Regulation of Iron Regulatory Protein 1 during Hypoxia and Hypoxia/Reoxygenation*

Eric S. Hanson and Elizabeth A. LeiboldDagger

From the Eccles Program in Human Molecular Biology and Genetics and the Department of Medicine, University of Utah, Salt Lake City, Utah 84112

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Given the important relationship between O2 and iron (Fenton chemistry) a study was undertaken to characterize the effects of hypoxia, as well as subsequent reoxygenation, on the iron-regulatory proteins 1 and 2 (IRP1 and IRP2) in a rat hepatoma cell line. IRP1 and IRP2 are cytosolic RNA-binding proteins that bind RNA stem-loops located in the 5'- or 3'-untranslated regions of specific mRNAs encoding proteins that are involved in iron homeostasis. In cells exposed to hypoxia, IRP1 RNA binding was decreased ~2.8-fold after a 6-h exposure to 3% O2. Hypoxic inactivation of IRP1 was abolished when cells were pretreated with the iron chelator desferrioxamine, indicating a role for iron in inactivation. IRP1 inactivation was reversible since re-exposure of hypoxically-treated cells to 21% O2 increased RNA binding activity ~7-fold after 21 h with an increase in activity seen as early as 1-h post-reoxygenation. IRP1 protein levels were unaffected during hypoxia as well as during reoxygenation. Whereas the protein synthesis inhibitor cycloheximide did not block IRP1 inactivation during hypoxia, it completely blocked IRP1 reactivation during subsequent reoxygenation. Reactivation of IRP1 during reoxygenation was also partially blocked by the phosphatase inhibitor okadaic acid. Finally, reactivated IRP1 was found to be resistant to inactivation by exogenous iron known to down-regulate its activity during normoxia. These data demonstrate that IRP1 RNA binding activity is post-translationally regulated during hypoxia and hypoxia/reoxygenation. Regulation of IRP1 by changing oxygen tension may provide a novel mechanism for post-transcriptionally regulating gene expression under these stresses.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Mammalian cells employ multiple mechanisms that allow them to adapt to changes in oxygen concentration. During hypoxia, a cellular response is mounted aimed at counteracting the effects of limited O2 by the increased expression of specific genes such as erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and inducible nitric oxide synthase (reviewed in Ref. 1). The transcriptional regulation of these and other hypoxia-induced genes is dependent on the activation of hypoxia-inducible transcription factor-1 (reviewed in Refs. 1 and 2), although post-transcriptional processes have been described (1). The mechanism by which O2 concentration is sensed by cells is not known. One clue comes from studies showing that iron chelation mimics the hypoxic response (3, 4), suggesting that O2 concentration may be sensed by a hemoprotein (1, 5).

When O2 is restored to hypoxic cells, reactive oxygen species (ROS),1 including superoxide (Obardot 2), hydrogen peroxide (H2O2), and hydroxyl radical (HO·) increase (6). The elevated levels of ROS produced during oxidative stress can damage DNA, proteins, and lipids. Iron is central in contributing to ROS-induced cell damage due to its ability to catalyze the formation of the highly reactive HO· via Fenton chemistry. This notion is supported by studies showing that iron chelation can protect cells from H/ReO2 injury (7-10).

Although free iron is toxic, it is required by most cells for survival, and consequently, cells have developed mechanisms to regulate iron uptake and sequestration. In eukaryotes, iron levels are post-transcriptionally regulated by the iron regulatory proteins 1 and 2 (IRP1 and IRP2) (reviewed in Refs. 11 and 12). IRPs exert their regulatory functions by binding to RNA stem-loop structures known as iron responsive elements (IREs). IREs are located in either the 5'- or 3'-untranslated regions of specific mRNAs encoding proteins involved in iron uptake, storage, or utilization. IREs are found in the 5'-untranslated regions of the iron storage protein ferritin mRNA, as well as in the mRNAs for the iron utilization proteins of mitochondrial aconitase, erythroid 5-aminolevulinate synthase, and succinate dehydrogenase. IREs are found in the 3'-untranslated regions of mRNAs encoding iron uptake proteins, such as the transferrin receptor and the metal-ion transporter (13). IRP1 binding to the 5'-IRE of ferritin mRNA inhibits translation initiation, whereas IRP1 binding to IREs located in the 3'-untranslated regions of the transferrin receptor stabilizes this mRNA.

IRP1 is similar to mitochondrial aconitase with the features that both can form a [4Fe-4S] cluster (14) and are active aconitases (15, 16). When cells are iron-replete, [4Fe-4S] cluster formation converts IRP1 to the non-RNA-binding cytosolic aconitase (c-aconitase) (15, 16). The ability of IRP1 to switch between a RNA-binding form and a c-aconitase form allows for the coordinated iron-dependent regulation of IRE mRNAs. By controlling the amount of iron taken up, stored, and utilized by cells, iron levels are maintained, and iron toxicity is avoided.

IRP2 shares 61% identity to IRP1 and binds IRE mRNAs (17, 18). Like IRP1, IRP2 RNA binding activity is modulated by iron (19, 20), but is thought not to contain a [4Fe-4S] cluster similar to IRP1 nor possess aconitase activity (20, 21). In iron-replete cells, IRP2 RNA binding activity is decreased due to iron-targeted degradation by the proteasome (19, 20, 22-24).

The ROS H2O2 (25-28), nitric oxide (NO·) (27, 29-33), and Obardot 2 (34-38) inactivate mitochondrial and c-aconitases by the disassembly of the [4Fe-4S] cluster. In addition, signaling pathways have been implicated in IRP1 regulation in H2O2-treated cells (26-28). The post-translational regulation of IRP1 activity by ROS couples the regulation of iron homeostasis with the oxidative environment of the cell.

Since cellular production of ROS is related to O2 concentration, and since there exists a relationship between iron and O2, we investigated the effects of hypoxia and H/ReO2 on IRP1 and IRP2 RNA binding activity. Our experiments demonstrate that IRP1, but not IRP2 RNA binding activity, decreased during hypoxia, and was reversible upon subsequent reoxygenation. The regulation of IRP1 during hypoxia and H/ReO2 occurred post-translationally without changes in protein levels. IRP1 inactivation during hypoxia required iron, indicating a role for the [4F4-4S] cluster. Interestingly, reoxygenation-induced IRP1 activation was not abrogated in the presence of exogenous iron.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture and Hypoxia/Reoxygenation Conditions-- Rat hepatoma cells (FTO2B) were cultured at 37 °C in atmospheric air plus 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin. Primary cardiac myocytes were prepared from the ventricles of 1-day-old rats (39) and cultured under similar conditions.

For hypoxia experiments, 1.2 × 106 hepatoma cells and 5.0 × 105 cardiac myocytes were plated in 30-mm dishes in 1.2 ml of media. After 8 h, the cells were then placed in a sealed, humidified modular chamber (Billups-Rothenberg, del Mar, CA) and flushed for 15 min at 4 p.s.i. with 3% O2, 5% CO2, balance N. Normoxic cells were similarly treated in a second chamber flushed with 21% O2, 5% CO2, balance N. For time course experiments, cells were rapidly moved from one chamber to another with the chambers immediately flushed with the appropriate gas. Reoxygenation of hypoxic cultures took place at 37 °C in a chamber flushed with 21% O2, 5% CO2, balance N.

For iron experiments, cells were treated with 50 µg/ml ferric ammonium citrate (FAC) or with 100 µM desferrioxamine mesylate (Df) (Sigma). Protein synthesis was inhibited with 20 µg/ml cycloheximide (22) and phosphatases were inhibited with 100 nM okadaic acid (Life Technologies, Inc.). All experiments were repeated at least three times with representative results shown.

Cytosolic Extract Preparation and RNA-Band Shift Assays-- Cells were washed twice with 1 ml of phosphate-buffered saline and cytosolic extracts were prepared by incubating the cells in 150 µl of lysis buffer (20 mM Hepes, pH 7.6, 25 mM KCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) for 5 min. Cell debris was removed by centrifugation at 15,000 × g for 30 min at 4 °C and protein concentration was determined by the Coomassie assay (Pierce).

RNA-band shift assays were performed as described previously (40) using 12 µg of cytosolic extract and a 32P-labeled ferritin IRE probe (50,000 cpm). RNA-protein complexes were resolved on 5% nondenaturing polyacrylamide gels in 0.5 × TBE and visualized by autoradiography. Quantification of RNA binding activity was performed using a Molecular Dynamics PhosphorImager.

Immunoblot Analysis-- Cytosolic extracts (130 µg) were separated on an 8% SDS-polyacrylamide gel and the protein was transferred to nitrocellulose membranes. IRP1 was detected by incubation of the membranes with IRP1-specific chicken polyclonal antibodies (41) and detected with a horseradish peroxidase-conjugated secondary antibody followed by visualization by chemiluminesence (NEN Life Science Products Inc.).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Hypoxia Inactivates IRP1 but Not IRP2 RNA Binding Activity-- To determine the effect of hypoxia on IRP1 and IRP2 RNA binding activity, we subjected a rat hepatoma cell line (FTO2B) to normoxic (21% O2) and hypoxic (3% O2) conditions followed by quantification of IRP1 and IRP2 RNA binding activity by RNA-band shift assays. IRP1 RNA binding activity decreased ~1.5-fold after 1 h of hypoxic exposure with a maximum decrease of ~2.8-fold by 6 h (Fig. 1, A and C). No significant changes in IRP2 RNA binding activity were observed. To determine the total amount IRP1 RNA binding activity, beta -mercaptoethanol was added to the binding reactions prior to addition of the 32P-labeled IRE RNA (Fig. 1, B and D). beta -Mercaptoethanol activates "latent" IRP1 RNA binding activity thus giving the total amount of IRP1 activity in an extract (42). Addition of beta -mercaptoethanol resulted in a similar increase in IRP1 RNA binding activity in all extracts, suggesting that the inactivation of IRP1 activity was not due to decreased levels of IRP1 protein nor to modifications that irreversibly inactivate IRP1 RNA binding. Immunoblot analysis using anti-IRP1 antibodies demonstrated that IRP1 levels did not change during hypoxia (Fig. 1E). The decrease in IRP1 RNA binding activity during hypoxia cannot be mimicked by purified recombinant IRP1 (data not shown). Furthermore, hypoxic inactivation of IRP1 RNA binding activity was not affected by inhibition of transcription by actinomycin D or translation by cycloheximide (data not shown). These data indicated that hypoxia inactivated IRP1 RNA binding activity by a post-translational mechanism not requiring de novo transcription or translation.


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Fig. 1.   Hypoxia inactivates IRP1 but not IRP2 RNA binding activity. A, rat FTO2B hepatoma cells were exposed for 21 h to normoxia (C) or hypoxia for 1-21 h and 12 µg of cytosolic extract was subjected to RNA binding assays. B, extracts in A were preincubated with 2% beta -mercaptoethanol which activates latent IRP1 (42), resulting in total IRP1 RNA binding activity. C and D, RNA binding activity of IRP1 and IRP2 complexes in A and B were quantified by PhosphorImager analysis and plotted as percent of control (100% = normoxic IRP1 activity without beta -mercaptoethanol). Note the differences in scales between C and D. E, extracts in A were immunoblotted and probed with anti-IRP1 polyclonal antiserum and detected by chemiluminesence. Molecular mass standards are indicated.

Iron Is Required for Hypoxia-induced IRP1 Inactivation-- Since IRP1 inactivation during hypoxia was regulated post-translationally, this suggested that iron might be responsible for the inactivation of IRP1 during hypoxia by promoting the formation of the [4Fe-4S] cluster which precludes RNA binding. To determine if iron played a role in IRP1 inactivation by hypoxia, FTO2B cells were treated with the iron chelator Df for 18 h under normoxia prior to hypoxic exposure in the continued presence of Df. Iron chelation prior to hypoxia abolished hypoxia-induced IRP1 inactivation (Fig. 2). As expected, the normoxic control cells treated with Df showed an increase in IRP1 and IRP2 RNA binding activity (Fig. 2) (20, 43). These data showed that chelatable iron was required for IRP1 inactivation by hypoxia, suggesting involvement of the [4Fe-4S] cluster in hypoxic regulation of IRP1.


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Fig. 2.   Iron chelation abolishes hypoxia-induced IRP1 inactivation. A, FTO2B cells were grown in 21% O2 in the absence or presence of 100 µM desferrioxamine (-/+Df) for 18 h prior to exposure to normoxia (C) or hypoxia (H) for 6 h in the continued presence of Df as indicated. Cytosolic extracts (12 µg) were subjected to RNA binding assays. Shown are IRP1 (black bars) and IRP2 (gray bars) activities plotted as percent of control (100% = IRP1 normoxic activity without Df) as determined by PhosphorImager analysis.

One possibility to explain the decrease in IRP1 activity during hypoxia is that lowered O2 tension gave rise to an increase in cellular free iron. If this were the case it would be expected that IRP2 RNA binding activity would also diminish during hypoxia since its activity is similarly decreased by increased iron (19, 20, 22). However, IRP2 was not affected in cells exposed to 3% O2 for up to 21 h (Fig. 1). To determine if iron is capable of decreasing IRP2 RNA binding activity during hypoxia, FAC was added to the medium during the last 6 h of a 12-h hypoxic exposure. IRP2 RNA binding activity decreased in hypoxic cells treated with FAC (Fig. 3A), indicating that the mechanism responsible for IRP2 iron-mediated degradation remained intact during hypoxia.


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Fig. 3.   Iron sensitivity of IRP1 and IRP2 during hypoxia. A, FTO2B cells were grown under normoxia (C) or exposed to hypoxia (H) for 12 h. Ferric ammonium citrate (50 µg/ml) was added to a parallel dish for the last 6 h of a 12-h hypoxic exposure (H + FAC). Cytosolic extracts (12 µg) were then subjected to a RNA-band shift assays. B, relative sensitivity of IRP1 and IRP2 to iron was determined by growing cells under normoxia in the presence of 0-1.0 µg/ml FAC for 6 h. Cytosolic extracts (12 µg) were subjected to RNA binding assays and relative amount of RNA binding activity in arbitrary units (AU) of IRP1 and IRP2 complexes were quantified by PhosphorImager analysis.

One explanation for the differential regulation of IRP1 and IRP2 during hypoxia might be due to differences in sensitivity of these proteins to iron. To determine if IRP1 and IRP2 respond to similar concentrations of iron, cells were treated with FAC ranging from 0 to 1.0 µg/ml for 6 h under normoxic conditions, and RNA binding activity of IRP1 and IRP2 was measured. Fig. 3B shows that IRP1 and IRP2 had equal sensitivities to FAC. These data suggested that the decrease in IRP1 RNA binding activity during hypoxia was not due to an increase in the concentration of cellular free iron.

IRP1 Inactivation by Hypoxia Is Reversible-- To test whether IRP1 inactivation by hypoxia was reversible, cells were first exposed to 3% O2 for 6 h to achieve maximum IRP1 repression (Fig. 1) and then re-exposed to 21% O2 for 1-21 h followed by quantification of IRP1 RNA binding activity (Fig. 4, A and B). Within 1 h post-reoxygenation, IRP1 activity increased ~0.5-fold and by 3 h IRP1 activity equaled that of normoxic-treated cells. At 18 h, IRP1 activity increased ~6-fold relative to hypoxic, non-reoxygenated cells. No significant changes in IRP2 RNA binding activity were observed during reoxygenation, indicating that any changes in iron levels were not sensed by IRP2 (Fig. 4, A and B). Immunoblot analysis of IRP1 demonstrated that the increase in IRP1 activity during reoxygenation was not due to increased protein levels (Fig. 4C).


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Fig. 4.   Hypoxia-induced inactivation of IRP1 RNA binding is reversible. FTO2B cells were exposed to hypoxia for 6 h followed by re-exposure to normoxia for 1-21 h as indicated. A, cytosolic extracts (12 µg) were subjected to RNA binding assays. B, relative IRP1 RNA binding was quantified by PhosphorImager analysis and plotted as percent of control. C, extracts in A (130 µg) were immunoblotted as in Fig. 1E. Molecular mass standards are indicated.

To determine if IRP1 reactivation during reoxygenation was sensitive to iron, FAC was added to the medium of a hypoxic culture at the onset of reoxygenation. Fig. 5 shows the surprising result that exogenous iron had no effect on IRP1 reactivation during reoxygenation, unlike the affect of FAC on IRP1 under normoxic conditions (Fig. 3B). In contrast, IRP2 RNA binding activity was appropriately decreased during reoxygenation, indicating that reoxygenation did not affect iron uptake.


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Fig. 5.   IRP1 RNA binding is not influenced by iron during reoxygenation. FTO2B cells were exposed to normoxia (C), 9 h hypoxia (H), 9 h hypoxia with FAC (50 µg/ml) present for last 3 h (H + FAC), or FAC (50 µg/ml) added after 6 h of hypoxia and present during 3 h of reoxygenation (H/Re02 + FAC). Cytosolic extracts (12 µg) were then subjected to RNA binding assays.

To determine if reoxygenation induced an activity required for post-translational activation of IRP1, hypoxic cells were treated with cycloheximide for 30 min prior to reoxygenation for 3 h. Cycloheximide completely blocked reactivation of IRP1 RNA binding activity during reoxygenation, indicating that H/ReO2 induced the synthesis of a protein required for IRP1 reactivation (Fig. 6). It is possible that the requirement for protein synthesis might be due to re-synthesis of IRP1 during reoxygenation, however, the lack of change in IRP1 levels during H/ReO2 make this less plausible (Fig. 4C). IRP1 RNA binding activity has been shown to increase in cells subjected to H2O2, and this increase was sensitive to okadaic acid (25, 26, 28). During reoxygenation, it is expected that cells are oxidatively stressed, and that ROS would be increased. Okadaic acid blocked IRP1 reactivation during reoxygenation by ~50%, suggesting that IRP1 reactivation may fall downstream of a phosphorylation/dephosphorylation event stimulated by H/ReO2. Whether the mechanism regulating IRP1 reactivation during H/ReO2 is similar to the mechanism operating during H2O2 exposure is unknown.


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Fig. 6.   Cycloheximide and okadaic acid block IRP1 reactivation. Cycloheximide (Cyx) at 20 µg/ml in ethanol or okadaic acid (OA) at 100 nM in Me2SO were added to FTO2B cells during the last 45 min of a 14-h hypoxic exposure with vehicle added in parallel to control dishes (V). Cells were then reoxygenated for 3 h in the continued presence of cycloheximide, okadaic acid, or vehicle. The first two lanes are normoxically (C) and hypoxically (H) grown cells (17 h) without any treatment. Cytosolic extracts (12 µg) were then subjected to RNA binding assays. Similar results were obtained repeating the experiment 4 times with a representative gel shown.

IRP1 RNA Binding Activity Is Inactivated in Primary Cardiac Myocytes during Hypoxia and H/ReO2-- To determine if IRP1 was regulated during hypoxia and H/ReO2 in other cells, primary cardiac myocytes, which have been shown to be sensitive to O2 concentration (44, 45), were exposed to hypoxia for 1-6 h and hypoxia for 14 h followed by reoxygenation for 3 and 6 h. Fig. 7 shows that IRP1 RNA binding activity was inactivated during hypoxia, and subsequently, reactivated during H/ReO2.


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Fig. 7.   IRP1 from primary cardiac myocytes is O2 regulated. Rat primary cardiac myocytes were exposed to hypoxia for 1-18 h or hypoxia for 6 h followed by reoxygenation for 3 and 6 h as indicated. Cytosolic extracts (12 µg) were then subjected to RNA binding assays and the relative IRP1 RNA binding activity was quantified by PhosphorImager analysis and plotted as percent of control.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We investigated the effects of O2 on the regulation of IRP1 and IRP2, and showed that IRP1 is post-translationally regulated by hypoxia and H/ReO2. During hypoxia, IRP1 RNA binding activity from rat hepatoma cells and primary cardiac myocytes decreased. Hypoxic inactivation of IRP1 was reversed during subsequent reoxygenation. Furthermore, our data suggested that regulation was due in part to the lability of the [4Fe-4S] cluster, indicating that the cluster could serve as a target for regulating IRP1 during hypoxia and H/ReO2. IRP2 RNA binding activity did not significantly change under our conditions of hypoxia and H/ReO2, presumably because it does not contain a [4Fe-S] cluster similar to IRP1 (21).

Iron chelation abrogated the decrease in IRP1 RNA binding activity during hypoxia, indicating a requirement for IRP1 to form and/or maintain its [4Fe-4S] cluster. By virtue of iron's ability to convert IRP1 from its RNA-binding form to its [4Fe-4S] c-aconitase form, an increase in iron during hypoxia is a reasonable explanation for the decrease in IRP1 activity. It would be expected that IRP2 activity would decrease if cellular iron levels increased during hypoxia. This, however, was not the case. IRP2 can responded appropriately to exogenous iron, indicating that hypoxic FTO2B cells can sense iron. In addition, the sensitivities of IRP1 and IRP2 to iron were found to be similar. Using IRP2 as an indicator of cellular iron status, we suggest that hypoxic inactivation of IRP1 may be due to factors other than increased cellular iron.

A model to explain hypoxia-induced inactivation of IRP1 activity is one in which the stability of the [4Fe-4S] cluster is enhanced in an O2 limiting environment. In this model, IRP1 from normoxic cells would exist in a dynamic state interconverting between the [4Fe-4S] cluster c-aconitase form and the apo-IRP1 RNA-binding form. When O2 is limiting, the equilibrium would favor stabilization of the [4Fe-4S] cluster, leading to a decrease in total IRP1 RNA binding and a slight (~15%) increase in c-aconitase activity (data not shown). The modest increase in c-aconitase activity observed in our experiments is not surprising since under normoxic conditions FTO2B cells contain ~15% of IRP1 in the RNA-binding form and ~85% in the c-aconitase. Hypoxia-induced IRP1 inactivation might result in increased ferritin synthesis similar to that observed in hypoxic oligodendrocytes (46).

How could the [4Fe-4S] cluster be stabilized during hypoxia? One possibility is the involvement of ROS, especially Obardot 2. Both prokaryotic and eukaryotic aconitases are reversibly inactivated by Obardot 2 via [4Fe-4S] cluster disassembly (34, 35, 37, 38), and IRP1 activity was increased in cells treated with the Obardot 2 generator paraquat (27). Based on these studies it would seem likely that a decrease in Obardot 2 during hypoxia could account for IRP1 inactivation by stabilization of its non-RNA-binding [4Fe-4S] form.

Since H/ReO2 has been shown to generate ROS in a variety of cell types, and since IRP1 is sensitive to ROS (25-28), the question arose whether IRP1 activity was affected during reoxygenation of hypoxic cells. IRP1 RNA binding activity increased in FTO2B cells and in primary cardiac myocytes without an increase in IRP1 protein levels. The increase in IRP1 activity during reoxygenation may result from an increase in ROS, leading to [4Fe-4S] cluster disassembly. Surprisingly, reactivation of RNA binding activity of IRP1 during reoxygenation was insensitive to exogenous iron. That iron was being taken up into cells is demonstrated by the decrease in IRP2 activity in iron-treated reoxygenated cells. One explanation for the inability of exogenous iron to decrease IRP1 RNA binding activity is that ROS-induced disassembly of the [4Fe-4S] cluster is more rapid than iron-stimulated cluster assembly resulting in the accumulation of apo-IRP1. The increase in IRP1 RNA binding during reoxygenation could increase transferrin receptor levels, leading to increased iron uptake. In this scenario, ferritin synthesis might be repressed, contributing further to increases in cellular iron. Whether IRP2 can compensate for IRP1 dysregulation during reoxygenation remains to be determined. The aberrant iron regulation of IRP1 during reoxygenation could contribute to cellular damage seen during ischemia-reperfusion injury.

In addition to the above model where IRP1 activation during H/ReO2 may occur by direct attack of ROS on the [4Fe-4S] cluster, signaling pathways are likely to be involved, as has been described for H2O2-induced IRP1 activation (28). Support for this comes from data demonstrating that cycloheximide completely blocked, while okadaic acid partially blocked, IRP1 reactivation during reoxygenation. These data suggested the possibility that H/ReO2 stimulates the synthesis of a protein(s) which is in a pathway ultimately leading to IRP1 activation. One potential candidate may be a kinase. Phosphorylation of apo-IRP1 at serine residues predicted to be near the active site cleft have been shown to stimulate RNA binding activity (47, 48). It is possible that phosphorylation, or some other modification, of IRP1 stimulated by H/ReO2 precludes formation of the [4Fe-4S], thus increasing apo-IRP1 levels. We cannot, however, rule out the possibility that cycloheximide might have affected ROS generation during H/ReO2. Collectively, our data support a model where IRP1 activation during H/ReO2 require a signal that modulates IRP1 activity, perhaps by phosphorylation, in addition to the disassembly of the [4Fe-4S] cluster.

The lability of the IRP1 [4Fe-4S] cluster to the oxidation status of the cell could serve as a mechanism for post-transcriptionally regulating gene expression during hypoxia and H/ReO2. Whether this regulation occurs in other cells types remains to be determined. Furthermore, identification of downstream mRNA targets (e.g. ferritin and transferrin receptor) will be an important next step in understanding the biological role of IRP1 regulation by changing O2 tension. The consequences of O2 regulation of IRP1 may prove to be far reaching, both in terms of cellular adaptation to hypoxia as well as cellular damage seen during H/ReO2.

    ACKNOWLEDGEMENTS

We thank Drs. Christi Terry and Don Ayer for helpful comments during the preparation of this manuscript and Jackie Thorburn for providing the cardiac myocytes.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM45201 (to E. A. L.) and T32 DK07115 (to E. S. H.) and a grant from the Huntsman Cancer Institute (to E. A. L.).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: University of Utah, 15 N. 2030 E., Bldg. 533, Rm. 2100, Salt Lake City, UT 84112. Tel.: 801-585-5002; Fax: 801-585-3501; E-mail: betty.leibold{at}genetics.utah.edu.

1 The abbreviations used are: ROS, reactive oxygen species; IRP, iron regulatory protein; IRE, iron responsive element; c-aconitase, cytosolic aconitase; FAC, ferric ammonium citrate; Df, desferrioxamine mesylate.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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