Roland H. Wenger

Integration of Oxygen Signaling at the Consensus HRE

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFα subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli–activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system. Oxygen availability regulates many physiological and pathophysiological processes, including embryonic development, high-altitude adaptation, wound healing, and inflammation, as well as contributing to the pathophysiology of ischemic diseases and cancer. Central to our understanding of these processes is an elucidation of the molecular mechanisms by which cells react and adapt to insufficient oxygen supply (hypoxia). The last few years have brought a wealth of novel insights into these processes. Oxygen-sensing protein hydroxylases have been discovered that regulate the abundance and activity of three hypoxia-inducible transcription factors (HIFs) and thereby the activity of at least 70 effector genes involved in hypoxic adaptation. In addition to the increase in HIF abundance in response to a decrease in tissue oxygenation, it became evident that HIF abundance is also proactively increased, even under normoxic conditions, in response to stimuli that lead to cell growth and thus ultimately require higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli–activated signaling pathways that influence the abundance and activity of HIFs include pathways that involve the activation of kinases and liberation of reactive oxygen species. All of these pathways converge at the hypoxia-response elements (HREs) of effector genes, to which the HIFs bind, thereby enabling HIF-dependent induction of gene expression.

HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia

Adaptation to hypoxia is regulated by hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐regulated α subunit and a constitutively expressed β subunit. Although HIF‐1 is regulated mainly by oxygen tension through the oxygen‐dependent degradation of its α subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF‐1 activation in vivo, we determined the spatial and temporal distribution of HIF‐1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohisto‐chemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF‐1α is present in mice kept under normoxic conditions and is further in‐creased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF‐1 α expression varies among different organs. In liver and kidney, HIF‐1 α reaches maximal levels after1hand gradually decreases to baseline levels after4hof continuous hypoxia. In the brain, however, HIF‐1α is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF‐1 β is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF‐1 α. Overall, HIF‐1 α expression in normoxic mice suggests that HIF‐1 has an important role in tissue homeostasis.—Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A. H., Bauer, C., Gassmann, M., Candinas, D. HIF‐1 is expressed in normoxic tissue and displays an organ specific regulation under systemic hypoxia. FASEB J. 15, 2445–2453 (2001)

Erythropoietin Gene Expression in Human, Monkey and Murine Brain

The haematopoietic growth factor erythropoietin is the primary regulator of mammalian erythropoiesis and is produced by the kidney and the liver in an oxygen‐dependent manner. We and others have recently demonstrated erythropoietin gene expression in the rodent brain. In this work, we show that cerebral erythropoietin gene expression is not restricted to rodents but occurs also in the primate brain. Erythropoietin mRNA was detected in biopsies from the human hippocampus, amygdala and temporal cortex and in various brain areas of the monkey Macaca mulatta. Exposure to a low level of oxygen led to elevated erythropoietin mRNA levels in the monkey brain, as did anaemia in the mouse brain. In addition, erythropoietin receptor mRNA was detected in all brain biopsies tested from man, monkey and mouse. Analysis of primary cerebral cells isolated from newborn mice revealed that astrocytes, but not microglia cells, expressed erythropoietin. When incubated at 1% oxygen, astrocytes showed >l OO‐fold time‐dependent erythropoietin mRNA accumulation, as measured with the quantitative reverse transcription‐polymerase chain reaction. The specificity of hypoxic gene induction in these cells was confirmed by quantitative Northern blot analysis showing hypoxic up‐regulation of mRNA encoding the vascular endothelial growth factor, but not of other genes. These findings demonstrate that erythropoietin and its receptor are expressed in the brain of primates as they are in rodents, and that, at least in mice, primary astrocytes are a source of cerebral erythropoietin expression which can be up‐regulated by reduced oxygenation.

Induction of HIF-1α in response to hypoxia is instantaneous

Despite the pivotal role the hypoxia‐inducible factor‐1α (HIF‐1α) plays in physiological and pathological processes, little is known regarding the timeframe and mechanisms involved in its regulation. We determined the onset, accumulation, and degradation of HIF‐1α and a number of redox‐sensitive nuclear factors over a range of pathophysiological oxygen concentrations. Experiments were carried out on nonadherent human HeLaS3 cells placed in tonometers to achieve rapid equilibration between the cell suspension and the various hypoxic/reoxygenation conditions. Exposure to hypoxia for less than 2 min already revealed nuclear HIF‐1α protein induction on Western blots and HIF‐1 DNA binding in EMSAs. One hour after anoxic/hypoxic exposure, nuclear HIF‐1α proteins reached maximal levels, which were maintained for 4 h. Reoxygenation reduced HIF‐1 DNA binding within 2 min, and nuclear HIF‐1α protein levels within 4 to 8 min, down to a level below the detection limit within 32 min. Western blot analysis of the redox sensitive nuclear factors NF‐κB, c‐Fos, c‐Jun, Ref‐1, and thioredoxin showed no alteration in their nuclear levels in response to anoxia/hypoxia, but reoxygenation rapidly caused a transient increase in nuclear NF‐κB and thioredoxin protein levels. The instant initiation of HIF‐1α accumulation shown here limits the hypoxic signaling pathway to below 2 min.

Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1β resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1β mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1β-deficient subline Hepa1C4, as well as antibodies raised against HIF-1α and ARNT/HIF-1β confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5′ HBS was more efficient in binding HIF-1 than the 3′ HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer.

Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.

Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) α-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1α which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1α. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1α and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Cutting edge: hypoxia-inducible factor 1alpha and its activation-inducible short isoform I.1 negatively regulate functions of CD4+ and CD8+ T lymphocytes.

To evaluate the role of hypoxia-inducible factor 1α (HIF-1α) and its TCR activation-inducible short isoform I.1 in T cell functions, we genetically engineered unique mice with: 1) knockout of I.1 isoform of HIF-1α; 2) T cell-targeted HIF-1α knockdown; and 3) chimeric mice with HIF-1α gene deletion in T and B lymphocytes. In all three types of mice, the HIF-1α-deficient T lymphocytes, which were TCR-activated in vitro, produced more proinflammatory cytokines compared with HIF-1α-expressing control T cells. Surprisingly, deletion of the I.1 isoform, which represents <30% of total HIF-1α mRNA in activated T cells, was sufficient to markedly enhance TCR-triggered cytokine secretion. These data suggest that HIF-1α not only plays a critical role in oxygen homeostasis but also may serve as a negative regulator of T cells.

General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1alpha.

Avian embryos and neonates acquire passive immunity by transferring maternal immunoglobulins from serum to egg yolk. Despite being a convenient source of antibodies, egg yolk immunoglobulins (IgY) from immunized hens have so far received scant attention in research. Here we report the generation and rapid isolation of IgY from the egg yolk of hens immunized against the α subunit of the human hypoxia‐inducible factor 1 (HIF‐1α). Anti‐HIF‐1α IgY antibodies were affinity purified and tested for their performance in various applications. Abundant HIF‐1α protein was detected by Western blot analysis in nuclear extracts derived from hypoxic cells of human, mouse, monkey, swine, and dog origin whereas in hypoxic quail and frog cells, the HIF‐1α signal was weak or absent, respectively. In electro‐phoretic mobility shift assays, affinity‐purified IgY antibody was shown to recognize the native HIF‐1 (but not the related HIF‐2) complex that specifically binds an oligonucleotide containing the HIF‐1 DNAbinding site. Furthermore, IgY antibody immunoprecipitated HIF‐1α from hypoxic cell extracts. Immunofluorescence experiments using IgY antibody allowed the detection of HIF‐1α in the nucleus of hypoxic COS‐7 cells. For comparison, the application of a mouse monoclonal antibody raised against the identical HIF‐1 α fragment was more restricted. Because chicken housing is inexpensive, egg collection is noninvasive, isolation and affinity purification of IgY antibodies are fast and simple, and the applicability of IgY is widespread, immunization of hens represents an excellent alternative for the generation of polyclonal antibodies. —Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J., Spielmann, P., Wenger, R. H., Gassmann, M. General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia‐inducible factor 1α. FASEB J. 13, 81–88 (1999)

Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.

Hypoxia-inducible factor (HIF) α subunits are induced under hypoxic conditions, when limited oxygen supply prevents prolyl hydroxylation-dependent binding of the ubiquitin ligase pVHL and subsequent proteasomal degradation. A short normoxic half-life of HIF-α and a very rapid hypoxic protein stabilization are crucial to the cellular adaptation to changing oxygen supply. However, the molecular requirements for the unusually rapid mechanisms of protein synthesis, folding and nuclear translocation are not well understood. We and others previously found that the chaperone heat-shock protein 90 (HSP90) can interact with HIF-1α in vitro. Here we show that HSP90 also interacts with HIF-2α and HIF-3α, suggesting a general involvement of HSP90 in HIF-α stabilization. The PAS B domain, common to all three α subunits, was required for HSP90 interaction. ARNT competed with HSP90 for binding to the PAS B domain since an excess of either component inhibited the activity of the other. HSP90 as well as the heterocomplex members HSP70 and p23, but not HSP40, were detected in immunoprecipitations of endogenous cellular HIF-1α. While HSP90 and HSP70 bound to HIF-1α predominantly under normoxic conditions, ARNT bound to HIF-1α primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1α following nuclear translocation. Hypoxic accumulation of HIF-1α was delayed in a novel cell model deficient for HSP90β as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1α protein induction.