From the Lady Davis Institute for Medical Research,
Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada,
¶ Division of Experimental Medicine, Faculty of Medicine,
McGill University, Montreal, Quebec H3A 1A3, Canada, and
§ Department of Internal Medicine, University Hospital,
Anichstrasse 35, A-6020 Innsbruck, Austria
Received for publication, January 11, 2001, and in revised form, March 12, 2001
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ABSTRACT |
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Cellular iron uptake and storage are coordinately
controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding
transferrin receptor (TfR) and ferritin. Under conditions of iron
starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs,
thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H2O2 that leads to
rapid activation of IRP1. Here we show that treating murine B6
fibroblasts with a pulse of 100 µM
H2O2 for 1 h is sufficient to alter
critical parameters of iron homeostasis in a time-dependent
manner. First, this stimulus inhibits ferritin synthesis for at least
8 h, leading to a significant (50%) reduction of cellular
ferritin content. Second, treatment with H2O2
induces a ~4-fold increase in TfR mRNA levels within 2-6 h, and
subsequent accumulation of newly synthesized protein after 4 h.
This is associated with a profound increase in the cell surface
expression of TfR, enhanced binding to fluorescein-tagged transferrin,
and stimulation of transferrin-mediated iron uptake into cells. Under
these conditions, no significant alterations are observed in the levels
of mitochondrial aconitase and the Divalent
Metal Transporter DMT1, although both are
encoded by two as yet lesser characterized IRE-containing mRNAs.
Finally, H2O2-treated cells display an
increased capacity to sequester 59Fe in ferritin, despite a
reduction in the ferritin pool, which results in a rearrangement of
59Fe intracellular distribution. Our data suggest that
H2O2 regulates cellular iron acquisition and
intracellular iron distribution by both IRP1-dependent and
-independent mechanisms.
To satisfy metabolic needs for iron, mammalian cells utilize
transferrin (Tf),1 the iron
carrier in plasma. Cellular iron uptake involves binding of Tf to the
cell-surface Tf receptor (TfR), followed by endocytosis. Within the
acidified endosome, iron is released from the Tf-TfR complex and
transported, most likely by the Divalent Metal
Transporter DMT1, across the endosomal membrane to the
cytosol, where it becomes bioavailable for the synthesis of iron
proteins. Excess iron is stored in ferritin, a multisubunit protein
consisting of H- and L-chains, that serves as the major intracellular
iron storage device (reviewed in Refs. 1-3). Sequestration of iron in
ferritin is viewed as a detoxification step to reduce the risk of
iron-mediated cell damage, which is based on the capacity of iron to
catalyze the generation of toxic oxygen radicals (4). Balanced iron homeostasis is critical for health, and both iron deficiency as well as
iron overload are associated with severe disorders (5).
At the cellular level, iron homeostasis is accomplished by the
coordinate regulation of iron uptake and storage. The expression of TfR
and ferritin is mainly controlled post-transcriptionally by iron
regulatory proteins, IRP1 and IRP2. Under conditions of iron
starvation, IRP1 and IRP2 are activated for high affinity binding to
multiple "iron-responsive elements" (IREs) in the 3'-untranslated region (UTR) of TfR mRNA and to a single IRE in the 5'-UTR of the
mRNAs encoding both H- and L-ferritin chains. This stabilizes TfR
mRNA (6) and inhibits ferritin mRNA translation (7). Conversely, failure of IRPs to bind to cognate IREs in iron-replete cells leads to degradation of TfR mRNA and synthesis of ferritin (reviewed in Refs. 8-10). The identification of additional
IRE-containing mRNAs suggests that the functional significance of
the IRE/IRP system stretches out beyond the control of cellular iron
uptake and storage. The mRNAs encoding the enzymes
5-aminolevulinate synthase-2 (involved in erythroid heme synthesis),
mammalian mitochondrial aconitase (m-aconitase), and the insect Ip
subunit of succinate dehydrogenase (both catalyzing reactions in citric
acid cycle) contain a "translation-type" IRE in their 5'-UTRs
(11-16). The mRNAs encoding the more recently discovered iron
transporters DMT1 (17, 18) and ferroportin/IREG1 (19-21) contain a
single and, in terms of function, incompletely characterized IRE in
their 3'- or 5'-UTR, respectively.
IRP1 and IRP2 share extensive homology and belong to the family of
iron-sulfur cluster isomerases that also includes m-aconitase. However,
their activities are controlled by distinct mechanisms. In iron-loaded
cells, IRP1 assembles a cubane 4Fe-4S cluster that converts it to a
cytosolic aconitase (c-aconitase) and prevents IRE-binding, whereas
IRP2 is oxidized and degraded by the proteasome. Iron starvation
increases IRE- binding activity by disassembly of the 4Fe-4S cluster in
IRP1 and stabilization/de novo synthesis of IRP2 (reviewed
in Refs. 8-10, 22). Iron regulatory proteins are subjected to
regulation by additional iron-independent signals, including nitric
oxide, hypoxia, and oxidative stress (reviewed in Refs.
23-25).
Of particular interest is the rapid induction of IRE binding activity
of IRP1 in response to hydrogen peroxide (H2O2)
(26, 27), because this "reactive oxygen intermediate" is implicated in iron toxicity. In the presence of catalytic amounts of ferrous iron,
H2O2 yields highly aggressive hydroxyl radicals
(Fenton reaction) that readily attack membranes, proteins, and nucleic acids (4). Exposure of different cell types to micromolar
concentrations of H2O2 is sufficient to induce
a rapid conversion of IRP1 from c-aconitase to the IRE-binding protein
within 30-60 min (26, 27) by an incompletely characterized mechanism
that involves signaling (28, 29). In contrast to this,
H2O2 does not affect the activity of IRP2 (30).
It should be noted that reactive oxygen species, including
H2O2, are widely viewed as participants in a
multitude of signaling pathways. These involve calcium signaling, mitogen-activated protein kinase cascades, tyrosine phosphorylation, regulation of phosphatases and phospholipases, or activation of transcription factors (reviewed in Refs. 31 and 32).
The effects of H2O2 on cellular iron metabolism
have been as yet only partially studied. We have previously utilized
mouse B6 fibroblasts, a cell line predominantly expressing IRP1 and negligible levels of IRP2, to characterize the mechanism of IRP1 induction by H2O2 (26, 28, 30, 33). We also
showed that a treatment of these cells with 100 µM
H2O2 for 1 h inhibits ferritin synthesis,
whereas longer treatments (4-6 h) increase TfR mRNA levels, as a
result of IRP1 activation (26). However, these responses have not been
correlated with the biological activity of TfR and ferritin, in terms
of iron uptake and sequestration. Here we extend the previous studies
and investigate the effects of H2O2 in the
expression and function of several IRE-containing mRNAs, as
reflected in the uptake of 59Fe-transferrin and
intracellular management of 59Fe.
Materials and Cell Culture--
Desferrioxamine (DFO) was
purchased from Novartis (Dorval, Canada), and
H2O2 was from Merck. Hemin, human apo- and
holo-Tf, fluorescein isothiocyanate (FITC)-conjugated holo-Tf, and
lactoferrin were from Sigma. B6 fibroblasts were grown and treated with
H2O2 as described (26).
Metabolic Labeling with [35S]Methionine/Cysteine
and Immunoprecipitation--
Cells were metabolically labeled for
2 h with (50 µCi/ml) Trans35S-label (ICN, a mixture
of 70:30 [35S]methionine/cysteine) and solubilized in
lysis buffer (50 mM Tris-Cl, pH 7.4, 300 mM
NaCl, and 1% Triton X-100). Cytoplasmic lysates (1 mg) were subjected
to quantitative co-immunoprecipitation with 5 µl of rabbit polyclonal
ferritin (Roche Molecular Biochemicals) and 2 µl of mouse monoclonal
TfR (Zymed Laboratories Inc.) antibodies. Sam68 was
then immunoprecipitated from supernatants by addition of 0.5 µl of
Sam68 antiserum (kindly provided by Dr. Stephane Richard).
Immunoprecipitated material was analyzed by SDS-PAGE/autoradiography (11).
Ferritin Assay--
Cells were solubilized in RIPA lysis buffer
(50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% (v/v)
Nonidet P-40, 0.5% (w/v) deoxycholate, and 0.1% (w/v) SDS). Insoluble
material was removed by centrifugation, and ferritin content was
analyzed by an immunoturbidimetric assay with the Tine-quant® kit
(Roche Molecular Biochemicals), according to the manufacturer's
recommendations, in a Hitachi 917 turbidimeter.
Northern Blotting--
RNA prepared with the Trizol® reagent
(Life Technologies, Inc.) was analyzed by Northern blotting (26) with
32P-radiolabeled mouse TfR, human ferritin H-chain, mouse
Western Blotting--
Total cell extracts (in RIPA lysis buffer)
were analyzed by Western blotting (34) with antibodies against TfR
(Zymed Laboratories Inc.), m-aconitase (a generous
gift of Dr. Rick Eisenstein), actin (Sigma), or DMT1 (raised in rabbits
against the peptide VFAEAFFGKTNEQVVE, which corresponds to amino acids
260-275 in human DMT1). Dilutions for antibodies are indicated in the
respective figure legends.
Fluorescence-activated Cell Sorting (FACS)--
To determine
cell surface expression or the Tf-binding capacity of TfR, cells were
scraped, suspended in medium, and tumbled with either 5 µl/ml
FITC-conjugated mouse TfR antibody (PharMingen) or with 50 µg/ml
FITC-conjugated human Tf (Sigma), respectively. Where indicated, a
50-fold molar excess human holo-Tf or lactoferrin was added prior to
FITC-Tf. Excess FITC label was removed by washing twice with
phosphate-buffered saline containing 0.1% bovine serum albumin. Cells
were fixed with 3.7% formaldehyde and analyzed for fluorescence on a
cell sorter (Beckman Coulter).
Generation of
59Fe-Tf--
59FeCl3 (PerkinElmer
Life Sciences) was mixed with sodium citrate (1:50 molar ratio in a
total volume of 1 ml) and incubated for 1 h at room temperature.
The resulting 59Fe-citrate was mixed with apo-Tf (2:1 molar
ratio); the volume was brought up to 4 ml in 0.6 M
NaHCO3, and incubation was continued overnight.
59Fe-Tf was separated from 59Fe-citrate on a
Centricon Plus-20 filter (Amicon), and its concentration was calculated
spectrophotometrically at 465 nm ( Cellular Uptake of 59Fe-Tf and Immunoprecipitation of
59Fe-Ferritin--
Cells were labeled with
59Fe-Tf in minimal essential medium containing 25 mM Hepes, pH 7.4, 10 mM NaHCO3, and
1% bovine serum albumin. Labeling was terminated by washing with
ice-cold phosphate-buffered saline, and cells were monitored for
radioactivity on a H2O2 Elicits a Time-dependent
Stimulation of TfR and Inhibition of Ferritin Synthesis--
We have
shown previously that treatment of cells with micromolar concentrations
of H2O2 results in rapid induction of IRP1 to
bind to IREs and that IRE binding activity remains elevated for at
least 4 h following removal of the inducer (30, 33). This
observation prompted us to study the effects of
H2O2 on the expression of TfR and ferritin, two
crucial proteins of iron metabolism under the control of the IRE/IRP
system. Our analysis covers intervals of up to 8 h following
exposure of cells to a bolus of 100 µM H2O2, allowing IRP1 activity to peak and
decrease to basal levels (30). No apparent toxicity was observed by the
trypan blue exclusion assay, under all experimental conditions employed
in this study, in line with earlier observations that exogenous
H2O2 is very rapidly degraded by these cells
(33). Nevertheless, a single bolus of 100 µM
H2O2 is sufficient to sustain a threshold of
~10 µM H2O2 for about 15 min,
which is the minimum concentration required to elicit IRP1 activation
(33). Thus, we established experimental conditions to activate IRP1 and
study the effects of H2O2 on cellular iron
metabolism in the absence of potential toxic side effects of
H2O2. B6 fibroblasts were first treated with
100 µM H2O2 for 1 h and
metabolically labeled with [35S]methionine/cysteine for
2 h either immediately or at different time points after
treatment, and TfR and ferritin synthesis were assessed by
immunoprecipitation (Fig. 1, top
panel). In cells previously treated with the iron chelator DFO
(100 µM), TfR synthesis is stimulated 3.3-fold compared
with untreated control cells, whereas synthesis of ferritin H- and
L-chains is strongly inhibited (11 and 12% of control, respectively,
lanes 1 and 2). Treatment with
H2O2 initially does not affect TfR expression
(lanes 2 and 3) but clearly stimulates TfR
synthesis by 2- and 2.1-fold, within 4 and 6 h after its
withdrawal, respectively (lanes 4-7). Soon afterward, TfR synthesis declines to almost control (1.1-fold) levels
(lanes 8 and 9). In contrast to TfR, ferritin
expression is affected immediately after H2O2
treatment; synthesis of ferritin H- and L-chains is reduced to 29 and
22% of control (lanes 2 and 3), in agreement
with earlier observations (26). Ferritin synthesis remains at low
levels even after 4 (28% for H- and 26% for L-chain) and 6 h
(35% for H- and 31% for L-chain) following
H2O2 withdrawal (lanes 4-7). After
8 h, ferritin synthesis only partially (60%) recovers, even
though TfR synthesis has essentially returned to basal levels
(lanes 8 and 9). As a control, the
non-iron-regulated protein Sam68 (68-kDa Src substrate
associated during mitosis) was
immunoprecipitated from TfR/ferritin-immunodepleted supernatants. Synthesis of Sam68 essentially remains unchanged during the course of
the treatment (Fig. 1, bottom panel).
Effects of H2O2 on the Steady-state Levels
of TfR and Ferritin mRNAs--
Analysis by Northern blotting (Fig.
2A) reveals that exposure of
cells to 100 µM H2O2 for 1 h
leads to a 3.4-, 4.0-, and 4.5-fold increase in steady-state levels of
TfR mRNA 2, 4, and 6 h after the treatment, respectively
(top panel, lanes 2-5). TfR mRNA levels drop after
8 h but are still 1.7 times higher than control (lane 6). As expected, iron chelation with DFO leads to a profound
(5.7-fold) induction of TfR mRNA (lane 1). In contrast
to TfR, ferritin (at least H-chain) mRNA levels are not affected by
iron chelation or H2O2 (middle
panel). The same holds true for non-iron-regulated H2O2-mediated Reduction of Ferritin Pool
and Accumulation of TfR--
We employed an immunoturbidimetric assay
to measure ferritin levels in cell extracts and to assess the effects
of H2O2 on total cellular ferritin content
(Fig. 2B). As expected, iron perturbations are strongly
reflected in the ferritin pool; exposure of cells to hemin increases
ferritin levels 3-fold, whereas iron chelation dramatically reduces
ferritin to 6% of control levels (lanes 1-3). Treatment
with 100 µM H2O2 for 1 h
initially decreases the ferritin content to 69% (lanes 3 and 4). Further reductions to 55 and 42% are evident 2 and
4 h after H2O2 withdrawal, respectively
(lanes 5 and 6). Ferritin concentration tends to
increase very slightly to 49 and 47% after 6 and 8 h (lanes
7 and 8), in line with the partial recovery in de
novo ferritin synthesis at these time points (Fig. 1). We conclude
that H2O2 leads to a marked reduction in the
ferritin pool for at least 8 h after the treatment.
To examine whether stimulation of TfR synthesis by
H2O2 is associated with an increase in TfR
concentration, we analyzed steady-state levels of TfR by Western
blotting (Fig. 2C). Treatment of cells with 100 µM H2O2 for 1 h leads to
gradual accumulation of TfR after 2-8 h (lanes 3-8).
H2O2-mediated induction of TfR reaches a
maximum 6 and 8 h after the treatment (1.9- and 1.8-fold,
respectively). As expected, treatments with DFO or hemin result in
2.2-fold increase and 0.6-fold decrease of TfR, respectively
(lanes 1-3). In this experiment, cells were solubilized in
RIPA lysis buffer, to extract membrane-bound TfR efficiently, but
similar results were obtained with cytoplasmic extracts (not shown).
H2O2 Leads to Increased Expression of
Functional TfR on the Cell Surface--
The data shown in Fig.
2C suggest that H2O2 stimulates TfR
expression. We next designed experiments to address whether this is
accompanied by increased Tf binding activity. The fraction of TfR
expressed on the cell surface is crucial for Tf binding. In a previous
report it was shown that H2O2 negatively
affects the size of this fraction, at least in human hematopoietic K562 and HL-60 cells (35). In light of these findings, we analyzed relative
changes in cell surface expression of TfR in mouse B6 fibroblasts by
means of FACS, using FITC-conjugated TfR antibodies (Fig.
3A). The levels of TfR on the
cell surface essentially remain unaltered within 2 h after
exposure of cells to H2O2 (100 µM
H2O2 for 1 h) (lanes 3-5), but
increase by 1.4-, 1.5- and 1.9-fold within 4, 6, and 8 h,
respectively (lanes 6-8). A profound cell surface
expression of TfR is achieved by treatment with DFO, whereas administration of hemin does not appear to cause any notable
alterations (lanes 1-3).
By having established that exposure of cells to
H2O2 is associated with increased expression of
TfR, including its cell surface fraction, we then employed a functional
assay to evaluate the effects of H2O2 on Tf
binding activity. Cells were incubated with FITC-conjugated Tf under
conditions allowing its binding to TfR. Changes in relative
fluorescence were then monitored by FACS (Fig. 3B).
Following treatment with H2O2 (100 µM H2O2 for 1 h), cells were
mixed with 50 µg/ml FITC-Tf, either at 4 °C for 2 h or at 37 °C for 40 min. Incubation at 4 °C inhibits recycling of TfR and thus serves to evaluate binding of FITC-Tf on the cell surface. Conversely, incubation at 37 °C is preferable to examine both cell
surface-bound and internalized (endosomal) FITC-Tf levels. To
facilitate displacement of serum-derived Tf from TfR, incubation at
4 °C was prolonged to 2 h. Under both experimental settings, FITC-Tf binding to TfR gradually increased 4-8 h following exposure of
cells to H2O2 (Fig. 3B, bars 3-8).
The increase was slightly elevated when incubations were performed at
37 °C (compare 1.2-, 1.4-, and 1.7-fold at 4 °C with 1.4-, 1.7-, and 1.8-fold increase at 37 °C, 4, 6, and 8 h after treatment,
respectively). Consistent with the data described above, iron chelation
with DFO elicits stronger effects on FITC-Tf binding to TfR than
H2O2 (up to 3.8-fold induction, Fig. 3B,
bar 2). As expected, the effects of hemin are inhibitory
(bar 1).
The specificity of the FITC-Tf binding assay is illustrated in Fig.
3C. Co-incubation of FITC-Tf with 50-fold excess non-labeled Tf competitor strongly reduces fluorescence intensity to 24.4% in
untreated and to 10% in DFO pretreated cells. In contrast, addition of
50-fold excess lactoferrin as a nonspecific competitor only slightly
interferes with FITC-Tf binding (~15% reduction). Incubations with
these competitors were performed at 37 °C, and similar results were
obtained at 4 °C (not shown). Taken together, our findings suggest
that exposure of B6 cells to H2O2 leads not only to an increase in TfR steady-state levels but also stimulates its
cell surface expression and the Tf-binding capacity. These conditions
are predicted to favor enhanced cellular iron uptake from Tf.
H2O2 Stimulates Uptake of
59Fe-Tf and Storage of 59Fe in Ferritin,
Leading to Alterations in the Relative Intracellular Distribution of
59Fe--
To determine directly the effects of
H2O2 on iron uptake, we incubated B6 cells with
5 µM 59Fe-Tf for 2 h and measured
cell-associated radioactivity on a
Under the conditions of the iron uptake experiment (e.g.
6-8 h following H2O2 treatment), ferritin
synthesis is still partially repressed (Fig. 1), whereas cellular
ferritin content has dropped to <50% of control levels (Fig.
2B). Since ferritin plays a major role in iron
detoxification as an iron-storage sink, we wondered how cells respond
to increased iron uptake when ferritin levels are reduced. To address
this question, B6 fibroblasts were labeled with 5 µM
59Fe-Tf (as in Fig. 4A) for 15 and 30 min and 1 and 2 h. Cytoplasmic extracts were analyzed by quantitative
immunoprecipitation with ferritin antibodies, and ferritin-associated
59Fe was plotted against the time of labeling (Fig.
4B). Ferritin immunoprecipitates from
H2O2-treated cells display a marked increase in
59Fe content compared with untreated control cells. After
2 h of labeling, ~8.16 pmol of 59Fe/mg protein in
extracts of control cells are associated with ferritin, whereas this
value increases to ~14.85 pmol of 59Fe/mg protein (181%)
in extracts from H2O2-treated cells.
Ferritin-associated 59Fe in extracts of cells pretreated
with DFO is very low (~0.9 pmol of 59Fe/mg of protein
after 2 h of labeling, representing 11% of control), most likely
due to sequestration of iron by the chelator. When cells were
prelabeled with 59Fe-Tf for 2 h and then left
untreated or treated with H2O2, no differences
in the amount of ferritin-associated 59Fe were observed
(not shown).
These data suggest that H2O2-treated cells have
an increased capacity to store newly internalized iron in ferritin,
despite the reduction in the translation and in the intracellular pool of ferritin. They also imply that their fraction of ferritin-associated 59Fe is significantly enriched. To calculate the
distribution of 59Fe in control,
H2O2-, and DFO-treated cells, we also measured radioactivity in the ferritin-immunodepleted extracts and in the insoluble cell fraction (similar methodology has been employed by
others (36)) and depicted the results in form of pie charts (Fig.
4C). Treatment with H2O2 leads to
alterations in intracellular distribution of 59Fe with a
notable increase in the fraction of ferritin-associated 59Fe from 15.8 to 26%. Considering that within 2 h,
106 B6 cells take up ~10.5 pmol of 59Fe if
untreated and ~11.74 pmol of 59Fe if treated with
H2O2 (Fig. 4A), the former store
~1.66 and the latter ~3.05 pmol of 59Fe in ferritin.
This represents an almost 2-fold increase under conditions where only
half the amount of ferritin is available (Fig. 2B).
The Steady-state Levels of m-Aconitase and DMT1 Are Not Affected by
H2O2--
By having established that
H2O2 modulates the expression (and the
function) of TfR and ferritin, we asked whether
H2O2 also affects the abundance of m-aconitase
and DMT1, both encoded by IRE-containing mRNAs. Western blotting
analysis at different time points after treatment of B6 cells with
H2O2 does not show any significant alterations
in steady-state levels of m-aconitase (Fig.
5A, top panel, lanes 3-8).
Overnight iron perturbations with DFO or hemin yield a similar outcome
(lanes 1 and 2). Probing with an antibody against
The effects of H2O2 on DMT1 mRNA were
assessed by Northern blotting. Probing with a mouse DMT1 cDNA
reveals two hybridizing bands of 3.1 and 2.3 kilobases (Fig. 5B,
top panel) that possibly correspond to the non-IRE and
IRE-containing isoforms of DMT1 mRNAs, respectively (37, 38). By
normalizing to the H2O2 as a Signal to the IRE/IRP Regulatory
System--
We show that a transient exposure of cells to
H2O2 stimulates TfR and decreases ferritin
translation in a time-dependent manner. TfR synthesis peaks
4-6 h following exposure of cells to 100 µM H2O2 (Fig. 1) as a result of the accumulation
of TfR mRNA (Fig. 2A). In contrast, ferritin (at least
H-chain) mRNA levels remain unaltered for up to 8 h after
H2O2 challenge (Fig. 2A), but
ferritin (H- and L-chains) synthesis is strongly inhibited immediately after H2O2 withdrawal and slowly recovers
afterwards (Fig. 1). As IRP1 is known to stabilize TfR mRNA and
inhibit ferritin mRNA translation by binding to their respective
IREs, these responses underlie the causal relationship of IRP1
induction by H2O2. In kinetic terms, the
activation of IRP1 is in perfect agreement with the regulatory effects
on its downstream targets. The translational inhibition of ferritin is
rapid and temporally coincides with the increase in IRE binding
activity (30), whereas accumulation of TfR mRNA is delayed
and follows its stabilization by binding of IRP1. The decline of IRE
binding activity to basal levels >4 h after
H2O2 treatment (30) is associated with a
decrease in TfR synthesis, as a result of TfR mRNA destabilization,
and gradual recovery of ferritin mRNA translation (Figs. 1 and 2).
We conclude that H2O2 modulates the expression
of ferritin and TfR via activation of IRP1.
We have also studied the effects of H2O2 (and
iron donors/chelators) on the abundance of m-aconitase and DMT1. Both
proteins are encoded by IRE-containing mRNAs. However, under our
experimental conditions, we did not observe any iron- or
H2O2-dependent alterations in their
steady-state levels (Fig. 5). The IRE in m-aconitase mRNA is
located in the 5'-UTR and is functional as a translational regulator
in vitro (13, 14). However, the range of
iron-dependent regulation of m-aconitase translation
in vivo lags orders of magnitude behind the respective range
of ferritin regulation (14, 15, 39). A potential explanation for this
is offered by the structural differences between m-aconitase and
ferritin IREs. The former contains a C-bulge and the latter an internal
loop/bulge that confer to them differential binding specificity toward
IRP1 and IRP2 in vitro (40). The functionality of
m-aconitase IRE in de novo synthesis of m-aconitase has been
demonstrated by sensitive immunoprecipitation assays following iron
perturbations and metabolic labeling of several cell lines with
[35S]methionine (15). Relatively small but significant
effects of iron on the steady-state levels of m-aconitase have been
documented by Western blotting analysis of mouse (14) and rat (39)
tissues following long term (over several weeks) modulation of dietary iron intake. In light of these data, we conclude that short term (<12
h) iron perturbations or treatments with H2O2
are not sufficient to lead to any detectable alterations in m-aconitase
steady-state levels (Fig. 5A).
The IRE in DMT1 mRNA is located in the 3'-UTR and has as yet only
partially been characterized. The levels of DMT1 mRNA
(IRE-containing isoform) are increased in iron-deficient enterocytes
from duodenal samples of hemochromatosis patients (41) or
HFE H2O2 as a Modulator of Cellular Iron
Metabolism Beyond the IRE/IRP Regulatory System--
Reactive oxygen
species are implicated in a wide array of signaling pathways (31, 32).
We thus wondered whether exposure of cells to
H2O2 affects the expression and function of
genes of iron metabolism at different levels, either upstream or
downstream of the IRE/IRP regulatory system (for example
transcriptionally or post-translationally). There is evidence that
ferritin synthesis is transcriptionally activated in response to
various forms of oxidative stress as part of a homeostatic antioxidant
defense mechanism (45-47). More recently, a functional "antioxidant
response element" has been identified in the promoters of L- (48) and H-ferritin (49). This element is shared in promoter regions of several
phase II detoxification genes and functions as a transcriptional enhancer in response to pro-oxidant stimuli. Treatment of mouse BNL
CL.2 normal liver cells or Hepa1-6 hepatoma cells with >250 µM H2O2 stimulated a delayed
(after 8 h) transcriptional activation of H- and L-ferritin
mRNAs via the antioxidant response element, which gradually
overcame the initial IRP1-mediated translational inhibition of ferritin
synthesis (49). The data presented in Fig. 2A suggest that
in B6 cells H2O2 fails to increase ferritin mRNA levels over 8 h (at least that of ferritin H-chain).
Although it is conceivable that application of more stringent
conditions of oxidative stress may stimulate ferritin mRNA
transcription, it is apparent that low micromolar concentrations of
H2O2 exhibit solely inhibitory effects on
ferritin expression. These are reflected in the decrease in ferritin
synthesis (Fig. 1) and the reduction of ferritin pool (Fig.
2B). In preliminary pulse-chase experiments, H2O2 did not appear to affect ferritin
half-life (not shown), suggesting that
H2O2-mediated translational inhibition of
ferritin synthesis suffices to reduce dramatically (<50% of control
levels) the intracellular ferritin pool for at least 8 h.
Previous studies in human hematopoietic K562 and HL-60 cells showed
that oxidative stress (either in the form of menadione or extracellular
H2O2) results in a rapid (within 30 min)
redistribution of TfR in intracellular compartments without alterations
in TfR levels (35, 50). These results appear to be IRP1-independent and
are in contrast to the stimulatory effects of
H2O2 on the synthesis, accumulation, cell
surface expression, and Tf binding activity of TfR observed in B6 cells
(Figs. 1-3). It is well established that the regulation of TfR
expression is more complex in erythroid cells, the major iron consumers
in the body, and involves transcriptional as well as
post-transcriptional mechanisms (51-54). Thus, it is conceivable that
additional, IRP1-independent pathways regulate TfR in various cell
types in response to H2O2. Nevertheless, it would be interesting to investigate the effects of
H2O2 on IRP1 activity in K562 and HL-60 cells.
In B6 fibroblasts, the H2O2-mediated increase
in TfR expression correlates with a modest (~11.5%) but significant
increase of 59Fe-Tf uptake (Fig. 4A), despite
the fact that Tf binding activity is stimulated 1.7-fold (Fig.
3B). Similarly, iron-starved cells (treated with DFO) take
up ~24.9% more 59Fe-Tf than untreated controls, despite
a 3.1-fold induction in Tf binding activity (Figs. 4A and
3B). These findings imply that intracellular iron release
during the Tf-TfR cycle may be controlled at additional checkpoints but
are also compatible with the idea that subtle perturbations in
intracellular iron balance may be sufficient to elicit significant
pathophysiological responses.
The experiments with 59Fe-Tf yielded another unanticipated
result. H2O2-treated cells have an increased
capacity to store 59Fe in ferritin, at a time point where
the ferritin pool is dramatically reduced (Figs. 4B and
2B), leading to changes in intracellular 59Fe
distribution (Fig. 4C). The reason for this is not clear,
but it is tempting to hypothesize that H2O2
signaling interferes with the incompletely defined mechanism of iron
sequestration in ferritin. It has been proposed that ferritin subunits
may be arranged in a flexible and dynamic structure allowing iron
entry/release by localized unfolding. In this sense, it is conceivable
that changes in iron entry in response to extracellular stimuli may be
associated with post-translational modification of ferritin that could
affect such localized unfolding (55). In fact, there is evidence in older literature that ferritin can be phosphorylated in
vitro (56). Along these lines, it will be interesting to examine
the phosphorylation status of ferritin in cells, following a treatment with H2O2. From a physiological point of view,
the increased capacity of ferritin to sequester iron may have evolved
for protection against iron-mediated injury and certainly adds to the
complexity of cellular responses to oxidative stress. In summary, by
utilizing B6 cells as a model system, we conclude that
H2O2 elicits complex effects on cellular iron
metabolism. These are both dependent and independent from the IRE/IRP
regulatory system and may be further complicated in various cell types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin, or mouse DMT1 cDNA probes.
= 4620 M
1
dm
1).
-counter. For immunoprecipitation of
59Fe-ferritin, cytoplasmic lysates were prepared in the
same way as lysates of 35S-labeled cells (see above), and 1 mg was tumbled at 4 °C with 5 µl of rabbit polyclonal ferritin
antibodies (Roche Molecular Biochemicals). Following addition of
protein A-coupled Sepharose CL-4B beads (Amersham Pharmacia Biotech),
immunoprecipitated material was washed twice in lysis buffer, and
radioactivity was monitored on a
-counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
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Fig. 1.
Time-dependent stimulation of TfR
and inhibition of ferritin synthesis by
H2O2. B6 cells were treated overnight with
100 µM DFO (lane 1), left untreated
(lanes 2, 4, 6, and 8), or treated for 1 h
with 100 µM H2O2 (lanes 3, 5, 7, and 9). Subsequently, cells were metabolically
labeled for 2 h with [35S]methionine/cysteine
(lanes 1-3) or washed, further incubated for another 2 (lanes 4 and 5), 4 (lanes 6 and
7), or 6 h (lanes 8 and 9), and
metabolically labeled for 2 h. Cytoplasmic cell extracts (1 mg)
were subjected to quantitative immunoprecipitation with 5 µl of
ferritin (Roche Molecular Biochemicals) and 2 µl of TfR
(Zymed Laboratories Inc.) antibodies. Ferritin and
TfR-immunodepleted supernatants were incubated with 0.5 µl of Sam68
antiserum (kindly provided by Dr. Stephane Richard). Immunoprecipitated
materials were analyzed by SDS-PAGE on 12% gels (top panel,
TfR and ferritin; bottom panel, Sam68). Proteins were
visualized by autoradiography and quantified by densitometric scanning
(NIH Image software). The positions of TfR, ferritin (H- and L-chains),
and Sam68 are indicated by arrows. The positions of
molecular mass standards are indicated on the right.
Induction for TfR and percentage inhibition of ferritin synthesis after
H2O2 treatment (lanes 3, 5, 7, and
9) is calculated compared with respective untreated controls
(lanes 2, 4, 6, and 8).
-actin
mRNA (bottom panel). Thus, the
time-dependent stimulation of TfR synthesis by
H2O2 (Fig. 1) correlates with an increase in
TfR mRNA levels, whereas H2O2-mediated
inhibition of ferritin synthesis appears to be translational.
View larger version (28K):
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Fig. 2.
Time-dependent increase in TfR
mRNA levels, accumulation of TfR, and decrease of ferritin content
in response to H2O2. B6 cells were treated
as indicated, and the mRNAs encoding TfR, ferritin (H-chain), and
-actin were analyzed by Northern blotting (A). 15 µg of
total RNA was resolved on an agarose gel (1%), electrotransferred onto
a nylon membrane, and hybridized to the respective
32P-radiolabeled cDNA probes. Radioactive bands were
visualized by autoradiography. Lane 1, 100 µM
DFO (overnight); lane 2, untreated control; lanes
3-6, 100 µM H2O2 (1 h),
following wash and further incubation for another 2, 4, 6, or 8 h.
B, ferritin content in cell extracts was analyzed by an
immunoturbidimetric assay (Tine-quant®, Roche Molecular Biochemicals).
Column 1, 100 µM hemin (overnight);
column 2, 100 µM DFO (overnight); column
3, untreated control; column 4, 100 µM
H2O2 (1 h), columns 5-8, 100 µM H2O2 (1 h), following wash and
further incubation for another 2, 4, 6, or 8 h. C, TfR
protein levels were analyzed by Western blotting. 30 µg of total cell
extracts were resolved by SDS-PAGE on a 10% gel and electrotransferred
onto a nitrocellulose membrane. The membrane was probed with 1:500
diluted monoclonal TfR antibody (Zymed Laboratories
Inc.), and TfR was detected by enhanced chemiluminescence
(Amersham Pharmacia Biotech). Lane 1, 100 µM
DFO (overnight); lane 2, 100 µM hemin
(overnight); lane 3, untreated control; lane 4,
100 µM H2O2 (1 h), lanes
5-8, 100 µM H2O2 (1 h),
following wash and further incubation for another 2, 4, 6, or 8 h.
TfR mRNA (A) and protein (C) levels were
quantified by densitometric scanning (NIH Image software).
View larger version (19K):
[in a new window]
Fig. 3.
H2O2 stimulates
expression of TfR on the cell surface and binding of Tf. A
and B, B6 cells were treated as indicated. Column
1, 100 µM hemin (overnight); column 2,
100 µM DFO (overnight); column 3, untreated
control; column 4, 100 µM
H2O2 (1 h); columns 5-8, 100 µM H2O2 (1 h), following wash and
further incubation for another 2, 4, 6, or 8 h. To assess the cell
surface expression of TfR (A), aliquots of 8 × 105 cells (duplicates) were suspended in medium and tumbled
for 30 min at 4 °C with 5 µl/ml FITC-conjugated TfR antibody
(PharMingen). To assess the binding of FITC-conjugated Tf to TfR
(B), aliquots of 6.5 × 105 cells
(duplicates) were suspended in medium and tumbled either at 37 °C
for 40 min (gray bars) or at 4 °C for 2 h
(hatched bars), with 50 µg/ml FITC-conjugated Tf (Sigma).
Following washing (to remove excess FITC label) and fixation,
fluorescent cells were analyzed by FACS. C, specificity
control for binding of FITC-conjugated Tf to TfR. B6 cells were either
left untreated (lanes 1-3) or treated overnight with 100 µM DFO (columns 4-6). Aliquots of 6.5 × 105 cells (duplicates) were suspended in medium, mixed
first with the indicated competitors and then with 50 µg/ml
FITC-conjugated Tf (Sigma), and tumbled at 37 °C for 40 min.
Following wash (to remove excess FITC label) and fixation, fluorescent
cells were analyzed by FACS. Columns 1 and 4 (gray bars), no competitor; columns 2 and
5 (white bars), 50-fold molar excess of holo-Tf;
columns 3 and 6 (black bars), 50-fold
molar excess of lactoferrin. Remaining FITC-Tf binding in the presence
of competitors is calculated compared with respective controls
(lanes 1-3 and 4-6).
-counter. Preliminary experiments
indicated that this concentration of 59Fe-Tf is saturating
(not shown). The results of the iron uptake experiment are depicted in
Fig. 4A. Untreated control
fibroblasts internalize ~10.5 pmol of
59Fe/106 cells during the time of labeling (2 h). Exposure of B6 cells to 100 µM
H2O2 for 1 h results in a modest
(~11.5%) but significant (p < 0.05 as estimated by
Student's t test) increase in 59Fe uptake, 6-8
h after the H2O2 treatment. Iron starvation by overnight treatment with 100 µM DFO leads to a more
pronounced (~24.9%) increase in 59Fe uptake. Considering
the profound effects of H2O2 and iron
starvation on the expression of TfR and its Tf binding activity (Fig.
3), the differences in 59Fe uptake in response to these
stimuli are not particularly strong, suggesting that the Tf-TfR cycle
may be subjected to additional controls. Nevertheless, these data show
that H2O2-treated cells have an increased
capacity to take up iron.
View larger version (34K):
[in a new window]
Fig. 4.
H2O2-mediated changes
in the uptake, storage, and intracellular distribution of
59Fe. B6 cells were left untreated or treated with 100 µM DFO overnight or with 100 µM
H2O2 for 1 h. Following
H2O2 treatment, cells were washed and further
incubated for another 6 h. Subsequently, cells received 5 µM 59Fe-Tf (349 cpm/pmol 59Fe).
A, labeling was stopped after 2 h; cells were washed
twice with ice-cold phosphate-buffered saline, and radioactivity was
measured on a -counter. The amount of radioactive iron taken up by
the cells, corresponding to triplicate samples, is expressed in pmol of
59Fe/106 cells in 2 h. B,
labeling was stopped after 15, 30, 60, or 120 min, and cells were
washed twice with ice-cold phosphate-buffered saline and lysed.
Cytoplasmic extracts (1 mg) were subjected to quantitative
immunoprecipitation with 5 µl of ferritin antibody (Roche Molecular
Biochemicals). Radioactivity in immunoprecipitated material was
measured on a
-counter. The amount of ferritin-associated
radioactive iron, expressed in pmol of 59Fe/mg protein in
cell extract, is plotted against the time of labeling (ctrl,
squares; H2O2, circles;
DFO, triangles). C, samples of B were
analyzed for radioactivity in the ferritin-immunodepleted supernatant
("soluble" fraction) and in the pellet obtained following cell
lysis and centrifugation ("insoluble" fraction). The relative
distribution of 59Fe in the ferritin-immunodepleted soluble
fraction (gray), in the insoluble fraction
(black), and in ferritin (white), analyzed in
untreated control cells and in cells treated with DFO or
H2O2, is plotted on a pie chart. The
percentages represent mean values of triplicates. Linear regression and
statistical analysis of triplicates was performed with GraphPad Prism
(version 2.0) software. *, p < 0.05 versus
control.
-actin (bottom panel) suggests that the slight reduction
in the intensity of the m-aconitase band on lane 7 is of no
functional importance and rather reflects unequal loading.
View larger version (35K):
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Fig. 5.
Treatment with H2O2
does not affect steady-state levels of m-aconitase, DMT1 mRNA, and
DMT1. A, 30 µg total cell extracts as in Fig.
2C were analyzed by Western blotting. The membrane was
probed with 1:500 diluted rabbit antibody raised against bovine heart
m-aconitase (a generous gift of Dr. Rick Eisenstein) and reprobed with
1:200 diluted rabbit antibody against -actin; m-aconitase and
-actin were detected by enhanced chemiluminescence (Amersham
Pharmacia Biotech). Lanes are as in Fig. 2C. B,
analysis of DMT1 and
-actin mRNAs by Northern blotting. 15 µg
of total RNA from B6 cells, treated as indicated, was resolved on an
agarose gel (1%), electrotransferred onto a nylon membrane, and
hybridized to the respective 32P-radiolabeled cDNA
probes. Radioactive bands were visualized by autoradiography.
Lanes 1-4, 100 µM
H2O2 (1 h), following wash and further
incubation for another 2, 4, 6 or 8 h; lane 5,
untreated control; lane 6, 100 µM hemin
(overnight); and lane 7, 100 µM DFO
(overnight). C, analysis of DMT1 by Western blotting. 30 µg total cell extracts from B6 cells, treated as indicated, were
resolved by SDS-PAGE on a 10% gel and electrotransferred onto a
nitrocellulose membrane. The membrane was probed with 1:200 diluted
rabbit antibody against DMT1, and DMT1 was detected by a chromogenic
(alkaline phosphatase) reaction. Lane 1, untreated control;
lanes 2 and 3, 100 µM
H2O2 (1 h and 4 h, respectively);
lane 4, 100 µM ferric chloride (4 h);
lane 5, 100 µM DFO (4 h); and lane
6, untreated control, probed in the presence of 10 ng of DMT1
immunogenic peptide.
-actin signal (bottom panel), no
obvious differences in the intensity of both bands are observed between
samples from untreated control (lane 5),
H2O2-treated (lanes 1-4) or
iron-perturbed cells (lanes 5-7). This finding is also
mirrored at the protein level; Western blotting with antibodies against
DMT1 (Fig. 5C) shows a faint band with an apparent molecular
mass of ~65 kDa that has the same intensity in samples from untreated
control, H2O2-treated, or iron-perturbed cells
(lanes 1-5). Since the sizes of polypeptides encoded by the
IRE- and non-IRE-DMT1 mRNAs differ by only 7 amino acids, the
~65-kDa band most likely corresponds to a mixture of both
isoforms. The specificity of the interaction is demonstrated on
lane 6. Probing the filter in the presence of excess DMT1
antigenic peptide does not produce the 65-kDa signal. Thus, in B6
cells, the expression of both non-IRE- and IRE-containing isoforms of DMT1 mRNAs does not appear to respond to iron or
H2O2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (42). In addition, a radiolabeled
DMT1 IRE probe is functional in gel retardation assays with cell
extracts (38).2 Taken
together, these results would argue for a role of the IRE in
controlling the stability of DMT1 mRNA. However, whereas a single
IRE is sufficient to function as a translational control element,
earlier experiments showed that the minimum requirement for regulating
the stability of TfR mRNA is defined by a combination of more than
one IRE together with additional non-IRE sequences (43). According to
the findings in Ref. 43, the single IRE in the 3'-UTR of DMT1 mRNA
would not qualify in its own right as a regulator of its stability via
IRE/IRP interactions. This view is supported by the data presented in
Fig. 5B. The Northern analysis does not reveal any
significant differences in the abundance of the 2.3- and 3.1-kilobase
transcripts, following treatments with H2O2
(over 8 h) or iron donors/chelators (overnight). The lack of iron
responsiveness in the abundance of DMT1 mRNA is in agreement with
recent data (38, 44). We speculate that the iron-dependent
regulation in the expression of the IRE-containing isoform of DMT1
mRNA in enterocytes may involve additional factors that are not
present in B6 cells. However, it should be noted that we do not have
sufficient information on the relative distribution of DMT1 encoded by
the IRE- or non-IRE forms of DMT1 mRNA, which may be an important
factor for the overall interpretation of the data. Along these lines,
it is not unexpected that under our experimental conditions iron or
H2O2 essentially has no effect on DMT1
steady-state levels (Fig. 5C).
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ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. Rick Eisenstein for providing us the antibody against m-aconitase. We also thank Dr. Stephane Richard for Sam68 antibody; Franca Sicilia, Lourdes Jipos, and Sabine Engl for technical assistance; and Drs. Joan Buss, Antonis Koromilas and Prem Ponka for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the Cancer Research Society Inc. (to K. P.) and by Grant FWF-14125 from the Austrian Research Fund (to G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Scholar of the Canadian Institutes of Health Research and a
researcher of the Canada Foundation for Innovation. To whom
correspondence should be addressed. Tel.: 514-340-8260 (Ext. 5293);
Fax: 514-340-7502; E-mail: kostas.pantopoulos@mcgill.ca.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M100245200
2 A. Caltagirone, G. Weiss, and K. Pantopoulos, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Tf, transferrin; IRP1, iron regulatory protein 1; IRE, iron-responsive element; UTR, untranslated region; TfR, transferrin receptor; m-, mitochondrial and c-, cytosolic aconitase; DMT1, divalent metal transporter 1; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; DFO, desferrioxamine.
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REFERENCES |
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