Hypoxia-inducible Factor and Its Biomedical Relevance (2003)

Oxygen homeostasis is a cornerstone of both the physiology and intermediary metabolism systems. Despite the explosion of knowledge brought about by recombinant DNA technology, links between classic physiology and molecular biology are often fragmentary and tenuous. However, during the past decade, there has been enormous progress in understanding adaptation to hypoxia at the molecular level. A growing number of physiologically relevant genes are up-regulated by falling intracellular oxygen tension via a novel mechanism for oxygen sensing and signaling that triggers the activation of the hypoxia-inducible transcription factor HIF. 1The abbreviations used are: HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; bHLH, basic helix-loop-helix; NAD, N-terminal activation domain; CAD, C-terminal activation domain; ODD, oxygen-dependent degradation domain; PAS, Per/Arnt/Sim; IPAS, inhibitory PAS; VHL, von Hippel-Lindau; E3, ubiquitin-protein isopeptide ligase; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; PI3K, phosphatidylinositol 3-kinase; FRAP, FK506-binding protein-rapamycin-associated protein; mTOR, mammalian target of rapamycin; MAP, mitogen-activated protein; E, embryonic day; VEGF, vascular endothelial growth factor; PTEN, phosphatase and tensin homolog deleted from chromosome 10. The importance of this pathway is suggested by its presence in virtually all cells within virtually all higher eukaryotes from flies and worm to man. In this Minireview we summarize a large body of recent information on the oxygen-dependent regulation of the α subunit of HIF, by both ubiquitin-proteasomal degradation and by transcriptional activation. In addition we review some of the biomedical aspects of HIF-dependent gene expression, particularly those that impact angiogenesis and tumor biology. HIF-1 is a transcription factor that binds specifically in hypoxia to a 5′-RCGTG-3′ hypoxia-responsive element (HRE) in the promoter or enhancer of various hypoxia-inducible genes (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1701) Google Scholar), which include erythropoietin, vascular endothelial growth factor, glucose transporters, and glycolytic enzymes, as well as genes involved in iron metabolism and cell survival (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1701) Google Scholar, 2Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar, 3Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1030) Google Scholar). HIF-1 is a heterodimer composed of a 120-kDa HIF-1α subunit and a 91–94-kDa HIF-1β/ARNT subunit, both of which are members of the basic helix-loop-helix (bHLH)-PAS family (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1701) Google Scholar, 3Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1030) Google Scholar). PAS is an acronym for the three members first recognized (Per, ARNT, and Sim). Accordingly, HIF-1α and HIF-1β each contain a bHLH domain near the N terminus preceding the PAS domain (Fig. 1). Whereas the basic domain is essential for DNA binding, the HLH domain and N-terminal half of the PAS domain are necessary for heterodimerization and DNA binding (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1701) Google Scholar). Moreover, there are two transcriptional activation domains in HIF-1α; one is referred to here as N-terminal activation domain (NAD) and the other as C-terminal activation domain (CAD). In contrast, HIF-1β contains only one transcriptional activation domain at the C terminus. Furthermore, HIF-1α possesses a unique oxygen-dependent degradation domain (ODD) that controls protein stability. A portion of the ODD overlaps with the NAD. In addition to the ubiquitous HIF-1α, the HIF-α family contains two other members, HIF-2α (also called EPAS1, MOP2, or HLF) and HIF-3α, both of which have more restricted tissue expression (3Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1030) Google Scholar). HIF-2α and HIF-3α contain domains similar to those in HIF-1α and exhibit similar biochemical properties, such as heterodimerization with HIF-1β and DNA binding to the same DNA sequence in vitro (1Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1701) Google Scholar). Despite these similarities, neither HIF1α–/– nor HIF2α–/– embryos can survive (see below), suggesting the lack of functional complementation in vivo within the family. In addition, several HIF-1α variants have been detected (3Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1030) Google Scholar). Of particular interest are splice variants HIF-1α516, HIF-1α557, and HIF-1α735 that terminate respectively at codons 516, 557, and 735, resulting in the absence of both NAD and CAD or of CAD only. However, the biological significance of these isoforms is unclear. Mouse HIF-3α has a splicing variant, termed as inhibitory PAS domain protein (IPAS, Fig. 1), that contains only bHLH and PAS domains (4Makino Y. Cao R. Svensson K. Bertilsson G. Asman M. Tanaka H. Cao Y. Berkenstam A. Poellinger L. Nature. 2001; 414: 550-554Crossref PubMed Scopus (537) Google Scholar, 5Makino Y. Kanopka A. Wilson W.J. Tanaka H. Poellinger L. J. Biol. Chem. 2002; 277: 32405-32408Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). IPAS dimerizes preferentially with HIF-1α instead of HIF-1β, thereby forming an abortive complex that is unable to bind to the HRE. As a result, strong expression of IPAS in the hypoxic corneal epithelium of the eye accounts for the suppression of HIF-mediated expression of angiogenic genes and consequently an avascular cornea (4Makino Y. Cao R. Svensson K. Bertilsson G. Asman M. Tanaka H. Cao Y. Berkenstam A. Poellinger L. Nature. 2001; 414: 550-554Crossref PubMed Scopus (537) Google Scholar). By contrast, in the mouse heart and lung tissues IPAS mRNA is hypoxia-regulated, indicating a negative feedback mechanism that controls HIF-1α activity (5Makino Y. Kanopka A. Wilson W.J. Tanaka H. Poellinger L. J. Biol. Chem. 2002; 277: 32405-32408Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Hypoxia is the physiologic trigger that activates HIF. However, HIF is also up-regulated by certain transition metals (Co2+, Ni2+, Mn2+) and by iron chelation. In addition, as discussed below, certain growth factors and cytokines can also activate HIF. Both HIF-1α and HIF-1β mRNAs and proteins are constitutively expressed. Only HIF-1α responds to changes in oxygen tension, although HIF-1β, despite its apparent insensitivity to hypoxia, is required for HIF-1 activity. The process of HIF-1α activation, including enhanced protein stability and transcriptional activity, has been studied extensively in the past few years. Mechanisms for HIF-2α regulation are similar, unless otherwise noted. HIF-1α Degradation—The half-life of HIF-1α is <5 min under normoxia (6Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (5208) Google Scholar, 7Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1035) Google Scholar), yet its hypoxic induction is instantaneous (8Jewell U.R. Kvietikova I. Scheid A. Bauer C. Wenger R.H. Gassmann M. FASEB J. 2001; 15: 1312-1314Crossref PubMed Scopus (462) Google Scholar). Although the term "hypoxia-inducible factor" implies increased production at low oxygen tension, hypoxia, in fact, slows destruction of HIF-1α. Under normoxia, HIF-1α undergoes continuous proteolysis through the ubiquitin-proteasome pathway (9Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1870) Google Scholar, 10Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar), which specifically targets the ODD (9Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1870) Google Scholar). The ODD-deleted HIF-1α is stable and constitutively active, corroborating the critical role of the ODD in HIF-1α stability. HIF-1α is also stable in cells lacking functional von Hippel-Lindau (VHL) protein, and expression of wild-type VHL gene restores HIF-1α instability (11Maxwell P.H. Wiesener M.S. Chang G.-W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4234) Google Scholar). HIF-1α degradation requires binding of VHL, which, in a complex with elongin B, elongin C, and Cul2 (12Ivan M. Kaelin Jr., W.G. Curr. Opin. Genet. Dev. 2001; 11: 27-34Crossref PubMed Scopus (187) Google Scholar), acts as an E3 ubiquitin ligase for HIF-α polyubiquitination (13Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (558) Google Scholar) by targeting the ODD (14Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar, 15Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.-Y. Huang L.E. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1289) Google Scholar, 16Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar). Oxygen-dependent hydroxylation of HIF-1α Pro-564 (Fig. 1), present in a highly conserved sequence Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp (codons 562–569) (17Srinivas V. Zhang L.-P. Zhu X.-H. Caro J. Biochem. Biophys. Res. Commun. 1999; 260: 557-561Crossref PubMed Scopus (127) Google Scholar), enables specific binding to VHL (18Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3981) Google Scholar, 19Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar, 20Yu F. White S.B. Zhao Q. Lee F.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (671) Google Scholar). VHL also binds to hydroxylated Pro-402 of HIF-1α (21Masson N. Willam C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (874) Google Scholar). The HIF-1α-VHL interaction strictly requires hydroxylation (19Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar). Hydroxylation of both prolines is catalyzed by a family of prolyl 4-hydroxylases that belong to the 2-oxoglutarate-dependent oxygenase superfamily (22Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2788) Google Scholar, 23Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2153) Google Scholar) and depend not only on O2 but also iron and 2-oxoglutarate (Fig. 2). Enzymatic activity is inhibited by hypoxia, iron chelation, cobaltous ions, and the 2-oxoglutarate analog N-oxalyl glycine. These properties can explain the hitherto puzzling activation of HIF by transition metals and iron chelation. Structures of HIF-1α-VHL complexes support the strict requirement for HIF-1α hydroxyproline in VHL binding, i.e. a central role for proline hydroxylation in oxygen sensing and signaling (24Hon W.C. Wilson M.I. Harlos K. Claridge T.D. Schofield C.J. Pugh C.W. Maxwell P.H. Ratcliffe P.J. Stuart D.I. Jones E.Y. Nature. 2002; 417: 975-978Crossref PubMed Scopus (595) Google Scholar, 25Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (613) Google Scholar). Both Pro-402 and Pro-564 occur in the sequence Leu-X-X-Leu-Ala-Pro (21Masson N. Willam C. 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Structural studies show that unbound CAD is intrinsically disordered and that the CH1 domain serves as a scaffold for CAD folding through extensive hydrophobic and polar interactions (44Dames S.A. Martinez-Yamout M. De Guzman R.N. Dyson H.J. Wright P.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5271-5276Crossref PubMed Scopus (352) Google Scholar, 45Freedman S.J. Sun Z.Y. Poy F. Kung A.L. Livingston D.M. Wagner G. Eck M.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5367-5372Crossref PubMed Scopus (382) Google Scholar). The CAD per se is stable, but its transcriptional activity is hypoxia-inducible. This response is, at least in part, attributable to hypoxia-induced p300/CBP binding, which is governed by hydroxylation of Asn-803 in HIF-1α (46Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1296) Google Scholar). The asparaginyl hydroxylation is catalyzed by another Fe(II)- and O2-dependent enzyme (47Lando D. Peet D.J. 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Because HIF-1α is a complex process is that these on HIF-1α proteins than HIF-1α to In fact, there is evidence that hypoxia HIF-1α or that transcriptional activity. Although in CAD has been to be necessary for CAD and p300/CBP binding, apparently is not a target of or K. C. Fujii-Kuriyama Y. Sogawa K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the that of with transcriptional activity that the is required not HIF-1 and essential in embryonic of and results in embryonic In embryos HIF-1α expression increases between and L.E. F. Leung S.W. E. Wenger R.H. Gassmann M. Semenza G.L. Genes Dev. 1998; PubMed Scopus Google Scholar). HIF1α–/– embryos by L.E. F. Leung S.W. E. Wenger R.H. Gassmann M. Semenza G.L. Genes Dev. 1998; PubMed Scopus Google Scholar, J. Johnson R.S. EMBO J. 1998; 17: PubMed Scopus Google Scholar). there lack of in the with in the