Hypoxia-Inducible Factor 1 (1998)

Hypoxia-inducible factor 1 (HIF-1) accumulates in the nuclei of mammalian cells exposed to reduced O2 tension in a time- and O2 concentration-dependent manner. HIF-1 plays an important role in O2 homeostasis by activating transcription of genes whose products mediate essential cellular and systemic responses to hypoxia, including erythropoiesis (erythropoietin), glycolysis (glucose transporter-1, aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase L, and phosphoglycerate kinase 1), vasculogenesis (vascular endothelial growth factor), and vasodilation (inducible nitric oxide synthase and heme oxygenase 1). Recent studies suggest that HIF-1 represents a vital link between O2 sensing, gene transcription, and physiological adaptation to chronic hypoxia in vivo. The response to hypoxia is complex and is determined by the nature of the inciting stimulus. Only a few of the many homeostatic adaptations will be described herein (Table 1). A lowlander who flies from Baltimore to Aspen, Colo, and takes a gondola ride to the top of Aspen Mountain (11,000 feet) will undergo a variety of physiologic adaptations to maintain homeostasis in the presence of approximately one third less O2 in the inspired air. Under these conditions, the stimulus is chronic in nature (as long as the lowlander remains at high altitude), and as in the case of any chronic stimulus (lasting longer than a few minutes), the responses involve changes in gene expression. One of the classic adaptations to hypobaric hypoxia is an increased rate of erythropoiesis1Faura J Ramos J Reynafarje C et al.Effect of altitude on erythropoiesis.Blood. 1969; 33: 668-676PubMed Google Scholar that is mediated by the glycoprotein growth factor/hormone erythropoietin (EPO). EPO is produced by peritubular interstitial cells in the kidney that sense O2 tension and respond to hypoxia by increasing transcription of the EPO gene.2Ratcliffe PJ Molecular biology of erythropoietin.Kidney Int. 1993; 44: 887-904Abstract Full Text PDF PubMed Scopus (65) Google Scholar In addition to representing a hazard of recreational travel, systemic hypoxia is also a feature of more ominous medical conditions, including those associated with the following: decreased O2 exchange, such as COPDs; decreased cardiac output, such as cardiomyopathy and congestive heart failure; and right-to-left circulatory shunts, such as congenital heart disease and pulmonary hypertension.Table 1Cellular and Systemic Homeostatic Responses to Hypoxia Mediated by HIF-1ResponseGene ProductErythropoiesisErythropoietin (EPO)GlycolysisAldolase A (ALDA)Enolase 1 (ENOl)Glucose transporter-1 (GLUT1)Lactate dehydrogenase A (LDHA)Phosphofructokinase L (PFKL)Phosphoglycerate kinase 1 (PGK1)VasodilationInducible nitric oxide synthase (iNOS)Heme oxygenase-1 (HO1)AngiogenesisVascular endothelial growth factor (VEGF) Open table in a new tab Hypoxia can also occur as a local disorder in the context of ischemia due to hypoperfusion, most notably of the cerebral and coronary circulations. When hypoxia is local and vascular in nature, a variety of vasoactive molecules are produced that function either to increase flow through existing vessels (vasodilation) or to promote the growth of new blood vessels (angiogenesis). Examples include the production of the vasodilators nitric oxide and carbon monoxide by the enzymes inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), respectively, and the production of vascular endothelial growth factor (VEGF), the primary regulator of angiogenesis. Increased expression of the iNOS, HO-1, and VEGF genes in response to hypoxia and/or ischemia has been demonstrated both in cultured myocardial and vascular cells and in the heart and lungs in vivo.3Hashimoto E Ogita T Nakaoka T et al.Rapid induction of vascular endothelial growth factor expression by transient ischemia in rat heart.Am J Physiol. 1994; 267: H1948-H1954PubMed Google Scholar, 4Ladoux A Frelin C Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart.Biochem Biophys Res Commun. 1993; 195: 1005-1010Crossref PubMed Scopus (311) Google Scholar, 5Lee PJ Jiang B-H Chin BY et al.Hypoxia-inducible factor 1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.J Biol Chem. 1997; 272: 5375-5381Crossref PubMed Scopus (659) Google Scholar, 6Levy AP Levy NS Loscalzo J et al.Regulation of vascular endothelial growth factor in cardiac myocytes.Circ Res. 1995; 76: 758-766Crossref PubMed Scopus (198) Google Scholar, 7Liu Y Cox SR Morita T et al.Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells.Circ Res. 1995; 77: 638-643Crossref PubMed Scopus (839) Google Scholar, 8Melillo G Musso T Sica A et al.A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.J Exp Med. 1995; 182: 1683-1693Crossref PubMed Scopus (546) Google Scholar, 9Tuder RM Flook BE Voelkel NF Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia: modulation of gene expression by nitric oxide.J Clin Invest. 1995; 95: 1798-1807Crossref PubMed Scopus (542) Google Scholar Regardless of whether hypoxia is systemic or local in nature, individual cells must undergo metabolic adaptation in order to maintain homeostasis under conditions of limited oxygen availability. The classic intracellular response to hypoxia is the transition from oxidative phosphorylation to glycolysis as the principal pathway for adenosine triphosphate generation. Under hypoxic conditions, increased expression of messenger RNA (mRNA) and protein for both glucose transporters and glycolytic enzymes has been documented in a number of different tissue culture cell types.10Ebert BL Firth JD Ratcliffe PJ Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct cis-acting sequences.J Biol Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 11Firth JD Ebert BL Pugh CW et al.Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3− enhancer.Proc Natl Acad Sci USA. 1994; 91: 6496-6500Crossref PubMed Scopus (456) Google Scholar, 12Firth JD Ebert BL Ratcliffe PJ Hypoxic regulation of lactate dehydrogenase A: interaction between hypoxia-inducible factor 1 and cAMP response elements.J Biol Chem. 1995; 270: 21021-21027Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 13Robin ED Murphy BJ Theodore J Coordinate regulation of glycolysis by hypoxia in mammalian cells.J Cell Physiol. 1984; 118: 287-290Crossref PubMed Scopus (106) Google Scholar, 14Webster KA Gunning P Hardeman E et al.Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts.J Cell Physiol. 1990; 142: 566-573Crossref PubMed Scopus (101) Google Scholar, 15Semenza GL Jiang B-H Leung SW et al.Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1.J Biol Chem. 1996; 271: 32529-32537Crossref PubMed Scopus (1413) Google Scholar, 16Semenza GL Roth PH Fang H-M et al.Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1.J Biol Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar Underlying the systemic and cellular physiologic adaptations to hypoxia such as erythropoiesis, angiogenesis, and glycolysis described above are changes in gene expression. Changes in the steady-state levels of the mRNA product of a given gene may reflect changes in the rate of either RNA transcription or RNA degradation. Analysis of the transcriptional regulation of hypoxia-inducible genes revealed the presence of short (<100 bp) DNA sequences within promoter or enhancer sequences that have been designated hypoxia response elements.17Semenza GL Transcriptional regulation by hypoxia-inducible factor 1: molecular mechanisms of oxygen homeostasis.Trends Cardiovasc Med. 1996; 6: 151-157Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar Representative exam- ples of hypoxia response elements from the genes encoding EPO, VEGF, HO-1, and the glycolytic enzymes enolase-1 (ENO-1), lactate dehydrogenase A (LDHA), and phosphoglycerate kinase-1 (PGK-1) are shown in Figure 1. These elements all have in common the presence of one or more binding sites for hypoxia-inducible factor-1 (HIF-1). HIF-1 was originally identified as a nuclear factor that was induced by hypoxia and bound to a site in the EPO hypoxia-response element.18Semenza GL Wang GL A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.Mol Cell Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2290) Google Scholar Nucleotide substitutions in the hypoxia-response element that eliminated HIF-1 binding also eliminated transcriptional activation in response to hypoxia. HIF-1 DNA-binding activity was shown to be tightly regulated by cellular O2 tension.19Wang GL Semenza GL Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.J Biol Chem. 1993; 268: 21513-21518Abstract Full Text PDF PubMed Google Scholar Biochemical purification by ion exchange and DNA affinity chromatography revealed that HIF-1 was a heterodimer consisting of HIF-lα and HIF-1β subunits.20Wang GL Semenza GL Purification and characterization of hypoxia-inducible factor 1.J Biol Chem. 1995; 270: 1230-1237Crossref PubMed Scopus (1772) Google Scholar Protein microsequence analysis and complementary DNA (cDNA) cloning revealed the primary structure of both subunits.21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar The amino terminal half of both subunits contained two important protein motifs (Fig 2). The basic helix-loop-helix (bHLH) motif is found in a large number of transcription factors. Protein dimerization is mediated by the helix-loop-helix domain that juxtaposes basic amino acids from each monomer to form an intact DNA-binding domain. In a subset of bHLH proteins, a second domain is required for efficient dimerization, which was named the PAS domain by virtue of its identification within the PER, ARNT, and SIM proteins.22Nambu JR Lewis JO Wharton Jr., KA The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development.Cell. 1991; 67: 1157-1167Abstract Full Text PDF PubMed Scopus (446) Google Scholar Analysis of protein and cDNA sequences21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar revealed that the HIF-1β subunit was identical to ARNT, the aryl hydrocarbon receptor nuclear translocator protein, a subunit of the aryl hydrocarbon receptor complex (dioxin receptor), a ligand-activated transcription factor involved in cellular responses to xenobiotic agents.23Hoffman EC Reyes H Chu F-F et al.Cloning of a factor required for activity of the Ah (dioxin) receptor.Science. 1991; 252: 954-958Crossref PubMed Scopus (906) Google Scholar ARNT was shown to be synthesized as isoforms of 774 and 789 amino acids based on alternative splicing of the primary RNA transcript.23Hoffman EC Reyes H Chu F-F et al.Cloning of a factor required for activity of the Ah (dioxin) receptor.Science. 1991; 252: 954-958Crossref PubMed Scopus (906) Google Scholar In contrast to HIF-1β, HIF-1α was shown to represent a novel bHLH-PAS protein of 826 amino acids.21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar The helix-loop-helix-PAS domains of HIF-1α were required for dimerization to HIF-1β, which was a necessary prerequisite for DNA binding via the basic domains of HIF-1α and HIF-1β.24Jiang B-H Rue E Wang GL et al.Dimerization, DNA binding, and trails activation properties of hypoxia-inducible factor 1.J Biol Chem. 1996; 271: 17771-17778Crossref PubMed Scopus (922) Google Scholar Forced expression of recombinant HIF-1α and HIF-1β in tissue culture cells resulted in a marked increase in transcription of reporter genes containing a wild-type 50-bp hypoxia-response element from the human EPO gene in cells exposed to 20% O2 and a superinduction in cells exposed to 1% O2.24Jiang B-H Rue E Wang GL et al.Dimerization, DNA binding, and trails activation properties of hypoxia-inducible factor 1.J Biol Chem. 1996; 271: 17771-17778Crossref PubMed Scopus (922) Google Scholar A mutation in the HIF-1 binding site of the hypoxia-response element eliminated the transcriptional response to hypoxia and the response to recombinant HIF-1. Forced expression of HIF-1α alone was sufficient to elicit maximal transactivation of reporter genes, indicating that HIF-lp was present in excess and that the levels of HIF-1α determined the transcriptional response to hypoxia (Fig 3). Truncation of HIF-1α after amino acid 390 resulted in a specific loss of transcriptional activation under hypoxic conditions.24Jiang B-H Rue E Wang GL et al.Dimerization, DNA binding, and trails activation properties of hypoxia-inducible factor 1.J Biol Chem. 1996; 271: 17771-17778Crossref PubMed Scopus (922) Google Scholar In addition, the truncated protein was expressed at constitutively elevated levels compared with the full-length recombinant HIF-1α that was present at much higher levels in hypoxic compared with nonhypoxic cells. These results suggested that the transcriptional activity of HIF-1 is regulated by changes in the steady-state levels of HIF-1α as well as changes in its transcriptional activity.24Jiang B-H Rue E Wang GL et al.Dimerization, DNA binding, and trails activation properties of hypoxia-inducible factor 1.J Biol Chem. 1996; 271: 17771-17778Crossref PubMed Scopus (922) Google Scholar Subsequent studies have confirmed these hypotheses by the identification of two transcriptional activation domains within the carboxyl terminal half of HIF-1α, the activity of which is regulated independently of the protein levels.25Jiang B-H Zheng JZ Leung SW et al.Transactivation and inhibitory domains of hypoxia-inducible factor la: modulation of transcriptional activity by oxygen tension.J Biol Chem. 1997; 272: 19253-19260Crossref PubMed Scopus (558) Google Scholar Thus, the level of transcriptional activation mediated by HIF-1 is determined by the steady-state level of HIF-1α protein and the specific activity of the HIF-1α transactivation domains, both of which are modulated by cellular O2 tension. In human Hep3B hepatoblastoma cells exposed to 20% O2, HIF-lα protein was undetectable, but after exposure of cells to 1% O2, HIF-1α was detectable in the nucleus within 30 min and levels peaked at 4 to 8 h of continuous hypoxia,21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar similar to the kinetics of induction as determined previously for HIF-1 DNA-binding activity.19Wang GL Semenza GL Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.J Biol Chem. 1993; 268: 21513-21518Abstract Full Text PDF PubMed Google Scholar When cells were exposed to 1% O2 for 4 h and then returned to 20% O2, HIF-lα levels decayed rapidly with a half-life of <5 min, indicating that the protein is exquisitely unstable in posthypoxic cells.21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar HIF-lα contains two 20-amino-acid sequences (residues 499–518 and 581–600) that each contain 15 (75%) PEST (proline, glutamic acid, serine, threonine) residues that have been implicated in protein instability.21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar HIF-1β protein was present in low levels, primarily within the cytoplasm of nonhypoxic cells, and nuclear protein levels were modestly induced by hypoxia with kinetics of induction and decay similar to those of HIF-1α, suggesting that HIF-1β protein may be stabilized by heterodimerization with HIF-1α. To determine the relationship between O2 concentration and HIF-1 expression, human HeLa cells were exposed to 20%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, or 0.5% O2 for 4 h in a tonometer either in the absence or presence of 1 mM potassium cyanide to block oxidative phosphorylation and eliminate O2 consumption and any extracellular or intracellular O2 gradients such that the O2 concentration in the medium was identical to the O2 concentration within the cell.26Jiang B-H Semenza GL Bauer C et al.Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant of O2 J Physiol. 1996; 271: Google Scholar in the presence or absence of the levels of HIF-lα protein, HIF-1β protein, and HIF-1 DNA-binding activity increased exponentially as O2 concentration (Fig The a of at approximately O2, were maximal at and at to When O2 have been the or of at the O2 have been found to vary with an of to to H et of blood flow and oxygen in the during coronary or hypoxic Exp Biol. Scopus Google Scholar, Y of Acad 1994; PubMed Scopus Google Scholar, et in the heart in and of the J Physiol. 1994; Google Scholar, H et for a in rat J Physiol. 1990; Google Scholar, P of O2 or O2 and of of on the of cerebral 1: PubMed Scopus Google Scholar that HIF-1 is expressed in response to hypoxia in in a similar to that in HeLa cells, then any in O2 tension in occur the of the a molecular for a transcriptional response to cellular hypoxia. HIF-1α and HIF-1β mRNA levels were in all and rat G Semenza GL In expression of encoding hypoxia-inducible factor Biophys Res Commun. 1996; PubMed Scopus Google Scholar In exposed to O2 for 30 to min, increased levels of HIF-lα mRNA were in the and in Hep3B cells transient induction of HIF-1α mRNA in response to hypoxia,21Wang GL Jiang B-H Rue EA et al.Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.Proc Natl Acad Sci USA. 1995; 92: 5510-5514Crossref PubMed Scopus (5219) Google Scholar suggesting that a transient increase in mRNA levels is sufficient to the large increase in protein levels in response to hypoxia. In HIF-1 mRNA levels are constitutively expressed and increase in mRNA levels is required for induction of protein expression. Thus, analysis of HIF-1α protein expression is most Analysis of the and rat VEGF genes revealed the presence of a hypoxia response element in the of the gene approximately 1 of the transcription Y Cox SR Morita T et al.Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells.Circ Res. 1995; 77: 638-643Crossref PubMed Scopus (839) Google Jiang B-H et of vascular endothelial growth factor gene transcription by hypoxia-inducible factor Cell Biol. 1996; PubMed Scopus Google Scholar, E G et transcriptional activation and mRNA of vascular endothelial growth factor in cells.J Biol Chem. 1995; 270: PubMed Scopus Google Scholar, AP Levy NS et al.Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia.J Biol Chem. 1995; 270: PubMed Scopus Google Scholar, et gene for vascular endothelial growth of the transcriptional and characterization of transcriptional and sequences.J Biol Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar The that HIF-1 VEGF transcription in hypoxic expression of recombinant HIF-1α and increased transcription of a reporter gene containing a hypoxia-response element from the human VEGF promoter in cells exposed to 20% or 1% O2 in a an HIF-1 binding site was identified in the VEGF hypoxia-response a mutation in the hypoxia-response element that eliminated HIF-1 binding also eliminated transcriptional responses to hypoxia and recombinant of a form of HIF-1α that with HIF-1β but to DNA in a the transcriptional response to hypoxia mediated by the VEGF hypoxia-response and cells that HIF-1 due to a mutation that expression of HIF-1β protein, a reduced induction of VEGF mRNA in response to hypoxia compared with wild-type cells or to cells by of a HIF-1β expression Jiang B-H et of vascular endothelial growth factor gene transcription by hypoxia-inducible factor Cell Biol. 1996; PubMed Scopus Google Scholar To link these molecular in tissue culture cells to physiologic responses in a of chronic by and and blood flow in to chronic J Physiol. 1991; Google Scholar In are in and to on a for which results in to in and a to in oxygen These undergo physiologic 1 cardiac is increased by associated with myocardial as by a increase in the blood flow is increased in with approximately half of the increase due to decreased blood that the increase in cardiac and an increase in to the with demonstrated in the and myocardial and a in in the of compared with control C Jiang B-H et in increased is associated with increased myocardial expression of vascular endothelial growth factor and hypoxia-inducible factor J Full Text Full Text PDF PubMed Scopus Google Scholar VEGF VEGF protein, and HIF-1α protein levels were each increased to in response to HIF-1β levels a These suggest the physiologic results in which is by the to the heart myocardial and necessary for the increased and cardiac that systemic for the in blood the rate of myocardial is sufficient to maintain under these conditions, in a local myocardial hypoxia that HIF-1α expression. Increased levels of HIF-1 are associated with increased VEGF gene transcription, increased levels of VEGF mRNA and protein, and increased angiogenesis. To the kinetics of HIF-1 induction in a and in which the pulmonary was in and the lungs were with either or HIF-1β protein was constitutively expressed and levels vary as a function of inspired O2 HIF-1α protein was in lungs with O2 and was induced exposure to O2 with maximal expression at 4 h of continuous hypoxia and kinetics similar to those in tissue culture cells GL et HIF-1α protein levels decayed within 1 min, posthypoxic of HIF-1α also as a function of inspired O2 concentration in vivo. Analysis of HIF-1α protein, HIF-1β protein, and HIF-1 DNA-binding activity in either primary or of a variety of pulmonary and vascular cell revealed low or levels in cells exposed to 20% O2 and a marked induction in cells exposed to 1% O2 for 4 h GL et the cell were the following: and pulmonary and pulmonary endothelial and and pulmonary cells. cells from all cell in high levels of HIF-1α protein, HIF-1β protein, and HIF-1 DNA-binding activity exposed to 20% is an that with the following: and of for oxygen and of for studies of the VEGF hypoxia-response and and for studies of