From the Division of Hematology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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It has been proposed that cells sense hypoxia by
a heme protein, which transmits a signal that activates the
heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1),
thereby inducing a number of physiologically relevant genes such as
erythropoietin (Epo). We have investigated the mechanism by which two
heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen
sensing and signaling. Two concentrations of CO (10 and 80%)
suppressed the activation of HIF-1 and induction of Epo mRNA by
hypoxia in a dose-dependent manner. In contrast, CO had no
effect on the induction of HIF-1 activity and Epo expression by either
cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane
coefficient, ~0.5). Parallel experiments were done with 100 µM sodium nitroprusside, a nitric oxide donor. Both
NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced
accumulation of HIF-1 Adaptation to hypoxia is of fundamental importance in
developmental, physiological, and pathophysiological processes (1, 2).
Humans and other mammals respond to low oxygen tension in part by fine
tuning the expression of a group of physiologically relevant genes.
Erythropoietin (3, 4) and tyrosine hydroxylase (5) act in concert to
raise blood oxygen levels by enhancing erythropoiesis and ventilation,
respectively. Hypoxic induction of genes encoding vascular endothelial
growth factor (6) and inducible nitric oxide synthase (7, 8) leads to
increased angiogenesis and vasodilation. Up-regulation of genes
encoding glucose transporters and specific glycolytic isoenzymes
(9-11) maximize ATP production in the setting of reduced oxygen
supply. Remarkably, the hypoxic induction of all of these diverse genes appears to depend on a common mode of oxygen sensing and signal transduction, triggering the activation of a critical transcription factor, hypoxia-inducible factor 1 (HIF-1)1 (for review, see
Ref. 1).
HIF-1 is a widely expressed heterodimeric protein composed of HIF-1 The activation of HIF-1 by hypoxia depends on a sensing and signaling
process that is poorly understood. There is strong, albeit
circumstantial, evidence that the oxygen sensor is a heme protein (1,
27). Support for this claim derives largely from experiments
demonstrating that heme binding ligands suppress the hypoxic induction
of HIF-1 activation and expression of the genes mentioned above. The
hypoxic induction of erythropoietin (27), vascular endothelial growth
factor (28, 29), and other genes (30) was markedly blunted in the
presence of carbon monoxide (CO), a molecule whose only established
biological function is binding to ferrous atoms on heme groups.
Subquently, another heme ligand, nitric oxide (NO), was shown to exert
a similar effect (29, 31, 32). In addition to hypoxia, HIF-1 can be
activated by the transition metal Co2+ as well as by iron
chelation (33). The mechanisms underlying the activities of these
agonists are unknown, although there is circumstantial evidence that
they affect the level of reactive oxygen species (34), which may serve
as signaling molecules (1). To gain insight into hypoxic sensing and
signaling, we have made an in-depth comparison of the downstream
effects of CO and NO on HIF-1 activation, HIF-1 Reagents and Cell Culture--
Sodium nitroprusside (SNP),
8-bromo-cGMP, cobaltous chloride, and desferrioxamine were all
purchased from Sigma.
Hep3B cells were incubated in alpha-modified Eagle's medium (Life
Technologies, Inc.), and 293 cells were incubated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with
10% fetal bovine serum (Hyclone), both at 37 °C. For experiments involving CO treatment, cells were seeded in 75-cm2 tissue
culture flasks (Becton Dickinson, Franklin Lakes, NJ) fitted with
autoclaved rubber stoppers punctured with sterile inflow (3.5-inch,
17-gauge) and outflow (1.5-inch, 18-gauge) needles. Gas flow was
maintained at 300 ml/min. In some experiments, cobaltous chloride,
desferrioxamine, or SNP was added to the culture medium at final
concentrations of 100, 130, and 100 µM, respectively. Cells were exposed to one of the following gas mixtures: normoxia (95%
air and 5% CO2), hypoxia (1% oxygen, 94% N2,
and 5% CO2), normoxia with CO (21% O2, 10%
CO, 5% CO2, and balance N2), or hypoxia with
CO (1% O2, 10 or 80% CO, 5% CO2, and balance
N2). Gases were mixed with a Series 200 computerized gas
standards generator (Environics, Tolland, CT) and humidified by being
passed through 0.15 M NaCl.
RNA Preparation and Analysis--
Total RNA was extracted by the
TRIzol method (Life Technologies, Inc.) according to the
manufacturer's protocol and dissolved in 100% formamide. A plasmid,
containing a portion of exon 5 of the erythropoietin (Epo) gene (bp
2650-2890) inserted into pBluescript, was linearized with
XhoI and in vitro transcribed in the presence of
[32P]CTP (DuPont NEN) with T7 polymerase (Promega). RNase
protection assays were performed with 40-100 µg of RNA hybridized at
55 °C overnight with the Epo riboprobe. For quantification of gels, a PhosphorImager was used with ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). Results are presented as the mean ± S.E. from
independent experiments.
Plasmids--
Both p(HA)HIF-1 Transient Transfection--
Human embryonic kidney 293 cells and
human hepatoma Hep3B cells were transfected in suspension using the
calcium phosphate method as described elsewhere (19). In general, 293 cells were transfected with 2 µg of each expression vector plus 0.25 µg of pCMV- Electrophoretic Mobility Shift Assay (EMSA) and Western
Blotting--
Whole cell extracts were analyzed for HIF-1 or Gal4 DNA
binding activity or were subjected to Western blot analysis as
described previously (19, 25). Endogenous HIF-1 Calculation of the Haldane Coefficient for Oxygen versus CO
Binding--
To calculate the fractional saturation of the oxygen
sensor with ligand (O2 versus CO), we used a
comprehensive and accurate set of earlier experimental data from our
laboratory (37) on the relationship between pO2
and level of Epo mRNA. These data, in combination with experimental
quantitation of Epo mRNA, enabled the estimation of pOs
values, the pO2 simulated by O2 + CO
ligation. Because the relevant experiments were carried out in 1%
O2 (=7 torr), the Haldane coefficient M was
calculated from the equation: M = (pOs Effect of Carbon Monoxide and Nitric Oxide on the Induction of Epo
mRNA--
Of all the genes up-regulated by hypoxia, Epo
shows the most robust response. Therefore we began by examining the
effect of CO and NO on the induction of Epo mRNA in Hep3B cells.
Epo mRNA, protected by a 240-bp riboprobe during ribonuclease
protection assays, displayed a doublet banding pattern (Fig.
1, A and B). As
expected, hypoxia, cobalt, and desferrioxamine markedly induced Epo
mRNA expression. CO suppressed hypoxic induction of Epo mRNA, almost to the level of normoxic controls (Fig. 1A, lanes 3 and 4). In determining the Haldane coefficient for the
oxygen sensor, we assumed that CO and O2 bind to the same
site, similar to other heme proteins. At 10 and 80% CO, the calculated
Haldane coefficients were 0.8 and 0.2, respectively. Interpretation of
these values may be confounded by the possibility that, like cytochrome
c oxidase, CO is likely to bind reversibly to the heme group
of the oxygen sensor, whereas oxygen binding may be irreversible if it
undergoes reduction to superoxide (1, 38). However, the coefficients that we obtained were reproducible and probably valid, owing to the
high flow of gas used in the incubations and the likelihood that the
pO2 was maintained at a constant level. Cobalt
and hypoxia had an additive effect on Epo mRNA levels, whereas
desferrioxamine and hypoxia did not. Combined cobalt and hypoxia
induction was decreased by 10 and 80% CO treatment, to the levels of
normoxic cobalt induction. Cobalt induction of Epo mRNA under
normoxic conditions was not suppressed by 10% CO (Fig. 1A, lane
14). Likewise, CO had no effect on desferrioxamine-induced Epo
mRNA levels (Fig. 1A, lane 11). These results indicate
that CO does not suppress Epo mRNA expression in a nonspecific
toxic manner.
Parallel experiments were done with 100 µM SNP as a
source of NO. As shown in Fig. 1B, lane 3, this
concentration of SNP mimicked CO in suppressing the induction of Epo
mRNA by hypoxia. However, in contrast to CO, SNP suppressed
induction of Epo mRNA by cobalt (Fig.
2, lanes 5, 7, and
9). These results with SNP are consistent with those of
Sogawa et al. (32), who tested a hypoxia-inducible reporter
gene with SNP and two other NO donors.
Effect of CO and NO on the Activation of HIF-1--
To delineate
the mechanism by which CO and NO suppressed Epo mRNA expression, we
examined how these agents affected the activation of HIF-1, the
transcription factor responsible for the regulation of Epo
and a number of other genes induced by hypoxia. Extracts prepared from
hypoxic cells, when incubated with radiolabeled wild-type HIF-1 binding
sequence, displayed doublet bands in gel shift assays. In contrast,
single bands were observed in extracts prepared from cells treated with
cobalt (Fig. 2, lane 2) or desferrioxamine. Similar results
have been reported by others (12, 33, 39). Hypoxia-induced HIF-1
binding was attenuated by treatment with CO in a
dose-dependent manner (Fig. 2, lanes 5 and
6). In contrast, cobalt and desferrioxamine induction of
HIF-1 binding was not affected by CO treatment (Fig. 2, lanes
7 and 8).
Similar to what was observed with CO, 100 µM SNP
significantly suppressed hypoxia-induced HIF-1 DNA binding in Hep3B
cells, whereas neither constitutive nor nonspecific binding was
affected (Fig. 2, lane 14). To investigate the mechanism
underlying the inhibitory effects of CO and SNP, 293 cells were
transfected with the CMV-driven vectors overexpressing HIF-1 (HA-tagged
HIF-1
Because HIF-1 activity is primarily determined by the abundance of
HIF-1 The Oxygen-dependent Degradation Domain Is Responsible
for Inhibition by CO and NO--
We recently identified an ODD domain
within HIF-1
Gal4 is normally a highly stable protein. However, when fused to the
ODD domain, it undergoes oxygen-dependent degradation (25).
To confirm that abrogation of HIF-1
In an effort to elucidate further the mechanism underlying the effect
of nitric oxide on the ODD domain, we tested the possibility that NO
could be forming an SNO adduct with a cysteine sulfhydryl group on the
ODD. Accordingly, Cys-520, the only cysteine residue present in the ODD
domain, was mutated to serine. As in experiments depicted in Figs. 2
and 3, HIF-1 binding activity was analyzed after cells were transfected
with ARNT and either wild-type HIF-1 CO and NO Inactivate HIF-1
These results raised the possibility that, in addition to impacting on
the protein stability of HIF-1 NO Suppression Is Not Mediated by Guanylate Cyclase--
Because
the predominant physiological action of nitric oxide is mediated
through an increase in cGMP by activation of soluble guanylate cyclase
(40), we also examined whether cell membrane-permeable 8-bromo-cGMP, an
analog of cGMP, simulates the effect of SNP. No loss of HIF-1 binding
was detected with up to 1 mM 8-bromo-cGMP (Fig. 7,
lane 3). This result makes it very unlikely that the inhibitory effect of SNP is mediated through a pathway involving soluble guanylate cyclase.
We have compared the effects of carbon monoxide and nitric oxide
on the sensing of oxygen and the signaling pathway leading to HIF-1
activation. Although CO and NO both bind to ferrous iron in heme
proteins, the two ligands differ in major respects. CO is chemically
inert and undergoes no known chemical modifications in biological
systems other than perhaps oxidation to CO2 coupled with
reduction of cytochrome c oxidase (41) and other heme
proteins (42). In contrast to CO, the biochemistry of NO is much more complex. NO binds to and activates its primary biological target, soluble guanylate cyclase, much more readily than does CO (43). Unlike
CO, NO can also bind to ferric heme. Moreover, unlike CO, NO is capable
of reacting with oxygen and superoxide (44), possibly affecting the
signaling pathway for HIF-1 activation. Finally, unlike CO, NO can form
S-nitroso derivatives of a broad repertoire of membrane,
cytosolic, and nuclear proteins (44).
Our experiments show that CO and NO have similar effects on oxygen
sensing and signaling: they both suppress expression of target genes
through inhibition of HIF-1 activation, and both trigger
destabilization of HIF-1 Heme proteins vary markedly in their relative affinities for CO
versus O2. Human hemoglobin binds CO ~210
times more tightly than O2. In contrast, the yeast
flavohemoglobin binds CO and O2 with approximately equal
affinity.2 Likewise,
b-type cytochromes, which have been proposed to function in
oxygen sensing (1, 38), are likely to have very low affinity for CO. We
estimate that the oxygen sensor in Hep3B cells has a Haldane
coefficient of ~0.5, a value significantly different from the
coefficient of ~10 that Warburg (46) obtained for yeast cytochrome
oxidase, which pertains to a wide range of higher organisms. This
difference is difficult to reconcile with a recently proposed mitochondrial model of oxygen sensing (47, 48). Despite the inherent
inaccuracies in our measurement of a "functional" Haldane coefficient for oxygen sensing, the value we have obtained may prove
useful in screening candidate genes suspected to encode the oxygen
sensor. An agreement between the "functional" value that we have
obtained and a coefficient obtained from spectra of overexpressed
protein would lend support to a candidate, whereas a significant
discrepancy would rule it out.
The low affinity of the oxygen sensor for CO may have adaptive
significance. The primary toxicity of CO in higher organisms is
attributable to its high affinity binding to hemoglobin, locking the
tetramer in the "oxy" conformation and thereby increasing oxygen
affinity and decreasing oxygen unloading, resulting in tissue hypoxia.
The organism needs intact oxygen sensors to adapt to this hypoxic
stress. These sensors would be unresponsive to CO-induced hypoxia if
they had high affinity for CO.
Our conclusion that CO and NO are acting at the same site is supported
by experiments that rule out other known effects of NO. The fact that a
mutation of the single cysteine in the ODD domain did not affect the
function of HIF-1 (Fig. 4) implies that the NO effect is not
attributable to S-nitrosylation of HIF-1 An alternate explanation for the suppressive effect of NO is via
binding to its most prominant target, guanylate cyclase. In contrast to
findings of Liu et al. (29) but similar to those reported by
Sogawa et al. (32), we found no effect of 8-bromo-cGMP on
HIF-1 activation (Fig. 5), making it unlikely that suppression of HIF-1
activation depends on up-regulation of guanylate cyclase.
It is possible that NO and CO act independently of one another with NO
generating reactive oxygen species (ROS) directly, whereas CO could
generate ROS indirectly through redox cycling of cytochrome
c (46) and other electron acceptors (42), including the
oxygen sensor. Cytochrome c participates in the oxidation of
CO to CO2 by reduction of its heme iron. Oxidation of
cytochrome c back to the ferric state would reduce oxygen to
superoxide, enabling the generation of ROS via the iron-catalyzed
Fenton reaction.
Our work supports and extends the paradigm that HIF-1 activation
correlates with the abundance of HIF-1 protein. Moreover, both NO and CO specifically
targeted the internal oxygen-dependent degradation domain
of HIF-1
, and also repressed the C-terminal transactivation domain
of HIF-1
. Thus, NO and CO act proximally, presumably as heme ligands
binding to the oxygen sensor, whereas desferrioxamine and perhaps
cobalt appear to act at a site downstream.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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and ARNT subunits, both of which belong to the basic helix-loop-helix
periodic-arylhydrocarbon receptor-simultaneous family (12, 13). Its
biological importance is underscored by recent observations that both
HIF-1
(14, 15) and ARNT (16) null mutant mouse embryos exhibit
developmental arrest and lethality resulting essentially from
impairment of vascularization. At the mRNA level, both HIF-1
and
ARNT genes are constitutively expressed and not significantly
up-regulated by hypoxia (17-20). Whereas changes in oxygen tension
fail to affect ARNT protein abundance (19, 20), hypoxia markedly
increases levels of HIF-1
protein (13, 19, 20). In fact, in normoxic
cells HIF-1 is barely detectable (21). Moreover HIF-1
is remarkably
unstable, with a half-life of <5 min (19). Thus, hypoxia-induced
activation of HIF-1 depends in part on posttranslational stabilization
of HIF-1
. In addition, hypoxia markedly enhances transactivating function of HIF-1
(22-24). The oxygen-dependent
degradation (ODD) of HIF-1
is governed by an internal 200-residue
ODD domain (25) via the ubiquitin-proteasome pathway (25, 26). Removal
of the ODD domain renders HIF-1
stable under normoxia, resulting in
autonomous heterodimerization, DNA binding, and transactivation in the
absence of hypoxia signaling (25).
stability, and the
expression of oxygen-responsive endogenous and reporter genes.
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ABSTRACT
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DISCUSSION
REFERENCES
and p(HA)HIF-1
(401
603)
are (CMV)-driven expression vectors expressing hemagglutinin
(HA)-tagged HIF-1
and the ODD domain-deleted HIF-1
(25).
p(HA)HIF-1
(C520S) contains a replacement of cysteine at codon 520 with serine generated by the Altered Sites II in vitro
mutagenesis system (Promega). pGal4-ODD and the human erythropoietin 3'
enhancer reporter pEpoE-luc were constructed as described previously
(19, 25). pGal4-luc and pSB92 (Gal4-CAD) were kindly provided by S. Bhattacharya from David Livingston's Laboratory.
gal, whereas Hep3B cells were transfected with 2 µg
of pCMV-
gal and 2 µg of reporter plasmid. Cells were treated with
100 µM sodium nitroprusside as indicated. After 24 h
of incubation, whole cell extracts were prepared as described
previously (19) for electrophoretic mobility shift assays and Western
blotting. For reporter gene assays, cells were harvested 48 h
after transfection and assayed for luciferase activity as described
elsewhere (19).
protein was detected by anti-HIF-1
monoclonal antibodies: a mix of monoclonal antibodies OZ12 and OZ15 directed against the C-terminal half (residues 530-826; Ref. 35) (kindly provided by Dr. David Livingston), monoclonal antibody
28b raised against residues 329-530 (36) (kindly provided by Dr.
Patrick Maxwell), and anti-HIF-1
(Novus Biologicals, Inc., Littleton, CO. All three monoclonal antibodies gave internally consistent results. The monoclonal anti-HA 12CA5 (Boehringer Mannheim) antibody was used to determine HA-tagged HIF-1
expression.
Polyclonal anti-ARNT antibodies were used as before (19). The
antigen-antibody complexes were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
7)/COtorr.
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ABSTRACT
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DISCUSSION
REFERENCES
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Fig. 1.
Effect of CO and NO on induction of Epo
mRNA expression in Hep 3B cells. A, cells were exposed
to 20% O2 (N) or 1% O2
(H) and incubated in either control medium (lanes
1-4) or in media containing 100 µM cobalt
(lanes 5-8) or 130 µM desferrioxamine
(lanes 9-11). Aliquots of cells were exposed to 10% carbon
monoxide (lanes 3, 7, and 11-13), and 80% CO
(lanes 4 and 8). Ribonuclease protection assays
were performed using radiolabeled wild-type Epo riboprobe (250 bp of
the fifth exon, which includes the 3' untranslated region). The
bar graph below shows densitometric quantitation of
replicate experiments: means ± S.E. of with the number of
experiments indicated above each bar. B,
experiments similar to A tested the effect of 100 µM SNP on induction of Epo mRNA by hypoxia and
cobalt.
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Fig. 2.
Effect of CO and NO on induction of HIF-1
binding and HIF-1 protein in Hep 3B cells. A and
B, effect of CO; C and D, effect of
SNP as a source of NO. A and C, results obtained
on endogenous HIF-1 in Hep3B cells; B and D,
results with overexpression of HIF-1
and ARNT subunits after
transfection of p(HA)HIF-1
and pARNT into 293 cells. Cells were
exposed to normoxia (N, 20% O2) or hypoxia
(H, 1% O2) in the absence or presence of 10%
CO, 80% CO, or 100 µM SNP. Cells were incubated either
in control medium or in media containing 100 µM cobalt
(lanes 2 and 4). Whole cell extracts were
prepared 24 h after transfection and analyzed with EMSAs using
32P-labeled oligonucleotides containing an HIF-1 binding
site. HIF-1, induced binding; C, constitutive
binding; N, nonspecific binding; F, free probe.
Shown below are Western blots of the same extracts used for the EMSAs,
probed with monoclonal anti-HIF-1
antibody (A and
C) or with anti-HA antibody (B and
D).
and ARNT, respectively). Both CO and NO abrogated DNA binding of overexpressed HIF-1 (Fig. 2, lanes 11 and 17). It is noteworthy that
under normoxia, despite exogenous overexpression of HIF-1, no HIF-1
binding activity was detected (Fig. 2, lanes 9 and
15). Together these results indicate that both endogenous
and exogenous HIF-1 are regulated by the same signaling pathway.
protein (19), we examined by Western blot whether CO and NO
affect HIF-1
accumulation in hypoxic cells. As shown in Fig. 2,
bottom, exposure of hypoxic cells to 10 and 80% CO resulted
in a dose-dependent reduction in the abundance of HIF-1
protein (Fig. 2, lanes 5 and 6), comparable with
the decrease in HIF-1 DNA binding on EMSA. Importantly, CO had no
effect on the abundance of HIF-1
induced by cobalt (Fig. 2,
lanes 7 and 8), again indicating that CO was not
exerting a nonspecific toxic effect. In a like manner, we examined
whether NO affects HIF-1
accumulation in hypoxic cells. Consistent
with the results obtained with CO, SNP blocked hypoxia-induced
accumulation of endogenous HIF-1
(Fig. 2, lane 14).
Moreover, both CO and NO suppressed accumulation of transfected
HIF-1
(Fig. 2, lanes 11 and 17). In all these
results, the level of HIF-1
closely paralleled that of HIF-1 binding activity.
that plays a regulatory role for hypoxia-induced
stabilization of HIF-1
. Internal removal of this domain rendered
HIF-1
stable irrespective of oxygen tension (25). The inhibitory
effect of CO and SNP on the accumulation of HIF-1
protein prompted
us to examine whether the ODD domain is targeted by treatment with
these agents. As in the experiment shown in Fig. 2, lanes
9-11 and 15-17, 293 cells were co-transfected with a
vector expressing ARNT and another vector expressing wild-type HIF-1
or the ODD-deleted HIF-1
(401
603; Fig.
3A). Consistent with previous
results (25), normoxic cell extracts prepared from cells transfected
with HIF-1
(401
603) gave rise to strong constitutive HIF-1 binding
(Fig. 3C, lane 4). In contrast to wild-type HIF-1
,
HIF-1
(401
603) was resistant to treatment with either CO (Fig.
3B, lane 4) or SNP (Fig. 3C, lane 6). These
results indicate that the ODD domain is required for the suppressive
effects of CO and SNP.
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Fig. 3.
The inhibitory effect of CO and NO is
mediated through the ODD domain. A, schematic representation
of three CMV-driven expression vectors: p(HA)HIF-1 ,
p(HA)HIF-1
(401
603), and pGal4-ODD. 293 cells were transfected
with p(HA)HIF-1
and pARNT (B, lanes 1 and 2;
C, lanes 1-3), with p(HA)HIF-1
(401
603) and
pARNT (B, lanes 3 and 4; C, lanes
4-6), or with pGal4-ODD (C, lanes 7-9). Twenty-four
hours after transfection, the cells were then subjected for 4 h to
normoxia (N) or hypoxia (H) in the absence or
presence of 80% CO (B) or 100 µM SNP
(C). Cells extracts were then prepared and analyzed with
EMSAs using 32P-labeled oligonucleotides containing an
HIF-1 binding site (B, lanes 1-4; C, lanes 1-6)
or Gal4 binding site (C, lanes 7-9). The endogenous HIF-1
complexes are marked with an arrowhead, whereas those
composed of HIF-1
(401
603) and ARNT are marked with
asterisks, and Gal4 binding is marked with an
arrow. PAS, periodic aryl hydrocarbon
receptor-simultaneous.
abundance by SNP is specifically
mediated by the ODD domain, a plasmid expressing Gal4-ODD was
transfected into 293 cells and assayed for Gal4 DNA binding activity.
Like wild-type HIF-1, Gal4-ODD exhibited very weak binding activity
under normoxia (Fig. 3C, lane 7) and robust induction with
hypoxia (Fig. 3C, lane 8). Of note, hypoxia-induced binding
was markedly reduced by SNP treatment (Fig. 3C, lane 9).
or the mutant HIF-1
(C520S).
Interestingly, the mutant HIF-1
was still sensitive to SNP treatment
(Fig. 4A, lane 3), which is in
agreement with the loss of HIF-1
protein accumulation demonstrated by Western blotting (Fig. 4B, top panel). As shown in Fig.
4B, bottom panel, probing of the same blot with anti-ARNT
antibodies demonstrated that ARNT protein levels remained
unchanged.
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Fig. 4.
Cysteine nitrosylation of the ODD domain is
not responsible for the effect of SNP. A, 293 cells were
transfected with p(HA)HIF-1 (C520S) and pARNT (lanes 1-3)
or p(HA)HIF-1
and pARNT (lanes 4-6), and were subjected
to the same treatment as described in the legend to Fig. 3. EMSAs were
performed with 32P-labeled oligonucleotides containing an
HIF-1 binding site. B, the above cell extracts were also
subjected to Western blot analyses with monoclonal anti-HA antibody
(top panel) and polyclonal anti-ARNT antibody.
HIF-1, induced binding; C, constitutive binding;
N, nonspecific binding; F, free probe.
Transactivating
Activity--
Previously Sogawa et al. (32) showed that SNP
specifically inhibited hypoxia-induced activity of a luciferase
reporter containing four copies of the HIF-1 binding site. Because we
found that expression of HIF-1
(401
603) gave rise to stable HIF-1
DNA binding activity that was insensitive to CO and SNP treatment (Fig.
3, B and C), we wondered whether this stable
HIF-1 activity could transactivate an HIF-1 target reporter gene in the
presence of SNP. A luciferase reporter EpoE-luc was used to test the
effect of SNP in Hep3B cells co-transfected with wild-type HIF-1
(HIF-1
and ARNT) or the ODD domain-deleted HIF-1
(HIF-1
(401
603) and ARNT) as mentioned above. The reporter
EpoE-luc contains a native HIF-1 binding site within a 50-bp region of
the human erythropoietin 3' enhancer (19). In agreement with the
results of Sogawa et al. (32), SNP drastically inhibited
hypoxia-induced luciferase activity when wild-type HIF-1 was
transfected (Fig. 5, left).
Surprisingly, a similar result was obtained when HIF-1
(401
603)
was tested (Fig. 5, right). It is noteworthy that HIF-1
contains two transactivation domains; one is within the ODD domain, and
the other is at the C terminus (23, 24), in contrast to
HIF-1
(401
603), which has only the latter. Under normoxic
conditions the transactivating activity of HIF-1
(401
603) was
substantially higher than that of the wild type, as observed previously
(25). In addition, hypoxia further stimulated the reporter
activity.
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Fig. 5.
Stabilized HIF-1 fails to transactivate
HIF-1 target reporter in the presence of SNP. Reporter pEpoE-luc
was co-transfected into Hep3B cells with p(HA)HIF-1
and pARNT
(left) or p(HA)-HIF-1
(401
603) and pARNT
(right). Approximately 16-18 h before harvest, cells were
incubated under normoxic or hypoxic conditions in the presence or
absence of 100 µM SNP as indicated. Luciferase activities
were measured as described (25), and the mean and S.E.s were plotted
from three independent experiments.
, CO and NO may also affect its
C-terminal activation domain. To address this question directly, we
took advantage of a Gal4 fusion system in which the stable C-terminal
transactivation domain was fused to the Gal4 DNA binding domain
(Gal4-CAD, Fig. 6A) so that
transactivating activity of the fusion protein could be assessed by a
luciferase reporter containing five copies of the Gal4 binding site.
Transactivating activity of the C-terminal portion of HIF-1
in a
Gal4 fusion system can be enhanced by hypoxia without altering the
protein abundance (23, 24). To that end, Hep3B cells were
co-transfected with a reporter (Gal4-luc) and a vector expressing Gal4
alone (Gal4) or Gal4 fusion (Gal4-CAD) and subjected to normoxic or hypoxic treatment in the presence or absence of either CO or SNP. As
expected, Gal4 alone failed to stimulate reporter activity when
challenged by hypoxia (Fig. 7,
B and C). In contrast, the reporter activity was
markedly increased under hypoxia when the Gal4 fusion protein was
expressed, but this induction was suppressed by both CO (Fig.
7B) and SNP (Fig. 7C). Taken together, these data
indicate that CO and NO exerted their inhibitory effects by targeting
the C-terminal transactivating domain of HIF-1
as well as its
protein stability.
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Fig. 6.
CO and SNP specifically inhibit
hypoxia-induced HIF-1 transactivation. A, schematic
drawings of HIF-1
and Gal4-CAD. Reporter pGal4-luc was
co-transfected into Hep3B cells with plasmids expressing Gal4 alone
(left) or Gal4-CAD (right) and subjected to
either normoxia (N) or hypoxia (H) for 16-18 h.
Half of these cell culture plates were also treated with either 10% CO
(B) or 100 µM SNP (C).
B, mean luciferase activities of duplicate experiments;
C, mean and S.E.s of luciferase activities from three
experiments. PAS, periodic aryl hydrocarbon
receptor-simultaneous.
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Fig. 7.
The effect of SNP is independent of the
soluble guanylate cyclase pathway. Hep3B cells were incubated
under normoxic (N, lane 1) or hypoxic (H, lanes 2 and 3) conditions for 4 h in the absence (lane
2) or presence (lane 3) of 1 mM
8-bromo-cGMP. HIF-1 DNA-binding was determined with EMSA.
HIF-1, induced binding; C, constitutive binding;
N, nonspecific binding; F, free probe.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
via the ODD domain and inhibition of the
C-terminal activation domain. The most parsimonious mechanism is
binding of these ligands to the heme group of the oxygen sensor. Our
experiments with CO, along with those of Liu et al. (29), are at odds with a recent report by Srinivas et al. (45),
who concluded that CO was not effective in suppressing expression of a
hypoxia-inducible reporter gene. They tested only a single concentration of CO, 6%, and in fact observed ~17% suppression, which was statistically significant. We show in this report that the
oxygen sensor has a low affinity for CO. Thus, it is likely that
Srinivas et al. (45) would have obtained a more robust effect had they used a higher concentration of CO. Our finding that CO
abrogates hypoxic stabilization of HIF-1
protein, in a
dose-dependent manner (Fig. 2D), is in conflict
with a recent report by Liu et al. (29), who found that 5%
CO suppressed HIF-1 activation but did not affect the level of HIF-1
protein. In contrast to their experiments, we used the same cell
extract for both EMSA and HIF-1
Western blot analysis.
Alternatively, the discrepancy between our results and those of Liu
et al. (29) may be explained in part by their use of only a
single low concentration of CO.
. Moreover, the
fact that the C520S mutation has no effect on the activation of HIF-1
and its suppression by NO argues that SH-dependent redox
chemistry is not involved in oxidant-dependent degradation. This result contrasts strikingly with an analogous phenomenon, the
iron-dependent degradation of iron regulatory protein 2, the protein that regulates either the translation or the stability of
mRNAs encoding proteins critical in iron homeostasis. Iwai et
al. (49) have recently shown that a cluster of conserved cysteine
residues is required for the iron-dependent oxidative attack on the protein.
protein. In addition, both CO
and NO suppressed hypoxic induction of the C-terminal activation
domain, which is independent of HIF-1
stability. Our demonstration
of the inhibitory effect of CO and NO on HIF1
stability and
transactivation supports the notion of two distinct pathways that lead
to inhibition of HIF-1 activity. The conjoint destabilization of
HIF-1
and inhibition of C-terminal transactivation of HIF-1
are
both distal events in the signaling pathway, triggered by CO and NO,
which act appear to act proximally, very likely at the level of the
oxygen sensor. The only difference between the two ligands that we
could find was that the induction of Epo mRNA by cobalt was
suppressed by NO but not by CO (Fig. 1). This result is consistent with
our hypothesis that this transition metal can substitute for iron in
the oxygen sensor and mimic deoxy heme (27). Carbon monoxide cannot
bind to cobalt-substituted heme, whereas NO binds to cobalt heme with
high affinity (50). However, the action of cobalt may be independent of
the oxygen sensor, as others have suggested (48, 51). In contrast to
cobalt, the activation of HIF-1 by the iron chelator desferrioxamine is unlikely to directly affect oxygen sensing, consistent with the inability of CO to suppress induction by this agent. The most plausible
explanation of the effect of desferrioxamine is that free iron
functions as a Fenton reagent, catalytically increasing levels of ROS.
Therefore iron chelation will result in a lower level of ROS, which
appears to be critical in signaling the activation of HIF-1 (1).
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ACKNOWLEDGEMENTS |
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We thank Shoumo Bhattacharya, Zoltan Arany,
and David M. Livingston for providing pGal4-luc, pSB92, and
anti-HIF-1 antibodies, Patrick Maxwell for providing anti-HIF-1
antibody, and Maureen Schau for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants RO1-DK41234 (to H. F. B.) and RO1-DK45098 (to M. A. G.) and by National Institutes of Health Individual National Research Service Award F32-DK09365 (to L. E. H.).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.
Contributed equally to this work.
§ To whom correspondence should be addressed: Hematology-Oncology Division, LMRC-2, Brigham & Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5841; Fax: 617-739-0748; E-mail: bunn{at}calvin.bwh.harvard.edu.
2 H. Zhu, J. Olsen, and A. Riggs, personal communicacation.
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ABBREVIATIONS |
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The abbreviations used are: HIF-1, hypoxia-inducible factor 1; ODD, oxygen-dependent degradation; SNP, sodium nitroprusside; Epo, erythropoietin; CMV, cytomegalovirus; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; luc, luciferase; ROS, reactive oxygen species; ARNT, aryl hydrocarbon nuclear translocater.
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