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INTRODUCTION |
Cellular hypoxia is an important component of several
pathophysiological conditions, including tumorigenesis and
ischemia-related disorders. In these and other hypoxic situations
mammalian cells alter gene expression to counter the effects of limited
O2. Hypoxia induces the transcriptional activation of a
variety of genes, including erthyropoietin, vascular endothelial growth
factor, transferrin, tyrosine hydroxylase, and various glycolytic
enzymes, all of whose products are involved in cellular adaptation to
decreased O2 (reviewed in Ref. 1). Increased expression of
these genes is mediated primarily by the heterodimeric transcription
factor hypoxia-inducible factor 1 (HIF-1),1 which is composed
of HIF-1
and HIF-1
subunits (1-4). Decreased O2
tension stimulates HIF-1
protein accumulation by decreasing its
proteasomal degradation (5, 6). Hypoxic stabilization of HIF-1
is
blocked in the presence of H2O2, suggesting
that hypoxia-induced changes in the level of this reactive oxygen
species may be involved in HIF-1 activation (7). Stabilization of
HIF-1
allows for heterodimerization with constitutive levels of
HIF-1
, also known as aryl hydrocarbon nuclear translocator or ARNT.
In turn HIF-1 binds to specific enhancer elements resulting in
transcriptional activation.
The current understanding of the regulation of gene expression during
hypoxia is primarily at the level of transcriptional activation by
HIF-1. However, it is clear that post-transcriptional mechanisms are
also employed. These mechanisms involve the induction of
mRNA-binding proteins that interact with elements in
3'-untranslated regions (UTRs) as seen for the vascular endothelial
growth factor (8-10) and tyrosine hydroxylase (11-13) mRNAs. For
example, hypoxia induces the binding of the HuR protein to
adenylate-uridylate rich elements in the vascular endothelial growth
factor 3'-UTR, stabilizing this mRNA (14).
We have recently reported that hypoxia and subsequent reoxygenation
post-transcriptionally regulates the activity of IRP1 (15). IRP1 and a
second family member, IRP2, are iron-sensor proteins that
post-transcriptionally regulate the expression of genes whose products
are involved in regulating cellular iron homeostasis (reviewed in Ref.
16). IRP1 and IRP2 bind specific RNA stem-loop structures termed
iron-responsive elements (IREs), which are found in either the 5'- or
3'-UTRs of specific mRNAs, including ferritin (iron storage),
mitochondrial aconitase (energy metabolism), erythroid-aminolevulinate
synthase (heme biosynthesis), and transferrin receptor (iron
transport). IRP binding to a 5'-IRE inhibits translation of the
mRNA by inhibiting the 43 S small ribosomal complex from binding to
mRNA, whereas binding to a 3'-IRE protects the mRNA from
degradation. The RNA binding activity of the IRPs is regulated by
cellular iron: RNA binding activity decreases in iron-replete cells and
increases in iron-deplete cells. In addition to iron, IRP1 activity
increases in cells producing nitric oxide and in cells treated with
hydrogen peroxide (17). Thus, the regulation of IRPs by iron and
oxidative stress controls the expression of proteins involved in iron
sequestration, uptake, and utilization, thus maintaining cellular iron homeostasis.
IRP1 is a dual-function protein, because iron converts it from its
apo-RNA binding form to its [4Fe-4S] cytosolic aconitase form (18).
Unlike IRP1, IRP2 does not have detectable aconitase activity. Rather,
in iron-replete conditions, IRP2 is targeted for rapid degradation by
the proteasome (19, 20). Degradation of IRP2 is mediated by an
iron-dependent oxidation mechanism that requires a unique
73 amino acid domain containing three essential cysteine residues
(19-21).
Our continuing studies on IRP regulation during changing O2
tension revealed an overall increase in total IRP RNA binding activity
when human 293 cells and mouse Hepa-1 cells were exposed to hypoxia.
Because human IRPs co-migrate during bandshift analysis, it was not
readily apparent whether IRP1 or IRP2 was responsible for the increase
in total activity. We report here that in human 293 and mouse Hepa-1
cells, IRP2 RNA binding activity is increased as a result of hypoxia.
The hypoxia-induced increase in IRP2 RNA binding activity was found to
be caused by increased protein levels resulting from protein
stabilization. Furthermore, IRP2 protein levels were elevated during
normoxia in the presence of cobalt, a known activator of HIF-1
(6,
22). However, hypoxic activation of IRP2 was not dependent on HIF-1,
suggesting a similar, but divergent, signaling pathway in the hypoxic
activation of these two proteins. Consistent with our previous studies
(15), IRP1 activity decreased during hypoxia. The
differential regulation of IRP1 and IRP2 during hypoxia may have
implications for the regulation specific IRE-mRNAs
required for hypoxic adaptation.
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MATERIALS AND METHODS |
Cell Culture--
Rat FTO2B hepatoma, human 293 embryonic
kidney, and human HeLa cervical cancer cells were all maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). EcR-293 cells were
grown in the same medium containing 400 µg/ml zeocin (Invitrogen).
Hepa-1 c1c7 (wildtype HIF-1
) and Hepa-1 c4 (mutant HIF-1
) (kindly
provided by Dr. Oliver Hankinson, UCLA) were grown similarly except in
minimal essential medium with Earle's salts (Life Technologies, Inc.). All cells were grown at 37 °C in a 5% CO2 humidified
incubator. Cells were plated at a density of 1.2 × 106 (FTO2B) and 2.5 × 105 (293, HeLa, and
Hepa-1) respectively, in 35-mm culture plates 1 day before normoxic or
hypoxic exposure. Hypoxia was achieved by flushing a Modular Incubator
(Billups-Rothenburg, Del Mar, CA) for 20 min at a flow of 2 p.s.i
with 1% O2, 5% CO2, and a balance of
nitrogen. 1 h before normoxia/hypoxia, the medium was replaced
with 1.2 ml of fresh medium. Normoxic control cells were treated
identically except with 21% O2. The experiment shown in Fig. 1A was the only experiment in which 3% O2
was used for hypoxia. Expression of recombinant IRP2 was done by
transiently transfecting EcR-293 cells with 10 µg of the
pIND(sp1)IRP2myc vector using Superfect (Qiagen), and 24 h later
inducing with 10 µM pronasterone A (ProA) for 20 h
(Invitrogen). The ecdysone analog ProA induces the heterodimerization
of a modified ecdysone receptor with the retinoid X receptor, both
constitutively expressed in the EcR-293 cell line, in turn activating
transcription from the ecdysone response element found on pIND(sp1).
The pIND(sp1)IRP2myc vector was generated by subcloning the
3.0-kilobase EcoRV fragment from pXS.IRP2 (23) into
EcoRV linearized pIND(sp1) (Invitrogen).
Cytosolic Extract Preparation and RNA Bandshift
Analysis--
Cells were washed once with cold phosphate-buffered
saline and cytosolic extracts were prepared by addition of 125 µl of
lysis buffer (20 mM Hepes, pH 7.6, 25 mM KCl,
0.5% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride)
to the 35-mm dish and incubated at room temperature for 3 min. Cell
debris was removed by centrifugation at 15,000 × g for
30 min at 4 °C. Protein concentration was determined by the
Coomassie assay method (Pierce). RNA bandshifts were performed as
described previously (24). Quantification of IRP-RNA complexes was
determined by PhosphorImager analysis. When "supershift" assays were performed, antiserum that specifically recognizes the unique 73 amino acid insert of IRP2 was added for 20 min before electrophoresis (25). Dithiothreitol (DTT) was used at 10 mM where shown.
Immunoblot Analysis--
Cytosolic extracts were resolved on 8%
sodium dodecyl sulfate polyacrylamide gels and then transferred to
nitrocellulose. After blocking in phosphate-buffered saline containing
0.2% Tween and 5% dried milk, the blot was probed with rabbit
anit-IRP2 antiserum or chicken anti-IRP1 antiserum (both at 10,000-fold
dilution) (25). Appropriate horseradish peroxidase-conjugated secondary antibodies (Pierce) were used for detection by chemiluminescence (New
England Nuclear).
Northern Blot Analysis--
Human 293 cells were plated at
2 × 106 on a 10-cm plate ~24 h before use. Fresh
medium (8 ml) was added 1 h before an 18 h normoxia or
hypoxic exposure. Total RNA was harvested using Trizol (Life
Technologies, Inc.), and 20 µg were resolved on a formaldehyde gel
and transferred to a nylon membrane. A 900-base pair fragment corresponding to the immediate 5'-end of rat IRP2 (26) was
random-primed labeled with [
-32P]dCTP and used to
probe for IRP2 mRNA.
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RESULTS |
Hypoxia Regulates Both IRP1 and IRP2 RNA Binding
Activities--
Cultures of rat FTO2B and human 293 cells were exposed
in parallel to 6 h of normoxia or hypoxia, and IRP RNA binding
activity was measured by RNA bandshift analysis. Fig.
1A shows that total IRP (IRP1
and IRP2) RNA binding activity in human 293 cells is elevated following
hypoxic exposure. Hypoxia decreased IRP1 RNA binding activity in rat
FTO2B hepatoma cells as previously reported (15). Rodent IRP1 and IRP2
separate during bandshift analysis unlike human IRPs. The
hypoxia-induced increase in total IRP activity from 293 cells was more
notable after 18 h of hypoxia (Fig. 1B). Addition of
hemin decreased total IRP RNA binding activity in both normoxic and
hypoxic cells, demonstrating that the IRP1 and IRP2 response to iron is
not impaired during hypoxia.

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Fig. 1.
Hypoxic regulation of IRPs in rat FTO2B and
human 293 cells. A, rat FTO2B and human 293 cells were
exposed, in parallel, to normoxia (21% O2) or hypoxia (3%
O2) for 6 h followed by bandshift analysis using a
32P-labeled IRE RNA and 12 µg of cytoplasmic extract.
IRP1 and IRP2 (right panel) and co-migrating IRP1/2
(left panel) bands are indicated. B, in a
separate experiment, 293 cells were exposed to normoxia or hypoxia (1%
O2) for 7 or 18 h with or without 50 µM
hemin, and bandshift analysis was performed as in A.
N, normoxia; H, hypoxia. Note that human IRP1 and
IRP2 co-migrate in bandshifts. Representative experiments are
shown.
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To determine whether IRP1 and/or IRP2 was responsible for the increase
in total RNA binding activity in 293 cells during hypoxia, RNA
"supershift" analysis was performed. Different amounts of IRP2-specific antibodies were added to the binding reactions containing cytoplasmic extracts derived from 293 cells subjected to normoxia or
hypoxia for 18 h, and the complexes were analyzed by bandshift analysis. Fig. 2A shows that
there was an increase in supershifted IRP2-RNA complexes from
hypoxically treated cells compared with normoxic controls (compare
lanes 6 and 7 with lanes 10 and 11 in Fig. 2A). FTO2B extracts were run in
parallel with increasing amounts of IRP2 antibody to verify that the
IRP2 antiserum did not nonspecifically supershift IRP1, and that a
sufficient amount of antiserum was added to shift all of the IRP2 (Fig.
2A, lanes 2-4). Because the nonshifted complexes
represent IRP1, it can be seen that IRP1 RNA binding activity decreased
as a result of hypoxia (compare lanes 6-8 with
10-12 in Fig. 2A), which is similar to what is
seen in FTO2B cells (Fig. 1A). Furthermore, consistent with
our earlier studies, the hypoxic decrease in IRP1 RNA binding activity
was not because of decreased protein levels because IRP1 protein levels
did not change following a 20 h hypoxic exposure (Fig.
2B) (15). These data indicate that IRP1 and IRP2 are both regulated during hypoxia but in opposite directions.

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Fig. 2.
Hypoxia increases IRP2 RNA binding by
increasing IRP2 protein levels concomitant with a decrease IRP1 RNA
binding. A, human 293 cells (lanes
5-12) were exposed to normoxia (N) or
hypoxia (H) for 18 h followed by supershift analysis
using 12 µg of cytosolic extract and anti-IRP2 antiserum as described
under "Materials and Methods." Either no antiserum or the indicated
amount of IRP2 antiserum was added to the extracts after the
RNA-protein complexes were allowed to form. Cytosolic extracts (12 µg) from FTO2B cells (lanes 1-4) were included
as controls to ensure that the amount of IRP2 antiserum used did not
shift IRP1. Rat IRP1 and IRP2 (rIRP1 and rIRP2),
co-migrating human IRPs (hIRP1/2) and
supershifted IRP2 are indicated. B, 293 cells were exposed
to 20 h of normoxia (N) or hypoxia (H), and
IRP1 levels were examined by immunoblot analysis. C,
determination of IRP2 protein levels by immunoblot analysis followed by
LumiImager analysis (Boehringer) for the indicated times of hypoxic
exposure was performed (see text). D, IRP2 supershift
analysis of extracts in A. PhosphorImager quantification was
then performed.
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The hypoxic increase in IRP2 RNA binding activity could result from
activation of IRP2 or from increasing IRP2 protein levels. Therefore,
we measured IRP2 protein levels following 4, 7, and 19 h of
hypoxic exposures. The immunoblot in Fig. 2C demonstrates that hypoxia increases IRP2 protein levels. Quantification of the bands
in Fig. 2C revealed a 1.5-, 2.9- and 3.9-fold increase in
IRP2 protein levels at 4, 7, and 19 h of hypoxia, respectively. The increase in IRP2 protein levels reflected a similar fold increase in IRP2 RNA binding activity as determined by PhosphorImager analysis (Fig. 2D). Therefore, we conclude that the increase in IRP2
RNA binding activity during hypoxia is because of an increase in IRP2 protein levels.
IRP2 Hypoxic Induction Is Mediated by a Post-transcriptional
Mechanism--
To determine whether the hypoxic increase in IRP2
protein is the result of increased IRP2 mRNA, mRNA levels were
measured by Northern blot analysis. Total RNA was isolated from 18-h
normoxic and hypoxic human 293 cells and hybridized with a radiolabeled IRP2 probe. Fig. 3A shows that
IRP2 mRNA levels are not elevated during hypoxia. In fact, IRP2
mRNA is slightly decreased, perhaps because of a general hypoxic
down-regulation of RNA synthesis (27). These results indicate that the
increase in IRP2 protein during hypoxia is not because of increased
transcription of the IRP2 gene nor to increased stability of the IRP2
mRNA.

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Fig. 3.
Hypoxic activation of IRP2 is
post-transcriptional. A, Northern blot analysis of
total RNA (20 µg) harvested from 293 cells exposed to hypoxia
(H) or normoxia (N) for 18 h was hybridized
with a 32P-labeled IRP2 probe. Ethidium bromide-stained rat
RNA (rRNA) is shown as a control for RNA loading. Molecular
size standards are indicated in kilobases. B, bandshift
analysis was performed using 12 µg of cytosolic extracts from 18-h
normoxically exposed (N) or hypoxically exposed
(H) wild-type Hepa-1 c1c7 cells containing a functional
HIF-1 and Hepa-1 c4 cells lacking a functional HIF-1 .
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Although we could eliminate the direct transcriptional activation of
the IRP2 gene during hypoxia, it was possible that an HIF-1 target gene
was required for hypoxic induction of IRP2. To analyze this
possibility, a mouse hepatoma cell line harboring a mutated HIF-1
subunit that is nonfunctional (28, 29) was used to determine whether
hypoxic induction of IRP2 was dependent on HIF-1 activation. Hepa-1 c4
cells, which contain a mutated HIF-1
and wild-type Hepa-1 c1c7 were
exposed to normoxia or hypoxia for 18 h, and IRP1 and IRP2 RNA
binding activities were measured. Fig. 3B shows that IRP2
RNA binding activity increased in both cell lines to a similar extend
as seen for 293 cells. Furthermore, IRP1 RNA binding activity decreased
in Hepa-1 c4c7 cells similar to hypoxic rat FTO2B and human 293 cells.
These data indicate that hypoxic activation of IRP2 is not downstream
of HIF-1.
Hypoxia Stabilizes IRP2--
Because hypoxic induction of IRP2
does not result from increased mRNA levels, we next examined
whether IRP2 is stabilized during hypoxia. EcR-293 cells were
transiently transfected with the pIND(sp1)IRP2 vector and allowed to
recover for 24 h before IRP2 expression was induced with ProA.
Following 20 h of induction, the cells were then either harvested
or the medium was replaced with medium lacking ProA and exposed to
normoxia or hypoxia for 12 h. Immunoblot analysis demonstrates
that recombinant IRP2 is induced in the presence of ProA (Fig.
4, lanes 1 and 2).
When expression of IRP2 is shut off by removal of ProA, the levels of
IRP2 decay after 12 h of normoxia, but remain elevated following 12 h of hypoxia (Fig. 4, lanes 3 and 4).
These data demonstrate that the increase in IRP2 protein during hypoxia
is because of protein stabilization. Furthermore, stabilization of IRP2
appears specific, because IRP1 protein levels were not significantly
altered following 20 h of hypoxia (Fig. 2B) (15).

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Fig. 4.
Hypoxia increases IRP2 protein
stability. EcR-293 cells were transiently transfected with the
pIND(sp1)-IRP2 vector and cells were then split equally to 4 dishes and
allowed to recover for 24 h before the addition of vehicle or 10 µM ProA to induce the expression of IRP2 as indicated.
Following 20 h with or without ProA cells were either immediately
lysed (lanes 1 and 2) or incubated for 12 h
under normoxia (N) or hypoxia (H) (lanes
3 and 4). IRP2 immunoblot analysis was then performed
on 25 µg of cytosolic protein.
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Cobalt Increases IRP2 Protein During Normoxia--
Addition of
cobalt to normoxic cells mimics hypoxia by the activation of expression
of several HIF-1 target genes. This is because of the cobalt-induced
increase in the HIF-1
protein and subsequent HIF-1 DNA binding (6,
22). HIF-1
protein stabilization has been suggested to be downstream
of a heme protein that functions as an O2 sensor (1, 3). It
is thought that cobalt, by displacing the heme iron, inactivates the
O2 sensor. A more recent report suggests that the
mitochondria may be the upstream O2 sensor for HIF-1
activation (42). Treatment of 293 cells with cobalt increased IRP2
protein levels in both a dose- and time-dependent manner (Fig. 5A and B).
Although 100 µM CoCl2 increased IRP2 protein levels, maximum stimulation was achieved at 1 mM, which is
~10-fold greater than that required for inducing HIF-1 activation in
HeLa cells (22). The requirement for a higher dose of cobalt in 293 cells is presumably because of differences in cobalt uptake, because IRP2 was induced in HeLa cells treated with 100 µM
CoCl2 for 8 h (Fig. 5C).

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Fig. 5.
CoCl2 induces IRP2 in 293 and
HeLa cells under normoxic conditions. CoCl2 was added
to 293 cells at the given concentrations for 22 h (A)
or at 1 mM for the indicated times followed by IRP2
immunoblot analysis (B). CoCl2 was added to HeLa
cells at the given concentrations for 8 h followed by immunoblot
analysis (C).
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To determine whether the cobalt-induced increase in IRP2 protein levels
reflects an increase in IRP2 RNA binding activity, 293 cells were
subjected to normoxia, 1 mM CoCl2, hypoxia, or hypoxia in the presence of 1 mM CoCl2 for
8 h followed by supershift and immunoblot analyses. Fig.
6A shows that in contrast to
hypoxia where IRP2 RNA binding activity increased, cobalt treatment
resulted in an inhibition of IRP2 activity (Fig. 6A, compare
lanes 5 and 6), even though both conditions
increased IRP2 protein levels (Fig. 6C). Furthermore, cobalt
treatment also inhibited the increase in IRP2 RNA binding activity
during hypoxia (Fig. 6A, compare lanes 7 and
8). To determine whether inactivation of IRP2 RNA binding
activity by cobalt could be reversed by a reductant, 10 mM
DTT was added to the binding reactions before supershift analysis. DTT
restored IRP2 RNA binding activity in both hypoxia- and
hypoxia/CoCl2-treated extracts (Fig. 6B).
Treatment with DTT also increased IRP1 RNA binding activity in all
extracts as previously shown (30). Because cobalt, hypoxia, and a
combination of both increased IRP2 protein levels to a similar extent
(Fig. 6C), their modes of action are not synergistic. These
results suggest that cobalt affects IRP2 by two distinct mechanisms:
first, by mimicking hypoxia, cobalt increases IRP2 protein levels, and
second, through a mechanism unrelated to hypoxia whereby cobalt
inhibits IRP2 RNA binding activity.

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Fig. 6.
Cobalt induces IRP2 lacking RNA binding
activity. A, human 293 cells were exposed for 8 h
to normoxia (N, lanes 1 and 5), 1 mM CoCl2 (Co, lanes 2 and
6), hypoxia (H, lanes 3 and
7), or both 1 mM CoCl2 and hypoxia
(Co + H, lanes 4 and 8). Cytosolic
protein (12 µg) was then subjected to bandshift analysis with
(lanes 5-8) or without (lanes 1-4) anti-IRP2
antiserum (1.5 µl). B, supershift analysis was carried out
as in A except that 10 mM DTT was present in the
binding reactions. C, immunoblot analysis using IRP2
antiserum was performed on the same extracts.
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DISCUSSION |
Mammalian cells employ adaptive responses when exposed to hypoxia
(1, 3). One such response is the regulated and specific alteration in
gene expression imposed by hypoxia-induced transcriptional activation.
To date HIF-1 is the most thoroughly characterized transcriptional
regulator of gene expression during hypoxia (reviewed in Refs. 31 and
32), whereas considerably less is known about post-transcriptional
mechanisms (9, 11, 12, 14). In this study we report that IRP2, in
addition to IRP1, is post-translationally regulated by hypoxia. These
data suggest that both proteins are components of a hypoxic-regulatory
pathway involved in post-transcriptional gene regulation.
Previously we demonstrated that hypoxia decreased IRP1 RNA binding
activity in rat hepatoma cells and primary cardiac myocytes (15). In
that study, IRP2 RNA binding activity increased slightly after
prolonged hypoxic exposure (18 to 21 h). We also observed an
increase in IRP2 activity in FTO2B cells during
anoxia.2 These results
prompted us to carry out the experiments reported here, which
investigated IRP2 regulation during hypoxia in other cell types,
including 293, Hepa-1, and HeLa cells. Both 293 and Hepa-1 cells
displayed significant hypoxia-inducible activation of IRP2 RNA binding
activity. The regulation of IRP2 by hypoxia appears to be
characteristic of many cell types with different cells varying in their
magnitude of IRP2 response.
IRP1 and IRP2 RNA binding activities responded to hypoxia differently:
hypoxia decreased IRP1 and increased IRP2 RNA binding activities. The
decrease in IRP1 RNA binding activity was paralleled by a ~35%
increase in its cytosolic aconitase activity with no change in IRP1
protein levels (data not shown). These results suggest hypoxia
stabilizes the [4Fe-4S] aconitase active form of IRP1 at the expense
of its RNA binding activity (15). Unlike IRP1, IRP2 regulation by
hypoxia occurs by accumulation of the IRP2 protein. Therefore, hypoxic
regulation of IRP1 and IRP2 RNA binding activities occurs by different mechanisms.
What are the functional consequences of the differential regulation of
IRP1 and IRP2 during hypoxia? One notion is that IRP1 and IRP2 regulate
different cellular IRE-mRNAs. Studies using in vitro
synthesized IREs (33, 34) showed that IRP1 and IRP2 can preferentially
bind to specific IRE-like structures. In addition, a recent study
showed that the range of IRE binding of IRP1 is more extensive than
IRP2. IRP1 binds with high affinity to different IREs, including those
of the ferritin, transferrin receptor, and erythroid-aminolevulinate
synthase mRNAs, whereas high affinity IRP2 binding is restricted to
ferritin IREs (35). The regulation of IRP1 and IRP2 activities in an
opposing manner during hypoxia may lead to the specific regulation of
different IRE-mRNAs whose expression/repression may be required for
adaptation during the hypoxia.
Hypoxic activation of IRP2 is not a result of an increase in mRNA
levels because of increased transcription or increased mRNA stability. Activation is also not dependent on HIF-1, because full IRP2
activation occurs in Hepa-1 c4 cells that do not contain a functional
HIF-1
. Furthermore, hypoxic accumulation of IRP2 is not because of
impaired proteasomal function, because hemin-treated cells resulted in
IRP2 degradation during hypoxia. Rather, our data suggest a model in
which the IRP2 protein is stabilized during hypoxia. This model is
consistent with the mechanism of iron-dependent regulation
of IRP2 (19, 20, 25). IRP2 is regulated by iron by a mechanism
involving metal-catalyzed oxidation of the protein followed by
ubiquitination and degradation via the proteasome (21). This process
requires the 73-amino acid degradation domain that potentially
coordinates iron via three conserved cysteines, which are required for
iron-mediated degradation (20). Iron coordination may provide a
"localized" Fenton reaction leading to IRP2 oxidation. Any model
for the regulation of IRPs during hypoxia must depart from one that
relies solely on changes in iron levels, because during hypoxia RNA
binding activity of IRP1 decreased, whereas IRP2 activity increased.
One possible mechanism for hypoxic regulation of IRP2 is that the
generation of H2O2, possibly from a
heme-containing O2 sensor, acts as an IRP2 degradation signal. A decrease in H2O2 output from such a
sensor during hypoxia would lead to a decrease in IRP2 oxidation and
consequently an increase in IRP2 stability. In this scenario, hypoxic
regulation of IRP2 would be dependent on the relative cytosolic levels
of both H2O2 and Fe2+. A similar
mechanism could potentially be involved in hypoxic HIF-1
protein stabilization.
Several important parallels can be drawn between hypoxic activation of
IRP2 and HIF-1
. First, both IRP2 and HIF-1
(5-7) proteins
accumulate during hypoxia by a post-translational mechanism involving
increased protein stability. Second, cobalt, which mimics hypoxia,
stimulated the accumulation of both IRP2 and HIF-1
(6, 22, 37),
perhaps by inactivating a heme-containing O2 sensor (38).
Third, iron chelation elevates the protein levels of both IRP2 (19, 23)
and HIF-1
(37, 39). Fourth, the activation of IRP2 and HIF-1
by
hypoxia and cobalt occurs in different cell types, suggesting that both
proteins are part of a global mechanism of gene regulation during
hypoxia. Collectively, these data suggest that the regulation of IRP2
and HIF-1
during hypoxia occurs, at least in part, by similar mechanisms.
An unexpected finding was that cobalt, in addition to increasing IRP2
protein levels, inactivated IRP2 RNA binding activity. Because IRP1
activity was not decreased, this suggested that the effect of cobalt
was specific for IRP2. IRP2 RNA binding activity could be restored by
the addition of DTT, suggesting that cobalt either directly or
indirectly resulted in the oxidation of critical cysteines required for
IRP2 RNA binding. Cobalt has been shown to cause a reduction of
cellular glutathione and oxidize thiols in lung cells (40). These
results are consistent with our previous studies showing that
inactivation of IRP2 RNA binding activity by oxidants in
vitro could be reversed by treatment with high concentrations of
reductants (41). Similar redox control of DNA binding activity of HIF-1
has also been reported (7). Whether IRP2 RNA binding activity can be
modulated by redox in vivo remains to be determined.