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Mitochondrial oxygen
sensing: regulation of
hypoxia-inducible factor by
mitochondrial generated
reactive oxygen species
Eric L. Bell* and Navdeep S. Chandel*†1
*Department of Medicine, Northwestern University Medical School,
Chicago, IL 60611, U.S.A. and †Department of Cell and Molecular
Biology, Northwestern University Medical School, Chicago, IL 60611,
U.S.A.
Abstract
Decreased oxygen availability (hypoxia) promotes physiological processes
such as energy metabolism, angiogenesis, cell proliferation and cell viability
through the transcription factor HIF (hypoxia-inducible factor). Activation
of HIF can also promote pathophysiological processes such as cancer and pulmonary hypertension. The mechanism(s) by which hypoxia activates HIF are
the subject of intensive research. In this chapter we outline the model in which
mitochondria regulate the stability of HIF through the increased production of
ROS (reactive oxygen species) during hypoxia.
Introduction
Oxygen is necessary for survival of most eukaryotes. Just as carbon sources
are used by glycolysis to generate energy, cells utilize oxygen to generate
1
To whom correspondence should be addressed (email [email protected]).
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the majority of ATP required for normal cellular processes through oxidative phosphorylation [1]. In the complete absence of oxygen (anoxia, 0–0.3%
oxygen), most organisms and cells cannot survive for long periods of time
[2,3]. However, in conditions of limited oxygen (hypoxia, 0.3–5% oxygen),
cells activate an adaptive response that is mediated by the transcription factor
HIF (hypoxia-inducible factor) [4]. This response facilitates survival through
the up-regulation of a number of genes involved in various physiological processes such as glycolysis and angiogenesis that promote survival in hypoxic
conditions by increasing the capacity for generating ATP and inhibiting the
depletion of oxygen. This adaptation response is not due to compromised
bioenergetics during hypoxia. The activity of the terminal complex in oxidative phosphorylation, cytochrome c oxidase, is not significantly decreased, and
therefore cellular ATP levels do not change in hypoxic conditions. The Km of
cytochrome c oxidase is less than 0.3% oxygen. In contrast with hypoxia, cytochrome c oxidase activity is substantially decreased during anoxia resulting in a
decrease in the generation of ATP [5].
The transcriptional activation of HIF is a stress response developed
through evolution to allow for cells to avoid a bioenergetic crisis in low
oxygen levels, making HIF a master regulator of oxygen homoeostasis.
An example of organismal maintenance of oxygen homoeostasis in higher
organisms is their ability to increase their capacity for systemic oxygen
delivery. This requires erythropoiesis, the process of increased generation
of erythrocytes. Erythropoiesis is increased under conditions of decreased
oxygen availability through an increase in expression of the protein erythropoietin, a HIF target gene [6]. In fact early examination of the regulation of
erythropoietin in response to oxygen led to the discovery of the transcription factor HIF.
Oxygen homoeostasis is also critical for proper development, making
HIF-mediated transcription necessary for normal organismal development.
The fact that HIF knockout mice are embryonic lethal at embryonic day 9.5
highlights the importance of HIF-mediated transcription in development
[7,8]. The lethality displayed by HIF knockout mice is due to impaired vascular development and abnormal placental development [9]. An important HIF
target gene is VEGF (vascular endothelial growth factor), which promotes
neovascularization and is required for development [10,11]. Unfortunately,
genes such as VEGF and other HIF target genes that are required for normal
maintenance of oxygen homoeostasis also promote pathophysiological processes such as pulmonary hypertension and cancer. Studies have indicated that
preventing HIF activation can suppress tumorigenesis or hypoxia-induced
pulmonary hypertension [12–15]. Therefore defining how a cell senses
decreased levels of oxygen to regulate the activity of HIF has broad implications for normal physiology as well as for numerous diseases associated with
hypoxia. Genetic studies from our laboratory and others support a model
in which hypoxia induces mitochondria to increase the generation of ROS
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(reactive oxygen species) that are both necessary and sufficient to activate the
HIF-mediated transcriptional programme for adaptation to hypoxia [16–18].
This chapter focuses on the role of mitochondrial ROS as a key component of
oxygen sensing.
Oxygen regulation of HIF
HIF is a heterodimer of two basic helix-loop-helix/PAS (Per-Arnt-Sim) proteins, HIF-␣ and the ARNT (aryl hydrocarbon receptor nuclear translocator)
or HIF-1␤ [19]. To date three HIF-␣ isoforms have been described, HIF-1␣,
HIF-2␣ and HIF-3␣. HIF-1␣ and HIF-2␣ are transcriptionally active [20–21].
Their target genes have been described to be both overlapping and distinct.
The function of HIF-3␣ is less well-defined, however it is thought to be an
antagonist for HIF-1␣- and HIF-2␣-mediated transcription [22]. Both HIF-␣
and ARNT protein subunits are expressed ubiquitously, but the stability of
each protein is differentially regulated by oxygen levels. Initial studies demonstrated that HIF-␣ protein levels are labile at normal oxygen conditions, whereas
HIF-␤ protein levels are constitutively stable [23]. Recent studies indicate that
oxygen levels regulate the hydroxylation of two proline residues, 402 and 564,
within the ODDD (oxygen-dependent degradation domain) of HIF-␣ [24]. This
hydroxylation reaction is catalysed by a family of PHDs (proline hydroxylation
enzymes) [25,26]. The PHDs require Fe2+, oxygen and 2-oxoglutarate to catalyse
the hydroxylation reaction. Hydroxylated proline serves as a binding site for
the VHL (von Hippel-Lindau) protein, the substrate recognition component
of the VBC-CUL-2 E3 ubiquitin ligase complex [27–29]. Once bound, pVHL
tags HIF-␣ with ubiquitin thereby targeting it for proteasomal degradation
[30]. HIF-␣ also contains two transactivation domains. The NAD (N-terminal
transactivation domain) is located within the ODDD. The CAD (C-terminus
transactivation domain) contains an asparagine residue that is hydroxylated by
FIH (factor inhibiting HIF) [31]. This reaction takes place under normoxic conditions (21% oxygen) and it inhibits the transactivation potential of HIF-␣ [32].
This is due to the inability of HIF-␣ to interact with the co-activator CBP/p300
as a result of the hydroxy group on Asn803 [33].
When oxygen levels decrease below 5%, HIF-␣ protein is stabilized due to
a lack of proline hydroxylation (Figure 1) in the absence of proline hydroxylation, VHL protein cannot bind HIF-␣ to initiate ubiquitin-proteasomal degradation. When stabilized, HIF-␣ translocates to the nucleus and dimerizes with
HIF-␤. Once in the nucleus, the HIF dimer binds to HREs (HIF response
elements) located throughout the genome [34]. The absence of a hydroxy
group on Asn803 allows HIF to associate with the co-activator CBP/p300 to
facilitate the transcription of various target genes. Therefore regulation of
HIF activity is tightly controlled by the hydroxylation of various amino acids.
Understanding how the hydroxylation enzymes are regulated by hypoxia
would provide the next step in elucidating the hypoxic response.
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Essays in Biochemistry volume 43 2007
21% O2
PHDs
HIF-α
OH
pVHL
HIF-α
HIF-α
FIH
1.5% O 2
Proteasomal
degradation
p300/CBP
ARNT
HIF-α
ARNT
vegf
Figure 1. HIF-1 is composed of two subunits, oxygen sensitive HIF-1␣ and HIF-1␤
(ARNT)
Under normoxia HIF-1␣ is hydroxylated at two different proline residues and an asparagine
residue. The hydroxylation of proline residues serves as a recognition motif for VHL protein
(pVHL). The binding of VHL protein targets the HIF-1␣ protein for ubiquitin-mediated degradation. HIF-1␣ contains two transactivation domains referred to as NAD (N-TAD; amino acids
531–575) and CAD (C-TAD; amino acids 786–826). The asparagine residue resides in the CAD.
The hydroxylation of asparagine prevents the binding of transcriptional co-activators such as
p300/CBP. Under hypoxia, the hydroxylation of the proline and asparagine residues is diminished, which allows for the protein to be stabilized and bind to HIF-1␤ as well as p300/CBP to
allow HIF-1-dependent gene transcription.
Mitochondrial electron transport and HIF
The mitochondria are responsible for the majority of the oxygen consumed
within the cell and evolutionarily poised to be a likely target in the hunt for
the cellular oxygen sensor. Mitochondria contain their own DNA (termed
mtDNA) which encodes 13 genes that are essential for the assembly of a functional electron transport chain. These genes encode subunits for complexes I,
III, IV and V. The major consumer of oxygen is complex IV. However, initial
studies indicated that pharmacological inhibition of complex IV by cyanide
did not activate or repress HIF [24]. Cells cultured with sub-lethal levels of
ethidium bromide, which inhibits the transcription and replication of mtDNA
resulting in the loss of a functional electron transport chain, were used to
genetically address whether mitochondria are involved in the HIF response
[35]. These cells devoid of mtDNA, ␳0 cells, are unable to activate HIF-␣ in
hypoxic conditions [36,37]. However, these cells are still able to stabilize HIF␣ in anoxic conditions, indicating that there is a fundamental difference in the
mechanism required to stabilize HIF-␣ protein in hypoxic compared with
anoxic conditions [3]. These data indicate that the ability of mitochondria to
perform electron transport is necessary for hypoxic stabilization of HIF-␣
but not anoxic stabilization. The mechanism utilized in anoxic conditions is
most likely the direct inhibition of the PHDs due to a lack of oxygen. Since
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the PHDs require oxygen as a co-substrate, they cannot hydroxylate HIF-␣ in
anoxic conditions to initiate degradation, thereby stabilizing HIF-␣ protein.
After initial studies demonstrating that ␳0 cells fail to activate HIF during
hypoxia, there were reports with conflicting data indicating that hypoxic activation of HIF is intact in ␳0 cells [38–40]. However these data were most like
due to the use of oxygen levels closer to anoxic conditions than hypoxic conditions. Alternatively, these data could be due to a difference in ␳0 cells generated
by ethidium bromide. Recently three independent reports utilized rigorous
genetic methods to manipulate mitochondrial electron transport system to
determine the requirement of a functional mitochondrial electron transport in
hypoxic stabilization of HIF-␣ protein. The first study used mouse genetics
to knockout cytochrome c [18]. In the electron transport chain, cytochrome
c accepts electrons from complex III and transfers them to complex IV to be
used to reduce molecular oxygen to water. Murine embryonic cells lacking
cytochrome c failed to stabilize HIF-␣ during hypoxic conditions. The other
two studies demonstrated that inhibiting complex III function by knocking down the Rieske Fe-S protein inhibits the ability of multiple cell lines to
stabilize HIF-␣ in hypoxic conditions [16,17]. However, cells that displayed
knockdown of the Rieske Fe-S protein were able to stabilize HIF-␣ in anoxic
conditions, further supporting the notion that different mechanisms allow for
the stabilization of HIF-␣ protein in hypoxic and anoxic conditions.
These data provide conclusive evidence that a functional electron transport
chain is required for the hypoxic stabilization of HIF-␣ protein. However, the
manner by which a functional electron transport chain activates HIF during
hypoxia remains controversial. The loss of a functional electron transport chain
prevents ROS generation as well as oxygen consumption and both have been proposed as potential mechanisms by which the mitochondrial electron transport chain
activates HIF during hypoxia. Both of these mechanisms are discussed below.
Mitochondria regulate HIF dependent on respiration
Molecular oxygen is used as the terminal electron acceptor in the mitochondrial
electron transport chain when cytochrome c oxidase converts oxygen into water
[1]. This property of mitochondria combined with the requirement of the PHDs
for molecular oxygen as a co-substrate led some groups to propose a model in
which mitochondrial oxygen consumption is the regulator of HIF activation
via the PHDs. These groups propose that during conditions of limited oxygen,
mitochondria create an oxygen gradient within the cells as a result of their ability
to consume oxygen [41,42]. This gradient would effectively sequester molecular
oxygen away from the cytosolic PHDs, thus inhibiting their ability to hydroxylate HIF-␣. In contrast, our studies have provided genetic experiments that demonstrate that HIF-␣ protein stability is independent of respiration in hypoxic
conditions. Cells lacking a functional complex IV due to a mutation in the surf
gene which encodes for a protein required for the proper assembly of complex
IV, retain the ability to stabilize HIF-␣ protein in hypoxic conditions [16].
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These cells that lack complex IV are respiratory deficient and cannot generate a
cytosolic oxygen gradient to sequester molecular oxygen from the PHDs. Thus
presently there is equivocal data on the role of mitochondrial respiration in the
regulation of HIF during hypoxia.
Mitochondria regulate HIF dependent on ROS
Initial studies in exploring the mechanism by which mitochondria regulate
the stability of HIF-␣ demonstrated that ROS levels paradoxically increase
in hypoxic conditions [36]. The increase in ROS production during hypoxia
is required and sufficient to stabilize HIF-␣ protein [37]. Cells deficient in
cytochrome c or Rieske Fe-S protein which are unable to stabilize HIF during
hypoxia also do not display an increase in ROS generation during hypoxia.
Incubating cells with pharmacological antioxidants such as ebselen and MitoQ
attenuates HIF activation in hypoxic conditions [17,43]. Expression of protein
antioxidants such as GPX (glutathione peroxidase) and catalase also attenuates
HIF-␣ protein stabilization and activation, but expression of SOD (superoxide
dismutase) has no effect [16]. SOD converts superoxide into H2O2, while GPX
and catalase converts H2O2 into water. The specificities of these antioxidants
for different forms of ROS tends toward the conclusion that H2O2 is the ROS
moiety required for stabilization of HIF-␣ protein. In fact HIF-␣ protein is
stabilized when cells are pulsed with 25␮ t-butyl H2O2, a more stable form
of H2O2, in normal oxygen conditions, indicating that H2O2 is also sufficient
to activate HIF-mediated transcription [37]. ROS are a normal by-product
of electron transport within the mitochondria, therefore the ability of cells
to increase the generation of ROS in hypoxic conditions provides the link
between mitochondrial electron transport and HIF activation.
A major controversy with the ROS model with respect to hypoxic activation of HIF is whether hypoxia increases ROS. There have been multiple
studies that have demonstrated both an increase and a decrease in ROS production during hypoxia [36,38,44–48]. All of these studies utilized dyes which
have limitations in their measurements of intracellular oxidative stress [49].
To resolve whether hypoxia increases or decreases ROS, Guzy et al. [17] have
utilized a sensitive FRET (fluorescence resonance energy transfer) probe to
assess redox status in the cytosol during hypoxia. This probe consists of a
fusion protein containing two fluorescent peptides (CFP and YFP) linked by
a redox-sensitive hinge that contains cysteine thiols that become cross-linked
by oxidant stress. When expressed in cells the redox-sensitive FRET probe
responds to hypoxia by producing a dose-dependent increase in FRET ratio.
FRET can be difficult to use, thus the recent development of redox-sensitive
GFP (green fluorescent protein) probes will help resolve whether hypoxia
increases or decreases ROS. Furthermore, Pan et al. [50] have shown that
MitoQ can inhibit respiration thus making it difficult to discern whether
MitoQ works as a respiratory inhibitor or an antioxidant. Future genetic studies need to uncouple ROS generation from oxygen consumption to address
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which function of the mitochondrial electron transport chain is the major
regulator of HIF during hypoxia.
Mitochondria and PHDs
The most proximal regulators of HIF activation are the PHDs. How mitochondrial-generated ROS would cross-talk to the PHDs is not presently
known. The addition of exogenous H2O2 to cells under normal oxygen conditions stabilizes the HIF-␣ protein, implying that ROS inhibit the hydroxylation of the HIF-␣ protein. Presently there are two mechanisms by which ROS
could prevent hydroxylation of HIF-1␣ protein. One possibility is that low
levels of oxygen decrease PHD activity while the ROS decrease the availability
of the co-factor Fe2+. Indeed Gerald et al. [51] found that loss of JunD caused
ROS to accumulate which decreased the availability of Fe2+ and reduced the
activity of HIF-PHDs [51]. A second possibility is that ROS activate signalling
pathways that catalytically make PHDs inactive. Multiple signalling pathways
have been implicated in hypoxic stabilization of HIF [52–55]. Finally, it could
be that the low oxygen levels decrease both PHD activity and increase the
ROS produced during hypoxia. These two events coupled together decrease
PHD activity to levels that prevent hydroxylation of HIF-␣ protein during
hypoxia (Figure 2). Further studies need to address how the mitochondria and
PHDs crosstalk.
O2
H2O2
ARNT
Hydroxylated
HIF
ARNT
Fe2+
PHDs
Fe3+
PHDs
(inactive)
Degradation
Figure 2. Hypoxia directly decreases the activity of PHDs which is coupled with an
increase in ROS generation; this depresses PHD activity therefore inhibiting hydroxylation of the HIF-␣ protein
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Concluding remarks
HIF is the master regulator of oxygen homoeostasis, with key roles in various
physiological processes especially during development. Aberrant HIF activation
leads to pathophysiological processes, most notably cancer, thereby demonstrating the importance of understanding how HIF is regulated by oxygen. Existing
literature clearly demonstrates that mitochondria regulate HIF activation during
hypoxia. However, it is not clear whether it is the ability of the mitochondria
to generate ROS or simply consume oxygen that is the major requirement in
regulation of HIF. Furthermore, additional data are required to understand
how to couple mitochondria to regulation of PHD activity during hypoxia.
Understanding this pathway will provide insight into how hypoxic signalling
can be modified in disease to promote physiological processes as well as how to
treat disease by hindering the pathophysiological processes governed by HIF.
Summary
•
•
•
•
•
•
•
Decreased oxygen availability (hypoxia) promotes physiological processes such as energy metabolism, angiogenesis, cell proliferation and cell
viability through the transcription factor HIF.
Activation of HIF can also promote pathophysiological processes like
cancer and pulmonary hypertension.
HIF-1 is composed of two subunits, oxygen sensitive HIF-1␣ and
HIF-␤. HIF-1␣ is hydroxylated at two different proline residues and
an asparagine residue under normoxia. The hydroxylation of proline
residues serves as a recognition motif for VHL protein. The binding of
VHL protein targets the HIF-1␣ protein for ubiquitin-mediated degradation under hypoxia, the hydroxylation of proline and asparagine
is diminished, which allows for the protein to be stabilized and bind
to HIF-1␤ as well as p300/CBP to allow HIF-1-dependent gene transcription.
Multiple studies have demonstrated that the mitochondrial electron transport chain regulates the activation of HIF during hypoxia.
Presently, there are two models to explain the mitochondrial regulation
of HIF.
The first model states that mitochondria create an oxygen gradient
within the cells as a result of their ability to consume oxygen. This gradient effectively sequesters molecular oxygen away from the cytosolic
PHDs, thus inhibiting their ability to hydroxylate HIF-␣.
The second model postulates that mitochondria generate ROS which
are required and sufficient to activate HIF during hypoxia.
Future genetic studies need to uncouple ROS generation from oxygen
consumption to address which function of the mitochondrial electron
transport chain is the major regulator of HIF during hypoxia.
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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Saraste, M. (1999) Oxidative phosphorylation at the fin de siecle. Science 283, 1488–1493
Graeber, T.G., Osmanian, C., Jacks, T., Housman, D.E., Koch, C.J., Lowe, S.W. & Giaccia, A.J.
(1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours.
Nature 379, 88–91
Schroedl, C., McClintock, D.S., Budinger, G.R.S. & Chandel, N.S. (2002) Hypoxic but not anoxic
stabilization of HIF-1␣ requires mitochondrial reactive oxygen species. Am. J. Physiol. Lung Cell Mol.
Physiol. 283, L922–L931
Semenza, G.L. (1998) Hypoxia-inducible factor 1 and the molecular physiology of oxygen homeostasis. J. Lab. Clin. Med. 131, 207–214
Chandel, N.S., Budinger, G.R. & Schumacker, P.T. (1996) Molecular oxygen modulates cytochrome c oxidase function. J. Biol. Chem. 271, 18672–18677
Semenza, G.L. (1994) Regulation of erythropoietin production. New insights into molecular
mechanisms of oxygen homeostasis. Hematol. Oncol. Clin. North Am. 8, 863–884
Ryan, H.E., Lo, J. & Johnson, R.S. (1998) HIF-1␣ is required for solid tumor formation and embryonic vascularization. EMBO J. 17, 3005–3015
Maltepe, E., Schmidt, J.V., Baunoch, D., Bradfield, C.A. & Simon, M.C. (1997) Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature
386, 403–407
Adelman, D.M., Maltepe, E. & Simon, M.C. (2000) Placental cell fates are regulated in vivo by HIFmediated hypoxia responses. Genes Dev. 14, 3191–3203
Forsythe, J.A., Jiang, B.H., Iyer, N.V., Agani, F., Leung, S.W., Koos, R.D. & Semenza, G.L. (1996)
Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.
Mol. Cell. Biol. 16, 4604–4613
Breier, G., Albrecht, U., Sterrer, S. & Risau, W. (1992) Expression of vascular endothelial growth
factor during embryonic angiogenesis and endothelial cell differentiation. Development 114,
521–532
Yu, A.Y., Shimoda, L.A., Iyer, N.V., Huso, D.L., Sun, X., McWilliams, R., Beaty, T., Sham, J.S.,
Wiener, C.M., Sylvester, J.T. & Semenza, G.L. (1999) Impaired physiological responses to chronic
hypoxia in mice partially deficient for hypoxia-inducible factor 1␣. J. Clin. Invest. 103, 691–696
Maranchie, J.K., Vasselli, J.R., Riss, J., Bonifacino, J.S., Linehan, W.M. & Klausner, R.D. (2002) The
contribution of VHL substrate binding and HIF1-␣ to the phenotype of VHL loss in renal cell carcinoma. Cancer Cell 1, 247–255
Kondo, K., Klco, J., Nakamura, E., Lechpammer, M. & Kaelin, Jr, W.G. (2002) Inhibition of HIF is
necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 1, 237–246
Kung, A.L., Wang, S., Klco, J.M. & Livingston, D.M. (2000) Suppression of tumor growth through
disruption of hypoxia-inducible transcription. Nat. Med. 6, 1335–1340
Brunelle, J.K., Bell, E.L., Quesada, N.M., Vercauteren, K., Tiranti, V., Zeviani, M., Scarpulla, R.C. &
Chandel, N.S. (2005) Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab. 1, 409–414
Guzy, R.D., Hoyos, B., Robin, E., Chen, H., Liu, L., Mansfield, K.D., Simon, M.C., Hammerling, U. &
Schumacker, P.T. (2005) Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. Cell Metab. 1, 401–408
Mansfield, K.D., Guzy, R.D., Pan Y., Young, R.M., Cash, T.P., Schumacker, P.T. & Simon, M.C.
(2005) Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen
sensing and hypoxic HIF-␣ activation. Cell Metab. 1, 393–399
Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. (1995) Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U.S.A. 92,
5510–5514
O’Rourke, J.F., Tian, Y.M., Ratcliffe, P.J. & Pugh, C.W. (1999) Oxygen-regulated and transactivating
domains in endothelial PAS protein 1: comparison with hypoxia-inducible factor-1␣. J. Biol. Chem.
274, 2060–2071
© The Authors Journal compilation © 2007 Biochemical Society
POIN003_C02[17-28].indd 25
7/23/07 6:11:46 PM
26
Essays in Biochemistry volume 43 2007
21.
Wiesener, M.S., Turley, H., Allen, W.E., Willam, C., Eckardt, K.U., Talks, K.L., Wood, S.M.,
Gatter, K.C., Harris, A.L., Pugh, C.W., Ratcliffe, P.J. & Maxwell, P.H. (1998) Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible
factor-1␣. Blood 92, 2260–2268
Makino, Y., Cao, R., Svensson, K., Bertilsson, G., Asman, M., Tanaka, H., Cao, Y., Berkenstam, A.
& Poellinger, L. (2001) Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible
gene expression. Nature 414, 550–554
Jiang, B.H., Semenza, G.L., Bauer, C., Marti, H.H. (1996) Hypoxia-inducible factor 1 levels vary
exponentially over a physiologically relevant range of O2 tension. Am. J. Physiol. 271, C1172–
C1180
Masson, N., Willam, C., Maxwell, P.H., Pugh, C.W. & Ratcliffe, P.J. (2001) Independent function of
two destruction domains in hypoxia-inducible factor-␣ chains activated by prolyl hydroxylation.
EMBO J. 20, 5197–5206
Bruick, R.K. & S.L. McKnight (2001) A conserved family of prolyl-4-hydroxylases that modify HIF.
Science 294, 1337–1340
Epstein, 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. et al. (2001) C. elegans EGL-9 and mammalian homologs
define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107, 43–54
Iwai, K., Yamanaka, K., Kamura, T., Minato, N., Conaway, R.C., Conaway, J.W., Klausner, R.D. &
Pause A. (1999) Identification of the von Hippel-Lindau tumor-suppressor protein as part of an
active E3 ubiquitin ligase complex. Proc. Natl. Acad. Sci. U.S.A. 96, 12436–12441
Jaakkola, P., Mole, D.R., Tian, Y.M., Wilson, M.I., Gielbert, J., Gaskell, S.J., Kriegsheim, A.v.,
Hebestreit, H.F., Mukherji, M., Schofield, C.J. et al. (2001) Targeting of HIF-alpha to the von
Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472
Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J.M., Lane, W.S. &
Kaelin, Jr, W.G. (2001) HIF␣ targeted for VHL-mediated destruction by proline hydroxylation:
implications for O2 sensing. Science 292, 464–468
Maxwell, 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. (1999) The tumour suppressor protein VHL targets
hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275
Mahon, P.C., Hirota K. & Semenza G.L. (2001) FIH-1: a novel protein that interacts with HIF-1␣
and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 15, 2675–2686
Lando, D., Peet, D.J., Whelan, D.A., Gorman, J.J. & Whitelaw, M.L. (2002) FIH-1 is an asparaginyl
hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes
Dev. 16, 1466–1471
Freedman, S.J., Sun, Z.Y., Poy, F., Kung, A.L., Livingston, D.M., Wagner, G. & Eck, M.J. (2002)
Structural basis for recruitment of CBP/p300 by hypoxia-inducible factor-1␣. Proc. Natl. Acad. Sci.
U.S.A. 99, 5367–5372
Jiang, B.H., Zheng, J.Z., Leung, S.W., Roe, R. & Semenza, G.L. (1997) Transactivation and inhibitory domains of hypoxia-inducible factor 1␣ modulation of transcriptional activity by oxygen tension. J. Biol. Chem. 272, 19253–19260
King, M.P. & Attardi G. (1988) Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA. Cell 52, 811–819
Chandel, N.S., Maltepe, E., Goldwasser, E., Mathieu, C.E., Simon, M.C. & Schumacker, P.T. (1998)
Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc. Natl. Acad. Sci.
U.S.A. 95, 11715–11720
Chandel, N.S., McClintock, D.S., Feliciano, S.E., Wood, T.M., Melendez, J.A., Rodriguez, A.M. &
Schumacker, P.T. (2000) Reactive oxygen species generated at mitochondrial complex III stabilize
hypoxia-inducible factor-1␣ during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 275, 25130–
25138
Vaux, E.C., Metzen, E., Yeates, K.M. & Ratcliffe, P.J. (2001) Regulation of hypoxia-inducible
factor is preserved in the absence of a functioning mitochondrial respiratory chain. Blood 98,
296–302
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
© The Authors Journal compilation © 2007 Biochemical Society
POIN003_C02[17-28].indd 26
7/23/07 6:11:46 PM
E.L. Bell & N.S. Chandel
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
27
Enomoto, N., Koshikawa, N., Gassmann, M., Hayashi, J. & Takenaga, K. (2002) Hypoxic induction
of hypoxia-inducible factor-1␣ and oxygen-regulated gene expression in mitochondrial DNAdepleted HeLa cells. Biochem. Biophys. Res. Commun. 297, 346–352
Srinivas, V., Zhu, X., Salceda, S., Nakamura, R. & Caro, J. (2001) Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway. J. Biol.
Chem. 276, 21995–21998
Hagen, T., Taylor, C.T., Lam, F. & Moncada, S. (2003) Redistribution of intracellular oxygen in
hypoxia by nitric oxide: effect on HIF1␣. Science 302, 1975–1978
Doege, K., Heine, S., Jensen, I., Jelkmann, W. & Metzen E. (2005) Inhibition of mitochondrial
respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF)
intact. Blood 106, 2311–2317
Sanjuan-Pla, A., Cervera, A.M., Apostolova, N., Garcia-Bou, R., Victor, V.M., Murphy, M.P. &
McCreath, K.J. (2005) A targeted antioxidant reveals the importance of mitochondrial reactive
oxygen species in the hypoxic signaling of HIF-1␣. FEBS Lett. 579, 2669–2674
Duranteau, J., Chandel, N.S., Kulisz, A., Shao, Z. & Schumacker, P.T. (1998) Intracellular signaling
by reactive oxygen species during hypoxia in cardiomyocytes. J. Biol. Chem. 273, 11619–11624
Killilea, D.W., Hester, R., Balczon, R., Babal, P. & Gillespie, M.N. (2000) Free radical production
in hypoxic pulmonary artery smooth muscle cells. Am. J. Physiol. Lung Cell Mol. Physiol. 279, L408–
L412
Wood, J.G., Johnson, J.S., Mattioli, L.F. & Gonzalez, N.C. (1999) Systemic hypoxia promotes leukocyte-endothelial adherence via reactive oxidant generation. J. Appl. Physiol. 87, 1734–1740
Fandrey, J., Frede, S. & Jelkmann, W. (1994) Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem. J. 303, 507–510
Michelakis, E.D., Hampl, V., Nsair, A., Wu, X., Harry, G., Haromy, A., Gurtu, R. & Archer, S.L.
(2002) Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ.
Res. 90, 1307–1315
Tarpey, M.M. & Fridovich, I. (2001) Methods of detection of vascular reactive species: nitric oxide,
superoxide, hydrogen peroxide, and peroxynitrite. Circ. Res. 89, 224–236
Pan, Y., Mansfield, K.D., Bertozzi, C.C., Rudenko, V., Chan, D.A., Giaccia, A.J. & Simon, M.C.
(2007) Multiple factors affecting cellular redox status and energy metabolism modulate hypoxiainducible factor prolyl hydroxylase activity in vivo and in vitro. Mol. Cell. Biol. 27, 912–925
Gerald, D., Berra, E., Frapart, Y.M., Chan, D.A., Giaccia, A.J., Mansuy, D., Pouyssegur, J., Yaniv M.
& Mechta-Grigoriou, F. (2004) JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118, 781–794
Turcotte, S., Desrosiers, R.R. & Beliveau, R (2003) HIF-1␣ mRNA and protein upregulation
involves Rho GTPase expression during hypoxia in renal cell carcinoma. J. Cell Sci. 116, 2247–
2260
Mottet, D., Dumont, V., Deccache, Y., Demazy, C., Ninane, N., Raes, M. & Michiels, C. (2003)
Regulation of hypoxia-inducible factor-1␣ protein level during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3␤ pathway in HepG2 cells. J. Biol. Chem.
278, 31277–31285
Hirota, K. & Semenza, G.L. (2001) Rac1 activity is required for the activation of hypoxia-inducible
factor 1. J. Biol. Chem. 276, 21166–21172
Emerling, B.M., Platanias, L.C., Black, E., Nebreda, A.R., Davis, R.J. & Chandel N.S. (2005)
Mitochondrial reactive oxygen species activation of p38 mitogen-activated protein kinase is
required for hypoxia signaling. Mol. Cell. Biol. 25, 4853–4862
© The Authors Journal compilation © 2007 Biochemical Society
POIN003_C02[17-28].indd 27
7/23/07 6:11:46 PM