Inactivation of Both RNA Binding and Aconitase Activities of Iron Regulatory Protein-1 by Quinone-induced Oxidative Stress*

Niels H. Gehring, Matthias W. Hentze, and Kostas PantopoulosDagger

From the European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

    ABSTRACT
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Abstract
Introduction
References

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.

    INTRODUCTION
Top
Abstract
Introduction
References

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 Obardot 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 Obardot 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 Obardot 2 to regenerate the quinone and to yield Obardot 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 gamma -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-1alpha 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.

    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).

    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.

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.

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.

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.

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.

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.

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.

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.

    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 Obardot 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.

    ACKNOWLEDGEMENTS

We thank Drs. Thomas Preiss and Dirk Ostareck for critical reading of the manuscript and for helpful suggestions.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 601).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.

Dagger To whom correspondence should be addressed. Present address: Lady Davis Inst. for Medical Research, Jewish General Hospital, 3755 Chemin de la Cote-Ste-Catherine, Montreal, PQ H3T 1E2, Canada. Tel.: 514-340-8260, Ext. 5293; Fax: 514-340-7502; E-mail: mbg1{at}musica.mcgill.ca.

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; IRP-1, iron-regulatory protein 1; IRE, iron-responsive element; TfR, transferrin receptor; MSB, menadione sodium bisulfite; EMSA, electrophoretic mobility shift assay; NAC, N-acetyl-L-cysteine; hGH, human growth hormone; BSO, L-buthionine-(S,R)-sulfoximine.

    REFERENCES
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Abstract
Introduction
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