine kinase 2 (Tie-2), tissueinhibitor of matrix metalloproteinase-1(TIMP-1), inducible nitric-oxide synthase(iNOS), endothelial nitric-oxide synthase(eNOS), atrial natriuretic peptide (ANP).


in the case of an abscess, complete avascularity of certainregions. Myeloid cells, key effectors of the innate immuneresponse, have evolved several HIF-dependent survival strat-egies to cope with this hypoxia, including enhanced glycol-ysis, inhibition of NF-kB dependent apoptosis and enhanceddiapedesis of neutrophils into hypoxic regions (Cramer et al.,2003). Within nephrology, this hypoxic upregulation of theimmune system is of importance not only in the modulationof autoimmune inflammation, but also in the regulation ofalloimmune inflammation, following the ischemic insultsustained during transplantation.

X. HYPOXIA INDUCIBLE-FACTOR (HIF)

Using DNA-affinity chromatography, HIF-1 was purified tohomogeneity allowing the determination of partial aminoacid sequence and identification of the encoding cDNAs.This revealed that HIF-1 is a heterodimer of the novel HIF-1a subunit (120 kDa) and a HIF-1b subunit (91–94 kDa)previously identified as the aryl hydrocarbon nuclear receptortranslocator (ARNT). In common with many transcriptionfactors, these proteins have distinct functional domains. Eachprotein was found to contain both a basic-helix-loop-helix(bHLH) motif (residues 17–70), common to many transcrip-tion factors, and a PAS domain, defined by its presence in theDrosophila Per and Sim proteins and in the mammalianARNT and AHR proteins (Wang et al., 1995). PAS domainscontain two internal homology units, the A and B repeats(Figure 1.3) and are implicated in protein–protein interac-tions. Residues 1–166 of HIF-1a are sufficient for heterodi-merization with HIF-1b, but residues 1–390 are required toeffect proper DNA binding. Consistent with the role of thebasic-helix-loop-helix (bHLH) domain in DNA binding oftranscription factors, deletion of the basic region (residues4–27) was found to abrogate DNA binding.

HIF-1b orARNTwas first identified frommutational andcomplementation studies, on mouse hepatoma (Hepa-1)cells, as essential for the transcriptional response to certainenvironmental hydrocarbons termed the xenobioticresponse. It is expressed constitutively and, with alternativedimerization partners, it functions in a range of transcrip-tional systems. Thus, in the xenobiotic response, ARNTforms a heterodimer with another basic helix-loop-helixPAS protein, the aryl hydrocarbon receptor (AHR), whichthen binds the xenobiotic responsive element, a controlsequence for genes such as CYP1A1, a cytochrome P450reductase that can convert aryl hydrocarbons to toxic orcarcinogenic metabolites.

In contrast, both HIF-1a and a closely related proteinHIF-2a, identified bioinformatically as a homologue ofHIF-1a, were found to be highly and specifically regulatedby hypoxia, a process shown to be mediated through rapiddegradation in the presence of oxygen (Salceda andCaro, 1997) and dependent on a central oxygen dependent

degradation domain. Further regulatory domains were iden-tified within the C-terminal portion of HIF-1a and HIF-2a,but not HIF-1b, that mediate hypoxia inducible transactiva-tion in a manner that is independent of protein level andinvolves regulated binding to p300/CBP. Thus, these studiesindicated that oxygen specifically regulates at least twoindependent properties of HIF-a subunits.

XI. THE ELUSIVE NATURE OF THEOXYGEN SENSOR

Although it was known that erythropoietin levels could beincreased several hundred-fold within hours of hypoxic stim-ulation, the exact nature of the oxygen-sensing mechanismthat regulated HIF remained elusive and the subject of muchdebate.

Interestingly, in mammals, it had long been observed thatcertain transition metal ions, such as cobalt(II), manganese(II) and nickel(II) ions, induced erythropoietin gene expres-sion both in vivo and in hepatoma cell lines (Goldberg et al.,1988). More recently, it had been demonstrated that HIF-1aitself and non-erythropoietin HIF target genes were alsoinduced by cobalt. Moreover, the efficacy of cobalt wasinversely related to the availability of iron, suggesting thatthe metal might be competing at an oxygen-sensing ironcenter.

Two broad categories of process were proposed as puta-tive oxygen sensors, either direct sensing of moleculardioxygen or indirect sensing through its effects on levels ofa number of metabolites, with proposals including the pro-ducts of oxidative metabolism such as ATP or heme, orreactive, oxygen-derived free radicals.

Since ATP is a major product of oxidative respirationand many systems respond to alterations in its level, it wasinitially attractive as a possible mediator of oxygen sensingin mammalian cells. However, early work on erythropoietinproduction and later studies on the HIF system clearlyindicated that the effects of hypoxia were not mimicked bymetabolic inhibitors of ATP production such as cyanide.

Another appealing model was that of a heme proteinsensor. In addition to the effects of transition metal ions onthe regulatory mechanism of erythropoietin production, itwas shown that carbon monoxide could influence the sys-tem. Based on the inhibition of hypoxia-inducible but notcobalt-inducible production of erythropoietin in Hep3B cellsby high concentrations of carbon monoxide (Goldberg et al.,1988), it was proposed that the oxygen sensor was a hemeprotein and in which cobalt substitution might generate aconstitutive ‘de-oxy’ form. However, the observation that theHIF system was also stimulated by highly selective ironchelators, desferrioxamine and hydroxypyridinones, wasdifficult to accommodate within the heme-sensing model.Heme-iron is not known to be affected by these chelatorsand, in fact, does not freely interchange with cobalt. It was


therefore necessary to propose that the sensing molecule wasitself turning over rapidly to allow incorporation of cobaltinto the nascent protein, or to permit removal of chelatableiron to affect heme synthesis and incorporation.

Despite uncertainties in the heme protein hypothesis, theperturbation of the system by iron chelators and transitionmetals strongly suggested that the oxygen-sensing processinvolved some form of iron center. Pharmacological studiesin which redox active chemicals have been found to perturb

the regulation of erythropoietin or HIF led to the proposal ofdifferent types of redox-sensing mechanism, in which a me-tabolite of oxygen, such as reactive oxygen species, might besensed. Several sources of such reactive oxygen species(ROS) have been suggested including the mitochondrialchain, specific NAD(P)H oxidases or the possibility of directattack by ‘metal-catalyzed’ oxidation (a local Fenton reac-tion) at the protein surface of the target molecule. However,conflicting results were obtained in different studies. Both

FIGURE 1.3 Oxygen-dependent regula-tion of HIF signaling. The figure shows thedomain structure for HIF-1a. The basic helix-loop-helix (bHLH) domains and the PASdomains (PAS domain = PER, AHR, ARNT,SIM domain; PER = periodic circadian pro-tein, AHR = aryl-hydrocarbon receptor,ARNT = aryl-hydrocarbon receptor nucleartranslocator, SIM = single-minded protein,A and B = A and B domains) are responsiblefor dimerization and DNA binding. The C-terminal portion of HIF-1a and HIF-2a con-tains the regulatory domains: the amino-ter-minal oxygen-dependent degradation domain(NODDD) and the carboxy-terminal oxygen-dependent degradation domain (CODDD), re-sponsible for regulating HIF-a stability andthe amino-terminal activation domain (NAD)and the carboxy-terminal activation domain(CAD) involved in regulating transactivatingability. When oxygen is available, enzymatichydroxylation of specific residues withinthese regulatory domains is effected by thePHD (prolyl hydroxylase domain) and FIH(factor inhibiting HIF) enzymes. Oxygen israte limiting for these modifications, whichalso require divalent iron (Fe2+) and 2-oxo-glutarate (2-OG) as co-factors. Hydroxylationof a specific asparagine (Asn) residue withinthe CAD blocks recruitment of the co-activa-tor p300, while hydroxylation of specificproline (Pro) residues in the NODDD andCODDD allows binding of the VHL E3ubiquitin ligase complex. This complex com-prises a specific recognition component,the von Hippel-Lindau protein (VHL) andelongin B (Elo B), elongin C (Elo C), Rbxand cullin-2 (Cul-2). Recognition of hydrox-ylated proline residues leads to covalent at-tachment of a polyubiquitin chain that thentargets the HIF-a subunit for proteosomal de-struction. Under hypoxic conditions HIF-asubunits dimerize with HIF-1b, bind DNAsequences containing hypoxia responseelements (HREs) and recruit transcriptionalco-activators such as p300 to effect transcrip-tion of target genes.


the very large number of biological interfaces of oxygen incells and even greater complexity of intracellular redoxchemistry make for extreme difficulty in distinguishingdirect from indirect responses to pharmacological or evengenetic probes. Together with methodological difficultyand controversy over the measurement of intracellular meta-bolites that might be postulated to be involved in oxygensensing, this creates substantial problems for such an‘outside- in’ approach to probe the oxygen-sensing process,leading several groups to favor an ‘inside-out’ approachfocusing on regulatory sequences in HIF-a.

The existence of distinct regulatory domains mediatingthe effects of oxygen on HIF defined specific polypeptidesequences that must interact with the upstream oxygen-sensitive signaling system. However, though these sequenceswere studied in detail, the nature of the signal transducinginteraction was not immediately apparent. Since HIF-asubunits are heavily phosphorylated and perturbationof protein phosphatase/kinase pathways can modulateHIF activity, it was generally expected that the oxygen-sensitive pathway would involve oxygen-regulated phos-phorylation of specific HIF-a residues. Unexpectedly,however, no sites of oxygen-regulated phosphorylation weredefined, suggesting the operation of a different mode ofsignal transduction.

XII. DEGRADATION OF HIF BY THEUBIQUITIN-PROTEOSOMALPATHWAY

Further progress into the regulation of HIF by oxygen wasmade by linking oxygen sensing to two related fields ofresearch. The first of these was the ubiquitin-proteosomepathway of protein degradation. Biochemical inhibitors ofthis pathwaywere found to induce HIF-1 DNA binding whenadded to normoxic cells and to prevent the degradation ofthe HIF-1 complex in cells transferred from hypoxia tonormoxia. Furthermore, cells with a temperature-dependentmutation of ubiquitylation manifested rapid accumulation ofHIF-1a at the non-permissive temperature (Salceda andCaro, 1997). Hence, it was demonstrated that HIF-1a isdegraded by oxygen regulated ubiquitin mediated proteoso-mal degradation.

The ubiquitin-proteosome system plays an important rolein a broad array of basic cellular processes, including cellcycle regulation, modulation of immune and inflammatoryresponses and control of signal transduction pathways,development and differentiation. In outline, degradation ofa protein by the ubiquitin system involves two successivesteps (for review see Hershko et al., 2000):

1. covalent attachment of multiple ubiquitin molecules tothe substrate

2. degradation of the tagged protein by the 26S proteasome.

Conjugation of ubiquitin proceeds via three stages: acti-vation, transfer and ligation. In the ligation step, the firstubiquitin moiety is attached to an e-amine group of an inter-nal lysine residue. Subsequent chain elongation proceeds bycross-linking to lysine 48 of the ubiquitinmolecule, althoughattachments to other lysine residues have been observed andmay have separate distinct functions. More recently, other,ubiquitin-like molecules (e.g. NEDD8, SUMO-1) have beenidentified which target substrates to other processes withinthe cell.

Within the pathway, specificity and control are conferredthrough recognition of substrate by the E3 ubiquitin ligasecomplex, as E1, E2s and the proteosome are all constitutive-ly active. However, it is rare that a single protein is targetedby a specific E3 ligase and, in most cases, an E3 recognizes asubset of proteins that contains similar structural motifs.Furthermore, some proteins are recognized by two differentE3 enzymes, via distinct recognition motifs. Some of thesemotifs are encoded within the protein itself and the proteinswhich harbor them are degraded constitutively. Stability ofother proteins may be regulated, depending on the state ofoligomerization or on post-translational modification, suchas phosphorylation.

The second field of research to provide important geneticinsight into the regulation of HIF evolved from observa-tions on the von Hippel-Lindau (VHL) syndrome and,in particular, the recognition that the von Hippel-Lindautumor suppressor gene product (pVHL) provides thespecific E3 ligase recognition component targeting HIF-a subunits for destruction in the presence of oxygen(Maxwell et al., 1999). Enhanced glucose metabolismand angiogenesis are classical features of cancer, involvingupregulation of HIF target genes. While partially attribut-able to the hypoxic microenvironment, genetic alterationsalso contribute to these effects, with VHL disease providinga striking example.

Affected individuals, bearing a germ line mutation in theVHL tumor suppresser gene, develop tumors resulting fromsomatic loss or inactivation of the remaining wild-type al-lele. A cardinal feature of these tumors is a rich supply ofblood vessels (e.g. hemangioblastomas, affecting the CNSand retina, and renal clear cell carcinomas) (Kaelin, 2002).Many of these tumors over-express hypoxia-inducible genes,such as VEGF and, more rarely, erythropoietin, providingearly evidence of a connection between the hypoxic responseand pVHL.

TheVHL gene encodes a 213 amino acid protein (pVHL)and was first isolated in 1993 (Latif et al., 1993). Althoughthe primary sequence did not immediately suggest a func-tion, protein association experiments defined a series ofpVHL interacting molecules, including elongins B and C,and CUL2. This complex showed homology to specific yeastubiquitin ligases referred to as SCF complexes (Skp1/Cdc53/F-box protein). In such complexes, the target protein des-tined for polyubiquitination and destruction is recognized or


bound by the F-box protein, suggesting an analogous,specific recognition, role for pVHL.

The definitive link between pVHL and HIF-a regulationwas established through the study of renal carcinoma celllines deficient in pVHL (Maxwell et al., 1999). In a series ofsuch cell lines, HIF-a subunits were found to be constitu-tively stabilized and HIF was activated, leading to increasedtranscription of reporter plasmids containing hypoxiaresponse elements (HREs) and dysregulation of a wide rangeof hypoxically regulable native genes. Re-expression ofwild-type pVHL in stable transfectants restored the normalpattern of oxygen-dependent instability demonstrating thecritical function of pVHL in HIF proteolysis. A physicalinteraction between pVHL and HIF-a was demonstratedby co-immunoprecipitation after blockade of HIF degrada-tion by proteosomal inhibitors. Further experiments formallyconfirmed the function of pVHL as part of an ubiquitin ligasecomplex targeting HIF-a subunits.

These findings provided a new focus for analysis of theoxygen-sensitive pathway through studies of the interactionbetween HIF-a polypeptides and pVHL. Further studiesdefined two short HIF-a sub-sequences that interact withpVHL corresponding to independently active proteolyticdegradation domains in HIF-a. These sequences interactdirectly with the b-domain of pVHLwhich, in turn, providesa link, through an interaction between its a-domain andelongin C, to the other components of the multi-ubiquitinligase complex (Ivan et al., 2001; Jaakkola et al., 2001;Masson et al., 2001).

Immunoprecipitation experiments using whole cellextracts revealed that capture of HIF-a by pVHL is sup-pressed by treatment of cells with iron chelators and cobal-tous ions (Maxwell et al., 1999) and (when oxygen isexcluded from the cell extraction buffers) by hypoxia (Ivanet al., 2001; Jaakkola et al., 2001), indicating that regulationof this interaction accurately reflects the properties of theoxygen-sensitive pathway.

Further studies showed that the HIF-a/pVHL interactioncould be reproduced in vitro using recombinant HIF-a andpVHL, but that the interaction required that the HIF-a poly-peptide was pre-incubated with a cell extract in the presenceof iron and oxygen. Temperature sensitivity and heat inacti-vation suggested that this process involved an enzymaticmodification and mutational analysis together with massspectrometry and functional testing of modified HIF-a poly-peptides showed that the critical modification was hydrox-ylation of a specific prolyl residue (P564 in human HIF-1a(Ivan et al., 2001; Jaakkola et al., 2001). Further studiesshowed that each of the two independent HIF-a degradationdomains contains a site of prolyl hydroxylation and defineda common LxxLxP motif at the hydroxylation site(Masson et al., 2001). Recognition of each hydroxyprolineby pVHL then targets the HIF-a molecule for rapiddegradation by the ubiquitin-proteosome pathway (seeFigure 1.3).

The mechanism by which addition of a single oxygenatom to a prolyl residue within a HIF-a degradation motifgoverns recognition by pVHL was subsequently studied byX-ray crystallography of a hydroxylated HIF-a peptidebound to the VCB (pVHL, elongins B and C). These studiesrevealed a single, well-defined hydroxyproline-bindingpocket on the surface of the pVHL b-domain. Highlyspecific discrimination between hydroxylated and non-hydroxylated HIF is achieved by an optimized hydrogen-bonding network between VHL residues, in the floor of thebinding pocket, and the HIF-a hydroxyproline residue thatwould be denied to proline.

Genetic analysis in model organisms defined the criticalprolyl hydroxylase and led to the identification of threehomologous and closely related mammalian enzymestermed PHD1, 2 and 3 (prolyl hydroxylase domain) thatcatalyze hydroxylation of prolyl residues within the twoindependent degradation domains of human HIF-a subunits(Epstein et al., 2001).

When assayed using synthetic peptides, kinetic analysisof HIF-a prolyl hydroxylation gives an apparent Km foroxygen (concentration of substrate that gives half maximalactivity) of 230–250 mM. Even though, when assayed usinglonger (and more physiological) HIF-a polypeptides, Km

values for oxygen are lower, they are still well above theoxygen concentration in tissues (believed to be 10–30 mM).Consequently, cellular availability of oxygen will limit therate of HIF-a hydroxylation, with the reaction rate varyingessentially linearly over the physiological range of tissueoxygen tensions. Because, in the presence of an intactpVHL/ubiquitin/proteosome pathway, the hydroxylationstep is rate limiting for HIF degradation, this allows HIF-alevels to reflect oxygen concentration in a graded mannerthat is essential for the system to act as an oxygen sensor.

Following the discovery of prolyl hydroxylation in theregulation of HIF-a stability, a further enzymatic hydroxyl-ation, that of an asparagine residue in the C-terminal ofHIF-a subunits, was defined by mass spectrometry anddemonstrated to direct non-proteolytic regulation of HIFtransactivation (Lando et al., 2002). In the presence ofoxygen, hydroxylation of this asparaginyl residue blocksthe interaction of HIF-a with the CH1 domain of p300 andCBP co-activators impeding its ability to initiate transcrip-tion. It was rapidly recognized, using bioinformatic analysis,that a molecule originally defined as a HIF interacting proteinthat inhibited HIF transactivation by unknown mechanisms(FIH, factor inhibiting HIF) was, in fact, the HIF asparaginylhydroxylase that targets this C-terminal asparagine residue.Thus, in the presence of oxygen, HIF is inactivated by a dualmechanism involving both proteolytic degradation and tran-scriptional inactivation of remaining protein HIF-a protein(Lando et al., 2003; Schofield and Ratcliffe, 2004).

Although to date only a single HIF asparaginyl hydrox-ylase has been identified, the presence of multiple HIFprolyl hydroxylases raises questions about their possible


redundancy and diverse roles. Inactivation of each PHDindividually and in combination using small interferingRNA has demonstrated that while all three contributeto the regulation of HIF, there are differences both in theirrelative importance and in effects on HIF-1a versus HIF-2a.Thus, while PHD2 appears to be the most important enzymein setting basal levels of HIF-1a in normoxic cells, PHD3has a more substantial effect on HIF-2a particularly in hyp-oxic cells. The importance of PHD2 under basal conditionsis strongly supported by the contrast between embryoniclethality seen in PHD2 knockout mice and the relativelyminor phenotypes of those lacking PHD1 and PHD3. Thethree PHD enzymes are conserved in mammals and a num-ber of other vertebrate species and show differential patternsof organ expression, intracellular localization and inducibil-ity by exogenous stimuli, suggesting that they have distinctfunctions (Schofield and Ratcliffe, 2004). Whether theseare discreet functions in the regulation of HIF or otherhydroxylation substrates is currently unclear.

Within a complex organism, levels of oxygenation aremarkedly heterogeneous. For example, the renal medulla hasa considerably lower oxygen tension than the renal cortex.Cells within these different environments have adapted theirHIF response to avoid inappropriate overactivation of thispathway. How this is achieved is incompletely understood,although may in part be due to hypoxic inducibility of PHD2and PHD3 expression (Epstein et al., 2001). These two PHDisoforms are under transcriptional regulation of HIF itself,providing a negative feedback loop in which HIF induces themechanism of its own destruction, permitting HIF levels toaccommodate to chronic changes in oxygen availability,while still responding to rapid perturbations.

Enzymatic hydroxylation of residues within proteinsdirectly connects the availability of molecular oxygen toprotein function by an entirely novel mechanism of signaltransduction, although such modifications are common inextracellular proteins and have structural roles, for instancein the formation of collagens. To date all prolyl and aspar-aginyl hydroxylases identified have been members of thesuperfamily of 2-oxoglutarate (a-ketoglutarate) dependentdioxygenases. In addition to oxygen, these enzymes alsorequire 2-oxoglutarate as a co-substrate and non-heme iron(Fe2+) as a co-factor. Furthermore, ascorbate (vitamin C) isrequired for full enzymatic activity, possibly to maintain thecatalytic iron center in the reduced state, or to facilitate theavailability of iron in a more general way (Schofield andRatcliffe, 2004). This raises important questions as to whatextent HIF-hydroxylase activity is affected by physiologicalor pathological variation in iron or ascorbate availability.Experiments in tissue culture certainly suggest that limitingiron availability could be pathologically important, particu-larly in cancer cells where rapid growth often depletescells of iron and HIF-a levels are commonly raised evenin the presence of oxygen (Gerald et al., 2004; Kaelin,2005). These co-factor requirements also raise the intriguing

possibility that physiological or pathophysiological changesin their levels in an appropriate cellular compartment mightprovide other links to the availability of oxygen. In theory, atleast, cellular disposition of Kreb’s cycle intermediates andthe redox status of iron/ascorbate might also be affectedby oxygen with the HIF hydroxylases effectively integratinga number of signals. Such influences appear likely to under-lie at least some of the effects of pharmacological andgenetic perturbations of oxygen radical metabolism on theHIF system.

XIII. DISRUPTION OF THE OXYGEN-SENSING PATHWAY IN CANCER

Inadequate tissue oxygen supply is a central component ofboth neoplastic and non-neoplastic ischemic vascular dis-ease. For instance, the essential physiological requirementof achieving a balance between oxygen supply and demandis not only integral to organized growth and development,but is recapitulated during the disorganized proliferationseen in the development of cancer. Many pieces of evidencenow link HIF to important aspects of tumor behavior. HIFactivation is commonly seen in many types of cancer andthere is a high degree of concordance between HIF targetgenes and those upregulated in cancerous tissue. For in-stance, the classical features of upregulated glycolysis andangiogenesis are driven, at least in part, by activation of HIFpathway in cancer. In many cancers, the degree of HIF-aimmunostaining correlates with tumors that are more aggres-sive and is seen as an independent marker of prognosis(Semenza, 2003).

Microenvironmental tumor hypoxia is clearly animportant mechanism of HIF induction in tumors and HIFimmunostaining is often most intense in hypoxic perinecro-tic regions of these growths. Nevertheless, other mechanismsclearly contribute to neoplastic upregulation of HIF and ithas become clear that a large number of pathways involvingoncogenic activation or tumor suppressor inactivationare linked to HIF activation, most probably reflecting thefundamental importance of linking growth to processesentraining an oxygen supply (Semenza, 2003).

These links include inactivation of PTEN or p53 path-ways and activation of ras, src and myc pathways as well asstimulation by exogenous growth factors such as epidermalgrowth factor, insulin, insulin-like growth factors 1 and 2,angiotensin II, thrombin and PDGF. Though the mechanismsunderlying some of these links remain poorly understood,evidence to date indicates that they involve both increasedHIF translational and impairment of oxygen-dependentdegradation.

The most direct link between genetic mutation in cancerand activation of HIF is in VHL-associated renal cellcarcinoma (RCC) (see above). In hereditary VHL disease,affected individuals develop multiple cystic lesions in their


kidneys and have a very high lifetime risk of (often multiple)RCC. These tumors are associated with somatic loss or in-activation of the remaining wild-type allele in individualswho are heterozygous for germline mutation in the VHLtumor suppressor gene, in accordance with the classicKnudson ‘two-hit’ hypothesis. Non-familial tumors mayresult from somatic loss or inactivation of both alleles withinthe same cell, a process that accounts for in excess of 80% ofsporadic RCC. However, on average it takes longer to accruetwo ‘hits’ within the same cell, accounting for the lowerprevalence and older age distribution of sporadic tumors.As outlined above, loss of functional pVHL in tumorcells blocks oxygen-dependent degradation of both HIF-asubunits. This direct influence on the HIF pathway has led tointense investigation of the causal role of HIF activation inVHL-associated RCC.

Genetic manipulation, involving both upregulation anddownregulation of HIF-a (particularly HIF-2a), in RCC celllines, and assessment of growth as tumor xenografts hasindicated that upregulation of HIF-2a is both necessaryand sufficient to promote growth, at least in these assays.This evidence for a causal role of HIF upregulation in RCC isbacked by functional genetic studies indicating, not only thatall RCC-associated VHL mutations result in HIF upregula-tion, both also that the severity of this HIF dysregulationcorrelates with risk of RCC in familial VHL disease.Interestingly, however, it appears that the two main HIF-aisoforms (HIF-1a and HIF-2a) have quite different actionsin the pathogenesis of VHL-associated RCC. In the kidneysof patients with VHL disease, selective HIF-1a upregulationis seen in the earliest dysplastic lesions, whereas HIF-2ais more abundant in more advanced dysplastic lesions.Concordant with this, many VHL-negative renal cancercell-lines preferentially express HIF-2a and, while overex-pression of HIF-2a in renal cancer xenografts enhances theirgrowth, overexpression of HIF-1a inhibits xenograft growth,at least in some RCC cell lines. Most probably, this reflectsdifferences in transcriptional selectivity between HIF-1aand HIF-2a, with HIF-2a preferentially activating pro-tumorigenic genes in RCC (Kaelin, 2002).

It is interesting that erythropoietin itself appears to beprincipally an HIF-2a target gene. Renal erythropoietin isnormally produced by the renal interstitial fibroblasts thatexpress HIF-2a rather than HIF-1a. Dysregulated erythro-poietin production and erythrocytosis is also, however, asso-ciated with RCC, particularly in the later stages of disease.In this situation, erythropoietin mRNA is produced by the(epithelial) RCC cells themselves (Da Silva et al., 1990).As described above, this is associated with a switch fromHIF-1a to HIF-2a expression in the neoplastic epitheliumthat might be related, in some way, to the epithelial-mesen-chymal transition that occurs during RCC progression.

Since the recognition of the importance of HIF in VHL-associated RCC, several other genetic tumor syndromes havebeen connected with dysregulation of the HIF pathway and,

interestingly, most of these include a predisposition to renalneoplasia.

Thus, certain nuclear genes encoding mitochondrialenzymes can also act as tumor suppressors. Succinate dehy-drogenase deficiency (SDHD) resulting from germlinemutation in genes encoding subunits B, C or D is associatedwith the formation of highly vascular tumors such as pheo-chromocytoma, paraganglioma of the carotid body (also anorgan with a specialized role in oxygen sensing), papillarythyroid carcinoma and renal cell carcinoma. Similarly, germ-linemutation in fumarate hydratase (FH) has been linked to asyndrome predisposing affected individuals to cutaneousand uterine leiomyomas and renal cell cancer. In each case,individuals are heterozygous for the enzyme loss, whiletumor cells have mutated or inactivated their second copy,resulting in high levels of upstream TCA cycle intermedi-ates, namely succinate and fumarate, which are able com-petitively to inhibit 2-oxoglutarate dependent dioxygenasesincluding the HIF hydroxylases, and result in elevated levelsof HIF-a within tumor cells. Nevertheless, though it is anattractive possibility that HIF activation contributes causallyto tumor development, as in VHL disease, this has not yetbeen assessed directly (Ratcliffe, 2007).

Tuberose sclerosis is well known to nephrologists,because of multiple renal angiomyolipomas and cysts, butclear-cell renal cell carcinomas are also observed. This andthe other familial hamartoma syndromes, Peutz–Jegherssyndrome, Cowdon syndrome and Bannayan–Riley–Ruval-caba syndrome are linked mechanistically through the TSC1and TSC2 tumor suppressors and their effects on the mTOR(mammalian target of rapamycin) pathway. Phosphorylationof TSC2 byAMPK, consequent to energy starvation, leads toinhibition of mTOR signaling. mTOR itself affects HIF-alevels independent of oxygen and may provide a means ofcoupling nutrient availability to oxygen sensing (Brugarolasand Kaelin, 2004). Inhibition of HIF also provides a possibleexplanation for some of the useful anti-cancer propertiesof the mTOR inhibitor and immunosuppressant agentrapamycin, in particular its actions on tumor angiogenesis.

XIV. DISRUPTION OF THE OXYGEN-SENSING PATHWAY IN HEREDITARYPOLYCYTHEMIA

Though most causes of erythrocytosis are either associatedwith appropriate depression of serum erythropoietin levels(e.g. polycythemia rubra vera) or an obvious defect inoxygen delivery (e.g. hypoxemia, or high affinity hemoglo-bins), some individuals have been identified with congenitalerythrocytosis and unexplained inappropriate high erythro-poietin levels, suggesting a defect in the oxygen-sensingprocess.

A genetic analysis of some of these syndromes hasindeed revealed defects in the oxygen-sensing pathways


outlined above. Thus, individuals affected by a rare form ofhereditary polycythemia, first described in patients fromwestern Russia (Ang et al., 2002) and termed ‘Chuvashpolycythemia’, have been found to be homozygous for aspecific mutation in the VHL tumor suppressor gene. Unlikecancer associated VHL mutations, the protein derived fromthis gene mutation remains partially active. While only mildactivation of the HIF pathways ensues, this is sufficient togenerate excessive erythropoiesis and elevation of the he-matocrit. Paradoxically, these patients do not developtumors, as seen in the VHL syndrome, raising the possibilitythat pVHL has a second function that is important intumor suppression. More recently, families have also beendescribed with different types of inactivating mutation in thePHD2 enzyme, leading to familial erythrocytosis.

XV. PHARMACOLOGICALMANIPULATION OF HIF

The central role of HIF in a wide range of common pathol-ogies, including cancer and ischemia, has made it an attrac-tive target for pharmacological manipulation. To date, suchapproaches have focused on downregulation of the HIF path-way in cancer and upregulation in ischemic/hypoxic/anemicdiseases, where the aim has been to promote erythropoiesis,angiogenesis or cytoprotective responses in differentsettings.

While pharmacological small molecule strategies forinhibition of transcriptional systems such as HIF arestill regarded as difficult to achieve, the enzymatic basis ofregulation by hydroxylation presents a classical small mol-ecule target for upregulation of HIF using small moleculeinhibitors of the HIF hydroxylases. Inhibition of theseenzymes by 2-oxoglutarate analogs was rapidly shown toblock the interaction of HIF-a with VHL, stabilize HIF-aand upregulate HIF target genes. Increasing numbers of suchagents are being developed (Hewitson and Schofield, 2004)and have been shown to stimulate erythropoietin productionand erythropoiesis and to afford protection in models ofrenal and other organ ischemia.

Despite this promise, achieving the specificity neededfor safe clinical intervention is likely to be challenging(Hewitson and Schofield, 2004). In addition to known2-oxoglutarate dioxygenases such as procollagen hydroxy-lases, bioinformatic predictions suggest that there may be upto fifty or so related and largely uncharacterized enzymes inthe human genome with potential to generate ‘off-target’effects from non-specific inhibitors (Elkins et al., 2003).The possibility of unwanted actions is increased still furtherwhen the pleiotropic actions of HIF are considered. This iswell illustrated by the ability of HIF activation to increaseboth erythropoiesis and angiogenesis. Promotion of erythro-poiesis, leading to polycythemia might be an unwanted sideeffect of treatments aimed at enhancing angiogenesis, while

safe treatment of anemia might be confounded by promotionof angiogenesis. However, dissociating these actions maysimply be a matter of delivering the drug to the requiredorgan. For example, systemic hypoxia at altitude predom-inantly affects erythropoiesis, with little in the way ofsystemic angiogenesis, whereas localized hypoxia, inhealing or neoplastic tissues incapable of expressingerythropoietin, stimulates localized angiogenesis. Never-theless, careful development will be needed to determinewhether and how safe therapeutic translation of thesefindings can be achieved.

XVI. SUMMARY

Following classical insights into the fundamental impor-tance of oxygen in living systems, physiological studies inthe 20th century demonstrated a circulating hormone, eryth-ropoietin, that was produced by the kidneys, in response totissue hypoxia and stimulated red blood cell production inthe bone marrow to complete a feedback loop contributingto oxygen homeostasis. The advent of molecular biology ledto identification of the erythropoietin gene in the 1980s,underpinning the immense therapeutic advance providedby recombinant erythropoietin in the treatment of renalanemia. In addition, study of the pathways underlyingregulation of erythropoietin gene has led to the discoveryof a highly conserved, widespread oxygen-sensing mecha-nism, regulated by novel signaling pathways involvingthe post-translation hydroxylation of specific amino acidresidues in the transcription factor HIF. This reaction iscatalyzed by a series of non-heme iron dioxygenases whoseabsolute requirement for molecular oxygen underlies theoxygen-sensing process.

Conflict of interest statement

PJ Ratcliffe is a founding scientist of the company ReOx Ltd.

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Further reading

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