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INTRODUCTION |
Iron is a key element in cellular growth and metabolism. The
element is part of the active site of many enzymes, often as a
component of heme or as part of an iron-sulfur complex (1). As an
enzyme prosthetic group, iron catalyzes redox reactions involving
proteins, lipids, carbohydrates, and nucleic acids. The ability of the
element to exist in either of two stable oxidation states (ferric,
Fe3+; ferrous, Fe2+) is the key to its
enzymatic activity. Unrestrained, however, iron can wreak havoc on
cells through the production of reactive intermediates (2), including
the deadly hydroxyl radical (OH
).
Cellular iron is closely regulated, in part through the actions of the
transferrin receptor and ferritin, the proteins of cellular iron uptake
and storage, respectively (3). Expression of these two proteins is
largely regulated at the post-transcriptional level (4). A conserved
28-base sequence termed the iron-responsive element
(IRE)1 exists in the ferritin
5'-UTR, whereas five such elements are located in the transferrin
receptor 3'-UTR (5). The two cytoplasmic iron-regulatory proteins 1 and
2 (IRP-1 and IRP-2) recognize and bind to the IRE (6, 7). IRE/IRP-1
binding in the 5'-UTR blocks message translation. Such binding in the
3'-UTR stabilizes the message against enzymatic degradation (8).
Regulation of IRE/IRP-1 binding is a complex affair (9). Iron modulates
IRP-1 binding to IRE elements by forming a 4Fe-4SH cluster within the
protein. When the cluster is intact, IRP-1 cannot bind to the IRE and
exists free in the cytosol (10). In this state, IRP-1 has aconitase
activity. In the absence of iron, the 4Fe-4SH cluster collapses,
aconitase activity is lost, and IRP-1 acquires IRE binding capacity.
Therefore, IRP-1 is a dual function protein, serving alternatively as a
cytoplasmic aconitase or as a transacting RNA-binding protein. In
contrast to IRP-1, the IRP-2 molecule lacks aconitase activity and is
susceptible to proteolytic degradation when it is free in the cytosol
(11, 12).
Investigators have described a number of other factors that modify
IRE/IRP-1 binding and ferritin synthesis such as
H2O2 and nitric oxide (13, 14). Cytoplasmic
IRP-1 appears to exist in equilibrium between forms that either possess
or lack aconitase activity. Ascorbic acid shifts the equilibrium toward
aconitase (+) IRP-1 (15). This creates a thermodynamic "sink" that
favors greater IRP-1 release when iron interacts with the IRE/IRP-1 complex.
The current work uses the human hepatoma cell line Hep3B to examine the
effect of hypoxia on cellular iron metabolism. These cells mimic fetal
hepatocytes in many respects and were the first cells shown to produce
human erythropoietin in response to hypoxia (16). Their response to
hypoxia has been studied extensively, making them an excellent model
system. Hypoxia induces the expression of HIF-1 in Hep3B cells, a
universal transcription factor that activates genes needed to adapt to
low oxygen tensions (17, 18). The expression of HIF-1 (and its DNA
binding capacity) increases as the oxygen tension falls, peaking at a
0.5-1% oxygen concentration (19). We now demonstrate that hypoxia
promotes powerful IRE/IRP-1 binding in these cells that resists even
iron-mediated dissociation. Hypoxia markedly alters the transcription
of several genes (17), including those encoding erythropoietin (epo)
and vascular endothelial growth factor (18-22). The promoters of these genes contain binding sites for HIF-1. Hypoxia changes gene
transcription by altering HIF-1 binding (23). In distinction to these
well-established effects of hypoxia, the effect on IRE/IRP-1
interaction is a post-transcriptional phenomenon. Hanson and Leibold
(24) reported previously that exposing rat hepatoma cells to a 3%
atmosphere decreased IRP-1 binding to the IRE but did not alter IRP-2
binding. We discuss possible reasons for the differences.
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MATERIALS AND METHODS |
Cell Culture--
Human hepatoma Hep3B cells were maintained in
-minimum essential medium supplemented with 10% fetal bovine serum
(Biowhittaker, Walkersville, MD), penicillin/streptomycin, and pyruvate
(15). Cells at 75% confluence were exposed to hypoxia (1%
02, 5% CO2, and 94% N2) at
37 °C in an Espec triple gas incubator (Tabai-Espec Corp., Osaka,
Japan) for different time periods (4 or 16 h) (23). Control cells
were cultured under normoxic conditions (21% 02, 5%
CO2, and 74% N2) in a humidified Napco
incubator. After hypoxia treatment, the cells were chilled, washed with
cold phosphate-buffered saline to preserve the hypoxic response,
harvested, and used immediately according to the experimental requirement.
K562 human erythroleukemia cells were maintained in RPM 1640 medium
supplemented with 10% fetal bovine serum (Biowhittaker) and
penicillin/streptomycin at a density of 5 × 105
cells/ml. Hypoxia treatment was as outlined above.
Electromobility Shift Assay--
IRP binding to RNA was assessed
by electromobility shift assay as described previously (4, 15).
Briefly, cytoplasmic cell extracts were prepared from Hep3B or K562
cells grown under conditions of hypoxia or normoxia in the presence or
absence of 100 µM desferrioxamine or 10 µg/ml ferric
ammonium citrate. An excess quantity of [32P]UTP-labeled
RNA transcript (from pSPT-fer, a 28-nucleotide fragment encoding the
human H-ferritin IRE; a generous gift of Dr. Kühn (25)) was
incubated at room temperature for 30 min with 3 µg of protein of
fresh cytoplasmic cell lysate. RNase T1 (1 unit/reaction) and heparin
(5 mg/ml) were added sequentially for 10 min each. IRE-IRP-1 complex
was analyzed on a 6% nondenaturing polyacrylamide gel, as detailed
previously (15).
Northern Blot Analysis--
RNA was isolated from control or
experimentally manipulated cells using a STAT-30 kit from TEL-TES B,
Inc. (Friendswood, TX), following the manufacturer's instructions.
Twenty µg of RNA were separated on an agarose/formaldehyde gel and
immobilized to Hybond-N nylon membrane (Amersham, Arlington Height, IL)
using a 0.05 N fixation (26). The RNA was hybridized with a 600-bp
pStI fragment of TfR cDNA labeled by the Random primer
method according to the supplier's protocol. After hybridization and
washing by the method of Church and Gilbert (27), the membrane was
exposed to x-ray film for 2 days. The TfR signal was stripped off by
boiling the membrane in distilled water for 1 min. The membrane then
was reprobed with human L-ferritin cDNA probe
(28).
Nuclear Run On Assay--
Nuclei were isolated from Hep3B cells
grown under normoxic or hypoxic conditions using a modified version of
the protocol of Greenberg and Ziff (29). Briefly, 5 × 107 cells were harvested, washed with phosphate-buffered
saline, and treated with 4 ml of hypotonic solution (5 M
NaCl, 1 M Tris-HCl, pH 7.4, 1 M
MgCl2, and 1 µl/ml
-mercapthoethanol) for 15 min on
ice, followed by 0.1% Triton and 140 mM KCl. The cells
were homogenized, and the nuclei were collected after repeated
centrifugation. The newly synthesized RNA was labeled with
[32P]UTP and hybridized to a nitrocellulose membrane
containing the coding region of ferritin L chains (28), TfR (25), or
epo (22). After a 72-h hybridization at room temperature, the membrane was washed twice with 2× SSC/0.2% SDS for 20 min and 0.2× SSC/0.2% SDS for 40 min at 68 °C and exposed to x-ray film for 3 days.
Cytosolic Aconitase Assay--
Cytosolic aconitase activity was
assessed by the consumption of cis-aconitate as measured by
the spectrophotometric absorbance at 240 nm (as detailed previously in
Ref. 15). Briefly, mitochondrial-free lysates from Hep3B cells grown
under conditions of hypoxia or normoxia (50 µg) were incubated with
200 µM cis-aconitate in 50 mM
Tris-HCl buffer, pH 7.2, 100 mM NaCl, and 0.02% bovine
serum albumin at room temperature in a volume of 1 ml. Specific
activity (µM substrate converted/mg protein/min) was
calculated as described by Emery-Goodman et al. (30).
Cytosolic Ferritin Content Determination--
The steady-state
level of cytosolic ferritin was determined using a Coat-A-Count
Ferritin IRMA assay kit whose limit of ferritin detectability is 0.1 ng/ml (Diagnostic Products Corp., Los Angeles, CA). Briefly,
106 cells/assay were harvested, washed, and solubilized in
a detergent buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, and 0.2 mM
phenylmethylsulfonyl fluoride). Ferritin was immunoprecipitated with
monoclonal antibody to the L subunit and detected with a second
antibody labeled with 125I.
Ferritin Biosynthesis--
To assess ferritin biosynthesis,
106 cells were incubated for 1 h at 37 °C in
methionine-free medium (RPMI 1640 or
-minimum essential medium,
without serum) supplemented with 250 µCi/ml [35S]methionine. The cells were washed and solubilized in
the lysis buffer (above), and the newly synthesized ferritin subunits
were immunoprecipitated with the polyclonal antibody against ferritin. The ferritin subunits were separated on SDS-phosphate-urea gel and
visualized by autoradiography (26).
Transferrin Receptor Expression--
After treatment, cells were
chilled to 4 °C and incubated for 1 h with
125I-labeled transferrin at concentrations ranging from 0.5 to 500 nM (30). Plates were washed with cold
phosphate-buffered saline and lysed in situ in a lysis
buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl,
and 0.1% Triton X-100), and the cell-associated radioactivity was
measured. A parallel set of cells was incubated with
125I-labeled transferrin along with a 100-fold excess of
unlabeled transferrin to assess nonspecific binding. Scatchard analysis provided the number of receptor sites/cell as well as the binding affinity. The total transferrin receptor pool was determined by solubilizing the cells in detergent buffer and removing the insoluble debris by centrifugation. A 150-µl aliquot of cell extract was incubated with 50 µl of 125I-labeled transferrin
(0.5-500 nM) for 30 min with or without a 100-fold excess
of unlabeled ligand at room temperature. The reaction was stopped by
adding 250 µl of ice-cold 60% ammonium sulfate. The precipitate
containing the transferrin/transferrin receptor complex was pelleted by
centrifugation, washed, and assessed for radioactivity using a gamma
counter (31).
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RESULTS |
Hypoxia Enhances IRE/IRP-1 Binding--
We used the
electromobility shift assay to assess IRE/IRP-1 interaction in control
Hep3B cells (21% O2) and cells exposed to 1%
O2 for 16 h. Fig. 1
shows that the baseline IRE/IRP-1 binding (C) in the hypoxic
cells exceeds that in the normoxic cells by at least 10-fold.

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Fig. 1.
Hypoxia promotes IRE/IRP-1 binding in Hep3B
cells. Hep3B cells (106 cells at 75% confluence) were
grown for 16 h under normoxic (21% O2) or hypoxic
conditions (1% O2). In addition to untreated cells for
each condition (C), cells were incubated with 100 µM desferrioxamine (D) or 10 µg/ml ferric
ammonium citrate (Fe). Three mg of fresh cell extract were
incubated with a 32P-labeled IRE probe. The IRE/IRP-1
complex was resolved on a 6% polyacrylamide gel that was subsequently
fixed, dried, and used to expose x-ray film. Parallel cell extracts
were used for IRE/IRP-1 binding in an incubation mixture containing 2%
-mercaptoethanol, which induces maximal IRE/IRP-1 binding. The
experiment was repeated several times, and one representative result is
shown.
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Consistent with a substantial body of previous information, IRE/IRP-1
binding rose dramatically when cells in a 21% O2
atmosphere were treated for 16 h with 100 µM
desferrioxamine (D). By contrast, desferrioxamine treatment
did not increase IRE/IRP-1 binding in cells in a 1% O2
atmosphere. Therefore, chelation of intracellular iron does not further
enhance hypoxia-stimulated IRE/IRP-1 binding. This suggests that
hypoxia promoted IRE/IRP-1 association to the maximum possible extent.
With each electromobility shift assay, a parallel incubation was
performed that included 2-mercaptoethanol. This agent promotes maximal
IRE/IRP-1 binding when added to the in vivo incubation mixture. As shown in Fig. 1, maximal IRE/IRP binding was equivalent for
cells in a 21% O2 or 1% O2 atmosphere.
Therefore, hypoxia increases the extent of IRE/IRP-1 binding but does
not change the overall IRE/IRP-1 binding capacity of the cells.
The expected decrease in IRE/IRP-1 binding that occurred with the
addition of iron in the form of ferric ammonium citrate to the normoxic
cells was paralleled in the hypoxic cells. Therefore, hypoxia enhances
IRE/IRP-1 binding without altering its physiological response to iron.
The 16-h period of hypoxia did not affect the viability of Hep3B cells
as assessed by trypan blue staining. Hypoxia enhanced IRE/IRP-1 binding
in cells exposed to 1% O2 for only 4 h (Fig. 2). The increase was only 2-fold over the
baseline, suggesting that the metabolic changes that augment IRE/IRP-1
binding develop over time.

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Fig. 2.
A brief period of hypoxia modestly increases
IRE/IRP-1 binding. Hep3B cells were treated as detailed in the
Fig. 1 legend, except that the period of hypoxia was 4 h. The
experiment was repeated several times, and one representative result is
shown.
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We also examined the effect of hypoxia on IRE/IRP-1 binding in K562
cells. Fig. 3 shows that 16 h of
hypoxia enhanced IRE/IRP-1 binding by only 2-fold (as assessed by
densitometric scanning). In contrast, incubating the cells with
erythropoietin at a concentration of 50 units/ml boosted IRE/IRP-1
binding by 5-fold, consistent with the observations by other
investigators (32). Therefore, K562 cells can shift IRE/IRP-1 binding
characteristics in response to environmental stimuli. The response of
Hep3B cells and K562 cells to hypoxia was qualitatively similar but
differed in magnitude. The standard electromobility shift assay
separates RNA/protein complexes by charge. The technique does not
separate IRE/IRP-1 and IRE/IRP-2 in extracts derived from human
hepatoma or erythroleukemia cells (in contrast to murine cells) (32).
Western blot analysis indicates that the level of IRP-2 protein is very
low in these cell lines (data not shown). Supershift experiments were
inconclusive.

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Fig. 3.
Hypoxia modestly increases IRE/IRP-1 binding
in K562 cells. K562 cells (106 cells in log-phase
growth) were treated with iron (Fe), desferrioxamine
(D), or human recombinant epo (10 and 50 units/ml) and grown
for 16 h in either a 21% O2 or 1% O2
atmosphere. Cells were harvested, and the electromobility shift assay
was performed as detailed in the Fig. 1 legend. The result shown is one
of three similar experiments.
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Hypoxia Decreases Cytosolic Aconitase Activity--
Cytosolic
aconitase activity and RNA binding capacity are mutually exclusive
properties of IRP-1. The hypoxia-induced increase in IRE/IRP-1 binding
should lessen the amount of free cytosolic IRP-1 protein. IRP-1 is an
aconitase enzyme when it is free in the cytosol, but it lacks this
activity when it is part of an IRE/IRP-1 complex. On this basis, we
predicted that hypoxia would lower cytosolic aconitase activity.
K562 cells and Hep3B cells exposed to 1% O2 for 16 h
were lysed, and the 100,000 × g supernatant was
collected. Cytochrome c oxidase activity was 1.5 unit in the
pellet and was undetectable in the supernatant, indicating no
contamination by this mitochondrial enzyme. The same should hold for
mitochondrial aconitase (15). As shown in Table
I, hypoxia markedly decreased cytosolic
aconitase activity in both cell lines. The magnitude of the decline
produced by hypoxia was similar to that seen with desferrioxamine
treatment. The significant decrease in enzyme activity correlates well
with the marked increase in the binding of IRP-1 to IREs. Although hypoxia shifts IRP-1 onto IRE-containing transcript, the aconitase that
remains in the cytosol still shows iron-dependent
regulation in a 1% oxygen atmosphere (Table I; Hep3B cells). Only
IRP-1 has aconitase activity. Our electromobility shift assay can not distinguish between IRP-1 and IRP-2. Consequently, we can not assess
the effect of hypoxia on IRP-2.
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Table I
The effect of hypoxia on cytosolic aconitase activity
Fresh cytosolic cell extracts (100,000 × g
supernatant, 50 µg of protein) from Hep3B and K562 cells grown under
conditions of normoxia or hypoxia (16 h) in the presence or absence of
100 µM desferrioxamine or 10 µg/ml ferric ammonium
citrate were analyzed for cytosolic aconitase activity as detailed
under "Materials and Methods." The extracts were free from
mitochondrial contamination, as judged by the lack of cytochrome
c oxidase activity. The experiments were repeated three
times in duplicate.
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Hypoxia Increases Transferrin Receptor mRNA
levels--
IRE/IRP-1 binding stabilizes mRNAs with IREs in the
3'-UTR. Therefore, hypoxia should increase the steady-state level of
the transferrin receptor transcript. To test this prediction, mRNA was isolated from Hep3B cells maintained at 1% O2 for
16 h.
Fig. 4 shows that hypoxia substantially
increases transferrin receptor mRNA levels relative to control as
assessed by Northern blot analysis (lane 1 versus lane 4).
The magnitude of the increase in transferrin receptor mRNA produced
by hypoxia is similar to that produced by desferrioxamine. In contrast
to the striking increase in transferrin receptor message produced by
16 h of hypoxia, a 4-h period of oxygen deprivation had little, if
any, effect on the level (data not shown). Fig. 4 also shows that
16 h of hypoxia did not affect the level of the ferritin L subunit
transcript. Therefore, the hypoxia-induced increase in transferrin
receptor message did not reflect a global change in mRNA
metabolism.

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Fig. 4.
Hypoxia substantially increases the
steady-state level of the transferrin receptor message. RNA was
isolated from untreated control (C) Hep3B cells, cells
treated with 100 µM desferrioxamine (D), or 10 µg/ml ferric ammonium citrate (Fe) for 16 h under
normoxia (21% O2). Parallel isolations were performed for
hepatoma cells exposed to hypoxia (1% oxygen for 16 h). 20 µg
of RNA were separated on a 1% agarose formaldehyde gel and immobilized
to a Hybond (Amersham) nylon membrane. The blot was consecutively
hybridized with a 600-bp PstI fragment of TfR and a 670-bp
PstI fragment of L-ferritin. The fragments were
radiolabeled using the random primer method, with the hybridization and
washing done according to Church and Gilbert (27). The figure shows a
representative result selected from several experiments.
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Nuclear Run On Assay Indicates No Effect by Hypoxia on Transferrin
Receptor Gene Transcription--
In both prokaryotic and eukaryotic
organisms, hypoxia can induce transcriptional activity of
physiologically important genes (for a review, see Ref. 17). Northern
blot analysis (Fig. 4) showed an increase of greater than 10-fold in
the steady-state level of TfR mRNA in Hep3B cells exposed to
hypoxia. Nuclear run on assay was performed to determine whether the
observed increase was due to enhanced transcription. We predicted that
the hypoxia-induced increase in transferrin receptor message
represented stabilization of the message due to binding of the IRP-1 to
the IREs in the 3'-UTR of the transcripts.
Nuclei were isolated from control Hep3B cells or from cells exposed to
1% O2 for 16 h. Hypoxia affected neither the cell
viability nor yield of isolated nuclei. Fig.
5 shows no effect by hypoxia on the
transcription of the transferrin receptor message. In contrast, hypoxia
increased the synthesis of erythropoietin message, as expected (16).
These data confirm that hypoxia increases TfR mRNA levels in Hep3B
cells by stabilizing the message.

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Fig. 5.
Hypoxia alters the transcriptional rate of
neither the transferrin receptor nor L-ferritin genes.
Nuclei were isolated from Hep3B cells grown under conditions of
normoxia or hypoxia (16 h). Viability was analyzed with trypan blue
staining, and 107 nuclei were used from each cell
population. cDNA (3 µg/lane) of the corresponding genes was
immobilized to the nitrocellulose membrane. The newly synthesized
mRNAs were labeled with [32P]UTP, and 107
cpm/ml probe were used for hybridization. After 72 h, the
membranes were washed as described by Greenberg and Ziff (29) and
exposed to x-ray film for 4 days. The experiment was repeated twice
with similar results.
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Hypoxia Increases Transferrin Receptor Surface Expression--
An
increase in IRP binding to the IREs in the 3'-UTR of the transferrin
receptor message raises the level of transferrin receptor mRNA and
secondarily increases transferrin receptor expression. This phenomenon
is seen most strikingly in cells treated with desferrioxamine. Because
hypoxia mimics the effect of desferrioxamine on IRE/IRP-1 binding and
transferrin receptor mRNA expression in these cells, we predicted
that hypoxia would also increase transferrin receptor protein expression.
Fig. 6 shows that surface expression of
transferrin receptors doubles in Hep3B cells exposed to hypoxia for
16 h (2.5 × 104 versus 4.9 × 104 receptors/cell). Scatchard analysis of the data shows
no change in ligand/receptor affinity (Kd = 8.6 nM). Binding studies also show that the total transferrin
binding capacity of the cells increased by more than 2-fold (5 versus 13.7 ng/106 cells). The increase in
transferrin receptor message produced by 16 h of hypoxia is
functionally significant in that it substantially raises transferrin
receptor surface expression. In contrast, a 4-h period of hypoxia had
no effect on transferrin receptor expression (data not shown).

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Fig. 6.
Hypoxia increases cell surface expression of
the transferrin receptor. Hep3B cells (5 × 105)
were exposed to hypoxia for 16 h. Cell surface
125I-labeled transferrin binding was measured as described
under "Materials and Methods." Briefly, cells were incubated in 25 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mg/ml
bovine serum albumin with the indicated concentrations of
125I-labeled transferrin (0.5-50 nM) with or
without 100-fold excess cold transferrin for 1 h at 4 °C. After
the binding, the buffer was removed, the cells were solubilized
in situ, and the radioactivity was measured by gamma
counting. The number of binding sites/cell (after correcting for
nonspecific binding) are as follows: control, 2.5 × 104; and hypoxia-treated cells, 4.9 × 104. The dissociation constant (Kd)
was 8.6 nM for both cell populations. The binding studies
were repeated several times, and the result of one representative
experiment is shown.
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Hypoxia Decreases Cellular Ferritin Synthesis and Content--
A
second functional consequence of IRE/IRP-1 binding is impaired
translation of messages containing an IRE element in the 5'-UTR. For
example, desferrioxamine chelates intracellular iron, increases
IRE/IRP-1 binding, and blocks translation of the ferritin message. We
predicted that the increase in IRE/IRP binding produced by hypoxia
would also block translation of the ferritin message.
We therefore assessed ferritin synthesis in control cells and in cells
exposed to hypoxia for 16 h. Fig. 7
is a polyacrylamide gel electrophoresis of
[35S]methionine-labeled ferritin immunoprecipitated with
polyclonal anti-ferritin antibody. Ferritin synthesis is diminished
modestly in the hypoxic cells relative to controls. Iron normally
increases ferritin synthesis by dissociating the IRP from the IRE. Fig. 7 shows that this occurred in control cells. However, hypoxic cells did
not increase ferritin synthesis in response to iron. This indicates
that the hypoxia-induced IRE/IRP-1 binding is very tight and resists
dissociation in response to iron.

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Fig. 7.
Hypoxia reduces baseline ferritin synthesis
and blocks iron-stimulated ferritin synthesis. Hep3B cells (5 × 106) at 75% confluence were treated with 10 µg/ml
ferric ammonium citrate (Fe) for 16 h under conditions
of normoxia (21% oxygen) or hypoxia (1% oxygen). Control and
iron-treated cells were washed with phosphate-buffered saline and
metabolically labeled with [35S]methionine in a
methionine-free medium for 2 h. The cells were washed and
solubilized in a buffer containing 1% Triton X-100, 0.15 M
NaCl, 0.02 M Tris-HCl buffer, pH 7.5, and 0.2 mM phenylmethylsulfonyl fluoride. The newly synthesized,
labeled ferritin molecules were immunoprecipitated with human
anti-ferritin antibody, and the complex was separated on a 15%
SDS-phosphate-urea gel. The experiments were repeated several times,
and one representative result is shown.
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Reduced ferritin synthesis in hypoxic cells should reduce the cellular
ferritin content. We assessed the ferritin content of control cells and
hypoxic cells using a commercially available radioimmunoassay kit.
Table II shows that the ferritin content of hypoxic cells is about one-third that of the control. Also consistent with the previous ferritin biosynthesis studies, the ferritin content of control cells treated with 10 mg/ml ferric ammonium
citrate was 10-fold greater than the baseline. In striking contrast,
iron salt increased the ferritin content of hypoxic cells by only about
20%.
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Table II
Effect of hypoxia on cytosolic ferritin content
106 K562 and Hep3B cells were treated with iron as ferric
ammonium citrate at a concentration of 10 µg/ml or desferrioxamine at
a concentration of 100 µM for 16 h under conditions
of hypoxia (1% O2) or normoxia (21% O2). Hypoxia did
not affect cell viability as judged by trypan blue staining. The cells
were harvested, washed with phosphate-buffered saline buffer three
times and lysed with the same buffer used for electromobility shift
experiments. The ferritin content was measured using a Coat-A Count
Ferritin IRMA kit (Diagnostic Products Corp.) according to the
supplier's manual. All treatments were performed in duplicate and
repeated several times.
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DISCUSSION |
The interplay of IRE/IRP-1 binding in the post-transcriptional
regulation of ferritin and transferrin receptor synthesis was first
described in the context of iron-mediated changes (1, 5). In the
presence of iron, the reactive center of the IRP-1 protein forms a
4Fe-4SH structure that confers aconitase activity to IRP-1, the first
of the two presently known IRP proteins to be described. In the absence
of iron, the active site collapses, obliterating aconitase activity.
This change is counterbalanced by the acquisition of RNA binding
capacity with the IRE sequence as the binding target.
Factors other than iron modulate IRE/IRP-1 interaction. Nitric oxide
promotes IRP-1 binding to the IRE (33, 34). The likely mechanism is an
attack by the free radical on the iron in the 4Fe-4SH cluster. The
result is a marked reduction in ferritin synthesis, along with a rise
in transferrin receptor expression. Ascorbic acid also strikingly
alters the synthesis profile of ferritin and the transferrin receptor.
Ascorbate alone does not change ferritin synthesis (15, 26). However,
the vitamin strikingly increases ferritin synthesis in response to
iron. Ascorbate does not directly alter the IRE/IRP-1 interaction but
rather shifts free IRP-1 between states with or without aconitase
activity (15). The result is that a larger fraction of IRP-1
dissociates from the ferritin message in response to iron.
The present report details our experience with the effects of hypoxia
on cellular iron metabolism using two human cell lines, K562 and Hep3B.
We focused on the latter because of the extensive studies of hypoxia in
the expression of epo and HIF-1-mediated gene responses (20-23).
Electromobility shift assay shows that IRE/IRP-1 binding increases
substantially in cells maintained for 16 h in a 1% O2
atmosphere. The phenomenon occurred in both Hep3B and K562 cells,
although the magnitude was much greater in the former. The
electromobility shift assay involves the addition of radiolabeled RNA
probe (in this case, IRE) to cytosol isolated from control and
hypoxia-treated cells. The increase in IRE/IRP-1 binding in this assay
means that the change in the IRP produced by the in vivo
manipulation of cellular oxygen status persists after the cells have
been lysed.
Several readouts of cell activity indicate that hypoxia enhances
IRE/IRP-1 binding in living cells as well. Messages with IRE elements
in the 3'-UTR resist enzymatic digestion once IRPs bind to the IREs
(5). The striking increase in transferrin receptor message levels in
cells exposed to hypoxia attests to IRE/IRP-1 binding in
vivo.
The decrease in ferritin biosynthesis in hypoxic cells is further
indication of significantly enhanced in vivo IRE/IRP-1
binding. Attachment of IRP-1 to IRE elements in the 5'-UTR blocks the
translation of the transcript. Hypoxia decreases ferritin translation
without altering message levels. The effect is most prominent in
hypoxic cells that are simultaneously exposed to iron. Hypoxia
completely abrogates the iron-mediated enhancement of ferritin synthesis.
IRE/IRP-1 binding as a biochemical event alters the biology of the
cell. Transferrin receptor expression doubles in Hep3B cells exposed to
hypoxia for 16 h. Scatchard analysis shows an identical
transferrin binding affinity for control and hypoxic cells. The
increase in transferrin surface binding therefore reflects an increase
in surface expression of transferrin receptors rather than a change in
the binding characteristics of a fixed number of receptors.
Hanson and Leibold (24) examined rat hepatoma cells in a 3% oxygen
atmosphere and found a time-dependent decrease in IRE/IRP-1 binding. These cells also contain substantial quantities of IRP-2, whose binding to the IRE was not affected by hypoxia. Several possibilities could account for the divergent results between their
experiments and our experiments.
The most apparent difference is that they used a rodent cell line,
whereas our cell lines were derived from humans. One difference in this
respect is that their cell line prominently expresses both IRP-1 and
IRP-2, whereas our cell lines produce less IRP-2. The metabolic
machinery may be set differently in cells from the two species with
respect to the hypoxia response owing to this difference in IRP-1 and
IRP-2 expression. Another example of a cell-specific response occurs in
rat oligodendroglial cells that increase ferritin production under
hypoxic conditions, whereas astrocytes and neurons do not (35).
Interestingly, mouse peritoneal macrophages that are of similar
derivation as oligodendroglial cells also show decreased IRE/IRP
binding and increased ferritin synthesis with hypoxia (36).
Pleiotrophic cell and tissue responses to a particular stimulus may be
due in part to the fact that IRP-1 and IRP-2 have distinct binding
characteristics for IRE structures with variations in the base
sequences of the loop structure (37). Differences in loops and
bulge/loops between IRE isoforms produce dramatic differences in the
relative binding affinity of IRP-1 and IRP-2 (38). IRP-2 has the
greatest variation in interactions with IRE isotypes, raising the
possibility that IRP-2 contributes substantially to differences in
IRE-dependent regulation in vivo. IRP-1 and
IRP-2 function independently as translational repressors in
vivo (39). This fact is driven home strikingly by a cell line that
expresses no IRP-1 and yet responds appropriately to all iron-mediated
stimuli solely through IRP-2 (40). The differences between IRP-1 and IRP-2 may allow fine tuning to a host of stimuli.
The relative expression of IRP-1 and IRP-2 varies greatly between
species and between tissues in a single species (37, 41). These
differences have likely contributed to some of the conflicting results
in the literature concerning changes in cellular iron metabolism in
response to various stimuli. A case in point involves nitric oxide
effects on cellular iron homeostasis. One group of investigators
reported that the effect of nitric oxide is slow and analogous to
desferrioxamine (42), whereas another group found the changes to be
rapid (43). The explanation for the divergent reports may be the fact
that in some cells, such as macrophages, differences in relative
expression of IRP-1 and IRP-2 bring the differences in their binding
specificities into greater relief (44).
Clearly, the cellular response to hypoxia is both important and
complex. Genes are activated, enzyme levels change, and the local
generation of free radicals is altered. This work raises the
possibility that the relative expression of IRP-1 and IRP-2 also
contributes to the adaptation of particular cells to hypoxia. More work
is needed to tease apart the many variables in these systems and to
understand this important biological response.