HIF-1 and mechanisms of hypoxia sensing Gregg L Semenza

Transcription

1 167 HIF-1 and mechanisms of hypoxia sensing Gregg L Semenza Hypoxia-inducible factor 1 (HIF-1) is an oxygen-regulated transcriptional activator that plays essential roles in mammalian development, physiology and disease pathogenesis. The HIF-1α subunit is subjected to oxygen-dependent ubiquitination and proteasomal degradation that is mediated by the von Hippel-Lindau protein. Interaction of HIF-1α transactivation domains with coactivators is induced by hypoxia. The signal transduction pathway remains enigmatic, but involves generation of reactive oxygen species. Nitric oxide induces HIF-1α under non-hypoxic conditions but inhibits hypoxia-induced HIF-1α expression. Addresses Johns Hopkins Hospital, CMSC-1004, 600 North Wolfe Street, Baltimore, Maryland , USA; gsemenza@jhmi.edu Current Opinion in Cell Biology 2001, 13: /01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. Abbreviations CO carbon monoxide FRAP FKBP-rapamycin-associated protein HIF-1 hypoxia-inducible factor 1 IRP-2 iron regulatory protein 2 MAP mitogen-activated protein NADPH nicotinamide adenine dinucleotide phosphate (reduced) NO nitric oxide P partial pressure PI3K phosphoinositol 3-kinase REF-1 redox factor 1 ROS reactive oxygen species TAD transactivation domain TAD-C carboxy-terminal TAD TAD-N amino-terminal TAD VEGF vascular endothelial growth factor VHL von Hippel-Lindau Introduction Although maintenance of oxygen homeostasis is an essential cellular and systemic function, it is only within the past several years that the molecular mechanisms underlying this fundamental aspect of cell biology have begun to be elucidated and their connections to development, physiology and pathophysiology have been established. Most basic cellular processes are modulated by the cellular oxygen concentration and a recent study suggests that important aspects of cell biology may be misinterpreted by performing studies under a non-physiologic (ambient) oxygen concentration [1 ]. This review will focus on HIF-1 (hypoxia-inducible factor 1), the transcriptional activator that functions as a master regulator of oxygen homeostasis. Recent advances in delineating upstream signal transduction pathways leading to the induction of HIF-1 activity and expression of downstream target genes that mediate its physiologic effects will be summarized. Among the recent major advances in this field are the discoveries of the role of reactive oxygen species (ROS) in hypoxia signal transduction; the role of ubiquitination in regulating HIF-1 activity; the regulation of HIF-1 activity by signals other than hypoxia, including nitric oxide (NO), cytokines and growth factors; and the involvement of HIF-1 in embryonic development, physiologic responses to hypoxia and the pathophysiology of common causes of mortality in the developed world, including heart attack, stroke, cancer and chronic lung disease. Here, the molecular mechanisms involved in the oxygen-dependent regulation of HIF-1 induction will be discussed. The physiologic and pathological consequences of HIF-1 induction have been reviewed recently [2]. HIF-1 structure, function and regulation HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits ([3]; Figure 1). Whereas HIF-1β is constitutively expressed, HIF-1α expression is induced in hypoxic cells with an exponential increase in expression as cells are exposed to O 2 concentrations of less than 6% [4], which corresponds to a partial pressure (P) of O 2 of approximately 40 mm Hg at sea level. Several dozen HIF-1-regulated target genes have been identified that play essential roles in cellular and systemic physiologic responses to hypoxia, including glycolysis, erythropoiesis, angiogenesis and vascular remodeling [2]. The amino-terminal half of HIF-1α (amino acids 1 390) is necessary and sufficient for dimerization with HIF-1β and for DNA binding ([5]; Figure 1). HIF-1α is ubiquitinated and subjected to proteasomal degradation in non-hypoxic cells [6 8]. Under hypoxic conditions, the fraction of HIF-1α that is ubiquitinated decreases dramatically, resulting in an accumulation of the protein [9 ]. A Pro Ser Thr rich protein stabilization domain is located between amino acids 429 and 608 of HIF-1α ([7,9 ]; Figure 1). The von Hippel-Lindau (VHL) protein binds to the protein stabilization domain and plays a critical role in the ubiquitination of HIF-1α [10,11,12,13,14 ]. Exposure of cells to cobalt chloride or iron chelators (e.g. desferrioxamine) induces HIF-1α expression [3,15] and inhibits HIF-1α ubiquitination [8] by dissociating VHL from HIF-1α [10 ]. However, hypoxia does not cause VHL to dissociate from HIF-1α [10 ] and thus the mechanism underlying the decreased ubiquitination under hypoxic conditions [9 ] remains undefined. VHL is unlikely to be the only ubiquitin protein ligase that regulates the half life of HIF-1α. Recent data indicate that p53 interacts with HIF-1α [16] and by doing so recruits the MDM2 ubiquitin protein ligase, which reduces the induction of HIF-1α expression under hypoxic conditions [17 ]. In tumor cells, loss of VHL or p53 activity results in increased

2 168 Cell regulation Figure 1 HIF-1α HIF-1β (ARNT) NLS-N bhlh PAS Dimerization + DNA binding bhlh PAS 429 VHL PSTD TAD- N 718 ID 721 NLS-C Transactivation + regulation p300 and CBP TRX REF TAD- C Current Opinion in Cell Biology Structure and function of HIF-1. The HIF-1α and HIF-1β subunits are shown with the basic helix-loop-helix (bhlh) and PER-ARNT-SIM (PAS) domains that are required for dimerization and DNA binding. Also shown for HIF-1α are the amino-terminal (N) and carboxyterminal (C) nuclear localization signal (NLS) and TAD; the Pro Ser Thr rich protein stabilization domain (PSTD; also known as the oxygen-dependent degradation domain); and sites of interaction with VHL, and p300 and CBP. The double-headed arrow indicates that reduction of Cys800, which is mediated by thioredoxin (TRX) and redox factor 1 (REF-1), is required for the interaction of TAD-C with cofactor p300 or CBP. The relevant amino acid residues are indicated numerically. See text and [29] for additional details and references. HIF-1α expression and increased transcription of downstream target genes such as VEGF [10,17 ]. Activation of the signal transduction pathway involving phosphoinositol 3-kinase (PI3K) and the serine/threonine kinases protein kinase B (AKT) and FKBP-rapamycin-associated protein (FRAP) has also been shown to induce expression of HIF-1α protein and vascular endothelial growth factor (VEGF) mrna under non-hypoxic conditions [18 ]. PTEN is a tumor suppressor with phosphoinositol phosphatase activity. PTEN negatively regulates the PI3K pathway and, therefore, loss of PTEN activity also leads to increased HIF-1α expression (see Wishart et al. pp of this issue for a discussion of PTEN) [19 ]. In addition to increased steady-state levels of HIF-1α protein in hypoxic cells, two transactivation domains (TADs) in the carboxy-terminal half of HIF-1α (amino acids and ) interact with coactivators such as CBP, p300, SRC-1 and TIF2 (Figure 1) to activate transcription [15,20,21,22,23 ]. Remarkably, cobalt chloride and desferrioxamine also induce TAD function [15,20]. Although the amino-terminal TAD (TAD-N, amino acids [15,20]) overlaps with the minimal VHL-binding domain (amino acids [12 ]; Figure 1), it does not appear that VHL regulates TAD function, as the transcriptional activity of a mutant TAD-N that cannot bind VHL is still hypoxia-induced [14 ]. Recent studies suggest that phosphorylation of HIF-1α by mitogen-activated protein (MAP) kinases increases its transcriptional activity independently of the effects of hypoxia [24,25 ], but the site(s) of phosphorylation and the mechanism by which transcriptional activity is enhanced have not been established. Hypoxia signal transduction The molecular mechanisms by which mammalian cells sense hypoxia and transduce this signal to HIF-1α have remained enigmatic [26]. Recent studies have provided experimental evidence in support of the hypothesis that mitochondrial generation of superoxide and, subsequently, hydrogen peroxide are required for induction of HIF-1 activity and transcription of downstream target genes in hypoxic cells [27,28,29,30,31 ]. This model thus proposes that ROS generation increases under hypoxic conditions, whereas an alternative model proposes the opposite, that hypoxia results in decreased production of ROS by nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidases [32,33]. The proponents of both models have obtained experimental support using various redox-sensitive dyes and pharmacologic inhibitors. The utilization of more sophisticated methods for the characterization of reactive oxygen and nitrogen species, such as electron paramagnetic resonance spectroscopy, might help resolve this issue. It is likely that HIF-1α is a direct target of redox regulation. The redox status of a cysteine residue in the carboxy-terminal TAD (TAD-C) has been shown to affect its interaction with CBP/p300 coactivators, and this interaction is positively regulated by redox factor 1 (REF-1) and thioredoxin (Figure 1; [22 ]). The interaction of the coactivator SRC-1 with HIF-1α is also redox regulated [23 ]. HIF-1α ubiquitination and degradation might also be regulated by redox modifications of the protein. Iron regulatory protein 2 (IRP-2) is targeted for ubiquitination and proteasomal degradation by oxidative modifications that occur in the presence of iron and oxygen [34]. A similar process might regulate HIF-1α protein stability except that rather than being modulated by iron levels, as is the case for IRP2, it is modulated by the cellular oxygen concentration. The regulation of HIF-1 activity by carbon monoxide (CO) and NO is also likely to be of physiologic relevance and may provide insight into the mechanisms of signal transduction leading to HIF-1 activity, but the connections at this point are not clear. The intriguing observation is that whereas CO or NO inhibits hypoxia-induced HIF-1α expression, HIF-1 DNA-binding activity and HIF-1 transcriptional activity [35 37], exposure of cells to NO under

3 HIF-1 and mechanisms of hypoxia sensing Semenza 169 non-hypoxic conditions paradoxically induces HIF-1α expression, HIF-1 DNA-binding activity and downstream gene expression [38,39 ]. One possible explanation is that reactive oxygen and nitrogen species might each be capable of (directly or indirectly) modulating HIF-1α expression, but when present together react to form compounds such as peroxynitrite that lack this property. In support of this hypothesis, superoxide has been shown to inhibit the activation of guanylate cyclase by NO in vascular smooth muscle cells [40]. One recent study, however, suggested that NO increased ROS production [41]. In addition, NO inhibits the activity of mitochondrial electron transport complex I, and pharmacologic inhibitors of complex I, such as rotenone and diphenylene iodonium, block hypoxia-induced HIF-1α expression [27,28,29,30 ]. To further complicate matters, expression of the genes encoding inducible nitric oxide synthase and heme oxygenase-1, which generate NO and CO, respectively, are induced by hypoxia in a HIF-1-dependent manner in some cell types [42 45]. NO has also been implicated in developmental and physiologic responses to hypoxia in Drosophila melanogaster [46], thus providing a further impetus to link these pathways in mammalian cells. Coupling of hypoxia and NO signaling has recently been demonstrated for the skeletal muscle calcium release channel/ryanodine receptor RyR1 [1 ]. Although some investigators in the field continue to search for the holy grail in the form of a monolithic oxygen sensor responsible for initiating a linear hypoxia signal-transduction pathway leading to HIF-1α, it is possible that multiple cellular processes, including, but not limited, to the mitochondrial electron transport chain and various NADPH oxidoreductases, might affect the concentration of specific reactive oxygen species capable of modulating HIF-1α expression and/or activity. In this model, HIF-1α would function to integrate multiple metabolic signals at the level of gene transcription. In addition, hypoxia signal-transduction pathways may be cell-type specific. For example, analyses of knockout mice lacking expression of the gp91 phox subunit of NADPH oxidase have revealed that the electrophysiologic responses to hypoxia are intact in the carotid body (arterial chemoreceptors) and pulmonary artery smooth muscle cells, whereas pulmonary neuroepithelial bodies (airway chemoreceptors) from these mice do not respond to hypoxia [47 49]. Conclusions HIF-1 is a master regulator of oxygen homeostasis that plays critical roles in a multitude of developmental and physiologic processes. Several dozen HIF-1 target genes have been identified to date that participate in responses to hypoxia [2], but these genes are unlikely to represent more than 10% of the total number that will eventually be identified by geneexpression profiling using DNA microarrays. Both the expression of HIF-1α and its transcriptional activity are modulated by the cellular O 2 concentration and, in both cases, there is evidence that the oxygen signal is converted to a redox signal, although the details remain far from clear. A variety of mechanisms have been proposed [26] involving either increased or decreased ROS generation and either direct ROS effects on HIF-1α or a ROS-initiated signal transduction pathway leading to altered phosphorylation of HIF-1α. Cytokines and growth factors may also induce HIF-1α expression via a redox signal and/or kinase cascades. As in other systems [1 ], a complex interplay between reactive oxygen and nitrogen species appears to regulate HIF-1 activity, and the mechanisms by which these molecules are generated may vary from one cell type to another. Oxygen homeostasis is of fundamental importance to the cell and complex relationships exist between O 2 concentration, energy metabolism, acid base status, redox state and the control of cell growth and proliferation. The regulation of HIF-1 may therefore best be viewed as a web a structure that is poorly defined by reductionist (i.e. linear) experimental approaches. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Eu JP, Sun J, Xu L, Stamler JS, Meissner G: The skeletal muscle calcium release channel: coupled O 2 sensor and NO signaling. Cell 2000, 102: The skeletal muscle calcium release channel/ryanodine receptor RyR1 mediates calcium release from the sarcoplasmic reticulum in response to an action potential, resulting in muscle contraction. This study demonstrates that at a physiological PO 2 (10 mm Hg or 1.5% O 2 ), RyR1 is in a reduced state in which a cysteine residue can be S-nitrosylated. The NO concentration required to activate the channel under ambient PO 2 (150 mm Hg or 21% O 2 ), however, exceeds physiologic levels; submicromolar concentrations of NO activate RyR1 efficiently at a physiologic PO 2. These results demonstrate the necessity of studying the activity of redox-sensitive ion channels at a physiologic PO 2 and the complex interplay between reactive oxygen and nitrogen species in vivo. 2. Semenza GL: HIF-1 and human disease: one highly involved factor. Genes Dev 2000, 14: Wang GL, Jiang B-H, Rue EA, Semenza GL: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-pas heterodimer regulated by cellular O 2 tension. Proc Natl Acad Sci USA 1995, 92: Jiang B-H, Semenza GL, Bauer C, Marti HH: Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O 2 tension. Am J Physiol 1996, 271:C1172-C Jiang B-H, Rue E, Wang GL, Roe R, Semenza GL: Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J Biol Chem 1996, 271: Salceda S, Caro J: Hypoxia-inducible factor 1α (HIF-1α) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions: its stabilization by hypoxia depends upon redox-induced changes. J Biol Chem 1997, 272: Huang LE, Gu J, Schau M, Bunn HF: Regulation of hypoxiainducible factor 1α is mediated by an O 2 -dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 1998, 95: Kallio PJ, Wilson WJ, O Brien S, Makino Y, Poellinger L: Regulation of the hypoxia-inducible transcription factor 1α by the ubiquitinproteasome pathway. J Biol Chem 1999, 274: Sutter CH, Laughner E, Semenza GL: HIF-1α protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. Proc Natl Acad Sci USA 2000, 97: This study demonstrates that the fraction of HIF-1α that is ubiquitinated (and thus targeted for proteasomal degradation) is markedly reduced in cells under hypoxic (4 h at 1% O 2 ) as compared with non-hypoxic (20% O 2 ) culture conditions.

4 170 Cell regulation 10. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe PJ: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999, 399: The von Hippel-Lindau syndrome is an autosomal dominant condition in which affected individuals have a greatly increased risk of developing clear cell renal carcinoma, cerebellar hemangioblastoma, retinal angioma and pheochromocytoma. It is caused by loss-of-function mutations in the VHL tumor suppressor gene; one allele is inactivated in the germ line and the second allele is inactivated by somatic mutation in the tumor. VHL-null renal carcinoma cells constitutively overexpress GLUT1, VEGF and other mrnas encoded by HIF-1 target genes as well as both HIF-1α and HIF-2α proteins, the latter being a structurally and functionally related protein product of the EPAS1 gene [50]. Introduction of a wild-type VHL coding sequence into these cells restores hypoxia-induced gene expression and oxygen-dependent degradation of HIF-1α and HIF-2α protein. 11. Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, Maher ER, Pugh CW, Ratcliffe PJ, Maxwell PH: Hypoxia inducible factor-α binding and ubiquitylation by the von Hippel- Lindau tumor suppressor protein. J Biol Chem 2000, 275: The authors develop an in vitro assay to demonstrate ubiquitination of HIF- 1α and HIF-2α (but not HIF-1β) that is dependent upon the presence of wild-type VHL protein. Tumor-associated missense mutations within the amino-terminal β domain of VHL (Arg82Pro, Pro86His, Asn90Ile, Gln96Pro) reduce the ability of VHL to interact with HIF-1α or HIF-2α in coimmunoprecipitation assays, to mediate HIF-1α and HIF-2α ubiquitination in vitro, and to regulate HIF-1α and HIF-2α protein expression or GLUT1 mrna expression in renal carcinoma cells. These mutations also prevent the binding of VHL to elongins B and C, which are required for ubiquitin protein ligase activity. Two other mutations (Tyr98Asn and Tyr112His), which only affect the ability of VHL to interact with HIF-1α and HIF-2α, result in a less severe dysregulation of HIF-1α and HIF-2α protein and GLUT1 mrna expression. VHL binds to amino acids of HIF-1α, which fall within the previously defined Pro Ser Thr rich protein stabilization or oxygendependent degradation domain [7]. VHL specifically mediates ubiquitination of HIF-1α and HIF-2α. IRP-2 is not a substrate for the VHL ubiquitin protein ligase complex despite the fact that, like HIF-1α, it is targeted for ubiquitination by an oxidative process that requires both oxygen and iron [34]. 12. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, Conaway JW: Activation of HIF1α ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci USA 2000, 97: The authors reconstitute a purified recombinant VHL complex that can function with E1 ubiquitin-activating and E2 ubiquitin-conjugating enzymes to mediate HIF-1α ubiquitination in vitro. 13. Ohh M, Park CW, Ivan M, Hoffman MA, Kim TY, Huang LE, Pavletich N, Chau V, Kaelin WG: Ubiquitination of hypoxia-inducible factor requires direct binding to the β-domain of the von Hippel- Lindau protein. Nat Cell Biol 2000, 2: These authors also demonstrate that interaction with the β domain of VHL is required for ubiquitination of HIF-1α. 14. Tanimoto K, Makino Y, Pereira T, Poellinger L: Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel- Lindau tumor suppressor protein. EMBO J 2000, 19: The authors show that forced expression of HIF-1α leads to its accumulation in non-hypoxic cells as previously reported [5]. When VHL is coexpressed, HIF-1α no longer accumulates in non-hypoxic cells, indicating that VHL had become a limiting factor for HIF-1α degradation. HIF-1α amino acids (encompassing TAD-N [15,20]) interact with VHL in cultured cells, whereas mutation of residues interferes with both ubiquitination and VHL binding. Ubiquitination occurs on lysine residues, and the mutation Lys532Arg increases TAD-N expression under non-hypoxic conditions, but it remains to be determined whether Lys532 is a site of ubiquitination. The authors also present evidence suggesting that nuclear localization of HIF-1α is necessary but not sufficient for hypoxia-induced protein stabilization. 15. Jiang B-H, Zheng JZ, Leung SW, Roe R, Semenza GL: Transactivation and inhibitory domains of hypoxia-inducible factor 1α: modulation of transcriptional activity by oxygen tension. J Biol Chem 1997, 272: An WG, Kanekal M, Simon MC, Maltepe E, Blagosklonny MV, Neckers LM: Stabilization of wild-type p53 by hypoxia-inducible factor 1α. Nature 1998, 392: Ravi R, Mookerjee B, Bhujwalla ZM, Sutter CH, Artemov D, Zeng Q, Dillehay LE, Madan A, Semenza GL, Bedi A: Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1α. Genes Dev 2000, 14: Loss of p53 expression in otherwise isogenic mouse embryo fibroblasts, human colon and ovarian cancer cells is associated with increased expression of HIF-1α protein, HIF-1 DNA-binding and transcriptional activity, and VEGF mrna in hypoxic cells. The MDM2 ubiquitin protein ligase is recruited to HIF-1α by p53. In this trimolecular complex, MDM2 appears to preferentially target HIF-1α for degradation, thus accounting for decreased p53 levels in HIF-1α-null cells [16] and increased HIF-1α levels in p53-null cells. 18. Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, Georgescu MM, Simons JW, Semenza GL: Modulation of hypoxiainducible factor 1α expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 2000, 60: Dysregulated signal transduction from receptor tyrosine kinases to PI3K, AKT and its effector kinase FRAP occurs via autocrine stimulation or inactivation of the tumor suppressor PTEN in many cancers. In human prostate cancer cells, basal, growth-factor-induced and mitogen-induced expression of HIF-1α and VEGF is blocked by inhibitors of PI3K or FRAP by overexpression of wild-type PTEN or by expression of dominant-negative PI3K or AKT mutants, thus identifying a pathway leading to HIF-1α expression that is independent of the cellular O 2 concentration. 19. Zundel W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, Gottschalk AR, Ryan HE, Johnson RS, Jefferson AB et al.: Loss of PTEN facilitates HIF-1-mediated gene expression. Genes Dev 2000, 14: Forced PTEN overexpression in human glioma cell lines inhibits hypoxiainduced HIF-1α expression. 20. Pugh CW, O Rourke JF, Nagao M, Gleadle JM, Ratcliffe PJ: Activation of hypoxia-inducible factor-1; definition of regulatory domains within the α subunit. J Biol Chem 1997, 272: Kallio PJ, Okamoto K, O Brien S, Carrero P, Makino Y, Tanaka H, Poellinger L: Signal transduction in hypoxic cells: inducible nuclear localization and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1α. EMBO J 1998, 17: Ema M, Hirota K, Mimura J, Abe H, Yodoi J, Sogawa K, Poellinger L, Fujii-Kuriyama Y: Molecular mechanisms of transcription activation by HLF and HIF1α in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBO J 1999, 18: The authors demonstrate interaction of the amino-terminal region (amino acids 1 452) of the coactivator CBP with TAD-C of HIF-1α (Figure 1). Hypoxia induces TAD-C function, an effect that is augmented by cotransfection of expression vectors encoding REF-1 and thioredoxin. The stimulatory effects of hypoxia, REF-1 and thioredoxin on TAD-C function and its interaction with CBP are lost when the missense mutation Cys800Ser is introduced into TAD-C. These results suggest that reduction of Cys800 is critical for the interaction of TAD-C with CBP that leads to increased transcription of HIF-1 target genes in hypoxic cells. 23. Carrero P, Okamato K, Coumailleau P, O Brien S, Tanaka H, Poellinger L: Redox-regulated recruitment of the transcriptional coactivators CREB-binding protein and SRC-1 to hypoxiainducible factor 1α. Mol Cell Biol 2000, 20: The authors demonstrate that two other coactivators SRC-1 and TIF2 also stimulate HIF-1-mediated transcription. SRC-1 functions synergistically with CBP and REF-1 to stimulate transcription mediated by TAD-N or TAD-C. They also show direct interaction between REF-1 and TAD-N or TAD-C in vitro. The functional and physical association of REF-1 with TAD-N, which contains no Cys residues, is surprising and suggests that in this case REF-1 has either another target for redox regulation (e.g. coactivators) or a nonredox related function. 24. Richard DE, Berra E, Gothie E, Roux D, Pouyssegur J: p42/p44 mitogen-activated protein kinases phosphorylate hypoxiainducible factor 1α (HIF-1α) and enhance the transcriptional activity of HIF-1. J Biol Chem 1999, 274: The authors provide data suggesting that the multiple isoforms of HIF-1α detected by immunoblot assay [3] represent varying degrees of phosphorylation by p42erk and p44erk MAP kinases. They demonstrate that overexpression of a constitutively active form of the upstream MAP kinase kinase kinase RAF results in increased HIF-1-dependent transcription but has no effect on HIF-1α protein levels, suggesting an effect on TAD function. The site(s) of phosphorylation remain to be determined, as mutating the only two sites in HIF-1α that matched the consensus for phosphorylation by MAP kinases (ProXSerPro) did not appear to prevent phosphorylation. 25. Sodhi A, Montaner S, Patel V, Zohar M, Bais C, Mesri EA, Gutkind JS: The Kaposi s sarcoma-associated herpes virus G protein-coupled receptor up-regulates vascular endothelial growth factor expression and secretion through mitogen-activated protein kinase and p38 pathways acting on hypoxia-inducible factor 1α. Cancer Res 2000, 60: The Kaposi sarcoma-associated herpes virus HHV8 encodes a constitutively active G-protein-coupled receptor that stimulates angiogenesis via increased VEGF expression. The authors demonstrate that increased HIF-1-mediated

5 HIF-1 and mechanisms of hypoxia sensing Semenza 171 VEGF gene transcription occurs as a result of p38 and ERK MAP kinase activity and that p38α, p38γ and ERK MAP kinases phosphorylate a GST fusion protein containing HIF-1α amino acids in vitro, whereas Richard et al. [24 ] found no evidence of HIF-1α phosphorylation by an unspecified p38 MAP kinase isoform. The sites of phosphorylation by p38 and ERK and their effects on HIF-1α transcriptional activity remain to be determined. 26. Semenza GL: Perspectives on oxygen sensing. Cell 1999, 98: Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT: Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 1998, 95: Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT: Reactive oxygen species generated at mitochondrial complex III stabilize HIF-1α during hypoxia: a mechanism of O 2 sensing. J Biol Chem 2000, 275: The authors provide pharmacologic evidence in support of their hypothesis that hypoxia results in the generation of superoxide at mitochondrial electron transport complex (ETC) III ( the O 2 sensor ) and that superoxide passes into the cytosol via anion channels and is converted to hydrogen peroxide, which induces PI3K and protein phosphatase activity leading to HIF-1α stabilization. 29. Semenza GL: Regulation of mammalian O 2 homeostasis by hypoxiainducible factor 1. Annu Rev Cell Dev Biol 1999, 15: Agani FH, Pichiule P, Chavez JC, LaManna JC: The role of mitochondria in the regulation of hypoxia-inducible factor-1 expression during hypoxia. J Biol Chem 2000, 275: The authors demonstrate that, similar to previous observations in transformed cells [27], inhibitors of electron transport complex I block HIF-1α expression in mice subjected to hypoxia. HIF-1α expression is also reduced in human-ape xenomitochondrial cybrids with a 40% reduction of ETC I activity and is restored when the ETC II substrate succinate is provided. 31. Haddad JJ, Olver RE, Land SC: Antioxidant/pro-oxidant equilibrium regulates HIF-1α and NF-κB redox sensitivity: evidence for inhibition by glutathione oxidation in alveolar epithelial cells. J Biol Chem 2000, 275: The authors report that exposure of fetal alveolar type II epithelial cells to the antioxidant N-acetyl cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) prevents the decay of HIF-1α levels observed upon reoxygenation [3]. These results are in contrast to those of Chandel et al. [27] who reported that NAC or PDTC block the induction of HIF-1α expression in hypoxic Hep3B cells. 32. Fandrey J, Frede S, Jelkmann W: Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem J 1994, 303: Ehleben W, Bolling B, Merten E, Porwol T, Strohmaier AR, Acker H: Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway. Respir Physiol 1998, 114: Iwai K, Drake SK, Wehr NB, Weissman AM, LaVaute T, Minato N, Klausner RD, Levine RL, Rouault TA: Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins. Proc Natl Acad Sci USA 1998, 95: Liu Y, Christou H, Morita T, Laughner E, Semenza GL, Kourembanas S: Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5 enhancer. J Biol Chem 1998, 273: Huang LE, Willmore WG, Gu J, Goldberg MA, Bunn HF: Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide: implications for oxygen sensing and signaling. J Biol Chem 1999, 274: Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, Fujii- Kuriyama Y: Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc Natl Acad Sci USA 1998, 95: Kimura H, Weisz A, Kurashima Y, Hashimoto K, Ogura T, D Acquisto F, Addeo R, Makuuchi M, Esumi H: Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxiainducible factor-1 activity by nitric oxide. Blood 2000, 95: The authors demonstrate that VEGF transcription is induced by NO in a cgmp-independent manner that is dependent upon the presence of an intact HIF-1 binding site in the promoter and is associated with induction of HIF-1α protein expression. 39. Palmer LA, Gaston B, Johns RA: Normoxic stabilization of hypoxia inducible factor 1 expression and activity: redox-dependent effect of nitrogen oxides. Mol Pharmacol 2000, 58: The authors show that exposure of cells to the NO donor NOC-18 induces HIF-1α protein expression. This effect of NOC-18 could be blocked by dithiothreitol but not by oxyhemoglobin, arguing against direct involvement of NO. HIF-1α expression was induced by the NO + donor S-nitrosoglutathione (GSNO) but not by the NO donor Angeli s salt. The effect of NOC-18 or GSNO may be mediated via S-nitrosylation or oxidation of protein thiols. 40. Gupte SA, Rupawalla T, Mohazzab-H KM, Wolin MS: Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD. Am J Physiol 1999, 276:H1535-H Genius J, Fandrey J: Nitric oxide affects the production of reactive oxygen species in hepatoma cells: implications for the process of oxygen sensing. Free Radic Biol Med 2000, 29: Melillo G, Musso T, Sica A, Taylor LS, Cox GW, Varesio L: A hypoxiaresponsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med 1995, 182: Lee PJ, Jiang B-H, Chin BY, Iyer NV, Alam J, Semenza GL, Choi AMK: Hypoxia-inducible factor 1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem 1997, 272: Palmer LA, Semenza GL, Stoler MH, Johns RA: Hypoxia induces type II NOS gene expression in pulmonary artery endothelial cells via HIF-1. Am J Physiol 1998, 274:L212-L Jung F, Palmer LA, Zhou N, Johns RA: Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res 2000, 86: Wingrove JA, O Farrell PH: Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila. Cell 1999, 98: Archer SL, Reeve HL, Michelakis E, Puttagunta L, Waite R, Nelson DP, Dinauer MC, Weir EK: O 2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase. Proc Natl Acad Sci USA 1999, 96: Fu XW, Wang D, Nurse CA, Dinauer MC, Cutz E: NADPH oxidase is an O 2 sensor in airway chemoreceptors: evidence from K + current modulation in wild-type and oxidase-deficient mice. Proc Natl Acad Sci USA 2000, 97: Roy A, Rozanov C, Mokashi A, Daudu P, Al-mehdi AB, Shams H, Lahiri S: Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca 2+ ] i and respiratory responses to hypoxia. Brain Res 2000, 872: Tian H, McKnight SL, Russell DW: Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev 1997, 11:72-82.