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RED CELLS
Effects of iron regulatory protein regulation on iron homeostasis during hypoxia
Brian D. Schneider and Elizabeth A. Leibold
Iron regulatory proteins (IRP1 and IRP2)
are RNA-binding proteins that affect the
translation and stabilization of specific
mRNAs by binding to stem-loop structures known as iron responsive elements
(IREs). IREs are found in the 5ⴕ–untranslated region (UTR) of ferritin (Ft) and
mitochondrial aconitase (m-Aco) mRNAs,
and in the 3ⴕ-UTR of transferrin receptor
(TfR) and divalent metal transporter-1
(DMT1) mRNAs. Our previous studies
show that besides iron, IRPs are regulated by hypoxia. Here we describe the
consequences of IRP regulation and show
that iron homeostasis is regulated in 2
phases during hypoxia: an early phase
where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis
increase, and a late phase where IRP2
RNA-binding activity increases and iron
uptake and Ft synthesis decrease. The
increase in iron uptake is independent of
DMT1 and TfR, suggesting an unknown
transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late
phase of hypoxia, IRP2 RNA-binding activ-
ity increases, becoming the dominant
regulator responsible for decreasing Ft
synthesis. During reoxygenation (ReO2),
Ft protein increases concomitant with a
decrease in IRP2 RNA-binding activity.
The data suggest that the differential regulation of IRPs during hypoxia may be
important for cellular adaptation to low
oxygen tension. (Blood. 2003;102:3404-3411)
© 2003 by The American Society of Hematology
Introduction
Periods of low O2 concentration or hypoxia occur during a broad range
of biologic conditions from early development to tumorigenesis and
heart disease. Under such situations, cells adapt to the low O2 environment by increasing the expression of a number of genes including
vascular endothelial growth factor, erythropoietin, glycolytic enzymes,1
and genes involved in iron homeostasis, such as ceruloplasmin, transferrin (Tf), and transferrin receptor (TfR).2-5 The expression of these
genes requires activation by hypoxia-inducible factor-1 (HIF-1), a
transcription factor consisting of hypoxia-inducible HIF-1␣ and constitutively expressed HIF-1␤ subunits. During normoxia, HIF-1␣ is
destabilized by a mechanism involving prolyl hydroxylation and
targeted for proteasomal degradation.6 During hypoxia, prolyl hydroxylase activity is reduced and the nonhydroxylated form of HIF-1␣ is
stabilized. HIF-1␣ then binds to the constitutively expressed HIF-1␤
subunit to activate transcription of genes that allow for adaptation
to hypoxia.
When the O2 concentration returns to normal, the production of toxic
reactive oxygen species (ROS), such as the hydroxyl radical ( 䡠OH),
superoxide, and hydrogen peroxide (H2O2) increases. Iron contributes to
ROS formation by catalyzing the generation of 䡠OH from H2O2 by
Fenton chemistry.7 ROS, especially 䡠OH, can damage proteins, DNA,
and lipids, and are thought to be responsible for much of the cellular and
tissue injury associated with reperfusion disorders.8,9 In both animal and
cell culture models, iron chelation has been shown to decrease the
damage caused during reperfusion.10-12
Due to the dual nature of iron as essential for both cellular
growth and survival, yet toxic when present in excess, cells have
evolved a mechanism to maintain iron homeostasis via iron
regulatory protein 1 (IRP1) and IRP2.13-15 When the cellular-free
iron pool is low, IRPs bind to specific RNA stem loop structures,
called iron-responsive elements (IREs), located in the 5⬘–
untranslated region (UTR) of mRNAs such as ferritin heavy chain
(FtH) and ferritin light chain (FtL) subunits and mitochondrial
aconitase (m-Aco), thereby preventing their translation. IRPs also
bind to IREs located in the 3⬘-UTR of TfR mRNA, stabilizing the
message from endonucleolytic cleavage and increasing the uptake
of Tf-bound iron. Conversely, when the cellular free iron pool is
high, IRP2 is degraded by the proteasome in an iron-dependent
manner, and IRP1 is converted from an RNA-binding protein to a
[4Fe-4S] cluster-containing protein that displays cytosolic aconitase (c-Aco) activity. These changes result in a decrease in TfR
synthesis with a corresponding increase in Ft translation, leading to
a decrease in the cellular-free iron pool. By constantly responding
to changes in the cellular-free iron pool, IRPs maintain iron
homeostasis by regulating iron uptake and sequestration.
In addition to iron, IRP activities are influenced by other effectors,
including ROS and reactive nitrogen species,16-19 phosphorylation,20,21
hypoxia/reoxygenation (ReO2),22-26 and ischemia/reperfusion.27,28 ROS
such as superoxide inactivate aconitases by disassembly of the [4Fe-4S]
cluster.29,30 Nitric oxide (NO), produced in cells stimulated with
cytokines or in cells treated with NO-generating compounds, activates
IRP1 RNA-binding activity by causing cluster disassembly19,31-33 while
decreasing IRP2 RNA-binding activity.33,34 Others have shown that
phosphorylation of a critical serine in IRP1 results in the destabilization
of the [4Fe-4S] cluster of IRP1, suggesting an iron-independent
mechanism of IRP1 regulation.21
Our previous work has shown that hypoxia regulates IRP
RNA-binding activities in opposing directions such that IRP2
From the Program in Human Molecular Biology and Genetics and the
Departments of Oncological Sciences and Medicine, University of Utah, Salt
Lake City.
University of Utah Graduate Research Fellowship (B.D.S.).
Submitted February 10, 2003; accepted June 24, 2003. Prepublished online as
Blood First Edition Paper, July 10, 2003; DOI 10.1182/blood-2003-02-0433.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by grants from the National Institutes of Health (grant GM45201)
and the American Heart Association Western States Affiliate (E.A.L.), and a
3404
Reprints: Elizabeth A. Leibold, University of Utah, 15 N 2030 E, Bldg 533, Rm
4220, Salt Lake City, UT 84112; e-mail: [email protected].
© 2003 by The American Society of Hematology
BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
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BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
RNA-binding activity increases while that of IRP1 decreases.23
However, the cellular consequences of this regulation remain
unclear. Here, we investigate the effects of IRP regulation during
hypoxia in human embryonic kidney 293 (HEK293) cells and
describe the mechanisms responsible for regulation of iron homeostasis during hypoxia.
Materials and methods
Cell culture and reagents
HEK293 cells were cultured in complete medium consisting of Dulbecco
modified eagle medium (DMEM) containing 10% heat-inactivated fetal
bovine serum (FBS; Invitrogen, Carlsbad, CA) supplemented with 100
units/mL penicillin (Invitrogen) and 100 ␮g/mL streptomycin (Invitrogen)
(pen/strep) at 37°C in 5% CO2 and atmospheric air (ie, normoxia). To
increase cellular adhesion, culture dishes were treated with 15 ␮g/mL
poly-lysine (Sigma, St Louis, MO). For hypoxia time-course experiments,
cells were plated in 60-mm culture dishes containing 2.5 mL complete
medium, and subsequently placed in a humidified incubator containing 1%
O2, 5% CO2, balance N2 for the indicated times. For iron and iron-chelation
experiments, cells were treated with 50 to 150 ␮g/mL ferric ammonium
citrate (FAC) for 5 hours or 100 to 200 ␮M deferoxamine mesylate (Df;
Sigma) for 16 hours under normoxic conditions as indicated. For experiments with high-molecular-weight (MW) Df (a gift from Dr Bo Hedlund,
Biomedical Frontiers), complete medium was treated overnight with 600
␮M high-MW Df before being added to cells for various lengths of time
under normoxic or hypoxic conditions as indicated. This concentration was
found to elicit the maximal increase in both IRP1 and IRP2 RNA-binding
activities after 16 hours under normoxic conditions (data not shown).
Cytosolic extract preparation and RNA supershift analysis
HEK293 cells were washed with phosphate-buffered saline (PBS) and lysed
with 140-␮L lysis buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N⬘2-ethanesulfonic acid], 25 mM KCl, 0.5% nonidet P-40 [NP-40], and 1 mM
dithiothreitol [DTT]). Cells were scraped and cell lysates cleared by
centrifugation at 15 000g for 15 minutes at 4°C. Protein concentration was
determined using Coomassie reagent (Pierce, Rockford, IL). RNA supershift assays were performed as described previously.23,35 Band intensity was
quantified with a PhosphorImager (Molecular Dynamics, Piscataway, NJ).
Immunoblot analysis
Cytosolic cell extracts (20 ␮g) of HEK293 cells were separated on 8%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
gels (15% for Ft detection), and proteins were transferred to a nitrocellulose
membrane. Blots were probed with the following primary antibodies:
chicken antirat IRP1 polyclonal antibody,36 rabbit antirat IRP2 polyclonal
antibody,37 sheep antihuman Ft antibody (The Binding Site, San Diego,
CA), and mouse antihuman TfR monoclonal antibody (Zymed, South San
Francisco). The m-Aco polyclonal antibody used was made by resolving
porcine heart aconitase (Sigma) over two 8% SDS-PAGE gels. The protein
band was excised and used as antigen for injection in rabbits. Proteins were
detected with a horseradish peroxidase–conjugated secondary antibody
followed by chemiluminescence (NEN Life Science Products, Boston, MA).
Northern analysis and preparation of RNA
HEK293 cells were grown in either hypoxic or normoxic conditions for the
indicated times, and total RNA was prepared using Trizol reagent (Invitrogen). Total RNA or poly(A)⫹ RNA, purified from total RNA using an
Oligotex purification system (Qiagen, Valencia, CA), was resolved on 0.9%
(poly(A)⫹ RNA) or 1.2% (total RNA) agarose formaldehyde gels. RNA
was transferred to a nylon membrane and hybridized with the following
32P-labeled DNA probes: human TfR,38 porcine m-Aco,39 rat FtH subunit,40
rat FtL pseudogene 66,35 human 18S rRNA, mouse skeletal muscle ␤-actin
(Stratagene, La Jolla, CA), and an IRP1 mouse/human hybrid.41 Blots were
EFFECTS OF IRP REGULATION DURING HYPOXIA
3405
prehybridized for one hour, hybridized overnight with 1 to 1.25 ⫻ 106
cpm/mL of the indicated probe, washed, and subjected to autoradiography.
Band intensity was quantified with a PhosphorImager (Molecular Dynamics).
Iron uptake
For uptake of 55Fe-Tf, HEK293 cells were washed with serum- and
pen/strep-free DMEM and incubated with 1 nM 55Fe-Tf in this medium for
the last one hour of normoxic or hypoxic treatment. Cells were then washed
with PBS and harvested in radioimmunoprecipitation assay (RIPA) buffer
(50 mM Tris [tris(hydroxymethyl)aminomethane, pH 8.0], 150 mM NaCl,
1% NP-40, 0.5% deoxycholic acid [DOC], 0.1% SDS) and the lysates
centrifuged at 15 000g for 15 minutes at 4°C. Protein concentration of cell
lysates was determined as described for cytosolic extract preparation, and
radioactivity was counted using a scintillation counter. Radioactive counts
were normalized to protein concentration and compared relative to the
normoxic control. For cold Fe-Tf experiments, 1 ␮M human holo-Tf
(Sigma) was added to cells immediately before 55Fe-Tf labeling.
55Fe-NTA was prepared by complexing 55Fe (55FeCl from NEN Life
3
Science Products) with a 4-fold molar excess of NTA (nitrilotriacetic acid)
in 20 mM HEPES, and the mixture was adjusted to pH 7 with 4 M NaOH.
To measure Tf-independent iron uptake, HEK293 cells were washed with
serum- and pen/strep-free DMEM or Hanks balanced salt solution (HBSS)
and incubated with 1 ␮M 55Fe-NTA in this medium for the last one hour of
normoxic or hypoxic treatment. As a control, 1 mM cold Fe-NTA was added
to cells immediately before 55Fe-NTA labeling. Cells were washed with
PBS and harvested in RIPA buffer. Cell lysates were centrifuged at 15 000g
for 10 minutes at 4°C. Protein concentration and radioactivity of cell lysates
was determined as described above. For the experiments in Figure 6A,
complete DMEM was treated with 600 ␮M high-MW Df overnight at 4°C,
while for Figure 6B, serum- and pen/strep-free DMEM was treated with
1 ␮M 55Fe-NTA and 600 ␮M high-MW Df overnight at 4°C before being
added to cells.
Separation of mitochondrial and cytosolic fractions and
aconitase assays
HEK293 cells were grown in DMEM (5% FBS, 100 units/mL penicillin,
100 ␮g/mL streptomycin) for the indicated times under normoxic or
hypoxic conditions. Cells were treated with 0.01% digitonin for 15 minutes,
scraped, and spun at 600g for 1 minute at 4°C to pellet cells. The
supernatant was removed and spun at 15 000g for 5 minutes at 4°C. The
cleared lysate was saved as the cytosolic fraction. The cell pellet was
washed with PBS and solubilized by the addition of Triton X buffer (0.2%
Triton X-100, 150 mM NaCl, 20 mM HEPES, pH 7.5) for 10 minutes on
ice. Cellular debris was removed by centrifugation at 15 000g, and the
cleared lysate was saved as the mitochondrial fraction. Protein concentration was determined as described for cytosolic extract preparation. Aconitase activity was determined in each fraction as described previously.42
Briefly, 0.2 mM cis-aconitate was added to 50 ␮g protein extract in 500 ␮L
of 50 mM Tris-HCl, pH 7.5. The disappearance of cis-aconitate was
followed at 240 nm over time.
35S-labeling
and Ft immunoprecipitation
HEK293 cells grown in 35-mm culture dishes were washed with DMEM
(Cys-, Met-, serum-, and pen/strep-free) followed by the addition of 100
␮Ci/mL (3.7 MBq/mL) 35S-Met/Cys (ICN Pharmaceuticals, Costa Mesa,
CA) in 500 ␮L of this medium for the last one hour of normoxic or hypoxic
treatment. Cells were lysed in RIPA buffer and cell extracts were
centrifuged at 15 000g for 25 minutes at 4°C. For immunoprecipitations,
200 ␮g cell lysate was incubated with 1.0 ␮L rabbit antihuman Ft
polyclonal antibody (Dako, Carpinteria, CA) in 1 mL RIPA buffer at 4°C for
one hour. Then, 50 ␮L of a 50% slurry of protein-A agarose beads
(Invitrogen) was added for one hour at 4°C. The protein-agarose beads were
spun and washed with 1 mL RIPA buffer and then boiled for 10 minutes in
SDS-loading buffer (350 mM Tris-base, pH 6.8, 10% SDS, 600 mM DTT,
36% glycerol, 0.01% bromophenol blue). Samples were centrifuged and
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3406
SCHNEIDER and LEIBOLD
BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
proteins resolved on 15% SDS-PAGE gels. Gels were dried and analyzed
by autoradiography.
Results
Iron homeostasis exhibits a biphasic response during hypoxia
Our previous studies showed that IRP1 and IRP2 RNA-binding
activities respond differently to hypoxia.22,23 To determine how this
affects iron homeostasis, we first carried out a detailed time course
to analyze IRP RNA-binding in HEK293 cells subjected to hypoxia
(1% O2) for 0 to 21 hours. Cells were harvested at various times
following hypoxic exposure, and protein extracts were subjected to
RNA supershift analysis using a 32P-labeled Ft IRE RNA. Supershift assays of IRP2 were necessary because human IRP1/IRP2
comigrate.43 Figure 1A demonstrates that IRP2 RNA-binding
activity increases more than 5-fold over control conditions by 21
hours of hypoxia. Immunoblot analysis shows that this increase
correlates with an increase in the amount of IRP2 protein (Figure
1B), which is due to protein stabilization.23 As controls, treatment
with Df, an iron chelator, and FAC increase and decrease,
respectively, the amounts of IRP2 protein (Figure 1B).
Unlike IRP2, IRP1 RNA-binding activity exhibits biphasic
regulation, decreasing to approximately 45% of control at 3 to 7
hours followed by an increase to approximately 70% of control by
21 hours of hypoxia (Figure 1A). The change in IRP1 RNAbinding activity cannot be accounted for by a decrease in protein
(Figure 1B). In fact, there may be a slight increase in the amounts
of IRP1 protein during this time course. These data are consistent
with studies in other cell lines,5,22 demonstrating the differential
regulation of IRPs during hypoxia.
Based on the mutually exclusive functions of IRP1 as either an
RNA-binding protein or c-Aco,44 we determined whether the
decrease in IRP1 RNA-binding activity during hypoxia resulted in
a corresponding increase in c-Aco activity. HEK293 cells were
grown under hypoxic conditions for 0, 1, 7, and 16 hours, and
aconitase activities were measured in fractionated cytosol and
mitochondria (Figure 2A). The purity of these fractions was
Figure 1. Hypoxia differentially regulates IRP RNA-binding activities. HEK293
cells were grown for the indicated times under hypoxic (1% O2) or normoxic (21% O2,
control) conditions. (A) RNA supershift analysis of cytosolic cell extracts using a
32P-labeled Ft IRE and anti-IRP2 antibodies. Experiment was performed 5 times with
a representative blot shown. (B) Immunoblot analysis of cytosolic cell extracts with
chicken anti-IRP1 antibodies36 or rabbit anti-IRP2 antibodies.37 Df, 100 ␮M for 16
hours; FAC, 50 ␮g/mL for 5 hours. The * represents a nonspecific band. IRP1 and
IRP2 Western analysis experiments were performed 3 and 4 times, respectively, with
representative blots shown.
Figure 2. Hypoxia increases c-Aco activity but does not regulate m-Aco protein
accumulation or activity. (A-C) HEK293 cells were grown for the indicated times
under hypoxic conditions or under normoxia with Df (200 ␮M for 16 hours) or FAC
(150 ␮g/mL for 5 hours). (A) Bar graph shows the mean aconitase activity of
mitochondrial and cytosolic cell fractions ⫾ SEM, n ⫽ 6 (0, 1, 7, and 16 hours
hypoxia) or n ⫽ 4 (FAC and Df). The * indicates points that differ from the control
group (0 hour hypoxia) with P ⬍ .007 using a Student t test. Aconitase activity was
assayed by measuring the change in absorbance at 240 nm over time as cisaconitate is converted to isocitrate.42 (B) Immunoblot analysis with chicken antiIRP136 or rabbit anti–m-Aco antibodies to determine the purity of the mitochondrial
and cytosolic fractions. Western analysis was performed for all experiments with a
representative blot of the 16-hour hypoxia time point shown. Cyto indicates cytosolic
fraction; M, mitochondrial fraction. (C) Immunoblot analysis of cytosolic cell extracts
with rabbit anti–m-Aco antibodies. Experiment was performed 3 times with a
representative blot shown.
determined by immunoblot analysis using m-Aco and IRP1
antibodies, respectively (Figure 2B). Figure 2A shows that hypoxia
increases c-Aco activity approximately 35% after 7 hours of
hypoxia, corresponding to a time when IRP1 RNA-binding activity
is at a minimum. By 16 hours of hypoxia, c-Aco activity returns to
about control levels, correlating with the increase in IRP1 RNAbinding activity (Figure 1A). m-Aco activity did not change
significantly over the hypoxia time course. As expected, FAC
increases c-Aco activity approximately 84% and Df decreases it by
approximately 87%. FAC did not significantly affect m-Aco
activity, although Df decreases its activity by approximately 90%.
These results suggest that m-Aco activity is not as sensitive as
c-Aco to hypoxia and excess intracellular iron. Because m-Aco
mRNA contains a 5⬘-IRE that is regulated by the IRPs,13 we asked
whether the differential regulation of IRP1 and IRP2 during
hypoxia influenced m-Aco regulation by measuring m-Aco protein.
Figure 2C shows that the amount of m-Aco protein does not change
during 21 hours of hypoxia, consistent with the lack of change in
m-Aco activity. Df and FAC also do not change the amounts of
m-Aco protein, although Df dramatically reduces m-Aco activity.
Taken together, these data show that the differential regulation of
IRPs during hypoxia does not affect the amounts of m-Aco protein,
despite the presence of a 5⬘-IRE.
Unlike m-Aco, Ft synthesis and protein levels are regulated
during hypoxia
Our data in Figure 2 show that m-Aco activity and protein do not
change during hypoxia. Since both IRPs bind to 5⬘-IREs in FtH and
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BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
FtL mRNAs, we wanted to determine how the differential regulation of IRP RNA-binding activities during hypoxia would influence
Ft synthesis. HEK293 cells were grown under hypoxic conditions
for 0 to 16 hours and radiolabeled for the last one hour of treatment
with 35S-Met/Cys. FtH and FtL subunits were immunoprecipitated
with an antihuman Ft antibody and subjected to SDS-PAGE and
autoradiography. Figure 3A shows that FtH and FtL subunit
synthesis exhibits a biphasic response, increasing by 2 hours of
hypoxia, peaking at 3 hours, and declining to barely detectable
levels by 16 hours of hypoxia. As a control, FAC treatment for 3
hours greatly enhances synthesis of both Ft subunits. The changes
in protein synthesis during hypoxia are not due to a decrease in
uptake of the radiolabel or a decrease in global protein synthesis as
determined by radioactive counts of cell lysates and trichloroacetic
acid (TCA)–precipitable protein, respectively (data not shown).
Western blot analysis shows that the steady-state amounts of both
Ft protein subunits remain constant through 7 hours but then
dramatically decrease by 16 to 21 hours of hypoxia (Figure 3B). As
expected, Df and FAC treatment of HEK293 cells decreases and
increases, respectively, the amounts of both Ft protein subunits
(Figure 3B). The increase in Ft synthesis at 3 hours of hypoxia
correlates with the maximal decrease in IRP1 RNA-binding
activity at 3 hours, while the decrease after 7 hours of hypoxia
correlates with the robust increase in IRP2 RNA-binding activity at
this time (Figure 1A). Figure 3C indicates that the biphasic
response of Ft synthesis during hypoxia correlates with IRP
regulation, since Northern blots show no change in the amounts of
FtH and FtL mRNA during 21 hours of hypoxia. This further
indicates that the changes in Ft synthesis and protein during
hypoxia occur by a posttranscriptional mechanism. Taken together,
Figure 3. Ft synthesis exhibits a biphasic response during hypoxia. (A-B)
HEK293 cells were grown for the indicated times under hypoxic conditions or under
normoxia with Df (16 hours) or FAC. (A) Synthetic rate of Ft during hypoxia. For the
last one hour of hypoxic or normoxic treatment, cells were labeled with 35S-Met/Cys in
serum-free DMEM. Cell extracts were immunoprecipitated with rabbit antihuman Ft
antibodies and proteins subjected to SDS-PAGE analysis and autoradiography. FAC,
50 ␮g/mL for 3 hours. Con indicates control immunoprecipitation lacking antibody.
Experiment was performed 4 times with a representative blot shown. (B) Immunoblot
analysis of cytosolic cell extracts with antihuman Ft antibodies. Df, 100 ␮M; FAC, 50
␮g/mL for 5 hours. Experiment was performed 4 times with a representative blot
shown. (C) Northern analysis of total cellular RNA. RNAs were transferred to a nylon
membrane and hybridized with a 32P-labeled FtH, FtL, or 18S rRNA probe (control).
Df, 200 ␮M; FAC, 150 ␮g/mL for 5 hours. Experiments were performed twice with
representative blots shown.
EFFECTS OF IRP REGULATION DURING HYPOXIA
3407
these data suggest that, unlike m-Aco, IRP2 does regulate Ft
translation during hypoxia.
Hypoxia increases Tf-independent iron uptake
To determine how the differential regulation of IRPs might alter the
expression of TfR and possibly the cellular free iron pool, we measured
TfR mRNA and protein during a 0- to 21-hour hypoxia time course in
HEK293 cells. Figure 4A shows that TfR mRNA levels increase
approximately 2-fold by 16 hours of hypoxia and are most likely a
combination of both transcriptional activation of TfR by HIF-1 as well
as increased mRNA stability via IRP2, although the contribution of each
cannot be discerned here. As expected, Df and FAC treatment result in
an approximately 5-fold increase and an approximately 3-fold decrease,
respectively, in the amounts of TfR mRNA. Although Df causes a slight
decrease in the amounts of actin mRNA, the mechanism for this is not
known. The increase in the amounts of TfR mRNA during hypoxia does
not result in a significant change in the amount of TfR protein (Figure
4B), although there is a slight increase in Df-treated cells. In FACtreated cells, TfR mRNA levels decrease, but this does not result in a
decrease in TfR protein, probably due to the more than 24-hour half-life
of the protein.45 These data indicate that an increase in TfR mRNA does
not always correlate with an increase in protein or Tf-dependent
iron uptake.
Although other studies have shown that TfR mRNA increases
during hypoxia,4,5 it was unclear in these studies if Tf-dependent
iron uptake also increased. To determine if Tf-dependent iron
uptake changed during hypoxia, we measured 55Fe-Tf uptake in
HEK293 cells exposed to hypoxia for 0, 1, 7, and 21 hours. Figure
4C shows that the uptake of 55Fe-Tf in serum-free DMEM was not
significantly altered during hypoxia, consistent with an undetectable change in TfR protein during this time course (Figure 4B). As
a control, labeling cells in the presence of 1000 ⫻ unlabeled Fe-Tf
significantly decreased the uptake of 55Fe-Tf during hypoxia.
Although this experiment does not address the possible mobilization of iron from internal stores, it suggests that the changes in
IRP1 RNA-binding activity and Ft synthesis cannot be accounted
for by an increase in Tf-dependent iron uptake.
Because cells can take up iron by both Tf-dependent and
Tf-independent mechanisms,46,47 we determined whether Tfindependent iron uptake was altered during hypoxia. Tf-independent iron uptake in the gut occurs through the proton-coupled Fe2⫹
transporter known as divalent metal transporter-1/divalent cation
transporter-1 (DMT1/DCT1), which functions optimally at acidic
pH.48-50 To determine if hypoxia altered Tf-independent iron
uptake, HEK293 cells were subjected to hypoxia for 0, 1, 7, and 21
hours, and the uptake of 55Fe-NTA in serum-free DMEM (pH 7.4)
or HBSS (pH 6.0) was measured in the presence of ascorbic acid.
Figure 5 shows that 55Fe-NTA uptake did not significantly change
during hypoxia when cells were assayed in serum-free HBSS at pH
6.0. When 55Fe-NTA uptake was measured in serum-free DMEM at
pH 7.4, in either the presence or absence of ascorbic acid,
approximately 2-fold increase in iron uptake was observed by one
hour of hypoxia, which remained elevated until 7 hours (Figure 5).
By 21 hours of hypoxia, 55Fe-NTA uptake returned to control
levels. Since DMT1 is more active in transporting iron at acidic
pH,49 these data indicate that the increase in 55Fe-NTA uptake is
independent of DMT1. Because there is relatively little change in
IRP RNA-binding activity by one hour of hypoxia, the data also
suggest that the initial increase is independent of IRP regulation yet
stimulated by hypoxia. Finally, these data indicate that Tfindependent iron uptake exhibits a biphasic response during
hypoxia that parallels Ft synthesis.
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analysis shows that IRP1 RNA-binding activity displays the same
biphasic regulation under hypoxia with high-MW Df compared
with hypoxia alone (compare Figure 6A, lanes 7-11 with Figure
1A). Figure 6A demonstrates that the high-MW Df was chelating
iron, since cells treated with the high-MW Df-containing medium
under normoxia increase both IRP1 and IRP2 RNA-binding
activities in a time-dependent fashion as expected (Figure 6A, lanes
1-6). To ensure that high-MW Df prevents Tf-independent iron
uptake, we labeled cells for one hour under normoxia or hypoxia
with 55Fe-NTA in only serum-free DMEM or with 55Fe-NTA–
containing serum-free DMEM that had been pretreated with
high-MW Df (Figure 6B). Figure 6B shows that pretreating
55Fe-NTA–containing serum-free DMEM with high-MW Df prevents the increase in Tf-independent iron uptake at one hour of
hypoxia. These data show that the increase in Tf-independent iron
uptake at one hour of hypoxia is not responsible for the decrease in
IRP1 RNA-binding activity during early hypoxia.
Hypoxic regulation of Ft protein is reversible upon ReO2
Figure 4. Hypoxia does not change Tf-dependent iron uptake. (A-B) HEK293
cells were grown for the indicated times under hypoxic conditions or under normoxia
with Df (16 hours) or FAC (5 hours). (A) Northern analysis of poly(A)⫹ mRNA. RNAs
were transferred to a nylon membrane and hybridized with a 32P-labeled TfR or actin
probe as a control. Df, 200 ␮M; FAC, 150 ␮g/mL. Experiment was performed 4 times
with a representative blot shown. (B) Immunoblot analysis of cytosolic cell extracts
with antihuman TfR antibodies. Df, 100 ␮M; FAC, 50 ␮g/mL. Experiment was
performed 5 times with a representative blot shown. (C) HEK293 cells were grown for
the indicated times under hypoxic conditions or under normoxia with 1000 ⫻ cold
Fe-Tf. Cells were incubated with 55Fe-Tf in serum-free DMEM for the last one hour of
treatment, washed, harvested, and radioactivity quantified by counting cell extracts.
The bar graph shows the mean percent radioactivity ⫾ SEM, n ⫽ 9 (0, 1, 7, and 21
hours hypoxia) or n ⫽ 3 (1000 ⫻ cold Fe-Tf) of hypoxia-treated cell extracts. The *
indicates point that differs from the control group with P ⬍ .005 using a Student t test.
Our previous work showed that hypoxic regulation of IRP1 RNAbinding activity is reversible upon ReO2.22 However, in that study, cells
were exposed to 3% O2 rather than 1% O2 used here. At 3% O2, IRP1
RNA-binding activity decreased, as shown here, but IRP2 RNAbinding activity was not affected. To determine if the hypoxic increase in
IRP2 RNA-binding activity is reversible upon ReO2, cells were grown
under hypoxia for 21 hours followed by growth under normoxia for 0 to
21 hours. RNA supershift analysis shows that IRP1 RNA-binding
activity starts to increase at 3 hours of ReO2, reaching normoxic
amounts by 16 hours without any changes in IRP1 protein (Figure
7A-B). In contrast, IRP2 RNA-binding activity decreases to normoxic
Extracellular iron is not responsible for the changes in IRP1
RNA-binding activity during hypoxia
The increase in Tf-independent iron uptake during early hypoxia
prompted us to determine whether extracellular iron is responsible
for the decrease in IRP1 RNA-binding activity at this time.
HEK293 cells were cultured in complete medium that had been
pretreated with 600-␮M high-MW Df (to chelate iron) and
subjected to a normoxia or hypoxia time course. RNA supershift
Figure 5. Tf-independent iron uptake displays a biphasic response during
hypoxia. HEK293 cells were grown for the indicated times under hypoxic conditions
or under normoxia with 1000 ⫻ cold Fe-NTA. Cells were incubated with 55Fe-NTA in
serum-free DMEM (pH 7.4) or HBSS (pH 6.0) for the last one hour of treatment,
washed, harvested, and radioactivity quantified by counting cell extracts. The bar
graph shows the mean percent radioactivity ⫾ SEM, n ⫽ 9 (0, 1, 7, and 16 hours
hypoxia) or n ⫽ 6 (1000 ⫻ cold Fe) of hypoxia-treated cell extracts. The * indicates
points that differ from the control group with P ⬍ .005 using a Student t test. The ⫾
refers to the presence or absence, respectively, of 20 ␮M ascorbic acid in serum-free
DMEM or HBSS.
Figure 6. Extracellular iron is not responsible for the changes in IRP1
RNA-binding activity during hypoxia. (A) RNA supershift analysis of cytosolic
HEK293 cell extracts as described in Figure 1A. Complete DMEM was treated with
600-␮M high-MW Df overnight to chelate iron. Cells were treated with this high-MW
Df-containing medium under normoxic or hypoxic conditions for the indicated times.
Experiments were performed 3 times with a representative blot shown. (B) Bar graph
shows the mean percent radioactivity ⫾ SEM, n ⫽ 3, of hypoxia-treated cell extracts
relative to the normoxic control. High-MW Df–containing serum-free DMEM was
treated with 55Fe-NTA overnight. Cells were labeled with 55Fe-NTA in serum-free
DMEM only or with high-MW Df (600 ␮M)/55Fe-NTA–containing serum-free DMEM
for one hour under hypoxic or normoxic conditions. The * indicates points that differ
from the control with P ⬍ .03 using a Student t test.
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BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
Figure 7. Hypoxic regulation of Ft and IRP2 protein levels is reversible upon
ReO2. (A-B) HEK293 cells were grown under hypoxic conditions for 21 hours
followed by ReO2 for the indicated times or under normoxia with Df (200 ␮M for 16
hours) or FAC (150 ␮g/mL for 5 hours). (A) RNA supershift analysis of cytosolic cell
extracts as described in Figure 1A. Con indicates normoxic control. Experiment was
performed 3 times with a representative blot shown. (B) Immunoblot analysis of the
cytosolic cell extracts used in panel A using antibodies against the indicated proteins.
Con indicates normoxic control. Experiments were performed 2 (IRP1, IRP2, TfR,
and m-Aco) or 3 (Ft) times with representative blots shown.
amounts after 7 hours of ReO2, paralleling a decrease in IRP2 protein
(Figure 7A-B). As controls, Df and FAC treatment of HEK293 cells
maximally increases and decreases, respectively, the RNA-binding
activity of both IRP1 and IRP2. To determine if the reversibility in both
IRP1 and IRP2 RNA-binding activity following ReO2 influenced the
expression of Ft, m-Aco, and TfR, cell extracts used in the supershift
analysis (Figure 7A) were used for immunoblotting. Figure 7B shows
that the amount of both Ft protein subunits increases by 7 hours of ReO2,
correlating with the decrease in IRP2 RNA-binding activity, while the
amounts of TfR and m-Aco protein remain unchanged throughout the
ReO2 time course, as during hypoxia.
Discussion
Here we report on the cellular consequences of the differential
regulation of IRPs during hypoxia and ReO2. Our studies show that
iron homeostasis is regulated in 2 distinct phases during hypoxia.
The early phase (0-7 hours) is marked by a decrease in IRP1
RNA-binding activity and an increase in Ft synthesis and Tf- and
DMT1-independent iron uptake. The late phase (16-21 hours) is
marked by greatly enhanced IRP2 RNA-binding activity, a corresponding decrease in Ft synthesis and protein levels, and a return of
Tf-independent iron uptake to control levels.
Our results indicate that Ft has a major role in iron homeostasis
during hypoxia. During early hypoxia, the decrease in IRP1
RNA-binding activity correlates with the derepression of Ft
synthesis, since Ft mRNA remains unchanged and IRP2 RNAbinding activity is only slightly increased during this time. The
increase in Ft synthesis during early hypoxia shown here is
consistent with studies in oligodendrocytes where Ft synthesis
increased after 6 hours of hypoxia.51 Because these investigators
EFFECTS OF IRP REGULATION DURING HYPOXIA
3409
did not perform a longer hypoxia time course, it is unknown if Ft
synthesis would be repressed at 16 hours as we report here. Others
have also detected an increase in Ft synthesis during hypoxia in
mouse peritoneal macrophages.25 However, Ft synthesis at 16
hours of hypoxia was not addressed and there was no detectable
IRP2 in these cells, making it difficult to draw conclusions as to the
contribution of IRP2 in Ft synthesis during hypoxia. Interestingly,
the initial increase in Ft synthesis is not reflected in a corresponding
increase in protein at this time. One explanation could be a
decreased half-life of Ft during hypoxia; however, we do not
believe this is the case since pulse-chase experiments showed that
the half-life of Ft during hypoxia was comparable with normoxia
(data not shown). We believe that the initial increase in Ft synthesis
is insufficient to detect by Western analysis. During the late phase
of hypoxia, IRP2 RNA-binding activity is greatly elevated and
represses Ft synthesis and protein. During this time, cells may have
limited iron-storage capacity, leaving them vulnerable to ironcatalyzed ROS production. Similar to hypoxia, H2O2 has been
shown to decrease the amount of Ft protein in cells yet increase its
capacity to store iron.52 Increased NADPH (nicotinamide adenine
dinucleotide phosphate) levels during hypoxia may also help
maintain iron in a reduced state, facilitating its incorporation into
Ft.53 Whether the reduced level of Ft during late-phase hypoxia has
an increased capacity to store iron has not been determined.
During ReO2, amounts of both Ft protein subunits increase to
above control levels, demonstrating the reversibility of Ft regulation. Such an increase would allow cells to sequester free iron, and
consequently, limit iron-catalyzed ROS production via Fenton
chemistry. Support for this includes studies in erythroid cells that
demonstrate a correlation between increasing FtH levels with lower
levels of ROS and cellular-free iron and protection from ROSinduced cell death.54 Several other studies also implicate a role for
Ft in response to oxidative stress and illustrate the significance of
IRPs in its regulation under such conditions. For example, Ft
synthesis is induced in response to phorone,55 H2O2,56 and NO.57
The increase in Ft synthesis by NO and phorone correlates with
decreasing IRP2 RNA-binding activity,55,57 while one study shows
that the response to H2O2 is biphasic.56 In this latter study, upon
H2O2 treatment, there is an initial decrease in Ft synthesis,
correlating with an initial increase in IRP1 RNA-binding activity,
followed by increasing Ft synthesis and a corresponding decrease
in IRP1 RNA-binding activity. Transcription of Ft genes is also
regulated by ischemia/reperfusion58 and other oxidants,55,56,59 further emphasizing the importance of both transcriptional and
translational regulation during oxidative stress.
Our data show that not all 5⬘-IRE mRNAs are regulated equally
by the IRPs during hypoxia. Whereas the synthesis and steady-state
amounts of Ft protein decrease after 7 hours of hypoxia, no change
is observed in the steady-state amounts of m-Aco protein. Although
we do not know whether m-Aco synthesis changes during hypoxia
because our antibody was not effective in immunoprecipitation, our
data are consistent with studies showing that structural differences
between Ft and m-Aco IREs influence the differential binding
affinities of IRP1 and IRP2.60,61 Other studies show that treatment
of HL-60 cells with hemin causes more than 2-fold increase in
m-Aco synthesis versus more than 20-fold increase in Ft synthesis,62 suggesting that IRPs can differentially regulate translation of
5⬘-IRE–containing mRNAs in response to iron. These data suggest
that an increased affinity of IRP2 for the 5⬘-IRE of Ft versus m-Aco
IRE is responsible for the differential regulation of Ft and m-Aco
during the late phase of hypoxia.
We show that Tf-dependent iron uptake does not significantly
change during hypoxia. Although both IRPs can bind to the
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3410
BLOOD, 1 NOVEMBER 2003 䡠 VOLUME 102, NUMBER 9
SCHNEIDER and LEIBOLD
3⬘-IREs of TfR mRNA,63 the early decrease in IRP1 RNA-binding
activity does not significantly alter TfR mRNA levels. As hypoxia
progresses, the increase in IRP2 RNA-binding activity may be
capable of stabilizing the message, although TfR is also transcriptionally regulated by HIF1.4,5 Interestingly, the stabilization of TfR
mRNA is not reflected in the steady-state amounts of TfR protein
during hypoxia. Similarly, others have shown that H2O2 treatment
of B6 fibroblasts increases the amount of TfR mRNA approximately 4-fold, although the amount of protein increases only
approximately 2-fold, suggesting additional mechanism(s) of TfR
regulation.52 Nonetheless, our result is consistent with the lack of
any significant change in Tf-dependent iron uptake during hypoxia.
Rather, hypoxia induces an early increase in Tf-independent iron
uptake, a response that has been shown to occur in rat myocardial
cells.64 DMT1 functions optimally at acidic pH.49,50 As shown here,
Tf-independent iron uptake shows little change when assayed at pH
6.0, but increases approximately 2-fold during hypoxia when
measured at pH 7.4 indicating the increase is DMT1 independent.
This increase in iron uptake during hypoxia is not blocked by
actinomycin D or cycloheximide, suggesting that transcription and
translation, respectively, are not required (data not shown). The
initial increase is also independent of IRP regulation since it occurs
before any significant change in IRP RNA-binding activity. These
data suggest that hypoxia may induce the rapid localization of an
unknown transporter to the plasma membrane. Iron uptake during
hypoxia may also occur through a recently defined pathway
involving a lipocalin.65 Such a rapid response to hypoxia may allow
cells to sequester sufficient iron to maintain enzyme function and
cellular survival during a potentially extended period of low
oxygen concentration.
What is the mechanism regulating IRPs during hypoxia? One
explanation to account for IRP1 regulation during hypoxia is an
increase in the cellular free iron pool. We show that Tf-independent
iron uptake increases by one hour of hypoxia. This increase,
however, is not responsible for the inactivation of IRP1 RNAbinding activity, since blocking iron uptake with high-MW Df did
not prevent the decrease in IRP1 RNA-binding activity during
hypoxia (Figure 6). Furthermore, this increase in iron uptake does
not signal IRP2 degradation, and in fact, IRP2 steadily accumulates
during hypoxia. This suggests that either the increase in iron is
insufficient to signal IRP2 degradation or that the iron is rapidly
incorporated into proteins or sequestered by Ft. Finally, we cannot
rule out the possibility of an increase in the cellular free iron pool
during hypoxia by mobilization of iron from internal stores that
could drive the formation of the [4Fe-4S] cluster in IRP1.
Our favored explanation for the regulation of IRP1/c-Aco
during hypoxia is reduced ROS production. Although there are
conflicting reports regarding how ROS levels change during
hypoxia,66,67 we believe our data are consistent with a decrease in
cytosolic ROS levels during hypoxia. We show that c-Aco activity
is increased by approximately 35% at 7 hours of hypoxia, while
FAC increases c-Aco activity approximately 84%, indicating that
this is the maximal amount of c-Aco activity that can be expected
under these conditions. Since [4Fe-4S] clusters are extremely
sensitive to ROS-induced disassembly,32,68,69 reduced ROS production would favor the stabilization of the [4Fe-4S] cluster and
increase c-Aco activity, consistent with our data shown here.
Surprisingly, m-Aco activity was unaffected by hypoxia. Some
studies have shown that mitochondrial ROS production increases
during hypoxia,66,70 which might prevent any hypoxic stabilization
of the m-Aco [4Fe-4S] cluster. Analysis of our data shows that
m-Aco activity is always lower than control by 16 hours of
hypoxia, although this did not reach statistical significance, suggesting that the effects on c-Aco and m-Aco activity during hypoxia
could be explained by the compartmentalization of ROS production. Although the physiologic relevance of elevated c-Aco during
hypoxia is unclear, one suggestion is that increases in c-Aco
activity would increase NADPH levels, thereby providing reducing
equivalents for regeneration of glutathione and allowing cells to
maintain redox balance.71 Increased NADPH levels may also favor
the Fe(II) state, aiding in the incorporation of iron into Ft.53 During
the late phase of hypoxia, IRP2 RNA-binding activity steadily
increases due to the accumulation of IRP2 protein.23 Although the
mechanism regulating IRP2 accumulation during hypoxia is not
clear, our studies suggest that this is due to an oxygen-dependent
decrease in IRP2 ubiquitination.72
Acknowledgments
We would like to thank Drs Jerry Kaplan, Dennis Winge, and Eric
Hanson for reading the manuscript. We also thank Dr Bo Hedlund
and Greg Hanson for technical assistance with the high-MW Df
and Dr Aniko Szabo at the HCI Biostatistics Core Facility for help
with the statistical analysis.
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2003 102: 3404-3411
doi:10.1182/blood-2003-02-0433 originally published online
July 10, 2003
Effects of iron regulatory protein regulation on iron homeostasis during
hypoxia
Brian D. Schneider and Elizabeth A. Leibold
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