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HEMATOPOIESIS
Inhibition of mitochondrial respiration elevates oxygen concentration but leaves
regulation of hypoxia-inducible factor (HIF) intact
Kathrin Doege, Sandra Heine, Inga Jensen, Wolfgang Jelkmann, and Eric Metzen
The transcription factor hypoxia-inducible
factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published
regarding the role of the mitochondrial electron transport chain (ETC) in the regulation
of HIF-1␣. We assessed cellular hypoxia by
pimonidazole staining and blotting of the
O2-labile HIF-1 ␣-subunit in human osteosarcoma cell cultures (U2OS and 143B). In
conventional, gas-impermeable cell culture
dishes, ETC inhibitors had no effect on
pimonidazole staining or HIF-1␣ abundance
in a 20% O2 atmosphere; both parameters
were undetectable. Pimonidazole staining
and HIF activity were substantial in 0.1% O2
irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O2) pimonidazole staining and HIF-␣ expression
were detectable but strongly reduced after
ETC inhibition in conventional cell cultures.
All effects of ETC inhibition on HIF-1␣ regulation were eliminated in gas-permeable
dishes. As shown in a 143B subclone deficient in mitochondrial DNA (206␳0), genetic
inactivation of the ETC led to similar responses with respect to HIF-1␣ regulation
as ETC inhibitors. Our data demonstrate
that reduction of oxygen consumption reduces the O2 gradient in conventional cell
cultures, causing elevation of the cellular O2
concentration, which leads to degradation
of HIF-␣. (Blood. 2005;106:2311-2317)
© 2005 by The American Society of Hematology
Introduction
Adaptation to low oxygen concentration leads to a series of
responses through the transactivation of more than 70 target genes1
mediated by the hypoxia-inducible factor-1 (HIF-1). The hematopoietic growth factor erythropoietin (EPO) and the angiogenic
peptide vascular endothelial growth factor (VEGF) are the most
prominent examples of HIF-1 target genes. Actually, the HIF
binding site embedded in the 3⬘ enhancer of the EPO gene allowed
purification and characterization of HIF-1.2 HIF is also known to be
involved in angiogenesis, tumor growth, and apoptosis,3,4 which
makes it a challenging target for therapeutic manipulation. HIF-1 is
a heterodimeric transcription factor composed of 2 subunits.2 Each
HIF-1 subunit contains an N-terminal basic helix-loop-helix domain responsible for dimerization and DNA binding followed by a
PAS domain (Per, aryl hydrocarbon nuclear translocator [ARNT],
and Sim were the first members of this protein family) that is
involved in protein interactions.5,6 Both subunits are constitutively
expressed. While the ␤-subunit is present in the nucleus at
detectable levels under normoxia and hypoxia, HIF-1␣ levels are
affected by changes in the cellular oxygen partial pressure (pO2).7,8
In hypoxia, HIF-1␣ accumulates and is translocated to the nucleus
where it dimerizes with HIF-1␤, forming an active DNA-binding
complex (reviewed by Semenza1). In contrast, atmospheric pO2
leads to a rapid destruction by the ubiquitin-proteasome system.9,10
Initially, HIF-1␣ is hydroxylated at Pro564 and Pro402 by 2-oxoglutarate–dependent dioxygenases,11,12 which have been termed
“prolyl hydroxylase domain,” containing protein 1, 2, and 3
(PHD-1, 2, 3)13 or HIF-1␣ prolyl hydroxylase 3, 2, and 1,14 which
prevent the stabilization. Furthermore, HIF-1␣ is hydroxylated at
Asn803 by an enzyme first identified as “factor inhibiting HIF-1”
(FIH-1),15 which abrogates the transactivation by preventing the
binding of transcriptional coactivators such as cyclic adenosine
monophosphate response-element binding protein (CREB) binding
protein (CBP)/p300.16,17 Thus, the hydroxylases, which regulate
HIF-1␣ by a direct oxygen-dependent mechanism, are oxygen
sensors in vivo.
In addition to the regulation of the HIF by oxygen supply, there
are different possibilities to either increase HIF-1␣ levels in
normoxia or attenuate HIF-1␣ expression in hypoxia. The stabilization in normoxia can occur by inhibition of the prolyl hydroxylases
by transition metals, iron chelators,18 nitric oxide,19,20 2-oxoglutarate analogs,21 or by an increased translational activity induced by
growth factors, hormones, and other factors (reviewed by Bilton
and Booker22). A destabilization in an hypoxic environment can be
caused by carbon monoxide and, again, nitric oxide.23,24 It has also
been reported that inhibitors of complex I of the mitochondrial
electron transport chain (ETC) (eg, rotenone) can compromise
normal up-regulation of HIF-1␣ in response to hypoxia.25,26
Interestingly, conflicting data have been published for antimycin A,
an inhibitor of the downstream part of complex III of the ETC:
While one group reported induction of HIF-␣,26,27 other investigators observed a pronounced down-regulation of HIF-␣ in
hypoxia.25 Consequently, the interpretation of the data was markedly different: The first group26,27 proposed that hypoxia leads to
an increase in the generation of reactive oxygen species (ROSs),
which in turn stabilize HIF-␣. The second group25 has provided
evidence for 2 important points: All inhibitors of the ETC have the
same effect on HIF-1␣ and, furthermore, the degradation caused by
inhibition of mitochondrial degradation is dependent on HIF-␣
prolyl hydroxylases. Thus, this group suggested that all inhibitors
of the ETC cause an intracellular redistribution of molecular
From the Institute of Physiology, University of Lübeck, Germany.
Lübeck, Germany; e-mail: [email protected].
Submitted March 21, 2005; accepted May 30, 2005. Prepublished online as
Blood First Edition Paper, June 9, 2005; DOI 10.1182/blood-2005-03-1138.
Supported by Deutsche Forschungsgemeinschaft (SFB367-C8) (W.J., E.M.)
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Eric Metzen, Institute of Physiology, Ratzeburger Allee 160, D23538
© 2005 by The American Society of Hematology
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
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BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
DOEGE et al
oxygen, which reactivates HIF-␣ prolyl hydroxylases. Two other
groups of investigators who used almost anoxic conditions (0.1%
O2) were unable to detect any effect of inhibition of the ETC on
HIF-␣ regulation.28,29 Here, we present a unifying hypothesis that
accommodates nearly all the observations reported.
Materials and methods
Reagents
The inhibitors of the respiratory chain were purchased from Sigma
(Steinheim, Germany) (rotenone, myxothiazol, antimycin A) and from
Merck (Darmstadt, Germany) (potassium cyanide [KCN]). Dimethyloxalylglycine (DMOG) was bought from Porphyrin Systems (Lübeck, Germany).
The Hypoxyprobe-1 Kit (Chemicon International, Temucula, CA) for the
detection of tissue hypoxia was a kind gift from Dr Klaus Wagner,
University of Lübeck, Germany.
Cell culture
Human hepatoma cells (HepG2) were maintained in RPMI medium (Gibco,
Karlsruhe, Germany). Two human osteosarcoma cell lines (U2OS and
143B-wt) were cultured in Dulbecco modified Eagle medium (DMEM;
Gibco). All growth media were supplemented with 10% (vol/vol) fetal calf
serum (FCS; Gibco), 2 mM glutamine (Gibco), and 50 IU/mL penicillin and
50 ␮g/mL streptomycin (Sigma). The mitochondrial DNA-deficient derivative 143B-206␳0 of 143B-wt cells30 was provided by Prof Rudolf Wiesner,
University of Cologne, Germany. For ␳0 cells DMEM was additionally
supplemented with 50 ␮g/mL uridine. The ␳0 status was proven by uridine
auxotrophy; all ␳0 cells failed to grow in the absence of uridine in the
growth medium.
The studies were carried out using conventional polystyrene dishes or
special gas-permeable dishes (Petriperm; Bachofer, Reutlingen, Germany)
with a hydrophilic fluoroethylene-propylene (FEP) copolymer Teflon
bottom membrane of 25 ␮m thickness on which the cells were grown.
Dishes of the same type are also available from another company (Lumox;
Greiner Bio-One, Frickenhausen, Germany). Cells were maintained in a
saturated humidified atmosphere at 37°C, 5% CO2 and 95% air. For these
culture conditions we use the term “normoxia” because we have experimentally demonstrated the absence of pimonidazole staining and of HIF
activation in our cells under these conditions (see “Results”). For hypoxic
stimulation, cultures were flushed in an InvivO2 400 hypoxia workstation
(Ruskinn Technologies, Leeds, United Kingdom) with a gas mixture of
0.1%, 1%, or 3% O2, 5% CO2, and balance N2.
Protein extraction and analysis
Whole cell extracts for HIF-1␣ analysis were prepared on ice using a
denaturing urea lysis buffer (8 M urea, 1% sodium dodecyl sulfate [SDS],
8.7% [vol/vol] glycerol, and 10 mM Tris [tris(hydroxymethyl)aminomethane] pH 6.8) supplemented with protease inhibitors (Protease InhibitorCocktail Set V; Calbiochem, Darmstadt, Germany). After repeated rinsing with
ice-cold phosphate-buffered saline solution (PBS), cells were harvested and
subjected to centrifugation (2500 rpm, 5 minutes, 4°C), lysed, and then
sonicated. Twenty micrograms of protein per lane was loaded onto 7.5%
SDS polyacrylamide gels, separated by electrophoresis, and blotted onto
polyvinylidene fluoride (PVDF) membranes (Schleicher and Schuell,
Dassel, Germany). Membranes were blocked with 5% skim milk in PBS
overnight at 4°C. As primary antibody for the detection of HIF-1␣ protein, a
monoclonal HIF-1␣ antibody (BD Transduction Laboratories, Heidelberg,
Germany) diluted 1:1000 in 5% skim milk/PBS for 2 hours was used,
followed by incubation with an anti–mouse horseradish peroxidase–
coupled antibody (DAKO, Hamburg, Germany) in a dilution of 1:2000 in
5% skim milk/PBS for 1 hour. A polyclonal antibody raised against SP-1
(PEP2; Santa Cruz, Heidelberg, Germany) was diluted 1:2000 in 5% skim
milk/PBS. SP-1 is not hypoxia inducible and was used to verify uniform
loading and transfer. In this case a goat anti–rabbit horseradish peroxidase–
coupled secondary antibody (1:2000, 5% skim milk/PBS; DAKO) was
used. Antibody binding was detected using the enhanced chemiluminescence (ECL) Western Blotting Analysis system (Amersham Biosciences,
Freiburg, Germany).
Assay of EPO and VEGF
For measurement of EPO and VEGF secretion into the growth medium,
HepG2 cells were plated in 24-well dishes. When the cells approached
confluence the dishes were incubated either in 20% O2 (“normoxia”) or in
3% O2 (“hypoxia”) for 24 hours. The growth medium was collected, and
EPO secretion was analyzed by enzyme linked immunosorbent assay
(ELISA; Medac, Hamburg, Germany). In the same samples VEGF secretion was assessed by ELISA (R&D Systems, Wiesbaden, Germany). Both
ELISAs were done exactly as recommended by the manufacturer.
Pimonidazole staining and fluorescence microscopy
U2OS cells were grown on coverslips and stimulated with antimycin A (1
␮g/mL) or rotenone (1 ␮M) in hypoxia (3% or 0.1% O2) or remained
untreated in normoxia (20% O2) for 4 hours. Pimonidazole was administered to cells in a concentration of 200 ␮M for 45 minutes. Cells were fixed
by methanol incubation for 10 minutes at ⫺20°C, rehydrated in PBS,
followed by blocking of nonspecific binding sites with 3% bovine serum
albumin (BSA) in PBS. Coverslips were incubated with the fluorescein
isothiocyanate (FITC)–labeled monoclonal Hypoxyprobe-1 monoclonal
antibody 1 (mAb1) (1:100 in 3% BSA) for 45 minutes at 4°C while BSA
alone served as negative control. Finally, nuclei were visualized using
bisbenzimide fluorochrome (Calbiochem). After washing 3 times with PBS
and distilled water, coverslips were mounted on object holders with Mowiol
(Calbiochem). The immunostained cells were examined by fluorescence
microscopy using an Axioplan 2 imaging microscope with Zeiss planNeofluar 20⫻/0.5 numeric aperture objective. An Axiocam camera was
used to capture images, which were digitally saved using Vision Axiovision
3.1 software (Carl Zeiss Vision, Mannheim, Germany).
Oxygen consumption assay
Cells were cultured in 75 cm2 flasks to approximately 90% confluence.
After trypsinization, cells were spun down (650g [2000 rpm]; 5 minutes)
and resuspended in 1 mL FCS-free cell culture medium. Cells were then
transferred into a temperature-controlled reaction chamber set to 37°C
(Hansa-Tech Instruments, King’s Lynn, United Kingdom; kindly provided
by Prof Joachim Fandrey, University Essen, Germany) and stirred magnetically. Measurement of the oxygen concentration was done polarographically against a platinum electrode, and values were recorded once per
second. To demonstrate maximal inhibition of the ETC, inhibitors of the
respiratory chain were added directly into the reaction chamber. All
experiments were repeated 4 times with cells from independent cultures.
After the experiment, cells were counted and the respiration rate was
calculated from the decrease of the oxygen concentration in the reaction
chamber during the experiment.
Statistical analysis
The data in bar graphs are presented as means ⫹ standard deviation (SD). A
significant difference between 2 group means was determined by Student t
test for unpaired data. The Dunnett post hoc test was applied to compare a
control mean with several treatment means. P ⬍ .05 was considered
statistically significant.
Results
Mathematic statement of oxygen diffusion in monolayer
cell culture
Cellular oxygen supply in conventional polystyrene cell-culture
systems is a 1-dimensional diffusion process through the growth
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BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
HIF REGULATION AFTER MITOCHONDRIAL INHIBITION
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Figure 1. Western blot analysis of HIF-1␣ in extracts from human osteosarcoma cells (U2OS) treated with various mitochondrial inhibitors. Cells were cultured for 4
hours in normoxia (NOX) or hypoxia (HOX) with or without inhibitors of the ETC. The transcription factor SP-1, which is not responsive to hypoxia, served as a control for
uniform loading and transfer. (A) Cells were cultured in intermediate hypoxia (3% O2) on conventional polystyrene dishes. (B) Cells were cultured in intermediate hypoxia (3%
O2) on gas-permeable dishes. (C) Cells were incubated with or without antimycin A (1 ␮g/mL) or rotenone (1 ␮M) for 4 hours under strong hypoxic conditions (0.1% O2) on
conventional cell culture supports.
medium, and a physical description is given by the Fick First Law
of Gas Diffusion:
dU O 2
dt
Cgas ⫺ Ccell
⫽ ⫺ DA
h
where
dU O 2
dt
is the quantity of oxygen transported in a period of time from the
top layer to the bottom (ie, the cellular layer); D is the diffusion
constant; and A is the area of diffusion. Assuming that the oxygen
solubility in growth medium is sufficient to lead to an equilibrium
between the incubator gas and the top layer of the growth medium,
Cgas is the oxygen concentration defined by the composition of the
incubator atmosphere. Ccell is the oxygen concentration at the
bottom of the dish, and h is the diffusion distance between the top
and the bottom layer of the growth medium and is defined by the
volume of the growth medium used. The area A, Cgas, and h are
constant over time in an individual experiment. In this setting
oxygen consumption of the cells will lead to a decrease of Ccell (ie,
Ccell must be lower than Cgas). The difference between Cgas and Ccell
will induce oxygen diffusion through the medium. Of note, it is
impossible to predict Ccell from Cgas. Clearly, Ccell depends on cell
density, with dense cells leading to a more pronounced decrease of
Ccell. Inhibition of oxygen consumption is expected to increase Ccell
as compared with the noninhibited state; the boundary condition
here is that Ccell is equal to Cgas in the absence of any oxygen
consumption by the cells.
Effect of mitochondrial inhibitors on HIF-1␣ protein
expression in hypoxia
Previous studies on the effects of the mitochondrial ETC on the
stabilization of HIF-1 have led to controversial results.25,26,28,29 In a
first set of experiments we incubated human osteosarcoma cells
(U2OS) in the presence of various inhibitors of the ETC in
conventional cell culture dishes and analyzed the expression of
HIF-1␣ protein by Western blotting. Cells were subjected to the
experimental conditions for 4 hours with either antimycin A (1
␮g/mL), myxothiazol (1 ␮M), rotenone (1 ␮M), or potassium
cyanide (1 mM). All concentrations used led to maximal inhibition
of the ETC as confirmed by oxygen consumption measurements
(data not shown). None of the inhibitors of the ETC induced
detectable levels of HIF-1␣ in an incubator atmosphere composed
of 20% oxygen, 5% CO2, balance N2. All substances inhibited the
accumulation of HIF-1␣ under moderately hypoxic conditions (3%
O2; Figure 1A). The effect was detectable after 4 hours and
persisted after 8 hours (data not shown). Consistent with previous
reports25 and in line with our mathematic analysis, all substances
showed the same effect independent of the complex inhibited.
According to the mathematic statement of O2 diffusion, HIF
activation in conventional cell culture dishes can be caused by an
oxygen gradient over a rather long diffusion distance, which is in
the range of 2 to 5 mm depending on the volume of the growth
medium used. This gradient is largely reduced when cells are
grown on very thin (25 ␮m) FEP Teflon membranes in which
oxygen is soluble.31 We used these dishes in a second set of
experiments to eliminate oxygen diffusion gradients. Strikingly,
HIF-1␣ levels were no longer affected by the treatment with
mitochondrial inhibitors—that is, all cells treated with inhibitors
showed HIF-1␣ protein expression comparable to the level in
control cells (Figure 1B). Again, no effect in normoxia could be
observed. Importantly, the exposition time of the x-ray film of
Figure 1B was significantly longer than in Figure 1A. A direct
comparison between U2OS cells cultured on gas-impermeable
versus gas-permeable supports showed that HIF-1␣ levels were
markedly lower in gas-permeable dishes (Figure 2A). When cells
were cultured in oxygen-impermeable dishes and the oxygen
concentration in the incubator was reduced to almost anoxic
Figure 2. Immunoblot analysis of HIF-1␣ protein expression in conventional, oxygen-impermeable culture dishes versus gas-permeable dishes. (A) Human
osteosarcoma cells (U2OS) were exposed to intermediate hypoxia (3% O2) for 18 hours on the indicated type of culture dish. (B) Human osteosarcoma cells (143B-wt) and
their mitochondrial DNA-deficient derivative 143B-206␳0 were grown on conventional or gas-permeable cell culture dishes and incubated in 20% O2, 1% O2, or 3% O2.
Confluent 143B-wt expresses detectable levels of HIF-1␣. (C) Comparison of 143B-wt and 143B-206␳0 cells cultured on conventional versus oxygen-permeable supports at
3% oxygen.
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DOEGE et al
BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
The effect of ETC inhibition on HIF-␣ protein levels is
antagonized by prolyl hydroxylase inhibitors
Figure 3. EPO secretion cells in response to variation of the diffusion distance.
Human hepatoma cells (HepG2) were cultured in 24-well polystyrene dishes. When
the cells approached confluence the growth medium was replaced with 0.5 mL (which
corresponds to a diffusion distance of 2.5 mm) or 1 mL (diffusion distance 5 mm). The
experiment was done in the absence or presence of rotenone (1 ␮M). EPO concentrations
determined by ELISA were corrected for medium volume and are therefore given as
secreted units. Bars represent mean of 3 independent cultures ⫹ SD, without rotenone (䡺)
or with rotenone (o). Inductions of EPO secretion by hypoxia, by an increase of the
diffusion distance and by combination of both, were all statistically significant (P ⬍ .05) as
calculated by the Dunnett post hoc test.
conditions (0.1% O2), the destabilizing effect of the ETC inhibitors
on HIF-1␣ was abolished (Figure 1C).
In a further experiment we used 0.5 mL or 1 mL growth medium
in 24-well oxygen-impermeable culture dishes at intermediate
oxygen concentration (3%) to analyze the effect of an increased
diffusion distance. As a model we used HepG2 cells, which show
hypoxia-inducible secretion of EPO and VEGF. As expected, the
increase of the EPO secretion, which was caused by reduction of
the oxygen fraction in the incubator from 20% to 3%, was
statistically significant, but the induction of EPO secretion induced
by an increase of the diffusion distance from 2.5 mm (which
corresponds to 0.5 mL growth medium) to 5 mm (which corresponds to 1 mL) appeared even larger. In line with our hypothesis,
rotenone eliminated the hypoxic induction (Figure 3). Secretion of
the angiogenic peptide VEGF was affected in the same way with
the exception that rotenone had a less pronounced effect on VEGF
induction by hypoxia (data not shown).
HIF-1␣ in cells depleted of mitochondrial DNA
A direct comparison of U2OS cells grown in gas-permeable dishes
to cells cultured in conventional, oxygen-impermeable supports
revealed that HIF-1␣ levels were generally lower in gas-permeable
dishes (Figure 2A). To further explore the role of a functioning
mitochondrial respiratory system, we used cells that had been
depleted of mitochondrial DNA (␳0 cells) and thus lack activity of
the ETC. Regarding HIF-␣ regulation in ␳0 cells, previous reports
have come to controversial conclusions.27-29 The 206␳0 cell line
has been derived from the human osteosarcoma cell line 143B.30
With respect to cellular oxygen consumption, genetic inactivation
of the ETC should have an effect comparable to pharmacologic
inhibition. When we exposed 206␳0 cells to a moderately
hypoxic atmosphere of 3% or 1% O2 in polystyrene dishes, the
cells showed attenuated HIF-1␣ protein levels in comparison to
the HIF-1␣ induction in 143B-wt cells (Figure 2B). In oxygenpermeable cell culture supports, 206␳0 cells displayed variable
hypoxic induction of HIF-1␣ when the experiments were
repeated under identical conditions (compare Figure 2B-C).
These results suggest that parameters are involved in the
regulation of HIF-1␣ in 206␳0 cells that were not under control
in our experimental settings.
The HIF-␣ prolyl hydroxylases PHD1-3 catalyze hydroxylation of
distinct proline residues in normoxia, which is the initial signal for
von Hippel-Lindau protein (pVHL)–dependent proteasomal degradation. Enzyme activity is dependent on oxygen, ferrous iron, and
2-oxoglutarate.13 As a consequence the hydroxylases are inactive in
hypoxia and in the presence of iron chelators18 or oxoglutarate
analogs21 in normoxia. To assess whether activity of the PHDs has
a role in HIF-␣ destabilization by ETC inhibitors, we studied the
response of cells treated with the oxoglutarate analog DMOG and
inhibitors of the respiratory chain. U2OS cells were incubated with
either antimycin A (1 ␮g/mL) or rotenone (1 ␮M) or a combination
of mitochondrial inhibitor and DMOG (Figure 4) in an atmosphere
of 3% oxygen. We demonstrated HIF-1␣ accumulation in response
to DMOG in normoxia and reproduced the destabilizing effect of
antimycin A and rotenone in moderate hypoxia. Strikingly, the
effect of ETC inhibition on HIF-1␣ accumulation was abrogated by
simultaneous application of an inhibitor of the ETC and DMOG.
This result suggests that the enhanced degradation of HIF-1␣ in
response to mitochondrial inhibition is mediated by increased
activity of the PHDs, which require molecular oxygen as a
cosubstrate.
Immunohistochemical analysis with pimonidazole as
an hypoxia marker
In an additional experimental approach we sought to determine
cellular hypoxia using pimonidazole as a surrogate marker for
hypoxia.32,33 This substance has been used previously in cell
culture,34 in animal studies,35 and in tumor patients36 to detect
hypoxic cells in tissues. Pimonidazole is activated by reduced
nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form a nitro
radical anion intermediate that is degraded in the presence of
oxygen. In the absence of oxygen further reduction leads to the
production of a hydroxylamine intermediate that binds to thiol
groups of low molecular weight substances (eg, glutathione) and to
cysteine residues in proteins. When FITC-labeled antibodies are
bound to the complexes produced in hypoxia, fluorescence microscopy can be used to detect hypoxic cells. Importantly, high levels of
NADH and NADPH, which are expected in hypoxia and after
inhibition of the ETC, are not sufficient to form pimonidazole
adducts.32 To analyze the effects of inhibitors of the mitochondrial
respiratory chain, cells were treated with mitochondrial inhibitors
in moderate hypoxia (3% O2) as described above, followed by the
incubation with pimonidazole. Confluent and sparse cell cultures
were used in the experiments because from our mathematic
analysis it is predictable that sparse cells will not create a large
oxygen gradient. In addition, the same experiment was conducted
Figure 4. HIF-1␣ protein expression in U2OS cells exposed to inhibitors of the
electron transport chain in combination with PHD inhibitors. Cells were exposed
to DMOG (100 ␮M) in normoxia, or to antimycin A (1 ␮g/mL) or rotenone (1 ␮M) in
normoxia (NOX) or in hypoxia (3% O2, HOX) for 4 hours. Additionally, a combination
of an ETC inhibitor and DMOG was applied to the cells.
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HIF REGULATION AFTER MITOCHONDRIAL INHIBITION
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Figure 5. Cellular hypoxia assessed by pimonidazole staining. Human osteosarcoma cells (U2OS) were grown in oxygen-impermeable dishes and incubated in the
absence or in the presence of either antimycin A (1 ␮g/ mL) or rotenone (1 ␮M) in hypoxia for 4 hours. Normoxic, untreated cells served as a control. Intracellular pimonidazole
complexes, which are formed under hypoxic conditions (pO2 less than 10 mm Hg), were detected by fluorescence microscopy using an FITC-labeled monoclonal antibody.
Pimonidazole was applied to the cells for 45 minutes. Cell nuclei were visualized by bisbenzimide staining. Cells were exposed to (A) intermediate hypoxia (3% O2) or (B) to
almost anoxic conditions (0.1% O2).
under almost anoxic conditions (0.1% O2). Representative results
of 3 independent experiments are illustrated in Figure 5. Confluent
cell cultures displayed a substantial pimonidazole signal that was
abrogated by incubation with ETC inhibitors (Figure 5A). In contrast,
pimonidazole staining was virtually absent in subconfluent cell cultures
in moderate hypoxia (data not shown). Furthermore, treatment of sparse
cells with antimycin A or rotenone did not have a detectable effect on
pimonidazole staining (data not shown). These results demonstrated that
inhibition of the ETC in moderate hypoxia leads to an increase in the
cellular oxygen concentration.
When U2OS cells were incubated under almost anoxic conditions (0.1% O2) pimonidazole staining was easily detectable in the
absence and in the presence of ETC inhibitors (Figure 5B). This
observation is in line with our equation, which predicts that the
cellular oxygen concentration cannot exceed the oxygen concentration of the incubator atmosphere. As the HIF-␣ prolyl hydroxylases
are inactive under these conditions, HIF-1␣ accumulates (Figure 1C).
Discussion
In the present work we analyzed the expression of the the ␣-subunit
of the transcription factor HIF-1 in response to inhibition of the
mitochondrial electron transport chain. The relationship between
HIF-1␣ protein expression and oxygen concentration was established in HeLa S3 cells grown in suspension culture. The cells were
exposed to defined oxygen concentrations in a tonometer.8 It was
observed that maximal HIF-1␣ activation occurs at oxygen concentrations between 0% and 1%. Cyanide was used in this study to
eliminate all oxygen gradients and, interestingly, this treatment had
no effect on HIF-1␣ expression and DNA binding between 0% and
6% oxygen. HIF-1␣ is a key regulator of oxygen homeostasis
known to be involved in erythropoiesis, angiogenesis, apoptosis,
vasomotor control, and many other physiologic and pathologic
situations (reviewed by Pugh and Ratcliffe3 and Semenza1).
Therefore, regulation of HIF-1␣ is currently regarded as an
objective of high biomedical relevance.4 Obviously, understanding
the regulation of HIF-␣ was the primary focus of our study, but our
results may be of importance whenever oxygen-dependent metabolic steps are to be studied.
The mitochondrial electron transport chain maintains the intracellular energy status by production of ATP while consuming most
of the oxygen delivered to cells. The ETC is also a major site of
ROS generation. It has been proposed that mitochondria act as O2
sensors in vivo. Specifically, it has been reported that in hypoxia
complex III of the ETC produces ROSs, which stabilize HIF1␣.26,27 Within this concept inhibitors that act upstream of complex
III (eg, rotenone) have been found to block ROS generation and
HIF stabilization while inhibitors that affect a site downstream of
complex III (eg, antimycin A) were reported to induce ROSs and
HIF-1␣. These differences were the rationale in our study to use
various inhibitors. Importantly, all substances had the same effect
in our hands, which is in line with a recent report.37 Other groups
were unable to detect any effect of inhibition of the ETC on HIF-␣
regulation in almost anoxic conditions of 0.1% oxygen.28,29
Our project was based on the idea that oxygen consumption of
cultured cells leads to a decrease of oxygen concentration in
conventional polystyrene dishes, which are not permeable for
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BLOOD, 1 OCTOBER 2005 䡠 VOLUME 106, NUMBER 7
DOEGE et al
oxygen.38 This drop in pO2 can cause cellular hypoxia as was first
recognized for cultured hepatocytes 40 years ago.39 According to
our mathematic analysis and to our data, cellular oxygen levels
depend on the cell type used, the state of confluence, the metabolic
activity of the cells, the diffusion distance, and the oxygen fraction
of the incubator atmosphere. We have reported previously that
confluent cultures with an active metabolism (eg, HepG2 cells)
experience a pO2 of less than 2 mm Hg when incubated in a gas
atmosphere containing 20% oxygen,40 which is usually termed
“normoxia.” HepG2 cells have been used to investigate hypoxiadependent regulation of the EPO gene.41 In the present study, an
increase of the diffusion distance augmented EPO and VEGF
secretion in response to hypoxia. An inverse correlation between
diffusion distance and cellular oxygen concentration is well in line
with the equation presented in “Results.” This effect was ablated by
inhibition of the ETC. This result suggests that mitochondrial
oxygen consumption can deplete the environment of oxygen.
Consequently, inhibition of oxygen consumption either by pharmacologic inhibition or by elimination of mitochondrial DNA leads to
an alleviation of the drop in pO2 as predicted from the mathematic
description of oxygen diffusion given in “Results.”
We compared HIF-1␣ regulation of cells cultured in conventional dishes versus O2-permeable dishes where the growth medium as an oxygen diffusion barrier is circumvented, thus eliminating the diffusion gradient.31 In these dishes chemical inhibition of
the ETC was without effect on the regulation of HIF-1␣. In
conventional (ie, gas-impermeable) dishes ␳0 cells showed HIF
induction only at lower oxygen concentrations than wild-type cells.
This difference was reduced in gas-permeable dishes in some
experiments although this finding showed some degree of variation. Importantly, inhibition of the ETC in conventional dishes only
had an effect at an intermediate oxygen concentration in the
incubator atmosphere. At higher oxygen levels HIF-1␣ is not
detectable; thus an increase of the cellular pO2 remains undetected.
At a very low O2 concentration of 0.1% in the incubator gas, the
inhibition of the ETC can only have a minor effect: The cells
remain hypoxic, and HIF is active. Reassuringly, the effects of ETC
inhibition on cellular oxygen concentration in conventional cell
culture dishes were confirmed by pimonidazole staining of the
cells. The chemical processes that lead to pimonidazole-protein
complex formation in hypoxia have been discussed previously.
Importantly, in this report it has been shown that pimonidazole
immunofluorescence is regulated by oxygen tension but not by
NADH or NADPH levels.32 Thus, pimonidazole seems suitable to
monitor the oxygen supply in cell cultures. In our study, this
approach was necessary because HIF-1␣ could not serve as the
prime subject of the investigation and as an indicator of cell
hypoxia at the same time.
Our analysis is in line with all reports where very low oxygen
concentrations have been used.28,29,42 For intermediate concentrations all investigators have reported a down-regulation of HIF-1␣
caused by inhibition of the ETC. However, an alternative explanation has been given for this phenomenon: intermediate hypoxia
(1.5% O2) has been reported to enhance production of ROSs, which
were proposed to stabilize HIF-␣. Although we have not determined ROSs in the course of our study it seems unlikely that ROSs
mediate hypoxic induction of HIF-1␣. Firstly, the HIF-␣ prolyl
hydroxylases have a Michaelis constant (Km) of approximately 200
␮M, which leads to an excellent regulation of enzyme turnover at
low oxygen concentrations as determined experimentally.43 No
other mediator was necessary for this regulation in an in vitro assay.
Secondly, our results indicate that HIF-1␣ degradation in the
presence of ETC inhibitors is PHD dependent. Clearly, these
enzymes require dioxygen as a cosubstrate. Thirdly, it has been
reported that ROSs can oxidatively damage the catalytic domain of
the HIF-␣ hydroxylases (eg, as a consequence of genetic inactivation of JunD).44 If this were also the mechanism of hypoxic PHD
inactivation, it would be difficult to explain how reoxygenation
could lead to rapid degradation of HIF-␣, especially because ROS
generation is increased by reoxygenation.45-47 In addition, it was
reported recently that the ROS scavenging agent Trolox eliminates
fluorescence of dichlorofluorescein (DCF), which has been used as
an ROS indicator but has no impact on HIF-1␣ protein expression,48 which is well in line with our results.
In summary, our results indicate that the cellular oxygen
concentration is profoundly affected by the oxygen consumption of
the cultured cells in conventional culture dishes. The oxygen
gradient that results from this process can be reduced by inhibition
of the mitochondrial electron transport chain. The elevation of
cellular oxygen concentration leads to an increased activity of
dioxygen-dependent HIF-␣ hydroxylases. In our view it is important that the cellular oxygen status cannot be predicted from the
composition of the incubator atmosphere. When experimental
confirmation is required, cellular hypoxia should be monitored by
pimonidazole staining.
Acknowledgments
The authors thank Prof J. Fandrey, University of Essen, Germany,
for provision of the equipment for oxygen consumption measurements and Prof R. Wiesner, University of Cologne, Germany, for
sending us 143B-wt and 206␳0 cells. Dr K. Wagner, University of
Lübeck, is acknowledged for help with the pimonidazole staining.
We are also grateful to Tanja Svensson and Patricia Rouina for
excellent cell culture work and to Dorothea Brennecke for preparation of the manuscript.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
2005 106: 2311-2317
doi:10.1182/blood-2005-03-1138 originally published online
June 9, 2005
Inhibition of mitochondrial respiration elevates oxygen concentration
but leaves regulation of hypoxia-inducible factor (HIF) intact
Kathrin Doege, Sandra Heine, Inga Jensen, Wolfgang Jelkmann and Eric Metzen
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