Biochem. J. (2007) 405, 1–9 (Printed in Great Britain)
1
doi:10.1042/BJ20070389
REVIEW ARTICLE
Oxygen-dependent regulation of mitochondrial respiration
by hypoxia-inducible factor 1
Gregg L. SEMENZA1
Vascular Biology Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A., Department of Pediatrics, The Johns Hopkins
University School of Medicine, Baltimore, MD 21205, U.S.A., Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A., Department of
Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A., Department of Radiation Oncology, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205, U.S.A., and the McKusick–Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.
The survival of metazoan organisms is dependent upon the utilization of O2 as a substrate for COX (cytochrome c oxidase),
which constitutes Complex IV of the mitochondrial respiratory
chain. Premature transfer of electrons, either at Complex I or at
Complex III, results in the increased generation of ROS (reactive oxygen species). Recent studies have identified two critical
adaptations that may function to prevent excessive ROS production in hypoxic cells. First, expression of PDK1 [PDH (pyruvate
dehydrogenase) kinase 1] is induced. PDK1 phosphorylates
and inactivates PDH, the mitochondrial enzyme that converts
pyruvate into acetyl-CoA. In combination with the hypoxiainduced expression of LDHA (lactate dehydrogenase A), which
converts pyruvate into lactate, PDK1 reduces the delivery of
acetyl-CoA to the tricarboxylic acid cycle, thus reducing the
levels of NADH and FADH2 delivered to the electron-transport
chain. Secondly, the subunit composition of COX is altered in
hypoxic cells by increased expression of the COX4-2 subunit,
which optimizes COX activity under hypoxic conditions, and
increased degradation of the COX4-1 subunit, which optimizes
COX activity under aerobic conditions. Hypoxia-inducible factor
1 controls the metabolic adaptation of mammalian cells to hypoxia
by activating transcription of the genes encoding PDK1, LDHA,
COX4-2 and LON, a mitochondrial protease that is required for
the degradation of COX4-1. COX subunit switching occurs in
yeast, but by a completely different regulatory mechanism, suggesting that selection for O2 -dependent homoeostatic regulation
of mitochondrial respiration is ancient and likely to be shared by
all eukaryotic organisms.
INTRODUCTION
The goal of oxidative phosphorylation is the transfer of electrons through a series of acceptor cytochromes in order to generate
a proton gradient within the inner mitochondrial membrane
(Scheme 2). The potential energy of this gradient is used to
synthesize ATP. O2 is utilized as the ultimate electron acceptor,
resulting in the production of water, a process that is catalysed
by COX (cytochrome c oxidase; Complex IV). This process is
not completely efficient, so that electron transfer to O2 may occur
either at Complex I or Complex III, resulting in the generation
of superoxide radicals, which are converted into H2 O2 by the
activity of superoxide dismutase. These ROS (reactive oxygen
species) have the potential to oxidize cellular proteins, lipids
and nucleic acids and, by doing so, may cause cell dysfunction
or death (Scheme 3). By contrast, physiological levels of mitochondrial ROS are utilized by mammalian cells as a means of
signal transduction. Most notably, increased mitochondrial ROS
production under hypoxic conditions is required for activation of
HIF-1 (hypoxia-inducible factor 1), as discussed below.
Several recent studies that will be the main subject of the
present review have revealed that mammalian cells utilize multiple
homoeostatic mechanisms to modulate O2 consumption, glucose
metabolism and mitochondrial respiration in response to changes
in cellular O2 availability. These mechanisms serve to strike a
balance between the properties of O2 as a facilitator of efficient
The evolution of multicellular organisms on Earth was dependent
upon two critical events. The first event, ∼ 2.5 billion years ago,
was the evolution of organisms that transduced solar energy into
the chemical energy of carbon bonds [1]. During photosynthesis,
CO2 , water and light are utilized to generate glucose and, as a
side product, O2 . The resulting progressive rise in atmospheric O2
levels led ∼ 1.5 billion years ago to the second critical event, which
was the establishment of a symbiotic relationship between singlecelled organisms and internalized primitive cells that became
specialized (as mitochondria) to perform a series of chemical
reactions in which glucose and O2 were utilized to generate
ATP with CO2 and water as side products. Compared with the
simple fermentation of glucose to lactate, this process of oxidative
phosphorylation greatly increased (∼ 18-fold) the net yield of
ATP per mol of glucose consumed. The creation of the glucose–
O2 –ATP cycle (Scheme 1) provided biochemical conditions that
were permissive for the evolution of higher organisms, as the
dramatic increase in energy production resulting from oxidative
phosphorylation provided the driving force required for the coordination of individual cells to act in concert as a multicellular
organism. However, as discussed below, the utilization of O2 was
not without its risks.
Key words: cytochrome c oxidase (COX), hypoxia-inducible
factor-1 (HIF-1), mitochondrial respiratory chain, oxidative phosphorylation, reactive oxygen species (ROS), tricarboxylic acid
cycle.
Abbreviations used: CBP, CREB (cAMP-response-element-binding protein)-binding protein; COX, cytochrome c oxidase; FIH-1, factor inhibiting HIF-1;
HIF-1, hypoxia-inducible factor 1; HPH1-3, HIF-1α prolyl hydroxylase; LDHA, lactate dehydrogenase A; PDH, pyruvate dehydrogenase; PDK1, pyruvate
dehydrogenase kinase 1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.
1
To whom correspondence should be sent, at the following address: Institute of Cell Engineering, The John Hopkins University School of Medicine,
Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD 21205, U.S.A. (email [email protected]).
c The Authors Journal compilation c 2007 Biochemical Society
2
G. L. Semenza
Scheme 1
The biochemical circle of life
Through the process of photosynthesis, plants and cyanobacteria (‘blue–green algae’) use solar
energy to synthesize glucose (C6 H12 O6 ) from CO2 and water, with O2 produced as a side product.
Glucose and O2 are used by most organisms to convert ADP into ATP, through the process of
oxidative phosphorylation, which yields CO2 and water as side products. ‘hv ’ represents solar
energy [where h is Planck’s constant (6.62 × 10−34 J · s) and v is the frequency of the photons
in Hz].
ATP production and O2 as a generator of ROS (Scheme 3). At
least one of these homoeostatic mechanisms is also utilized by
yeast, suggesting that selective pressure for adaptation to changes
in oxygenation has existed throughout eukaryotic evolution.
HIF-1: MASTER REGULATOR OF OXYGEN HOMOEOSTASIS
Oxygen homoeostasis represents a central organizing principle
of metazoan evolution and development. At the molecular level,
a critical event in metazoan evolution was the emergence of a
transcription factor that functions to regulate gene expression
in response to changes in O2 availability. HIF-1 consists of an
Scheme 2
Scheme 3
Oxygen homoeostasis
The O2 partial pressure (‘tension’; P O2 ) in each mammalian cell is determined by the supply
of O2 by diffusion from the vasculature and the local consumption of O2 by the cell and those
surrounding it. In each cell, a set point is established at which consumption of O2 and production
of ATP and ROS are optimized. Changes in P O2 result in decreased ATP production and/or
increased ROS production, which, if uncorrected by homoeostatic mechanisms, can result in
cell death.
oxygen-regulated HIF-1α (or HIF-2α) subunit and a constitutively
expressed HIF-1β subunit. HIF-1 activates the transcription of
target genes by binding to the core DNA sequence 5 -RCGTG-3
(where R is G or A) within cis-acting hypoxia response elements and recruiting the trans-acting co-activators p300 and
CBP [CREB (cAMP-response-element-binding protein)-binding
protein]. Hundreds of human genes are regulated by HIF-1 [2,3]
and dozens of these have been established as direct targets by
identification of critical HIF-1 binding sites (Table 1).
Mitochondrial oxidative phosphorylation
The respiratory-chain complexes are as follows: I, NADH:ubiquinone oxidoreductase; II, succinate:ubiquinone oxidoreductase; III, cytochrome bc 1 complex; IV, COX. IMM and OMM refer to the
mitochondrial (Mito) inner and outer membranes respectively. CoQ, coenzyme Q; Cyt C, cytochrome c ; O2 •− , superoxide anion; TCA, tricarboxylic acid. An animated version of this Scheme can be
found at http://www.BiochemJ.org/bj/405/0001/bj4050001add.htm
c The Authors Journal compilation c 2007 Biochemical Society
O2 -dependent regulation of mitochondrial HIF-1
Table 1
Genes that are directly regulated by HIF-1
Gene product
Reference(s)
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4
ABCB1 (multidrug resistance 1)
ABCG2 (ATP-binding cassette, subfamily G, member 2)
Adrenomedullin
Aldolase A
Atrial natriuretic peptide
Caeruloplasmin
Carbonic anhydrase 9
CD18 (a β 2 integrin)
CITED2*/p35srj
c-Met tyrosine kinase
Connective-tissue growth factor
COX4-1
CXCR4 [chemokine (C-X-C motif) receptor 4; fusin]
Cyclo-oxygenase 2
CYP3A6 (cytochrome P450 3A6)
DEC1 (differentiated embryo chondrocyte 1)
DEC2 (differentiated embryo chondrocyte 2)
Ecto-5 -nucleotidase (CD73)
EGLN1 (HIF-1α prolyl hydroxylase PHD2)
EGLN3 (HIF-1α prolyl hydroxylase PHD3)
Endocrine-gland-derived VEGF
Endoglin
Endothelin-1
Enolase 1
eNOS (endothelial nitric oxide synthase)
Erythropoietin
ETS-1 (avian erythroblastosis virus E2 oncogene homologue 1)
Glucose transporter 1
Glucose-regulated protein of 94 kDa
Glyceraldehyde-3-phosphate dehydrogenase
Haem oxygenase-1
HGTD-P (an HIF-1α-responsive pro-apoptotic molecule)
ID2 (inhibitor of differentiation/DNA binding proteins)
Integrin β 2
Intestinal trefoil factor
Lactase
LDHA
Leptin
Lon
Membrane type-1 matrix metalloproteinase
Midkine
MNL1 (myeloid cell factor 1)
Monocarboxylate transporter 4
NIP3 (nineteen kDa interacting protein-3)
Nitric oxide synthase 2
NOXA (a pro-apoptotic member of the Bcl-2 protein family)
Nur 77 (a nuclear orphan receptor)
Phosphoglycerate kinase 1
Plasminogen activator inhibitor 1
Procollagen prolyl-4-hydroxylase α(I)
RORα (retinoid-related orphan receptor α)
S100C (calgizzarin; a Ca2+ -binding protein of the S100 family)
Stromal-derived factor 1
Telomerase
Transferrin receptor
Transferrin
Transforming growth factor β 3
VEGF (vascular endothelial growth factor)
VEGF receptor-1 (Flt-1)
α 1B -Adrenergic receptor
[93,94]
[95]
[85]
[46]
[48]
[4,5]
[49]
[52]
[50]
[51]
[91]
[53]
[54]
[42]
[57–59]
[55]
[56]
[60]
[60]
[61]
[76]
[75]
[62]
[63]
[64]
[65]
[66]
[67,68]
[69]
[4,5,70]
[73]
[71,72]
[74]
[77]
[78]
[51]
[79]
[80]
[4,65]
[81]
[42]
[82]
[83]
[86]
[84]
[88]
[87]
[89]
[90]
[4,50,65,92]
[96]
[97]
[98,99]
[100]
[57,101]
[102]
[104,105]
[103]
[106]
[4–6,107]
[108,109]
[47]
* CBP/p300-interacting transactivator with Glu/Asp-rich C-terminal domain 2.
In mice and other mammals, HIF-1 is required for establishment
of all three components of the circulatory system (blood, heart and
vasculature) at mid-gestation, when sufficient O2 can no longer
3
be supplied to all cells of the developing embryo by simple
diffusion from the maternal circulation [4–6]. HIF-1 is present
in the simplest metazoan organisms, such as the nematode worm
Caenorhabditis elegans [7], which consists of ∼ 103 cells and has
no specialized physiological systems for O2 uptake or delivery.
As initially discovered in mammalian cells [4,5], HIF-1 induces
expression of genes encoding glucose transporters and glycolytic
enzymes when C. elegans is subjected to hypoxic conditions in the
laboratory [8], which may mimic conditions that are encountered
when worms burrow into the soil in search of food. Thus it is
likely that O2 -dependent regulation of glucose metabolism was a
primordial function of HIF-1.
O2 -DEPENDENT REGULATION OF HIF-1 ACTIVITY
HIF-1α is continuously synthesized and degraded by the proteasome under well-oxygenated conditions, so that the cell is
poised to respond instantaneously to reduced O2 availability
(hypoxia) by blocking the degradation of HIF-1α, allowing its
rapid accumulation [9–12]. The molecular mechanism by which
O2 regulates the half-life of HIF-1α involves the hydroxylation
of two proline residues (Pro402 and Pro564 in human HIF-1α)
by dioxygenases that utilize O2 and the tricarboxylic-acid-cycle
intermediate 2-oxoglutarate (α-ketoglutarate) as substrates for a
reaction in which one oxygen atom is inserted into a proline
residue and the other is used to convert 2-oxoglutarate into
succinate [7,13–16] (Scheme 4). Under hypoxic conditions, the
hydroxylation reaction is inhibited, owing to substrate (O2 )
limitation [7] and/or oxidation of the prolyl hydroxylases, which
contain Fe(II) in their active site, by ROS generated in the
mitochondria by electron-transport Complex III [17–19]. High
NO (nitric oxide) concentrations block the induction of HIF-1α
under hypoxic conditions, an effect that has been attributed to
the inhibition of electron-transport Complex IV activity and O2
consumption by NO, thus relieving substrate limitation of the
prolyl hydroxylases [20]. Hydroxylation of Pro402 and/or Pro564 in
human HIF-1α is required for the binding of pVHL (von Hippel–
Lindau protein), which is the recognition component of an E3
ubiquitin-protein ligase. Thus, under aerobic conditions, HIF-1α
is rapidly hydroxylated, ubiquitinated and degraded.
In addition to the negative regulation of HIF-1α protein stability
by prolyl hydroxylases, the transactivation function of HIF-1α is
regulated by FIH-1 (factor inhibiting HIF-1) [21]. FIH-1 hydroxylates an asparagine residue (Asn803 in human HIF-1α) in another
dioxygenase reaction that utilizes O2 and α-ketoglutarate [22,23].
Hydroxylation of Asn803 blocks the interaction of HIF-1α with
the co-activators p300 and CBP [24]. Thus, both the half-life and
specific activity of HIF-1 are directly regulated by the cellular O2
concentration through these hydroxylation reactions (Scheme 4).
This pathway represents the most completely defined metazoan
mechanism for oxygen sensing and signal transduction.
REGULATION OF GLUCOSE TRANSPORT AND GLYCOLYSIS BY HIF-1
Since the time of Louis Pasteur (1822–1895) it has been recognized that cells preferentially metabolize glucose by respiration
when O2 is available and by fermentation when O2 availability is
reduced. Under hypoxic conditions, mammalian cells increase
the expression of genes encoding glucose transporters as well as
virtually all of the glycolytic enzymes, including hexokinase, 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, aldolase,
phosphoglycerate kinase, enolase and LDHA (lactate dehydrogenase A) (Table 1). The increase in glycolytic flux functions to
compensate for the reduced efficiency of ATP production associated with reduced respiration. HIF-1, by co-ordinately regulating
c The Authors Journal compilation c 2007 Biochemical Society
4
G. L. Semenza
Scheme 4
O2 -dependent regulation of HIF-1
O2 -dependent dioxygenase reactions (red box), mediated by HPH1-3 (HIF-1α prolyl hydroxylase) and the asparaginyl hydroxylase FIH-1, each result in the transfer of one oxygen atom to HIF-1α
and one oxygen atom to 2-oxoglutarate (α-ketoglutarate) to form succinate (box at bottom left). Hydroxylation of proline residues Pro402 and Pro564 (P402 and P564) leads to binding of pVHL, which
promotes the ubiquitination and proteasomal degradation of HIF-1α under aerobic conditions. Hydroxylation of an asparagine residue Asn803 (N803) in the C-terminal transactivation domain (TAD
C) blocks the binding of the co-activators p300 and CBP. The hydroxylation reactions are inhibited under hypoxic conditions, leading to increased HIF-1α half-life and transcriptional activation.
bHLH, basic helix–loop–helix motif; PAS, PER-ARNT-SIM-like sensor domain; TAD, transactivation domain.
the expression of genes encoding each of the glucose transporters and glycolytic enzymes described above [4], functions as
an essential mediator of the ‘Pasteur effect’ in mammalian cells
[25]. Macrophages and granulocytes, which must function in the
hypoxic microenvironment associated with tissue inflammation
and infection, depend on glycolysis for ATP production and are
rendered non-functional and energy-depleted in the absence of
HIF-1 [26]. In response to stimuli other than hypoxia, several
other transcription factors have been shown to regulate genes
encoding glycolytic enzymes, including Myc, Sp1/Sp3 and USF2
(upstream stimulatory factor 2) [27,28]. The activity of glycolytic
enzymes is also acutely regulated by NO, AMP-dependent kinase
and other signal-transduction pathways [29].
REGULATION OF THE PYRUVATE DEHYDROGENASE REACTION
BY HIF-1
Although conventional wisdom held that reduced respiration in
hypoxic cells was simply a consequence of reduced substrate
(O2 ) availability, early molecular studies had revealed that the
increase in the levels of mRNAs encoding glycolytic enzymes was
accompanied by a co-ordinate decrease in the levels of mRNAs
encoding respiratory-chain proteins, suggesting that respiration
might be actively repressed in response to hypoxia [30]. A key
decision point in the metabolism of glucose occurs at pyruvate,
which can be converted either into lactate by LDHA or into acetyl
c The Authors Journal compilation c 2007 Biochemical Society
CoA by the mitochondrial enzyme PDH (pyruvate dehydrogenase). The activity of the catalytic subunit of PDH is negatively
regulated by phosphorylation, which is mediated by PDK (PDH
kinase). Expression of the PDK1 gene is induced by hypoxia in
a HIF-1-dependent manner [31,32]. Thus pyruvate metabolism is
dramatically altered in hypoxic cells by the co-ordinate stimulation of LDHA and inhibition of PDH. As a result, pyruvate is
shunted away from the mitochondria, reducing flux through the
tricarboxylic acid cycle and thereby decreasing the delivery of
reducing equivalents to the electron-transport chain.
These observations suggested that the reduction in tricarboxylic-acid-cycle activity is an adaptive response to hypoxia. When
HIF-1α-deficient mouse embryo fibroblasts were maintained
under hypoxic conditions for 72 h, ROS levels increased dramatically, leading to cell death [31]. However, ROS levels and
apoptosis were markedly reduced in subclones transfected with
an expression vector encoding PDK1. These data suggest that flux
through the tricarboxlic acid cycle and, presumably, the electrontransport chain, are reduced under hypoxic conditions as an adaptive response that prevents the generation of toxic levels of ROS.
REGULATION OF COX ACTIVITY BY HIF-1
The hypothesis that alterations in electron transport chain activity
might occur in mammalian cells under hypoxic conditions
was supported by data from the budding yeast Saccharomyces
O2 -dependent regulation of mitochondrial HIF-1
cerevisiae demonstrating O2 -dependent regulation of COX subunit composition. COX, which is located in the mitochondrial
inner membrane, is a dimer in which each monomer consists of 13
subunits [33]. Subunits I, II and III (COX1–COX3 respectively),
which are encoded by the mitochondrial genome and constitute
the catalytic core of the enzyme, are highly conserved in all
eukaryotes. The high-resolution crystal structure of bovine COX
revealed that subunit IV (COX4) interacts, via its transmembrane
domain, with COX1 and, via its C-terminal hydrophilic domain,
with COX2 [33]. COX4 binds ATP, leading to allosteric inhibition
of COX activity at high ATP/ADP ratios [34].
Molecular modelling studies indicate that the spatial relationships of the Saccharomyces homologues of COX1, COX2 and
COX4 (which, in yeast, is designated COX5) are remarkably
similar to the mammalian complex [35]. The yeast genome contains two genes encoding COX5 proteins, which show reciprocal
patterns of gene expression: at high O2 concentrations, COX5a
transcription is activated and COX5b transcription is repressed,
whereas at low O2 concentrations, COX5a transcription is inactivated and COX5b transcription is de-repressed [36–38]. Substitution of COX5b for COX5a increases the catalytic-centre activity
(‘turnover number’; CCAmax ) of COX by increasing the rate of
electron transfer from haem a to the binuclear reaction centre
within COX1 [39,40]. Thus yeast cells adapt to hypoxia by
assembling COX complexes that have higher CCAmax values at
low O2 concentrations.
Mammalian cells express a predominant COX4-1 isoform,
whereas an alternative COX4-2 isoform is also expressed in
certain tissues [41]. However, neither the molecular mechanisms
regulating expression of the COX4I1 and COX4I2 genes that encode these proteins, nor the functional significance of alternative
isoforms, was known. Analysis of cultured mouse and human
cells revealed that COX4-2 mRNA and protein expression were
induced by hypoxia [42]. HIF-1 heterodimers containing HIF-1β
and either HIF-1α or HIF-2α bound to hypoxia response elements
located in the 5 -flanking region and first intron of the COX4I2
gene within nuclear chromatin of human cells cultured under
hypoxic conditions [42].
In contrast with COX4-2, COX4-1 mRNA levels did not
change in response to alterations in the cellular O2 concentration
[42]. However, COX4-1 protein levels were markedly reduced
in response to hypoxia as a result of increased degradation of
COX4-1 protein, which was HIF-1-dependent, but independent
of proteasome activity. Instead, the expression of LON, a mitochondrial protease, was required for hypoxia-induced degradation
of COX4-1. Remarkably, LON gene expression was induced in
response to hypoxia, and multiple HIF-1 binding sites were identified in the 5 -flanking region of the human LON gene by chromatin
immunoprecipitation and reporter-gene transcriptional assays
[42]. Thus HIF-1 mediates co-ordinate regulation of the COX4-1
and COX4-2 subunit expression in response to changes in O2
availability.
Analysis of the effects of gain-of-function, loss-of-function and
loss-of-function-with-subunit-rescue experiments for COX4-1
and COX4-2 revealed that the regulated expression of these subunits optimized the efficiency of respiration in human cells under
aerobic and hypoxic conditions respectively [42]. When COX4-2
was replaced by COX4-1, there were significant decreases in O2
consumption, COX activity and ATP concentration under hypoxic
conditions. When COX4-2 replaced COX4-1 under non-hypoxic conditions, O2 consumption, COX activity and ATP
concentration were maintained at normal levels, but at the cost
of increased ROS production and caspase activation.
Taken together, the results of these experiments indicate that the
COX4 subunit switch constitutes a critical adaptive response of
Scheme 5
cells
5
COX subunit composition in regulated by O2 in yeast and human
Under aerobic conditions S. cerevisiae and Homo sapiens express COX5a and COX4-1 subunits,
whereas under hypoxic conditions they express COX5b and COX4-2 respectively. The changes
in subunit composition provide a mechanism by which mitochondrial respiration is optimized
according to the cellular O2 concentration. Heme = Haem.
mammalian cells to hypoxia. In a striking example of convergent
evolution, mammalian COX4 and yeast COX5 subunit expression
are both regulated in an O2 -dependent manner. However, the
molecular mechanisms underlying this common strategy for
regulating COX activity differ (Scheme 5). In Saccharomyces
cerevisiae, COX5a transcription is activated by the Hap2/3/4/5
protein and COX5b transcription is repressed by the Rox1 protein
under aerobic conditions, whereas, under hypoxic conditions,
COX5a transcription is no longer activated and COX5b is no
longer repressed [35]. The involvement of O2 in this yeast pathway
is indirect: the transcription factors Hap2/3/4/5 and Hap1 [the
transactivator of Rox1 (regulation by oxygen 1)] are activated by
binding haem, which is only synthesized under aerobic conditions. In hypoxic mammalian cells, HIF-1, which is negatively
regulated by O2 -dependent hydroxylation, activates transcription
of the COX4I2 and LON genes, which leads to increased COX4-2
mRNA and protein synthesis and increased COX4-1 proteolysis.
COX4-2 and LON mRNA expression were induced by hypoxia in
the liver and lungs of mice exposed to 10 % (v/v) O2 , indicating
that this pathway represents a physiological response to hypoxia
[42].
COX4 subunit switching provides a mechanism to maintain the
efficiency of respiration under conditions of reduced O2 availability and may represent the initial adaptive response to hypoxia.
When hypoxia is too prolonged or severe for COX subunit switching to maintain energy and redox homoeostasis, the induction
of PDK1 activity may occur to shunt pyruvate away from the
mitochondria and thereby reduce tricarboxylic-acid-cycle activity
and flux through the respiratory chain. The extent to which PDK1
expression and/or COX4 subunit switching is utilized as a strategy
for maintaining oxygen homoeostasis may be tissue-specific,
as evidenced by the induction of COX4-2 and LON mRNA
expression in several, but not all, tissues of chronically hypoxic
mice [42]. These results are consistent with those obtained in
previous studies demonstrating that the battery of HIF-1 target
genes expressed in response to hypoxia differs from one cell
type to another [43]. Further studies are required to investigate
c The Authors Journal compilation c 2007 Biochemical Society
6
G. L. Semenza
and glucose in response to changes in the availability of these substrates, in order to most efficiently generate ATP without excessive generation of ROS (Scheme 6). Although HIF-1α deficiency
results in embryonic lethality, HIF-2α-deficient mice are viable
on certain genetic backgrounds, but succumb to multi-organ
failure with evidence of severe oxidative damage [45]. Studies
reviewed above indicate that changes in oxygenation, relative
to the normal physiological set point for a given cell, result in
increased ROS production and provide evidence that oxygen and
redox homoeostasis are inextricably linked (Scheme 3). HIF-1
plays a critical role in the maintenance of oxygen homoeostasis
by mediating increased transcription of genes encoding each of
the O2 -regulated pathway components that have been described
in the present review (Scheme 6). The remarkable similarities in
the regulation of COX subunit composition in yeast and human
cells indicate that the selection for O2 -dependent homoeostatic
regulation of mitochondrial respiration is ancient and likely to be
shared by all eukaryotic organisms.
Scheme 6
HIF-1
Co-ordinate regulation of glucose and energy metabolism by
In response to cellular hypoxia, increased HIF-1 activity leads to: increased glucose transport
into the cell (1); increased glycolysis (2) and conversion of pyruvate into lactate (3); decreased
conversion of pyruvate into acetyl-CoA (AcCoA) (4); and altered COX subunit composition that
maintains efficient electron transport and minimizes superoxide (O2 •− ) production (5). ETC,
electron-transport chain; ext , external; int , internal; TCA, tricarboxylic acid.
the adaptive significance of tissue-specific metabolic responses to
hypoxia. In addition, these adaptive metabolic responses may play
important roles in hibernation states in which O2 consumption is
dramatically reduced.
CENTRAL CONCLUSIONS
The survival of metazoan organisms is dependent upon the utilization of O2 as a substrate for COX, which constitutes Complex
IV of the mitochondrial respiratory chain. Premature transfer of
electrons at Complex I or at Complex III results in the generation
of ROS. The efficiency with which electrons are transferred to
O2 at Complex IV (rather than at Complex I or Complex III) is
dependent upon the net availability of: (i) O2 , which is determined
by relative rates of O2 delivery and consumption; and (ii) reducing
equivalents (NADH and FADH2 ), which are generated through
the oxidation of acetyl-CoA in the tricarboxylic acid cycle
(Scheme 2). The studies reviewed here suggest that reduced O2
availability results in a reduced rate of electron transfer to O2 by
COX. This mismatch between the delivery of electrons from
Complex III and their transfer to O2 at Complex IV results in an
increased rate of superoxide formation at Complex III, which
leads to increased HIF-1 activity [17–19]. HIF-1-mediated transcription results in two potential adaptive responses to hypoxia
that serve to correct the putative mismatch: (i) increased PDK1
expression, leading to decreased tricarboxylic-acid-cycle activity,
which reduces electron flux through Complexes I, III and IV;
and/or (ii) increased COX4-2 and LON expression, leading to increased efficiency of electron transfer to O2 at Complex IV as
a result of the COX4-1-to-COX4-2 subunit switch. These dual
mechanisms may be important in responding to different physiological stimuli. For example, continuous hypoxia leads to increased ROS generation at Complex III [17], whereas intermittent hypoxia results leads to increased ROS generation at Complex
I [44].
The studies reviewed here demonstrate that HIF-1 modulates
multiple key metabolic pathways to optimize the utilization of O2
c The Authors Journal compilation c 2007 Biochemical Society
I am grateful to Dr Nanduri Prabhakar (Department of Medicine, Division of Biological
Sciences, University of Chicago, Chicago, IL, U.S.A.) for providing helpful comments on
the manuscript before its submission. Work in my laboratory was supported by funds
from the Johns Hopkins Institute for Cell Engineering and by Public Health Service grants
N01-HV28180, P50-CA103175 and R01-HL55338 from the National Institutes of Health.
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Received 20 March 2007/10 April 2007; accepted 12 April 2007
Published on the Internet 13 June 2007, doi:10.1042/BJ20070389
c The Authors Journal compilation c 2007 Biochemical Society