Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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The molecular mechanisms by which cells
detect hypoxia (1.5% O2), resulting in the stabilization
of hypoxia-inducible factor 1 (HIF-1
) protein remain
unclear. One model proposes that mitochondrial generation of
reactive oxygen species is required to stabilize HIF-1
protein.
Primary evidence for this model comes from the observation that cells
treated with complex I inhibitors, such as rotenone, or cells that lack
mitochondrial DNA (
0-cells) fail to generate reactive
oxygen species or stabilize HIF-1
protein in response to hypoxia. In
the present study, we investigated the role of mitochondria in
regulating HIF-1
protein stabilization under anoxia (0%
O2). Wild-type A549 and HT1080 cells stabilized HIF-1
protein in response to hypoxia and anoxia. The
0-A549
cells and
0-HT1080 cells failed to accumulate HIF-1
protein in response to hypoxia. However, both
0-A549 and
0-HT1080 were able to stabilize HIF-1
protein levels
in response to anoxia. Rotenone inhibited hypoxic, but not anoxic,
stabilization of HIF-1
protein. These results indicate that a
functional electron transport chain is required for hypoxic but not
anoxic stabilization of HIF-1
protein.
rho zero; rotenone; complex I
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INTRODUCTION |
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HYPOXIA
TRANSCRIPTIONALLY activates a multitude of gene products involved
in metabolism, angiogenesis, and erythropoiesis through the activation
of the transcription factor hypoxia-inducible factor 1 (HIF-1) (for
review, see Ref. 28). HIF-1 is a heterodimer of two basic
helix loop-helix/PAS proteins, HIF-1 and the aryl hydrocarbon
nuclear translocator HIF-1
(32). Aryl hydrocarbon nuclear translocator protein levels are constitutively expressed and
not significantly affected by oxygen, whereas HIF-1
protein is
present only in hypoxic cells. During normoxia (21% O2),
the von Hippel-Lindau tumor suppressor protein (pVHL) binds to the oxygen-dependent degradation domain located in the central region of
HIF-1
. This binding results in the subsequent degradation of
HIF-1
protein through the ubiquitin-proteasome pathway (24, 26). The targeted degradation of HIF-1
via pVHL binding is dependent on the hydroxylation of proline residues within HIF-1
(15, 16). In contrast, the degradation of HIF-1
is
suppressed under hypoxic conditions, and transcription of mRNAs
encoding hypoxically responsive genes can occur.
A fundamental question for understanding HIF-1 regulation involves
the mechanism by which cells sense the lack of oxygen and initiate a
signaling cascade that results in the stabilization of HIF-1
protein. Our laboratory has recently proposed that mitochondrial complex III (Bcl complex) serves as an oxygen sensor by increasing the
generation of reactive oxygen species (ROS) during hypoxia that are
required for HIF-1
protein stabilization (6). In support of this model, hypoxia does not induce an increase in ROS
production or stabilize HIF-1
in cells lacking mitochondrial DNA and
electron transport activity (
0-cells). Complex I
inhibitors, such as rotenone and the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, prevent the hypoxic stabilization of HIF-1
by blocking the electron flow proximally to
complex III, thereby ablating the ROS generation (1, 6). In contrast, the complex IV inhibitor cyanide is sufficient to activate
HIF-1-dependent transcription in wild-type Chinese hamster ovary cells
and HT1080 cells under normoxic conditions (34). Furthermore, hypoxic induction of HIF-1
was severely reduced in
human xenomitochondrial cybrids harboring a partial (40%) complex I
deficiency (1).
An alternative model for oxygen sensing comes from the observation that
the hydroxylation of proline residues within HIF-1 is catalyzed by
prolyl hydroxylases and requires molecular oxygen and iron (4,
9). In the absence of oxygen, HIF-1
would not undergo proline
hydroxylation and subsequent pVHL-mediated ubiquitin-targeted
degradation. Thus the prolyl hydroxylases have been suggested to be
oxygen sensors that regulate stabilization of HIF-1
protein.
Moreover, recent studies have demonstrated that
0-cells
and Chinese hamster lung fibroblasts that display a significant deficiency in complex I activity are still able to stabilize HIF-1
protein at oxygen levels in the range of 0.1-0.5% O2
(30, 31). Depending on the cell confluence and the
metabolic rate of the cells, intracellular oxygen tensions might have
been close to anaerobic conditions in these latter studies. Therefore,
the lack of molecular oxygen would prevent proline hydroxylation, and
HIF-1
protein would be stabilized independent of intracellular
signaling pathways. Accordingly, in the present study, we examined
HIF-1
protein levels under hypoxia (1.5% O2) or anoxia
(0% O2) in
0-cells, cells with reduced
complex I activity, and wild-type cells exposed to rotenone. Our
results indicate that mitochondrial-dependent oxidant signaling is
required for hypoxic but not anoxic stabilization of HIF-1
protein.
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METHODS |
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Cell culture.
Human lung epithelial A549 cells, human fibrosarcoma HT1080 cells,
human kidney epithelial HEK-293 cells, and the Chinese hamster lung
fibroblast lines CCL16-B2 and CCL16-NDI1 were cultured in DMEM
containing pyruvate. The medium was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% heat-inactivated fetal calf
serum. Dr. Immo Scheffler provided the CCL16-B2 cells (3).
Dr. Takao Yagi provided the CCL16-NDI1 cells (29).
Wild-type A549 cells and wild-type HT1080 cells were incubated in
medium containing ethidium bromide (50 ng/ml), sodium pyruvate (1 mM), and uridine (50 µg/ml) for 4-6 wk to generate
0-A549 cells and
0-HT1080 cells
(20). These cells were not treated with rotenone or
antimycin at any point in the preparation of becoming
0-cells. The
0 status of cells was
confirmed by the absence of cytochrome oxidase subunit II by polymerase
chain reaction and the failure to grow in the absence of uridine in the
medium. In all experiments, cells were plated at 50% confluence to
prevent the development of anaerobic conditions at 1.5%
O2.
Oxygen conditions. Hypoxic conditions (1.5% O2, 93.5% N2, and 5% CO2) were achieved in a humidified variable aerobic workstation (INVIVO O2, Ruskinn Technologies). The INVIVO O2 contains an oxygen sensor that continuously monitors the chamber oxygen tension. Anoxic conditions (0% O2, 85% N2, 10% H2, and 5% CO2) were achieved in a humidified anaerobic workstation at 37°C (BugBox, Ruskinn Technologies). An anaerobic color indicator (Oxoid) confirms anaerobicity of the chamber. Before experimentation, media were preequilibrated overnight at either oxygen level.
Measurement of ROS.
ROS generation in cells was assessed by using the fluorescent probe
5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate
acetyl ester (CM-H2DCFDA) (Molecular Probes). Cells were
washed with PBS and loaded with CM-H2DCFDA (10 µM) for 30 min in MEM- without phenol red. CM-H2DCFDA is a
cell-permeable indicator for ROS that is nonfluorescent until the
removal of the acetate groups by intracellular esterases and oxidation
occurs within the cell (23). Cells were exposed to
normoxia (21% O2), hypoxia (1.5% O2), or
anoxia (0% O2) for 4 h in the presence of the
CM-H2DCFDA (10 µM), as described above. All procedures
were carried out in the dark. After the exposure, the cells were
washed and removed from the plate by using PBS, without calcium or
magnesium, containing 1 mM EDTA, centrifuged (200 g for 5 min), and resuspendend in PBS. Fluorescence was measured at the flow
cytometry core facility at the Cancer Center of Northwestern
University. The graphs were generated by using the Beckman Coulter
Epics XL-MCL running System II software (version 3.0).
Analysis of HIF-1 protein by immunoblotting.
HIF-1
protein was analyzed in nuclear extracts prepared from cells,
as previously described (6). Nuclear extract (40 µg) was
mixed with an equal volume of electrophoresis buffer (1.0 ml glycerol,
0.5 ml
-mercaptoethanol, 3.0 ml 10% SDS, 1.25 ml 1.0 M
Tris · HCl, pH 6.7, and 1-2 mg bromophenol
blue). After heating, the protein was resolved on a 7.5%
polyacrylamide-SDS gel and transferred to Hybond-enhanced
chemiluminescence nitrocellulose paper (Amersham). After transfer, the
gel was stained with Ponceau stain to verify uniform loading and
transfer. Membranes were blocked with 5% milk in TBS-T (10 mM
Tris · HCl, 150 mM NaCl, 0.1% Tween-20, pH 8.0)
for 3 h at room temperature and subsequently incubated with 2.0 µg/ml of HIF-1
antibody (Novus Biolgical Sciences) overnight at
4°C. The membrane was washed three times with TBS-T and incubated for
1 h at room temperature with 1.0 µg/ml horseradish
peroxidase-conjugated secondary antibody (Cell Signaling).
Subsequently, the membrane was washed three times with TBS-T and
analyzed by electrochemiluminescence (Amersham).
Transfection and reporter gene assays.
Transfections of A549 and 0-A549 cells were carried out
on cells plated on 35-mm petri dishes at 30-50% confluence by
using LipofectAMINE reagent (Life Technologies,) according to the
manufacturer's protocol. A typical transfection was performed by using
0.5 µg of a luciferase reporter driven by a trimer of a hypoxic
response element (HRE). The DNA-lipofectamine was incubated with plated cells for 24 h. Subsequently, the media were replaced, and cells were exposed to various conditions. Cell lysis was performed by using a
reporter gene lysis buffer from Promega. Luciferase assays were
performed by using the Luciferase assay system (Promega). Data were
normalized by using total protein concentration as determined by the
Bio-Rad protein assay (Bio-Rad).
Measurement of ATP levels and oxygen consumption rates. ATP levels were measured by the luciferin/luciferase method by using an ATP bioluminescence assay kit HS II (Roche Molecular Biochemicals). Cells were lysed with lysis buffer provided by the manufacturer. Luciferase reagent (50 µl) was manually injected into 50 µl of cell lysate, and luminescence was analyzed after a 30-s delay with a 2-s integration on a SpectraMax Gemini microplate reader (Molecular Devices). A standard curve was generated from known concentrations of ATP and used to calculate the concentration of ATP in each sample. Luminescence increased linearly with the negative log of the ATP concentration in the samples over the range of concentrations measured. Data were normalized by using total protein concentration as determined by the Bio-Rad protein assay (Bio-Rad). Oxygen consumption was measured by using the Oxytherm respirometer (Eurosep instruments). The Oxytherm system provides a simple method for computer-controlled measurement of oxygen consumption from liquid suspensions of 200 µl to 2.5 ml.
Quantitation of apoptosis.
Cells were fixed in 100% methanol for 2 min at 20°C, and nuclei
were stained with 1 µg/ml Hoescht no. 33258 for 30 min. The percentage of apoptotic cells was determined by scoring for
fragmented and/or condensed nuclei, as visualized by fluorescence
microscopy. For each treatment condition, at least 200 nuclei were scored.
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RESULTS |
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Hypoxic but not anoxic stabilization of HIF-1 protein requires a
functional electron transport chain.
Previously, our laboratory has shown that a functional electron
transport chain is required for the hypoxic (1.5% O2)
stabilization of HIF-1
protein in human hepatoma Hep3B cells
(6). It is not known whether a functional electron
transport chain is required for the anoxic (0% O2)
stabilization of HIF-1
protein. To test this hypothesis, wild-type
and
0-cells of human lung epithelial A549 cells and
human fibrosarcoma HT1080 cells were exposed to hypoxic and anoxic
conditions for 4 h (Fig. 1,
A and B, respectively). Wild-type A549 cells and wild-type HT1080 cells stabilized HIF-1
protein in response to both
hypoxia and anoxia. The
0-A549 cells and
0-HT1080 cells failed to accumulate HIF-1
protein
levels in response to hypoxia. However, both
0-A549
cells and
0-HT1080 cells were able to stabilize HIF-1
protein levels in response to anoxia. These results indicate that
hypoxic but not anoxic stabilization of HIF-1
protein requires a
functional electron transport chain.
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Hypoxia stimulates mitochondrial ROS generation in Chinese hamster
fibroblasts deficient in complex I activity.
Recently, Vaux et al. (31) have reported that CCL16-B2
cells containing a significant loss of complex I activity (<10%) still exhibit HIF-1 protein stabilization at 0.1% O2,
suggesting that complex I inhibition does not affect hypoxic HIF-1
protein stabilization. We investigated HIF-1
protein levels in
CCL16-B2 cells and CCL16-NDI1 cells under hypoxic and anoxic
conditions. The CCL16-B2 cells require glucose for growth and survival
(3, 8). These cells undergo rapid death in media
containing galactose instead of glucose. The CCL16-NDI1 cells are
CCL16-B2 cells that have been stably transfected with the NDI1
gene that encodes the rotenone-insensitive internal NADH-quinone
oxidoreductase of Saccharomyces cerevisiae mitochondria
(29). The NDI1 gene restores the NADH oxidase
activity of complex I. Thus CCL16-NDI1 cells are respiratory competent
and can grow in either glucose or galactose media. CCL16-B2 cells and
CCL16-NDI1 cells were both able to stabilize HIF-1
protein levels
under hypoxic and anoxic conditions (Fig.
5A). A possible explanation
for the ability of CCL16-B2 cells to stabilize HIF-1
protein is that
these cells retain their ability to generate ROS during hypoxia.
Indeed, hypoxia stimulated ROS production, as measured by the oxidation
of the fluorescent dye CM-H2DCFDA in both CCL16-B2 cells
and CCL16-NDI1 cells (Fig. 5, B and C). These
results indicate that cells with decreased complex I activity are still
able to generate ROS during hypoxia, thereby stabilizing HIF-1
protein.
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Rotenone abolishes hypoxic but not anoxic stabilization of HIF-1
protein.
Complex I inhibitors such as rotenone have been shown to inhibit the
stabilization of HIF-1
protein at 1-1.5% O2,
presumably by blocking the electron flow proximally to complex III,
thereby ablating the ROS generation during hypoxia (6,
14). To determine whether rotenone abolishes both the
hypoxic and the anoxic stabilization of HIF-1
protein, HEK-293
cells were exposed to rotenone (0.5 µg/ml) for 30 min under normal
oxygen conditions followed by exposure to hypoxia (1.5%
O2) or anoxia (0% O2). Rotenone abolished the hypoxic stabilization of HIF-1
protein (Fig.
6). In contrast, rotenone failed to
affect the anoxic stabilization of HIF-1
protein (Fig. 6). To
confirm that rotenone prevented hypoxic stabilization of HIF-1
protein by inhibiting complex I, we investigated whether methyl-succinate would restore the ability of HEK-293 cells to generate
ROS and stabilize HIF-1
protein in the presence of rotenone. Methyl-succinate is a complex II substrate that can provide
electrons to complex III in the presence of rotenone, thus
allowing cells to generate ROS at complex III during hypoxia. HEK-293
cells increased ROS production and stabilized HIF-1
protein during
hypoxia, as measured by the fluorescent dye CM-H2DCFDA
(Fig. 7). Rotenone, but not antimycin,
prevented the increase in CM-H2DCFDA fluorescence and
HIF-1
protein stabilization during hypoxia (Fig. 7). Antimycin inhibits complex III distally to the site of ROS production, thus maintaining the generation of ROS during hypoxia. Rotenone also abolished the increase in CM-H2DCFDA fluorescence and
HIF-1
protein stabilization observed during hypoxia in the presence
of antimycin (Fig. 7). Methyl-succinate restored CM-H2DCFDA
fluorescence and HIF-1
protein stabilization during hypoxia in the
presence of rotenone and antimycin (Fig. 7). The complex II inhibitor
thenoyltrifluoroacetone abolished CM-H2DCFDA fluorescence
and HIF-1
protein stabilization during hypoxia in the presence of
rotenone, antimycin, and methyl-succinate (Fig. 7). These results
suggest that rotenone prevents HIF-1
protein stabilization during
hypoxia by inhibiting complex I in HEK-293 cells.
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Functional consequences of hypoxia and anoxia.
We further examined the difference between hypoxia and anoxia by
determining the effects of oxygen concentration on respiratory rates,
ATP levels, HIF-1-dependent transcription, and apoptosis. To
investigate the changes in respiratory rate as a function of oxygen
levels, fifteen million A549 cells were placed in a respirometer. As
seen in Fig. 8A, the oxygen
consumption rate of A549 cells was independent of oxygen levels until
the oxygen levels reached ~0.5%. Below 0.5% oxygen levels, the
respiratory rate was dependent on oxygen levels. Furthermore, ATP
levels in A549 cells did not change between 21 and 1.5% O2
over 8 h (Fig. 8B). In contrast, there was a ~60%
decrease in ATP levels at 0% O2 compared with 21%
O2. To examine HIF-1-dependent transcription, A549 cells
were transiently transfected with a luciferase reporter construct
driven by a trimer of the HRE and exposed to hypoxia or anoxia for
8 h (Fig. 9A). Both
hypoxia and anoxia were able to activate HIF-1-dependent transcription,
as measured by an increase in HRE-luciferase expression. To address
whether a functional electron transport chain was required for hypoxic
or anoxic stimulation of HIF-1-dependent expression, 0-A549 cells were transfected with a luciferase reporter
construct driven by the HRE and exposed to hypoxia or anoxia for 8 h. Hypoxia-induced HRE-luciferase expression was not observed in
0-A549 cells. However, anoxia induced HRE-luciferase
(Fig. 9A). Although both hypoxia and anoxia can activate
HIF-1-dependent transcription, the long-term consequences of hypoxia
and anoxia are different with regard to apoptosis. A549 cells
exposed to anoxia underwent apoptosis after 48 h, whereas
cells exposed to hypoxia did not (Fig. 9B). Collectively,
these findings indicate that there are physiologically different
outcomes in cells exposed to hypoxia compared with anoxia.
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DISCUSSION |
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The molecular events regulating HIF-1 protein stabilization
during hypoxia are important for understanding the mechanisms of
cellular oxygen sensing. Our laboratory has previously proposed a model
in which the increased generation of ROS at complex III of the
mitochondrial electron transport chain serves as the oxygen sensor for
HIF-1
protein stabilization during hypoxia (6). We
demonstrated that the stabilization of HIF-1
protein at oxygen concentrations of 1-2% required a functional electron transport chain. In the present study, we confirm these findings and also demonstrate that anoxic stabilization of HIF-1
protein does not require a functional electron transport chain. This observation is
consistent with the requirement of proline hydroxylation as a mechanism
for HIF-1
protein degradation under normal oxygen conditions. In the
absence of oxygen, hydroxylation of proline residues within HIF-1
by
prolyl hydroxylases cannot occur, and intracellular signaling events
are not required for the stabilization of HIF-1
protein. Thus prolyl
hydroxylases would effectively serve directly as the oxygen sensors
during anoxia.
Our present data are not consistent with the hypothesis that prolyl
hydroxylases serve as the primary oxygen sensor regulating the hypoxic
stabilization of HIF-1 protein. Rather, our data suggest that the
stabilization of HIF-1
protein under 1-2% O2 levels requires the activation of intracellular signaling pathways by a
mitochondrial-dependent ROS signal. Recently, Hirota and Semenza
(14) have shown that hypoxia (1% O2)
stimulates Rac1 activity, and Rac1 is required for the hypoxic
stabilization of HIF-1
protein. Both the hypoxic activation of Rac1
and the stabilization of HIF-1
protein were abolished by the complex
I inhibitor rotenone. These results indicate that Rac1 is downstream of
mitochondrial signaling. Additional intracellular signaling systems,
including phosphatidylinositol 3-kinase and diacylglycerol kinase, have been shown to be required for hypoxic (1% O2)
stabilization of HIF-1
protein (2, 37). Moreover,
mitochondrial-dependent oxidant signaling has been shown to regulate
HIF-1
protein accumulation after exposure to tumor necrosis
factor-
(13). Nonmitochondrial-dependent oxidant
signaling has also been shown to stabilize HIF-1
protein under
normoxia. For example, thrombin or angiotensin II stabilizes HIF-1
under normoxia through an increase in ROS generation from nonmitochondrial sources (12, 27). Therefore, if prolyl
hydroxylase is by itself the oxygen sensor for both hypoxic and
anoxia-induced HIF-1, then there would be no signaling required
upstream of prolyl hydroxylase. There should be no requirement for
activation of kinases or oxidant-dependent signaling upstream of prolyl
hydroxylase. We speculate that the ultimate target of the
oxidant-dependent signaling pathway originating from mitochondria
during hypoxia or nonmitochondrial sources such as angiotensin II or
tumor necrosis factor-
during normoxia is to inhibit proline
hydroxylation (Fig. 10).
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Two previous observations further support the premise that hypoxic
signaling is distinct from anoxia with respect to HIF-1 activation.
First, Gleadle et al. (10) have demonstrated that diphenylene iodonium (DPI), an inhibitor of a wide range of
flavoproteins, including complex I, prevents stabilization of HIF-1
protein and HIF-1 target genes at oxygen levels of 1%. However, DPI
fails to affect stabilization of HIF-1 in response to the iron chelator desferrioxamine (DFO). Iron and oxygen are required for prolyl hydroxylases to be catalytically active. Thus DFO or anoxia directly inhibit prolyl hydroxylase activity because of substrate limitations and stabilize HIF-1
protein. Interestingly, DPI can prevent a variety of other hypoxic responses, such as pulmonary vasoconstriction and carotid body nerve firing. Second, Jiang et al. (17a)
have demonstrated that HIF-1
protein levels stabilize half-maximally between 1.5 and 2% O2 and are maximally stabilized at
0.5%. If prolyl hydroxylase were the oxygen sensor over a wide
range of oxygen tensions, then HIF-1
protein levels should be
maximally stabilized at 0% O2. Taken together, these
previous findings and our present observations suggest distinct
pathways of initiating the hypoxic response compared with DFO or anoxia.
A more fundamental difference between oxygen levels of 1-2% and 0-0.5% is the metabolic state of the cells. Below an oxygen concentration of 0.5%, molecular oxygen begins to limit the respiratory rate, causing ATP levels to decrease (18, 35). Exposure of cells to 1-2% O2 compared with 0% O2 can have varying consequences for intracellular signaling, cellular metabolism, and survival. For example, cells do not undergo apoptosis or growth arrest at 1-2% O2 (7). However, oxygen concentrations closer to 0% cause cells to undergo apoptosis that requires a functional electron transport chain and the Bcl-2 family members Bax or Bak (25). Gleadle and Ratcliffe (11) have also demonstrated that src kinase is activated at oxygen levels of 0.1 or 0% O2 but not at 1% O2 (11).
Our results also provide an explanation for the differing results
obtained from using 0-cells to examine mitochondrial
regulation. Previously Vaux et al. (31) and Srinivas et
al. (30) have demonstrated that
0-cells are
still able to stabilize HIF-1
protein at oxygen levels of 0.1 or
0.5% O2. The use of antimycin and rotenone as a selection procedure in the generation of
0-cells has been cited as
a potential explanation for the difference in our previous results
compared with those of Vaux et al. and Srinivas et al. In the present
study, we generated
0-cells by exposing cells to
ethidium bromide for 4-6 wk in the absence of any mitochondrial
inhibitors. A possible explanation for the conflicting results is that
cells exposed to oxygen levels of 0.1 or 0.5% are likely to experience
anaerobic conditions within the cytosol, because mitochondrial
cytochrome-c oxidase will consume residual molecular oxygen.
Taken together, these findings suggest that intracellular signaling
pathways are likely to be important for hypoxic but not anoxic
stabilization of HIF-1
protein.
An alternative genetic strategy to using 0-cells in
examining the role of mitochondria in the regulation of HIF-1
is to
use cells with diminished complex I activity. Agani et al.
(1) have demonstrated that cybrid cells containing a
partial defect in complex I activity have reduced HIF-1
protein
levels at 1% O2. Succinate, a mitochondrial complex II
substrate, restored the hypoxic response in cybrid cells, suggesting
that electron transport chain activity is required for the
stabilization of HIF-1
protein. In contrast, Vaux et al.
(31) have shown that Chinese hamster fibroblasts CCL16-B2
which contain complex I activity <10% of control cells are still able
to stabilize HIF-1
protein at 0.1% O2. In the present
study, we found that CCL16-B2 cells were still able to generate ROS
during hypoxia and stabilize HIF-1
protein under both hypoxic and
anoxic conditions. Mitochondrial generation of ROS has been estimated
to be 1-2% of the total electron flux through the respiratory
chain (17). The electron flux required for ROS
generation at complex III is likely to be significantly less than the
electron flux required to reduce molecular oxygen to H2O at
cytochrome-c oxidase. Thus a significant loss of complex I
activity might significantly inhibit oxygen consumption at
cytochrome-c oxidase but still provide sufficient electrons
to generate ROS at complex III, thus stabilizing HIF-1
protein
during hypoxia.
Our present observations are also consistent with the role of complex I
inhibitors in preventing the stabilization of HIF-1 protein.
Previously, our laboratory reported that a 1 µg/ml concentration of
rotenone inhibits the stabilization of HIF-1
protein at 1.5% O2 in Hep3B cells (6). These results have been
corroborated by Hirota and Semenza (14), who reported that
rotenone at a concentration of 1.0 µg/ml inhibited the stabilization
of HIF-1
protein at 1.0% O2 in HEK-293 cells. The
neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, another
complex I inhibitor, prevents hypoxic accumulation of HIF-1
protein
in dopaminergic cell lines (1). In the present study, we
found that rotenone (0.5 µg/ml) abolished ROS generation and the
stabilization of HIF-1
protein during hypoxia. The ability of
succinate to restore ROS generation and to stabilize HIF-1
protein
in the presence of rotenone during hypoxia and the abolishment of this
effect by complex II inhibitor suggest that rotenone exerts its effects
specifically by inhibiting mitochondrial complex I. Rotenone did not
abolish HIF-1
protein stabilization during anoxia. These results are
consistent with the hypothesis that rotenone affects hypoxic but not
anoxic stabilization of HIF-1
protein. Furthermore, these results
potentially explain why rotenone failed to inhibit HIF-1
protein
stabilization in previous studies that examined HIF-1
protein levels
at 0.1% O2 (31).
In summary, our present results demonstrate mitochondrial-dependent
signaling for the hypoxic stabilization of HIF-1. Data from a
variety of other investigators suggest that other signaling elements
are also likely to be required for the hypoxic stabilization of
HIF-1
. In contrast, the anoxic stabilization of HIF-1
does not
require mitochondrial-dependent signaling. We would predict that
HIF-1
stabilization under anoxic conditions occurs in the absence of
intracellular signaling upstream of proline hydroxylation. Mitochondria
might also serve as oxygen sensors for a variety of other hypoxic
events. For example, we and other investigators have demonstrated that
similar mitochondrial-dependent signaling is required for hypoxic
pulmonary vasoconstriction (21, 33). Future experiments
will have to address how hypoxia stimulates the production of
mitochondrial ROS and the downstream targets of ROS that result in a
diverse activation of responses to hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Mary Paniagua and Mehrnoosh Abshari at the Flow Cytometry Facility for technical assistance.
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FOOTNOTES |
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This study was supported by the Crane Asthma Center and National Institute of General Medical Sciences Grant GM-60472-03 (to N. S. Chandel).
Address for reprint requests and other correspondence: N. S. Chandel, Division of Pulmonary & Critical Care, Tarry Bldg. 14-707, 300 East Superior St., Chicago, IL 60611-3010 (E-mail: nav{at}northwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 24, 2002;10.1152/ajplung.00014.2002
Received 11 January 2002; accepted in final form 17 May 2002.
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