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INTRODUCTION |
As an essential constituent of many cellular macromolecules, iron
participates in numerous biochemical activities, such as oxygen
transport or electron transfer reactions (1). Excess of iron is,
however, toxic for cells, and pathologic conditions of iron overload
are associated with tissue injury and degeneration. The reactivity of
ferrous/ferric iron against H2O2 and
O
2 to yield hydroxyl radicals in the Fenton/Haber-Weiss
reactions provides a molecular basis for iron toxicity (2). Considering
that reactive oxygen species
(ROS),1 including
H2O2 and O
2, are inevitable byproducts
of aerobic respiration, cells have to tightly control iron homeostasis
to minimize the toxic effects of iron.
In vertebrates, iron uptake by the transferrin receptor (TfR) and
intracellular iron storage in ferritin are controlled by the IRE/IRP
regulatory system: TfR and ferritin mRNAs contain iron-responsive
elements (IREs) in their untranslated regions, which constitute the
binding sites for two related iron regulatory proteins, IRP-1 and
IRP-2. Both IRPs are activated by iron deficiency and NO to bind to
IREs. This activation results in stabilization of TfR mRNA, which
contains IREs in the 3'-untranslated region, and translational
inhibition of ferritin mRNA, which contains an IRE in the
5'-untranslated region (reviewed in Refs. 3 and 4).
IRP-1, which is considerably more abundant than IRP-2 in most tissues
examined so far, is a bifunctional protein that assembles a cubane
4Fe-4S cluster in response to increased cellular iron levels. This
cluster assembly renders it to be a cytosolic aconitase and inhibits
its IRE binding activity. The two mutually exclusive functions of IRP-1
as an IRE-binding protein and as an aconitase are controlled by a
reversible switch between 4Fe-4S-IRP-1 and apo-IRP-1 (for reviews see
Refs. 3-7). In addition to iron starvation and NO, which induce both
IRP-1 and IRP-2, IRP-1 is specifically activated to bind to IREs when
cells are exposed to low micromolar concentrations of
H2O2 (8-10).
H2O2 constitutes a distinct signal independent
from iron starvation and NO, which induces 4Fe-4S cluster disassembly
and activation of IRE binding (10-12). The physiology of oxidative
stress is intimately connected with the regulation of iron metabolism.
Treatment of cultured fibroblasts with H2O2
inhibits the synthesis of endogenous ferritin (9) and the translation
of a transfected hGH reporter under the control of an IRE (14).
Moreover, pharmacological treatment of cultured fibroblasts with
antimycin A, a respiratory chain inhibitor that stimulates generation
of ROS in mitochondria, also leads to IRP-1 activation (10). However,
treatment of rats with the drug phorone, which elicits oxidative stress
by depletion of glutathione pools, induces ferritin synthesis (13).
Thus, iron metabolism and oxidative stress are interconnected in
multiple ways, probably involving primary and compensatory responses
influenced by the chemical nature of the ROS and the time of treatment.
These considerations prompted us to study the effects of other
oxidative stress-inducing agents on cellular iron metabolism. Considerable attention was recently drawn to the so-called
"quinone-induced oxidative stress." Quinones are pro-oxidant
compounds that undergo redox cycling under aerobic conditions (15).
Inside the cell, quinones are reduced to semiquinone radicals by
one-electron transfer reductases and to hydroquinones by NADPH-quinone
reductases at the expense of NADPH. These intermediates readily react
with O2 and O
2 to regenerate the quinone and to
yield O
2 and H2O2, respectively. Apart
from this, some quinones engage in nucleophilic addition reactions that
deplete the intracellular glutathione pool.
Quinone-induced oxidative stress has been shown to elicit various
genetic responses: Menadione (2-methyl-1,4-naphthoquinone, vitamin
K3) activates the metallothionein gene CUP1 in
Saccharomyces cerevisiae (16) and aziridinyl-benzoquinones
activate the cell cycle inhibitor p21 in human colon carcinoma HCT116
cells (17). Menadione or 2,3-dimethoxy-1,4-naphthoquinone induce
-glutamylcysteine synthetase, the rate-limiting enzyme of
glutathione synthesis, in bovine pulmonary artery endothelial (18) and
rat lung epithelial L2 cells (19). In addition, menadione activates the
expression of the mRNA encoding the macrophage inflammatory
protein-1
in rat alveolar macrophages (20) and induces a heat shock
response in human hepatoma HepG2 and Chinese hamster lung V79 cells
(21). Interestingly, menadione also negatively modulates surface
expression and recycling of the insulin receptor in human erythrocytes
(22) and of the transferrin receptor in the human hematopoietic cells lines K562 and HL-60 (23). The latter result indicates that quinone-induced oxidative stress may affect cellular iron metabolism.
In this study, we have utilized menadione sodium bisulfite (MSB), a
water-soluble derivative of menadione, to assess the effects of
quinone-induced oxidative stress on cellular iron metabolism and, in
particular, on the activities of IRP-1 and the expression of
IRE-containing mRNAs.
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EXPERIMENTAL PROCEDURES |
Materials and Cell Culture--
MSB,
N-acetyl-L-cysteine (NAC),
L-buthionine-(S,R)-sulfoximine (BSO),
desferrioxamine, paraquat, and diamide were purchased from Sigma. The
fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate was from
Molecular Probes (Eugene, OR), and heme arginate was from Leiras Oy
(Turku, Finland). Polyclonal ferritin antibodies were obtained from
Boehringer Mannheim, and hGH antibodies were from the National Hormone
and Pituitary Program (Baltimore, MD). Murine B6 and Ltk
fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 100 units/ml penicillin,
0.1 ng/ml streptomycin, and 10% fetal calf serum. B6.IREhGH cells (10)
were grown in the same medium, additionally supplemented with
hypoxanthine/aminopterin/thymidine medium.
Detection of Intracellular H2O2 with
Fluorescence-activated Cell Sorting--
The redox-sensitive
fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate was
employed to assess levels of intracellular H2O2
by fluorescence-activated cell sorting as described (11). The dye is
oxidized to 2',7'-dichlorofluorescein by H2O2,
and thus 2',7'-dichlorofluorescein fluorescence intensity serves as a
measure for the intracellular H2O2 concentration.
EMSA and Aconitase Assay--
Detergent extracts were prepared
by lysis of cells in a buffer containing 1% Triton X-100, 40 mM KCl, and 25 mM Tris-Cl, pH 7.4. Lysates were
clarified by centrifugation at 10,000 × g for 10 min
and used for EMSAs, as described in Ref. 11. RNA-protein complexes were
quantitated by densitometric scanning of the depicted autoradiographs. Cytosolic aconitase activity in extracts from cells lysed with digitonin was determined as described (9, 24).
Northern and Western Blotting Immunoprecipitation--
Northern
and Western blottings, pulse-chase experiments and immunoprecipitations
were performed as described (9, 10, 25).
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RESULTS |
Control of the IRE Binding Activity of IRP-1 by MSB--
When
administered to cells, menadione, like other quinones, undergoes redox
cycling that leads to oxidative stress. In B6 fibroblasts, the
accumulation of ROS in response to the water-soluble menadione
derivative MSB is time-dependent: treatment of cells with
100 µM MSB for 15, 30, 60, or 120 min results in 1.8-, 2.6-, 3.1-, or 3.0-fold increases in intracellular
H2O2 levels, respectively, as measured by
2',7'-dichlorofluorescein fluorescence (Fig.
1). This prompted us to kinetically
analyze the effects of MSB on the IRE binding activity. After 15 min of
treatment of B6 cells with 100 µM MSB, IRP-1 is slightly
(1.8-fold) activated (Fig. 2A,
top panel, lanes 1 and 2). However,
IRE binding activity decreases to 1.3-fold within 30-45 min and
reaches basal levels after 1-2 h (lanes 3-6). This
analysis of cytoplasmic extracts by EMSA reveals the "spontaneous"
IRE binding activity in cells. The levels of total, "activable"
IRP-1 can be assessed after treatment of the cell extracts with
2-mercaptoethanol (26). Reducing agents compete at high concentrations
with the cysteines of IRP-1 for co-ordinating the 4Fe-4S cluster and
thus liberate the total IRE binding capacity (26-28). Analysis of
activable IRP-1 has become a common practice in many studies over the
last few years, first because it yields valuable information on IRP-1
content and second because it can serve as a control for equal protein
loading. In extracts of MSB-treated cells this assay yielded an
unexpected outcome: the activable IRE binding is significantly
(~80%) diminished after treatment of cells with 100 µM
MSB for >45 min (Fig. 2A, bottom panel, compare lanes 1-3 with lanes 4-6).

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Fig. 1.
Time-dependent increase in the
concentration of reactive oxygen species by menadione. B6
fibroblasts (107) received 5 µM
2',7'-dichlorodihydrofluorescein diacetate for 120 min and were
subsequently either left untreated or treated with 100 µM
MSB for 15, 30, 60, or 120 min. 2',7'-Dichlorofluorescein fluorescence
was monitored by fluorescence-activated cell sorting. The intensity of
2',7'-dichlorofluorescein fluorescence is plotted against the time of
treatment with MSB.
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Fig. 2.
Time-dependent effect of
menadione on IRE binding activity. A, B6 cells
(107) were left untreated (lane 1) or treated
with 100 µM MSB for 15, 30, 45, 60, or 120 min
(lanes 2-6). B, B6 cells (107) were
pretreated overnight with 100 µM desferrioxamine
(lanes 2-6) or left untreated (lane 1).
Subsequently, cells received no further treatment (lanes 1 and 2) or were treated with 100 µM MSB for 15, 30, 60, or 120 min (lanes 2-6). Cytoplasmic extracts (25 µg) were analyzed by EMSA with 25,000 cpm 32P-labeled IRE
probe in the absence (top panel) or presence (bottom
panel) of 2% 2-mercaptoethanol (2-ME). The positions
of the IRE-IRP-1 complexes and of excess free IRE probe are indicated
by arrows.
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To further investigate this unexpected response of IRP-1, cells were
pretreated overnight with the iron chelator desferrioxamine (100 µM) to increase the levels of spontaneous IRE binding.
Subsequently, the cells were treated with 100 µM MSB for
different time intervals. Analysis of IRE binding in cytoplasmic
extracts shows that the treatment with desferrioxamine results in a
complete (8-fold) activation of IRP-1 (Fig. 2B,
top and bottom panels, lanes 1 and 2). Administration of MSB to iron-starved cells for 15-60
min has no significant effects on IRP-1 (lanes 2-5).
Interestingly, treatment with MSB for 2 h results in a marked
decrease in both spontaneous as well as activable IRE binding of IRP-1
in desferrioxamine-pretreated cells (lane 6).
Thus, MSB elicits two distinct effects on IRP-1: brief (15-45 min)
incubations result in a slight IRP-1 activation, whereas longer (>1 h)
treatments lead to a reduction of activable IRE binding in control
cells and of spontaneous and activable IRE binding in iron-starved
cells. To determine the optimal conditions for these responses, control
and iron-starved B6 cells were treated with increasing concentrations
of MSB for 30 min or 2 h, respectively. Analysis of cytoplasmic
extracts for IRE binding shows that maximal IRP-1 activation is
achieved with relatively low concentrations of MSB (Fig.
3A, 2-fold activation with 10 µM and 2.5-fold with 25 µM MSB,
respectively). On the other hand, higher concentrations of MSB are
required to inactivate IRE binding: administration of 10 µM MSB to iron-starved B6 cells for 2 h fails to
elicit any effects on IRP-1, whereas higher doses of MSB (50 and 100 µM) lead to 60 and 90% inactivation of IRP-1,
respectively (Fig. 3B). To assess the effects of
menadione on IRP-2, we utilized murine Ltk
fibroblasts,
which express significantly higher levels of IRP-2 than B6 cells.
Analysis of IRE binding activity following treatment of iron-starved
Ltk
cells with 100 µM MSB for 2 h lead
to the inactivation of IRP-2 and, as expected, IRP-1 (Fig.
3C). This result suggests that menadione-induced oxidative
stress affects both iron regulatory proteins.

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Fig. 3.
Dose-dependent effects of
menadione on IRE binding. A, B6 cells (107)
were left untreated (lane 1) or treated for 30 min with 1, 2.5, 10, 25, 100, or 250 µM MSB (lanes 2-7).
B, B6 cells (107) were pretreated overnight with
100 µM desferrioxamine and either left untreated
(lane 1) or treated for 2 h with 10, 50, or 100 µM MSB (lanes 2-4). C,
Ltk cells (107) were pretreated overnight
with 100 µM desferrioxamine and either left untreated
(lane 1) or treated for 2 h with 100 µM
MSB (lane 2). Cytoplasmic extracts (25 µg) were analyzed
by EMSA with 25,000 cpm 32P-labeled IRE probe in the
absence (top panel) or presence (bottom panel) of
2% 2-mercaptoethanol (2-ME). The positions of the IRE-IRP
complexes and of excess free IRE probe are indicated by
arrows.
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The two effects of MSB on IRP-1 are antagonized by pretreatment of B6
cells with 30 mM NAC for 2 h. This antioxidant first inhibits the partial activation of IRE binding after a short (30-60 min) exposure to low (25 µM) concentrations of MSB (Fig.
4A, compare lanes
2-4 with lanes 5-7) and second protects the
spontaneous and activable IRE binding in iron-starved and control cells
from inactivation after a longer (2 h) treatment with higher (100 µM) concentrations of MSB (Fig. 4B, compare
lanes 7 and 8, upper and lower
panel). Treatment with 100 µg/ml BSO for 18 h, which
depletes the intracellular glutathione pools (29), does not elicit
significant changes in IRE binding activity (Fig. 4C,
lanes 1 and 2 and Ref. 11). Interestingly,
treatment with 100 µM MSB for 2 h following BSO
treatment leads to a more profound, almost complete loss of the
activable IRE binding compared with control cells (Fig. 4C, lanes 3 and 4).

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Fig. 4.
The antioxidant NAC antagonizes and the
glutathione-depleting drug BSO augments the effects of menadione on IRE
binding. A, B6 cells (107) were pretreated
with 30 mM NAC (lanes 5-7) or not (lanes
1-4). After 2 h, cells were either left untreated
(lane 1) or treated with 25 µM MSB for 30 min
(lanes 2 and 5), 60 min (lanes 3 and 6), or 120 min (lanes 4 and 7).
B, B6 cells (107) were incubated overnight with
100 µM desferrioxamine (lanes 5-8) or not
(lanes 1-4) and then pretreated with 30 mM NAC
(lanes 2, 4, 6, and 8) or
not (lanes 1, 3, 5, and 7).
After 2 h, cells received 100 µM MSB for another
2 h (lanes 3, 4, 7, and
8), or incubation was continued without MSB (lanes
1, 2, 5, and 6). C, B6
cells (107) were pretreated for 18 h with 100 µg/ml
BSO (lanes 2 and 4) or not (lanes 1 and 3). Subsequently, cells were washed and either left
untreated (lanes 1 and 2) or treated with 100 µM MSB for 2 h (lanes 3 and
4). Cytoplasmic extracts (25 µg) were analyzed by EMSA
with 25,000 cpm 32P-labeled IRE probe in the absence
(top panel) or presence (bottom panel) of 2%
2-mercaptoethanol (2-ME). The positions of the IRE-IRP-1
complexes and of excess free IRE probe are indicated by
arrows.
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Menadione-induced Inactivation of the Cytoplasmic Aconitase
Activity of IRP-1--
In iron-replete cells, IRP-1 acquires enzymatic
activity as a cytoplasmic aconitase at the expense of IRE binding. To
investigate the effect of MSB on the aconitase activity of IRP-1,
cytoplasmic aconitase activity was measured in the extracts of
MSB-treated and untreated control cells. Treatment of B6 fibroblasts
with 25 µM MSB for 30 min results in a partial (15%)
inactivation, whereas a higher concentration of MSB (100 µM) leads to 60 or 82% inactivation of cytoplasmic
aconitase within 30 or 120 min, respectively (Fig.
5). These results show that MSB
negatively modulates the aconitase activity of IRP-1. At the early
stages of the treatment, this effect correlates with the activation of IRE binding (Figs. 2A and 3A). However, at a
later stage (2 h), MSB leads to a decline of both the aconitase (Fig.
5) and IRE binding (Figs. 2 and 3B) activities of IRP-1.

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Fig. 5.
Menadione-mediated inactivation of
cytoplasmic aconitase activity. B6 cells (3 × 107) were either left untreated or subjected to treatment
with 25 µM MSB for 30 min or 100 µM MSB for
30 or 120 min. 400 µg of cytoplasmic extracts were used to assay
aconitase activity. The percentage of remaining aconitase activity is
plotted against the various treatments.
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Menadione-induced Inactivation of IRP-1 Is Not Due to Protein
Degradation--
Previous work by several laboratories established
that the aconitase and IRE binding activities of IRP-1 are reciprocally regulated by an Fe-S cluster switch. The simultaneous loss of both
activities by menadione is unusual and raises the possibility that
IRP-1 could be degraded. To evaluate this possibility, B6 cells were
metabolically pulse-labeled with [35S]methionine for
2 h. Subsequently, cells were either directly harvested or chased
with media containing excess unlabeled methionine in the presence of
100 µM MSB for 30 min, 1 h or 2 h.
Immunoprecipitation of IRP-1 from cell lysates shows that treatment
with 100 µM MSB over 2 h does not affect the
stability of newly synthesized IRP-1 (Fig.
6A). To assess the effects of
menadione on the steady-state levels of IRP-1, B6 cells were treated
with 100 µM MSB for up to 2 h, and IRP-1 was
analyzed by Western blotting. No differences in IRP-1 content were
found (Fig. 6B). We conclude that menadione-induced oxidative stress does not profoundly affect the stability of IRP-1.

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Fig. 6.
Menadione does not affect the stability of
IRP-1. A, B6 cells (107) were metabolically
labeled with [35S]methionine for 2 h and
subsequently chased with medium lacking [35S]methionine
in presence of MSB for the indicated time intervals. 500 µg of
cytoplasmic extracts were subjected to quantitative immunoprecipitation
with 30 µl of IRP-1 antiserum. Immunoprecipitated material was
analyzed by SDS-polyacrylamide gel electrophoresis on a 10% gel, and
IRP-1 was visualized by autoradiography. Lane 1, chase at
time 0; lanes 2-4, chase with 100 µM MSB for
30, 60, or 120 min, respectively. B, B6 cells
(107) were treated with 100 µM MSB for the
indicated time intervals, and IRP-1 was analyzed by Western blotting.
Cytoplasmic extracts (30 µg of protein) were resolved by
SDS-polyacrylamide gel electrophoresis on a 8% gel and
electrotransferred onto a nitrocellulose filter. The filter was probed
with 1:200 diluted IRP-1 antiserum, and proteins were detected by
enhanced chemiluminescence. IRP-1 is shown by arrows. The
positions of molecular mass standards are indicated on the
right.
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Menadione Modulates the Expression of IRE-containing
mRNAs--
How does the menadione-induced inactivation of IRP-1
affect the expression of IRE-containing mRNAs? We first examined
whether MSB modulates TfR mRNA expression. B6 cells were treated
with 100 µM desferrioxamine for 6 h to increase the
steady-state levels of TfR mRNA as a result of IRP-1 activation. To
assess the effects of MSB on TfR mRNA stabilization, 100 µM MSB was administered together with desferrioxamine for
2 h, in the presence or absence of the antioxidant NAC (30 mM). Following the removal of MSB, incubation with
desferrioxamine was continued for up to 6 h, with or without NAC.
This treatment is sufficient to allow a partial stabilization of TfR
mRNA (9). To induce complete stabilization or degradation of TfR
mRNA, as a control, cells were treated with 100 µM
desferrioxamine or heme arginate overnight. Northern blot analysis
(Fig. 7, upper panel) shows
that MSB down-regulates TfR mRNA to the levels of untreated cells,
and this effect is inhibited by NAC (lanes 1 and
4-6). Equal RNA loading was confirmed by hybridization with
a ferritin H chain probe (Fig. 7, bottom panel). Consistent with previous results (9), ferritin H chain mRNA levels are increased after treatment with heme arginate (lane 3),
likely due to heme-induced transcriptional activation (30). Preliminary experiments employing iron-starved cells (preincubated overnight with
desferrioxamine) did not show any significant menadione-induced changes
on TfR mRNA steady-state levels (data not shown). Considering that
under these conditions TfR mRNA molecules are protected by binding
of iron regulatory proteins to their IREs, it is conceivable that the
IRPs already engaged in complexes with IREs are less susceptible to
menadione-induced inactivation.

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Fig. 7.
Modulation of TfR mRNA levels by
menadione. B6 cells (107) were treated as indicated,
and the mRNAs encoding TfR (top panel) and ferritin H
chain (bottom panel) were analyzed by Northern blotting.
Lane 1, untreated control; lanes 2 and
3, treatment with 100 µM desferrioxamine or
100 µM heme arginate, respectively, for 12 h;
lane 4, treatment 100 µM desferrioxamine for
6 h; lane 5, treatment with 100 µM
desferrioxamine and 100 µM MSB for 2 h, following
wash and incubation for up to 6 h in the presence of 100 µM desferrioxamine; lane 6, pretreatment with
30 mM NAC for 30 min, following addition of 100 µM desferrioxamine and 100 µM MSB for
2 h, wash, and further incubation for up to 6 h in the
presence of 100 µM desferrioxamine and 30 mM
NAC. The sizes of TfR and ferritin H chain mRNAs are indicated in
parenthesis.
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The expression of ferritin is controlled at the translational level by
the IRE/IRP regulatory system. The switch of IRP-1 from aconitase to
IRE binding activity inhibits ferritin mRNA translation. To analyze
the effect of menadione on ferritin biosynthesis, B6 cells were
pretreated overnight with 5 µM ferric ammonium citrate to
increase basal levels of ferritin expression and subsequently subjected
to treatment with 100 µM MSB for 2 h in the presence or absence of the antioxidant NAC (30 mM). Following
metabolic labeling with [35S]methionine, ferritin
biosynthesis was assessed by immunoprecipitation. Iron manipulations
with 100 µM heme arginate or desferrioxamine result in a
profound stimulation or inhibition, respectively, of ferritin
translation (Fig. 8A,
lanes 1-3). Likewise, extracellular oxidative stress after
treatment with 100 µM H2O2, which
activates IRE binding (8, 9), inhibits ferritin synthesis (lane
7). Unexpectedly, menadione-induced oxidative stress, under
conditions where IRP-1 is completely inactivated, also inhibits
ferritin synthesis (compare lanes 1 and 4). This inhibitory
effect of MSB is antagonized by addition of NAC (lanes
5-6).

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Fig. 8.
Effects of menadione on the translation of
IRE-containing mRNAs. A, translational inhibition
of ferritin H and L chains by menadione. B6 cells (107)
were treated as indicated and metabolically labeled with
[35S]methionine for 2 h. Cell extracts were
normalized for trichloroacetic acid-insoluble radioactivity and
subjected to quantitative immunoprecipitation with 5 µl of ferritin
antibodies (Boehringer Mannheim) and 30 µl of U1A antiserum (internal
control, a generous gift of Dr. I. Mattaj). Immunoprecipitated material
was analyzed by SDS-polyacrylamide gel electrophoresis on a 15% gel,
and proteins were visualized by autoradiography. Lane 1,
untreated control; lanes 2 and 3, treatments with
100 µM heme arginate (H) or 100 µM desferrioxamine (D) for 4 h; lane 4,
100 µM MSB for 2 h; lane 5, pretreatments
with 30 mM NAC for 2 h and addition of 100 µM MSB for another 2 h; lane 6, treatment
with 30 mM NAC for 4 h; lane 7, 100 µM H2O2 for 1 h.
B, menadione-mediated translational regulation of an
IRE-containing hGH mRNA. B6.IREhGH cells were treated and
metabolically labeled as in A. hGH expression was analyzed
by quantitative immunoprecipitation with 5 µl of hGH antibodies
(National Hormone and Pituitary Program) as in A.
Lanes 1 and 2, treatments with 100 µM heme arginate or 100 µM desferrioxamine
for 4 h; lane 3, untreated control; lane 4,
treatment with 100 µM H2O2 for
1 h; lane 5, treatment with 100 µM MSB
for 2 h. All cells were pretreated overnight with 5 µM ferric ammonium citrate prior to subsequent
treatments. Ferritin, U1A, and hGH are shown by arrows. The
positions of molecular mass standards are indicated on the
right.
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It should be noted that oxidative stress results in an apparent
inhibition of global protein synthesis, as the radioactivity of
trichloroacetic acid-insoluble material in extracts of MSB- and
H2O2-treated cells constitutes only ~15-20%
of that in control extracts (this refers to Fig. 8, A and
B). To compare the specific effects of oxidative stress on
ferritin biosynthesis, immunoprecipitations were performed after
normalization for trichloroacetic acid-insoluble radioactivity. The
data presented in Fig. 8A show that the inhibitory effect of
MSB is not due to the global reduction of protein synthesis but
specific for ferritin, because the synthesis of an unrelated polypeptide, the spliceosomal protein U1A, is not affected by any of
the treatments. In addition, the results shown in Fig. 7 (bottom
panel) indicate that the steady-state levels of ferritin H chain
mRNA are not affected by MSB.
The apparent inhibition of ferritin mRNA translation by MSB is
unexpected in the light of the MSB-induced inactivation of IRE binding.
To further investigate this, the previously described stably
transfected cell line B6.IREhGH (10) was utilized to assess the effects
of MSB on the expression of an hGH indicator mRNA, which is under
the control of a ferritin IRE in its 5'-untranslated region. Cells were
subjected to iron manipulations with 100 µM heme arginate
or desferrioxamine for 4 h or oxidative stress by treatment with
100 µM extracellular H2O2 for
1 h or 100 µM MSB for 2 h. Following metabolic
labeling with [35S]methionine, biosynthesis of
(endogenous) ferritin and (transfected) hGH were analyzed by
immunoprecipitation. As in wild type B6 cells (Fig. 8A),
ferritin expression in B6.IREhGH cells was repressed by both
extracellular H2O2 and MSB (data not shown). In
contrast, hGH biosynthesis is inhibited by extracellular
H2O2 and even slightly enhanced by MSB (Fig.
8B). We conclude that the inactivation of IRP-1 by MSB
derepresses the translation of the IRE-regulated hGH reporter mRNA.
However, this does not apply to the translation of ferritin mRNA,
which appears to be specifically inhibited by a mechanism involving
non-IRE sequences in response to menadione-induced oxidative stress.
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DISCUSSION |
Menadione-induced oxidative stress regulates IRP-1 in a biphasic
mode. Initially, relatively low concentrations (10-100
µM) of MSB elicit a partial activation of IRE binding
within 15-30 min, which is accompanied by the inactivation of its
cytoplasmic aconitase activity. Subsequently, higher (50-100
µM) concentrations of MSB administered to cells for
2 h inhibit both the IRE binding and aconitase activities of
IRP-1.
The presence or absence of the 4Fe-4S cluster is a critical determinant
for the function of IRP-1 as an aconitase or as an IRE-binding protein.
On that basis, the early response of IRP-1 to menadione can be
interpreted as a "classical," though incomplete, Fe-S cluster
switch. The mechanism underlying the oxidative stress-mediated Fe-S
switch of IRP-1 has remained elusive. The sensitivity of the Fe-S
cluster in the bacterial transcription factor FNR under aerobic
conditions (31) and the inactivation of the 4Fe-4S cluster in bacterial
and mammalian mitochondrial aconitases by O
2 (32, 33) formed a
basis to propose a "direct attack" model to explain the oxidative
stress-mediated Fe-S switch in IRP-1. According to this, the Fe-S
cluster of IRP-1 is liable to direct chemical modification and/or
removal by ROS (34). Recent data have defined conditions of
intracellular oxidative stress that are not sufficient to activate
IRP-1 (11). Moreover, recent evidence suggests that the activation of
IRP-1 by extracellular H2O2 operates via a
stress-response signaling pathway and not solely by a direct
interaction of IRP-1 with H2O2 (10-12, 35).
The early responses of IRP-1 to MSB do not rule out the possibility
that quinone-induced oxidative stress may promote conditions favoring a
destabilization of the 4Fe-4S cluster by ROS. An alternative scenario
would be that menadione and extracellular H2O2
may activate the same stress-response pathway, at least in the early
stages of MSB treatment, considering the early similarities in their
effects on IRP-1 (partial activation of IRE binding within 15-30 min;
Fig. 2A and Refs. 8 and 9).
Under many experimental conditions, the functions of IRP-1 as a
cytoplasmic aconitase or an IRE-binding protein have been found to be
inversely correlated. Here we show that MSB treatment for >1 h leads
to a decline of both aconitase and IRE binding activities. Such an
inactivation of IRP-1 has not been elicited by other stimuli so far.
When B6 cells were subjected to prolonged incubations with 100 µM paraquat, another redox cycling drug, the IRE binding
activity was partially induced (9). On the other hand, treatment of
Ltk
cells with diamide, a strong oxidant, resulted in a
reversible inactivation of IRE binding, which could be recovered
in vivo by iron starvation after removing diamide from the
cells and in vitro by treatment of cell extracts with
2-mercaptoethanol (36). We have reproduced these data in parallel
treatments of cells with paraquat, diamide, and MSB and found that only
MSB leads to a nonreversible decrease in the IRE binding activity of
IRP-1 (data not shown). Thus, the inactivation of both IRP-1 functions appears to be a more specific response to menadione- (or quinone-) induced oxidative stress.
What is the molecular basis for this inactivation of IRP-1 (and IRP-2)?
The biological effects of menadione and other quinones are based on
their potential to undergo redox cycling and thus generate ROS, and on
their electrophile character in nucleophilic addition reactions,
resulting in depletion of glutathione (15). We suggest that the
inactivation of IRPs observed here could be a result of a nonreversible
oxidation of critical amino acid residue(s) in IRPs by ROS generated
via quinone-induced oxidative stress. This may lead to a rearrangement
of the protein folding associated with functional inactivation. The
pharmacological depletion of glutathione in B6 cells (Ref. 11 and Fig.
4C) and in rats (13) alone is not sufficient to elicit
significant effects on the IRE binding activity of IRP-1. However,
depletion of glutathione appears to render IRP-1 more susceptible to
the putative oxidative inactivation by menadione (Fig. 4C).
In agreement with this scenario, the antioxidant N-acetylcysteine antagonizes IRP-1 inactivation by
scavenging the ROS. The critical oxidation step may be preceded by a
partial or complete disassembly of the Fe-S cluster.
The targets of nonreversible oxidation of proteins under conditions of
oxidative stress are not necessarily specific, because all amino acid
residues are sensitive to oxidant attack by hydroxyl radicals,
generated via the Fenton/Haber-Weiss reactions (reviewed in Ref. 37).
Inside the cell, oxidized proteins are usually prone to degradation by
the proteasome (reviewed in Ref. 38). This implies that the putative
oxidation of IRP-1 by menadione-induced oxidative stress may affect its
half-life, which is otherwise relatively long (>12 h) (25, 39). Even
though we have not determined the half-life of IRP-1 in
menadione-treated cells, the data presented in Fig. 6 exclude the
possibility that IRP-1 inactivation by menadione is due to protein degradation.
The menadione-induced inactivation of IRP-1 modulates the expression of
IRE-containing mRNAs at the levels of mRNA stability and
translation. First, it inhibits the increase in TfR mRNA levels by
iron starvation in B6 cells. Second, it leads to a specific translational derepression of an IRE-controlled hGH reporter mRNA in stably transfected B6.IREhGH cells. Considering the inhibitory effects of oxidative stress on global translation (see "Results"), the stimulation of hGH mRNA translation is relative. On the other hand, in contrast to the apparent translational stimulation of the hGH
indicator (Fig. 8B), MSB appears to elicit a specific translational inhibition of endogenous ferritin mRNAs (Fig.
8A). The underlying mechanism remains to be defined.
However, technical reasons also need to be considered as explanations
for the observed failure to identify de novo synthesized
ferritin (Fig. 8A); a possibly oxidative modification of
newly synthesized ferritin polypeptides may lead to their degradation
or negatively affect their recognition by the ferritin antibodies.
We conclude that menadione-induced oxidative stress controls both IRP-1
activities and thereby controls cellular iron metabolism via the
IRE/IRP regulatory system. The effects of menadione are clearly
distinct from those of extracellular H2O2,
emphasizing the notion that IRP-1 responds differentially to diverse
oxidative stress stimuli.