From the Eccles Program in Human Molecular Biology and Genetics and the Department of Medicine, University of Utah, Salt Lake City, Utah 84112
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ABSTRACT |
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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.
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INTRODUCTION |
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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 (O2), 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 O2 (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.
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EXPERIMENTAL PROCEDURES |
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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.).
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RESULTS |
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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,
-mercaptoethanol was added to the binding reactions prior to
addition of the 32P-labeled IRE RNA (Fig. 1, B
and D).
-Mercaptoethanol activates "latent"
IRP1 RNA binding activity thus giving the total amount of IRP1 activity
in an extract (42). Addition of
-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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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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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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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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DISCUSSION |
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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 O2. Both prokaryotic and eukaryotic aconitases are reversibly inactivated by
O
2 via [4Fe-4S] cluster disassembly (34, 35, 37, 38), and
IRP1 activity was increased in cells treated with the O
2 generator paraquat (27). Based on these studies it would seem likely that a decrease in O
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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