Binding Of Iron Regulatory Proteins Research Articles

ANP-induced decrease of iron regulatory protein activity is independent of HO-1 induction.

Atrial natriuretic peptide (ANP)-preconditioned livers are protected from ischemia-reperfusion injury. ANP-treated organs show increased expression of heme oxygenase (HO)-1. Because HO-1 liberates bound iron, the aim of our study was to determine whether ANP affects iron regulatory protein (IRP) activity and, thus, the levels of ferritin. Rat livers were perfused with Krebs-Henseleit buffer [+/-ANP, 8-bromo-cGMP (8-Br-cGMP), and tin protoporphyrin, 20 min], stored in University of Wisconsin solution (4 degrees C, 24 h), and reperfused (120 min). IRP activity was assessed by gel-shift assays, and ferritin, IRP phosphorylation, and PKC localization were assessed by Western blot. Control livers displayed decreased IRP activity at the end of ischemia but no change in ferritin content during ischemia and reperfusion. ANP-pretreated livers showed reduced IRP activity, an effect mimicked by 8-Br-cGMP. Ferritin levels were increased in ANP-pretreated organs. Simultaneous perfusion of livers with ANP and tin protoporphyrin did not reduce ANP-induced action, arguing against a role for HO-1 in changes in IRP activity. ANP and 8-Br-cGMP decreased membrane localization of PKC-alpha and PKC-epsilon, but this modulation of PKC seems unrelated to inhibition of IRP binding. This work shows the cGMP-mediated attenuation of IRP binding activity by ANP, which results in increased hepatic ferritin levels. This change in IRPs is independent of ANP-induced HO-1 and reduced PKC activation.

Effects of sham air and cigarette smoke on A549 lung cells: implications for iron-mediated oxidative damage

Inhalation of airborne pollution particles that contain iron can result in a variety of detrimental changes to lung cells and tissues. The lung iron burden can be substantially increased by exposure to cigarette smoke, and cigarette smoke contains iron particulates, as well as several environmental toxins, that could influence intracellular iron status. We are interested in the effects of environmental contaminants on intracellular iron metabolism. We initiated our studies using lung A549 type II epithelial cells as a model, and we evaluated the effects of iron dose and smoke treatment on several parameters of intracellular iron metabolism. We show that iron at a physiological dose stimulates ferritin synthesis without altering the transferrin receptor (TfR) mRNA levels of these cells. This is mediated primarily by a reduction of iron regulatory protein 2. Higher doses of iron reduce iron regulatory protein-1 binding activity and are accompanied by a reduction in TfR mRNA. Thus, for A549 cells, different mechanisms influencing IRP-IRE interaction allow ferritin translation in the presence of TfR mRNA to provide for iron needs and yet prevent excessive iron uptake. More importantly, we report that smoke treatment diminishes ferritin levels and increases TfR mRNA of A549 cells. Ferritin serves as a cytoprotective agent against oxidative stress. These data suggest that exposure of lung cells to low levels of smoke as are present in environmental pollutants could result in reduced cytoprotection by ferritin at a time when iron uptake is sustained, thus enhancing the possibility of lung damage by iron-mediated oxidative stress.

Alterations in cellular IRP-dependent iron regulation by in vitro manganese exposure in undifferentiated PC12 cells

Manganese (Mn) may interfere with iron regulation by altering the binding of iron regulatory proteins (IRPs) to their response elements found on the mRNA encoding proteins critical to iron homeostasis. To explore this, the effects of 24-h in vitro manganese exposure (1, 10, 50, and 200 μM Mn) on: (i) total intracellular and labile iron concentrations; (ii) the cellular abundance of transferrin receptor (TfR), H- and L-ferritin, and mitochondrial aconitase proteins; and (iii) IRP binding to a [ 32P] - labeled mRNA sequence of L-ferritin were evaluated in undifferentiated PC12 cells. In vitro manganese exposure altered the cellular abundance of TfR, H-/L-ferritin, and m-aconitase, resulting in an increase in labile iron. This latter effect led to a decrease in IRP binding activity at the lower (10 and 50 μM) manganese exposures. In contrast, 200 μM manganese exposure increased IRP binding, in spite of the significant increase in labile iron. These data indicate that at lower exposures, manganese directly interfered with IRP-dependent translational events, producing an increase in labile iron, which in turn signaled a decrease in IRP binding at 24 h. At higher exposures, the intracellular burden of manganese resulted in overt cytotoxicity and appeared to compromise the normal compensatory response to increased labile iron, producing increased IRP binding. We conclude that low to moderate manganese exposure interferes with cellular iron regulation, and thus may serve as a contributory mechanism underlying manganese neurotoxicity.

Multiple, Conserved Iron-responsive Elements in the 3′-Untranslated Region of Transferrin Receptor mRNA Enhance Binding of Iron Regulatory Protein 2

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

Pathogenesis of Hyperferritinemia Cataract Syndrome

ABSTRACT Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder characterized by bilateral cataracts and increased serum L-ferritin, in the absence of iron overload. Under physiological conditions, ferritin synthesis is finely regulated at the translational level by iron availability. This regulation is achieved by the high-affinity interaction between cytoplasmic mRNA-binding proteins (iron regulatory proteins, IRPs), and mRNA stem-loop structures, known as iron responsive elements (IREs), located in the untranslated regions (UTRs) of the mRNAs. A single IRE is located on the 5′ UTR of a series of genes involved in iron metabolism, like L-ferritin, and the binding IRE-IRPs represses these genes translation. The deregulation of ferritin production responsible of HHCS is caused by heterogeneous mutations in the iron regulatory element (IRE) of L-ferritin that interfere with the binding of iron regulatory proteins, disrupting the negative control of l-ferritin synthesis and causing the constitutive up-regulation of ferritin l-chains. The HHCS families originate from different countries of Europe and North America, suggesting that HHCS may be distributed widely throughout the world and not sporadic, whereas its prevalence remains to be established. The lens seems to be particularly sensitive to the increased amount of L-ferritin and the alteration of the proteic equilibrium in this tissue can be responsible of the cataract. In spite of the elucidation of the genetic basis, the genotype phenotype correlation is not clear. Recently, a study based on the thermo-denaturation profile and dissociation constant of the IRE-IRP complex performed for several mutated IREs has provided evidence for a possible correlation between heterogeneous IRE mutations and serum ferritin levels. On the other hand, the in vivo relevance of these conclusions has not been determined completely. A clinical variability among subjects sharing the same mutation, whether they belonged to the same family or not, has also been demonstrated. These findings suggest that, besides the L-ferritin IRE genotype, additional factors are likely to modulate the lens involvement and the rate of progression to severe cataract in HHCS patients.

An Iron-responsive Element Type II in the 5′-Untranslated Region of the Alzheimer's Amyloid Precursor Protein Transcript

Iron-responsive elements (IREs) are the RNA stem loops that control cellular iron homeostasis by regulating ferritin translation and transferrin receptor mRNA stability. We mapped a novel iron-responsive element (IRE-Type II) within the 5'-untranslated region (5'-UTR) of the Alzheimer's amyloid precursor protein (APP) transcript (+51 to +94 from the 5'-cap site). The APP mRNA IRE is located immediately upstream of an interleukin-1 responsive acute box domain (+101 to +146). APP 5'-UTR conferred translation was selectively down-regulated in response to intracellular iron chelation using three separate reporter assays (chloramphenicol acetyltransferase, luciferase, and red fluorescent protein reflecting an inhibition of APP holoprotein translation in response to iron chelation. Iron influx reversed this inhibition. As an internal control to ensure specificity, a viral internal ribosome entry sequence was unresponsive to intracellular iron chelation with desferrioxamine. Using RNA mobility shift assays, the APP 5'-UTRs, encompassing the IRE, bind specifically to recombinant iron-regulatory proteins (IRP) and to IRP from neuroblastoma cell lysates. IRP binding to the APP 5'-UTR is reduced after treatment of cells with desferrioxamine and increased after interleukin-1 stimulation. IRP binding is abrogated when APP cRNA probe is mutated in the core IRE domain (Delta4 bases:Delta83AGAG86). Iron regulation of APP mRNA through the APP 5'-UTR points to a role for iron in the metabolism of APP and confirms that this RNA structure can be a target for the selection of small molecule drugs, such as desferrioxamine (Fe chelator) and clioquinol (Fe, Cu, and Zn chelator), which reduce Abeta peptide burden during Alzheimer's disease.

Lack of up-regulation of ferritin is associated with sustained iron regulatory protein-1 binding activity in the substantia nigra of patients with Parkinson's disease.

Dopaminergic neurones degenerate during Parkinson's disease and cell loss is most extensive in the subpopulation of melanized neurones located in the substantia nigra pars compacta. Iron accumulation, together with a lack of up-regulation of the iron-storing protein, ferritin, has been reported and may contribute to increased oxidative stress in this region. We investigated the binding activity of iron regulatory protein-1 (IRP1) to the iron-responsive element that precludes ferritin mRNA translation, in the substantia nigra of a group of parkinsonian patients who presented a statistically significant reduction in the number of nigral melanized-neurones and an increased iron content, together with unchanged H-ferritin and L-ferritin subunit levels as compared to matched controls. The levels of ferritin mRNAs and the binding activity of IRP1 to the iron-responsive element of ferritin mRNA did not differ significantly between the two groups. Moreover, there was no detectable contribution of the iron regulatory protein-2 (IRP2) binding activity. No change in IRP1 control of ferritin mRNA translation explains the lack of up-regulation of ferritin expression in cytoplasmic extracts of SNpc that would be normally expected with cytosolic iron accumulation. The data of this study do not favor changes in transcription and post-transcriptional regulation of ferritin expression in Parkinson's disease and suggest a 'compartmentalized' iron accumulation.

Erythroblast Iron Metabolism in Sideroblastic and Sideropenic States

Iron appears to exert self-regulatory control over erythroblast iron uptake, iron storage and its incorporation into haem. It does this via iron regulatory proteins (IRPs) which bind reversibly to the iron responsive elements (IREs) on the mRNA of transferrin receptor (TfR), erythroid 5-aminolaevulinic acid synthase (ALA-S2) and ferritin. Iron deficiency leads to the binding of IRP to IRE. This binding inhibits the translation of mRNA for ALA-S2 and ferritin but stabilizes mRNA for TfR expression.Sideroblastic erythropoiesis is highly ineffective and characterized by mitochondrial iron loading. The study of X-linked sideroblastic anaemia has shown that the entry of iron into the mitochondria is poorly controlled and able to occur when protoporphyrin production is reduced, as is seen with the ALA-S2 mutations, or when it is increased as has been seen with ABC7 transporter mutations.Sideropenia characterises both iron deficiency anaemia (IDA) and the anaemia of chronic disease (ACD). Erythroblasts in ACD seem doubly equipped to protect their iron supply with their ability to increase the efficiency of transferrin-iron uptake as well as to activate the IRP/IRE system to increase surface TfR production. This increase in efficiency restricts the need to increase surface TfR production and maintains serum soluble TfR (sTfR) values within the normal range in iron replete ACD. The coexistence of iron deficiency with chronic disease, however, is associated with an increase in both the efficiency and number and a highly significant rise in sTfR values.

The Intracellular Location of Iron Regulatory Proteins Is Altered as a Function of Iron Status in Cell Cultures and Rat Brain

Iron regulatory proteins (IRPs) are proteins involved in the regulation of intracellular iron homeostasis that bind to specific mRNA structures termed iron responsive elements (IREs). Because the target mRNAs for the IRPs are both cytosolic and membrane associated, we hypothesize that movement of IRPs between the cytosolic and the membrane associated subcellular fractions occurs in response to intracellular iron changes. We tested this hypothesis in a cell culture model, using mouse fibroblast cells (NIH 3T3) and macrophage cells (J774), and in a rat model of early iron deficiency and excess. This presented the first opportunity to examine IRP binding activity in rat brain during states of dietary iron deficiency and excess. Binding activity for IRPs was demonstrated in both membrane and cytosolic fractions in the cell lines and the rat brain homogenates. Although IRP binding activity is predominantly located in the cytosol (90%), there was increased IRP/IRE binding activity in both cytosolic and membrane fractions when the cells were treated with deferoxamine, and decreased binding activity after treatment with iron. In the rat study, brain cortex, hippocampus and striatum homogenates had more IRP binding activity in iron-deficient rats and less in iron-supplemented rats in a region- and time-specific manner. The intracellular distribution of IRPs also changed between the cytosolic and membrane fractions of the brain homogenates in conjunction with changes in iron. These in vivo studies are consistent with the cell culture analyses showing intracellular redistribution of IRPs as a function of iron status. The results of these experiments extend our understanding of cytoplasmic mRNA binding protein activity and raise questions regarding the mechanism by which mRNA binding proteins can locate their target mRNAs within cells. The elucidation of this mechanism will have a significant impact on our understanding of eukaryotic gene regulation.

IRP1 Activation by Extracellular Oxidative Stress in the Perfused Rat Liver

The expression of several proteins with critical functions in iron metabolism is regulated post-transcriptionally by the binding of iron regulatory proteins, IRP1 and IRP2, to mRNA iron responsive elements (IREs). In iron-deficient tissues and cultured cells, both IRP1 and IRP2 are activated for high affinity IRE binding. Previous work showed that IRP1 is also activated when cultured cells are exposed to H(2)O(2). The well established role of iron and H(2)O(2) in tissue injury (based on Fenton chemistry) suggests that this response may have important pathophysiological implications. This is particularly relevant in inflammation, where cytotoxic immune cells release large amounts of reactive oxygen species. Here, we describe a rat liver perfusion model to study IRP1 activation under H(2)O(2) generation conditions that mimic a physiological inflammatory response, using steady-state concentrations of H(2)O(2) produced by a glucose/ glucose oxidase/catalase system. We show first that stimulated neutrophils are able to increase serum levels of H(2)O(2) by a factor of 10, even in the presence of H(2)O(2)-degrading erythrocytes. We further show that perfusion of rat liver with glucose oxidase leads to a rapid activation of IRE binding activity in the intact organ. Mobility shift assays with liver extracts and IRP1 or IRP2-specific probes indicate that only IRP1 responds to H(2)O(2). Our study demonstrates a principal existence of iron regulation by oxidative stress at the intact organ level. It also provides a link between iron metabolism and the inflammatory response, as H(2)O(2) is a major product of the oxidative burst of neutrophils and macrophages.

Effects of hyperoxia and iron on iron regulatory protein-1 activity and the ferritin synthesis in mouse peritoneal macrophages

Ferritin is an intracellular iron storage protein and its translation is inhibited by binding of iron regulatory proteins (IRPs) to the iron-responsive element (IRE) located in the 5′ untranslated region of its mRNA. In this paper, we have investigated the effect of hyperoxia and iron on the binding activity of IRP-1 and the ferritin synthesis in mouse peritoneal macrophages. The binding activity of IRP-1 was increased and the ferritin synthesis was suppressed when the macrophages were cultured under hyperoxia, and the reverse occurred under hypoxia. Iron diminished the IRP-1-binding activity and the enhanced synthesis of ferritin. However, this effect was arrested under hyperoxia. Consistently, hypoxia-induced loss of binding activity of IRP-1 and the enhanced synthesis of ferritin were blocked in the presence of an iron chelator deferoxamine. These alterations of the binding activity of IRP-1 in response to oxygen and iron were not reproduced in the cell-free extract. The data suggest that in the macrophages oxygen and iron inversely act on the binding activity of IRP-1 and the ferritin synthesis, and that intracellular mechanism(s) to sense iron and/or oxygen is required for these actions.

Modulation of Cellular Iron Metabolism by Hydrogen Peroxide: EFFECTS OF H2O2 ON THE EXPRESSION AND FUNCTION OF IRON-RESPONSIVE ELEMENT-CONTAINING mRNAs IN B6 FIBROBLASTS

Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H(2)O(2) that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 microm H(2)O(2) for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H(2)O(2) induces a approximately 4-fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H(2)O(2)-treated cells display an increased capacity to sequester (59)Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of (59)Fe intracellular distribution. Our data suggest that H(2)O(2) regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms.

Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression.

The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.

Thyrotropin-releasing Hormone and Epidermal Growth Factor Regulate Iron-regulatory Protein Binding in Pituitary Cells via Protein Kinase C-dependent and -independent Signaling Pathways

Intracellular iron homeostasis is regulated, in part, by interactions between iron-regulatory proteins (IRP1 and IRP2) and iron-responsive elements (IREs) in ferritin and transferrin receptor mRNAs. In addition to iron, cellular oxidative stress induced by H(2)O(2), nitric oxide, and hypoxia, and hormonal activation by thyroid hormone and erythropoeitin have each been shown to regulate IRP binding to IREs. Hormonal signals, in particular mediated through protein kinase C (PKC), play a central role in the modulation of IRP/IRE interactions since phorbol esters were shown to activate IRP binding (Eisenstein, R. S., Tuazon, P. T., Schalinske, K. L., Anderson, S. A., and Traugh, J. A. (1993) J. Biol. Chem. 268, 27363-27370). In pituitary thyrotrophs (TtT97), we found that thyrotropin releasing hormone (TRH) and epidermal growth factor (EGF) increased IRP binding to a ferritin IRE, dependent on PKC and mitogen-activated protein kinase (MAPK) activity. In contrast, TRH and EGF decreased IRP binding in pituitary lactotrophs (GH3), despite activation of PKC and MAPK. IRP1 and IRP2 levels remained constant and IRP2 binding was predominant throughout. TRH and EGF markedly decreased IRP binding in MAPK kinase inhibitor-treated GH3 cells, whereas, they increased IRP binding in phosphatase inhibitor-treated GH3 cells. IRE-dependent CAT reporter translational expression closely reflected IRP binding to the ferritin IRE in both GH3 and TtT97 cells. Interestingly, ferritin protein levels were regulated similarly by TRH in both cell lines. These data link two different cell receptor systems to common signaling pathways that regulate IRP binding and ferritin expression. Remarkably, for TRH and EGF, these effects may be PKC-dependent or -independent determined by the cell type.

Post-transcriptional control via iron-responsive elements: the impact of aberrations in hereditary disease.

Tight regulation of iron metabolism is crucial to avoid formation of deleterious radicals and is mainly executed at the post-transcriptional level. The regulatory loops are exerted by trans-acting iron regulatory proteins (IRPs) and cis-acting stem-loop motifs, termed iron-responsive elements (IREs), located in the untranslated regions (UTRs) of target mRNAs. Iron scarcity induces binding of IRPs to a single IRE in the 5'-UTR of ferritin, eALAS, aconitase and SDHb mRNAs, which specifically suppresses translation initiation. Simultaneous interaction of IRPs with multiple IREs in the 3'-UTR of transferrin receptor (TfR) mRNA selectively causes its stabilization. The pattern is reverted under iron overload: IRP-mRNA binding affinity is reduced, which results in efficient protein synthesis of target transcripts harboring IREs in the 5'-UTR and rapid degradation of TfR mRNA. Although multiple evidences support this model, several studies reported massive alterations in the regulation of iron homeostasis under specific physiological conditions, raising the possibility for additional regulatory events. Intensive analysis of the palindromic IRE consensus sequence revealed the critical elements for the formation of a functional structure and demonstrated the consequences of IRE mutations in IRP binding. Recent investigations indicated the involvement of naturally occurring IRE mutations of the ferritin L subunit in the hyperferritinemia-cataract syndrome, a hereditary disorder. This review summarizes the apparent links between iron-dependent post-transcriptional control and its abnormalities, governed by the properties of a single mRNA stem-loop structure.

Induction of ferritin and heat shock proteins by prostaglandin A1 in human monocytes. Evidence for transcriptional and post-transcriptional regulation.

Prostaglandins of the A type (PGA) exert a cytoprotective activity during hyperthermia and virus infection. This effect is associated with induction of heat shock proteins (HSP) in mammalian cells. We now report that, in human monocytes, PGA1 is able to induce the synthesis of the iron-binding, redox-regulated protein ferritin. L-chain ferritin induction is consequent to a substantial increase in the accumulation of L-chain ferritin transcripts in PGA1-treated cells, whereas H-chain ferritin is regulated post-transcriptionally, consequently to reduction of iron-regulatory protein binding to iron-responsive elements in ferritin mRNA. Ferritin induction is specific for cyclopentenone prostaglandins (PGA1, PGA2, PGJ2, Delta12-PGJ2), whereas other arachidonic acid (AA) metabolites have no effect. In human monocytes, PGA1 also induces heat shock gene transcription via heat shock factor activation, as well as the synthesis of the oxidative-stress protein heme oxygenase (HOS). Differently from HSP, the induction of ferritin by PGA1 is specific for monocytes. Monocytes/macrophages play a pivotal role in inflammation, controlling iron metabolism and releasing a variety of mediators, including proinflammatory reactive oxygen species (ROS), cytokines and AA metabolites. As ferritin, together with hsp70 and HO, plays a key role in protection from oxidant damage, these results suggest that PGA1 may have cytoprotective activity also during oxidative injury.

The effect of intracellular iron concentration and nitrogen monoxide on Nramp2 expression and non-transferrin-bound iron uptake.

Recent studies have demonstrated that the protein product (natural resistance associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts as an Fe transporter involved in the uptake of Fe from transferrin (Tf) and low Mr Fe complexes. Interestingly, there are two splice variants of Nramp2, one with a putative iron-responsive element (IRE) in its 3' untranslated region (UTR) and another without. Due to the importance of Nramp2 in Fe transport, and the presence of an IRE in its 3'-UTR, we have examined the effect of Fe-deprivation, Fe-loading, and nitrogen monoxide on the expression of Nramp2 mRNA. These results were compared to the expression of transferrin receptor (TfR) mRNA which also has IREs in its 3'-UTR and is regulated by Fe and NO via the binding of iron-regulatory proteins (IRPs) to its IREs. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in lysates from a mouse fibroblast cell line (LMTK-). Moreover, reverse transcription-PCR (RT-PCR) demonstrated that both the IRE and non-IRE-containing transcripts were present within these cells. However, there was no change in Nramp2 mRNA expression in LMTK- cells after a 20-h incubation with either the Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrate (FAC), or the NO generator, S-nitroso-N-acetylpenicillamine (SNAP). In contrast, these agents caused a marked change in the RNA-binding activity of the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caused an appropriate change in [59Fe] uptake from [59Fe]Tf, viz., an increase in Fe uptake after exposure to DFO and a decrease after treatment with FAC. As Nramp2 can transport Fe from non-Tf-bound Fe, the effect of preincubation with DFO and FAC was also examined on Fe uptake from [59Fe]nitrilotriacetate and [59Fe]citrate. However, in contrast to the results found for [59Fe]Tf, incubation with DFO and FAC did not result in appropriate regulation of Fe uptake from [59Fe]nitrilotriacetate or [59Fe]citrate. These data demonstrate that non-Tf-bound Fe uptake was not under control of the IRP-IRE system in these cells. Collectively, the results indicate that in LMTK-fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA.

Human Cytoplasmic Aconitase (Iron Regulatory Protein 1) Is Converted into Its [3Fe-4S] Form by Hydrogen Peroxide in Vitro but Is Not Activated for Iron-responsive Element Binding

Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involved in iron homeostasis by binding to iron-responsive elements (IREs) of messenger RNA. IRP1 is a cytoplasmic aconitase when it contains a [4Fe-4S] cluster and an RNA-binding protein after complete removal of the metal center by an unknown mechanism. Human IRP1, obtained as the pure recombinant [4Fe-4S] form, is an enzyme as efficient toward cis-aconitate as the homologous mitochondrial aconitase. The aconitase activity of IRP1 is rapidly lost by reaction with hydrogen peroxide as the [4Fe-4S] cluster is quantitatively converted into the [3Fe-4S] form with release of a single ferrous ion per molecule. The IRE binding capacity of IRP1 is not elicited with H(2)O(2). Ferrous sulfate (but not other more tightly coordinated ferrous ions, such as the complex with ethylenediamine tetraacetic acid) counteracts the inhibitory action of hydrogen peroxide on cytoplasmic aconitase, probably by replenishing iron at the active site. These results cast doubt on the ability of reactive oxygen species to directly increase IRP1 binding to IRE and support a signaling role for hydrogen peroxide in the posttranscriptional control of proteins involved in iron homeostasis in vivo.

Hypoxia Alters Iron-regulatory Protein-1 Binding Capacity and Modulates Cellular Iron Homeostasis in Human Hepatoma and Erythroleukemia Cells

Ferritin and transferrin receptor expression is post-transcriptionally regulated by a conserved mRNA sequence termed the iron-responsive element (IRE), to which a transacting protein called the iron-regulatory protein (IRP) is bound. Our data demonstrate that hypoxia powerfully enhances IRE/IRP-1 binding in human cell lines. Using the human hepatoma cell line Hep3B as a model, we found that 16 h in a 1% oxygen atmosphere markedly increases IRE/IRP-1 binding as assessed by electromobility shift assay. Hypoxia also decreased cytosolic aconitase activity. The hypoxia-enhanced IRE/IRP-1 binding stabilized the transferrin receptor message, increased the cellular mRNA content by over 10-fold, and doubled surface receptor expression. Simultaneously, hypoxia suppressed ferritin message translation. Hypoxia's effect was most strikingly depicted by the absence of ferritin synthesis in cells challenged with inorganic iron. Our results contrast with previously reported data (Hanson, E. S., and Leibold, E. A. (1998) J. Biol. Chem. 273, 7588-7593) in which a 3% oxygen atmosphere reduced IRE/IRP-1 binding in rat hepatoma cells. We discuss some possible reasons for the differences. In aggregate with other investigations involving responses to hypoxia, iron, or nitric oxide, our data indicate that cellular iron metabolic responses are complex and that IRE/IRP-1 interactions vary between cell lines and perhaps between species.

Molecular Cloning of Mouse Glycolate Oxidase: HIGH EVOLUTIONARY CONSERVATION AND PRESENCE OF AN IRON-RESPONSIVE ELEMENT-LIKE SEQUENCE IN THE mRNA

Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells.