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The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 www.elsevier.com/locate/ijbcb Review Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation Andrew M. Thomson a,*, Jack T. Rogers b, Peter J. Leedman a a Laboratory for Cancer Medicine and University Department of Medicine, Royal Perth Hospital, GPO Box X2213, Perth, WA 6001, Australia b Division of Hematology-Oncology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA Received 29 March 1999; accepted 30 March 1999 Abstract Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. The TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by dierentially regulating ferritin mRNA translational eciency and TfR mRNA stability. Several dierent stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA±protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Iron-regulatory proteins; Iron-responsive element; Ferritin; RNA-binding proteins; Posttranscriptional regulation 1. Introduction Iron is an essential nutrient and a potential toxin. The cloning of the iron-regulatory proteins * Corresponding author. Tel.: +61-89-224-0336; fax: 61-89224-0246. E-mail address: [email protected] (A.M. Thomson) (IRPs) and understanding of their interaction with iron-responsive elements (IREs) has revealed a fascinating and exquisite system for controlling iron homeostasis in mammalian cells [1]. In vertebrate species, uptake of transferrin bound iron into cells occurs via transferrin-receptor (TfR) mediated endocytosis [2,3]. An uncharacterised low molecular weight iron chelate mediates distribution of iron within the cell and iron released 1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 8 0 - 1 1140 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 Nomenclature Cys eALAS FeATPase GOX H 2O 2 IL IRE IRP mt-acon MAPK NO nt cysteine erythroid 5-aminolaevulate synthase iron ATPase glycolate oxidase hydrogen peroxide interleukin iron-responsive element iron-regulatory protein mitochondrial aconitase mitogen activated protein kinase nitric oxide nucleotides from transferrin is incorporated into newly formed iron containing proteins and/or the iron storage protein, ferritin [4±6] (Fig. 1). Iron is found stored in cells in the ferric state as ferric oxyhydroxide (Fe(III)OOH) bound to ferritin [6]. Ferritin is a mixture of 24 heavy (H-, 21 kDa) and light (L-, 19 kDa) chain subunits and these subunits are coded for by their corresponding mRNAs [6]. Maintenance of iron homeostasis is obviously complex and a ®nely tuned system for the regulation and sensing of cellular iron requirements is, therefore, paramount to all cells. Central to this theme in iron homoestasis is the existence of two mammalian IRPs, IRP1 and IRP2, whose function is modulated by iron. IRPs behave as cytoplasmic trans-acting mRNA-binding proteins. At least one type of IRP has been found in every mammalian cell type studied, and more recently in the invertebrates, Drosophila melanogaster and Caenorhabditis elegans[7]. Posttranslational modi®cation of these RBPs alters their binding anity to their speci®c cognate mRNA hairpin structures, the iron responsive elements (IREs), and controls expression of target genes posttranscriptionally. IREs present in the 5'-untranslated region (UTR) of ferritin high (H) and low (L) chain [6], erythroid 5-aminolaevulinate synthase (eALAS) [8], mammalian mitochondrial aconitase (mt-acon) [9] and D.melanogaster succinate dehydrogenase subunit O2 OH PKC REMSA Ser Stats SUS Thr TNF T3 TRH Superoxide anion hydoxyl radical protein kinase C RNA electrophoretic mobility shift assay serine signal transducers and activators of transcription Sequences Upstream of Start codon threonine tumour necrosis factor thyroid hormone thyrotropin releasing hormone b [10] mediate the translational eciency of these mRNAs (Fig. 1). 5 individual IRE motifs present in the 3'-UTR of the transferrin receptor (TfR) [11] mediate mRNA stability (Fig. 2). The coordinate but divergent posttranscriptional regulation of IRP/IRE RNA±protein interactions in these genes governing iron uptake, storage and usage in cellular iron metabolism maintains intracellular iron at steady state levels (see Figs. 1 and 2). Much work has focussed on understanding how the IRP/IRE RNA±protein interaction is modulated as it forms the foundation of the cellular iron sensory and regulatory network. Recent evidence suggests that IRP binding is regulated dierently in dierent cell types and that there are a number of eectors that modulate IRP/IRE RNA±protein interactions. Indeed, if IRPs are central regulators of iron homeostasis and iron is required for multiple diverse cellular functions, then responses to multiple eectors, other than iron, should be apparent. Important modulators of IRP binding and/or function include nitric oxide (NO) [12], oxidative stress by H2O2 [13] and hypoxia [14], erythropoietin [15], hepatic a-antitrypsin [16], phosphorylation by protein kinase Cs (PKCs) [17], thyroid hormone (T3) [18] and heme [19]. We have also found that thyrotropin releasing hormone (TRH) and phosphatases also modulate IRP1 and IRP2 binding A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 1141 Fig. 1. Basic overview of cellular iron metabolism. Transferrin-bound iron is taken into the cell by TfR-mediated endocytosis, released from the endosome, processed into an inactive form, and sequestered as ferritin or used for immediate metabolic demands, especially in the mitochondrion. Iron and heme levels can modulate IRP/IRE interactions and coordinately regulate ferritin and TfR levels. The metabolic byproducts, H2O2/O2/OH, can also alter iron levels and IRP/IRE anity. Extracellular stimuli (e.g., erythropoietin, TRH, T3, IL-1b, NO, and H2O2) activate intracellular kinases and phosphatases that modulate IRP phosphorylation status, or cause subsequent redox changes, that alter IRP anity for IRE (AB=acute box sequence). in pituitary cells. As there is a major review in this issue (by Ponka and Lok) on the regulation of TfR gene expression, including the role of IRPs in controlling TfR mRNA stability, we will concentrate on the role of IRPs in the translational regulation of ferritin gene expression in this mini-review. 2. IREs, IRPs and translational control The IRE stem-loop structure (Fig. 3A) acts as both a repressor and an enhancer of translation. The position of the IRE relative to the 5 'UTR mRNA cap structure (<60 nt) has been shown to be a crucial factor regulating translation [20]. Binding of an IRP to the IRE is thought to sterically hinder the ability of the 43 S initiation complex (consisting of eIF4E, eIF4G, eIF3 and the 40 S ribosomal subunit) from forming (blocking recruitment of the 40 S ribosomal subunit to the mRNA) or from binding at the 5' cap, and so inhibits initiation of mRNA translation (Fig. 2) [21,22]. The IRE/IRP interaction is not suciently stable to prevent scanning by the small ribosome subunit after it has attached to the 5' cap site, and once translation has been initiated, the IRE stem-loop is melted by the advancing 43 S scanning ribosomal subunit (Fig. 2). The IRE sequence is thought to act as a translational 1142 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 Fig. 2. Posttranscriptional regulation of ferritin and TfR mRNAs by IRP1 and 2 interactions with IRE. IRP1 and 2, when bound to the 5'UTR IRE of ferritin mRNA block the binding of the ribosomal initiation complex and inhibit translation. The binding of an uncharacterised RNA-binding protein to the acute box (AB) stem loop also plays a role in translational initiation. Both stemloop structures may be important for steric presentation of the mRNA to the initiation complex. (Interaction of poly A tail and associated proteins in translation not shown in this diagram). When IRP1 and 2 are bound to the 5 IREs in the TfR mRNA 3'UTR, they block endonuclease attack which results in an increase in TfR mRNA stability. TRH and T3 can increase or decrease IRP1 and IRP2 binding activity to the ferritin IRE depending on the cell type. enhancer region by attracting the intiation complex preferentially to other 5'UTR sequences [23], though this is still controversial and other sequences, especially in the 5 'UTR of the H and L ferritin, may act as the enhancer regions [24]. The nucleotide sequence of IRE stem-loops is highly conserved in the genes in which they have been described within and across species (Fig. 3A) [25]. Elucidation of the structure by mutational analysis and nuclear magnetic resonance (NMR) indicates that IREs contain 2 regions, each of which is necessary for IRP binding [26]. The ®rst, a hairpin loop, comprises a 6 nt loop (5 'C1A2G3U4G5N6) at the end of a ®ve base pair stem and NMR data suggest important hydrogen bonding between C1 and G5 in a tertiary interaction across the loop [25]. Mutations within the ®rst 5 nt of the loop reduce IRP binding [25]. Altering the loop sequence in vitro can select for speci®c binding of IRP1 (UAGUAC) or IRP2 (CCGAGC) [27]. Interestingly, individuals suering from familial hyperferritinaemia carry a C2 4 G2 mutation in the ferritin L-chain gene which leads to excessive ferritin sysnthesis [28]. The second critical region of the IRE is the contiguous sequence containing either an internal loop/bulge (found in H- and L-chain ferritin A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 1143 Fig. 3. (A) Predicted structures of identi®ed IREs. Conserved regions of the IRE hairpin-loop (CAGUG) are shown in bold. The boxed region of the ferritin IRE indicates the speci®c internal loop/bulge which has an increased anity for IRP2 than the bulge structure of the other IREs. (B) Schematic comaparison of IRP1 and IRP2 sequences. Sequence comparison is high, and the position of the 73-amino acid (aa) IRP2 speci®c region is shown. The cysteine (C) residues important for IRE and iron-sulphur cluster binding are indicated. Percentages represent the level of identity of each domain of IRP2 compared to IRP1. (C) Postulated model of IRP1 apoprotein and haloprotein structure. High iron levels induce formation of the iron±sulphur cluster (4Fe±4S) which closes the putative cleft site and blocks IRE binding but allows citrate/iso-citrate to bind. Low iron levels causes iron±sulphur cluster disassembly which allows IRE entry and which facilitates IRP binding. The cysteine residues considered important in IRE and ironsulphur cluster formation are indicated. IRP1 is synthesised in its apoprotein form. Under certain high iron conditions the 3Fe±4S form of IRP1 is degraded. mRNAs) or a cytosine (C)-bulge (TfR, eLAS, mt-acon mRNAs) [29]. Recently, a thermodynamically unstable IRE-like motif with an A:A mismatch in the contiguous region was found in the 5'-UTR of the murine glycolate oxidase (GOX) mRNA [30] (Fig. 3A). However, translational eciency of the GOX mRNA was not controlled by IRP1 and IRP2 in vivo, although the sequence bound IRP1 and IRP2 in vitro [30]. IRP1 and IRP2 isoforms bind equally and with higher anity to the internal loop/bulge structure (ferritin) than to the C-loop. However, binding anity of IRP2 to the C-loop is much lower than for IRP1 [29]. The abundance of the two IRP isoforms varies considerably in dierent cell types and together with dierential binding to IREs by IRP1 and IRP2, this system provides for high precision and ®ne-tuning of IRE-dependent mRNA regulation (see below). Human IRP2 (105 kDa) has 61% sequence identity and 79% similarity to human IRP1 and this degree of homology is conserved across 1144 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 Fig. 4. Demonstration of IRP1 and IRP2 in rat pituitary cells. (A) RNA-gel shift (REMSA) of rat pituitary cytoplasmic protein extracts from the mammosomatroph GH3 cell line showing IRP1 and IRP2 binding to a 32P-radiolabeled ferritin IRE riboprobe. The ferritin IRE was incubated with 10 mg GH3 cell cytoplasmic protein extract at 228C for 30 min. RNase T1 (1 U/sample) and heparin (5 mg/ml ®nal concentration) was then added prior to resolving on a 4.2% PAGE gel. The gel was dried and exposed by PhosphorImage. The RNA-protein complexes were supershifted with isoform-speci®c antibodies. (B) GH3 cell cytoplasmic protein extract (20 mg) was incubated with 32P-ferritin IRE probe for 30 min at room temperature as above, RNaseT1 and heparin added, followed by UV-irradiation for 10 min. The samples were then resolved on a 7.5% SDS±PAGE gel, dried and analysed by PhosphorImage. This UV-crosslinking analysis (UCA) of a rat pituitary cytoplasmic protein extract shows IRP1 (97 kDa) and IRP2 (102 kDa). species. IRP2 (962 amino acids) is notably distinguished from IRP1 (889 amino acids) by a unique 73 amino acid segment which is responsible for mediating proteolytic cleavage of the protein in iron replete cells (Fig. 3B). The two IRPs are readily detectable in rat pituitary tissue using RNA-electrophoretic mobility shift assays (REMSA). Fig. 4A shows the IRP1 and IRP2 RNA±protein complexes with a ferritin IRE. In addition, each IRP was supershifted using isoform-speci®c antibodies. In UV-crosslinking assays the IRPs migrate at 95±105 kDa (Fig. 4B). Both IRPs comprise a 4 domain structure, with domain 4 linked (directly to domain 3) by a hinge region to the other 3 domains (Figs. 3B, 3C) [31]. Most of the recent research on the binding of the IRPs to IREs using site directed mutagenesis, UV-crosslinking and proteolytic analysis has shown that several regions within the protein moieties are important in RNA-binding, protein structural alterations and the modulation of binding [32,33]. The conservation of binding site structure helps explain why IREs demonstrate similar high anity binding to both IRP1 and IRP2 (Kd 1 10±30 pM) [17]. Cleft exposure is controlled by the positioning of domain 4 relative to domains 1±3 (see later). Although IRP1 and IRP2 are similar proteins that share 61% overall amino acid identity, they do seem to exhibit dierent biochemical properties and mechanisms of regulation which would allow for precise changes in the regulation of iron homeostasis within a cell subjected to many stimuli. It is of particular interest that the ratios of IRP1:IRP2 is cell type speci®c (e.g., IRP1>IRP2 in liver, kidney, intestine, brain; IRP1 < IRP2 in pituitary, Ba/F3 pro-B lymphocyte cell line [34]). This suggests that IRP2, as well as IRP1, plays an equally important role in governing iron homeostasis in specifc cell types. A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 3. Regulation of IRP1 binding activity 3.1. Iron induced modulation IRP1 was the ®rst IRP to be described and, until recently, was most intensively investigated. Its central importance in intracellular iron homeostasis was demonstrated when IRP1 overexpression rendered the cell unable to regulate its iron levels [35]. IRP1 is a bifunctional protein, acting either as a cytoplasmic aconitase (haloprotein) in iron replete cells or as a RNA-binding protein (apoprotein) in iron depleted cells. This functional switch is dependent upon the assembly of a iron-sulphur (4Fe±4S) cubane structure in iron replete cells that is bound by the 3 cysteines (Cys) in the cleft of the protein [36] and limits IRE access, but allows the binding of citrate [33,37]. It is hypothesised that the removal of the iron±sulphur cluster, a process which is still not fully understood, causes major structural rearrangements that increase the accessibilty of IREs to the putative cleft site between domains 1±3 and 4 (Fig. 3C) [38]. Indeed, IRP1 exists in 3 forms within cells; either as a cytoplasmic aconitase haloprotein (4Fe±4S, binds citrate, not an RNA-binding protein), devoid of aconitase activity (3Fe±4S, binds citrate, not an RNA-binding protein), or as an apoprotein (RNA-binding protein, little citrate binding). It is not clear at this point in time whether the 3Fe±4S form represents a physiological intermediate in cluster assembly/disassembly. However, it is evident that both functions of IRP1 (aconitase and RNAbinding) are mutually exclusive even though the putative cleft serves as a common locus for the two functions of the protein (Fig. 3C) [38]. Speci®c cysteine residues, C437, C503 and C506, have been identi®ed in the putative cleft of IRP1 that are critical for the formation of the stable iron±sulphur cluster and RNA-binding [36]. The oxidation state of these three residues is central in the IRP1 response to iron (Figs. 3B and C) [1]. Several studies have shown that other regions of IRP1 (amino acids 116±151, 480±623, and the C-terminal region) also contribute to the dual functionality of the protein [1,39]. The iron± sulphur cluster appears to limit the access to po- 1145 tential phosphorylation residues (Ser 138) and those residues important in RNA-binding located in and around the putative cleft [17,39]. Disruption of the Fe±S cluster alters structural con®gurations of both the putative cleft and hinge linker regions, increasing the accessibility of the cleft to RNA (Fig. 3C) [39]. There appears to be strict control of the steady state level of cellular IRP1. As is evident from above, iron induced alterations in IRP1-binding activity are due to posttranslational changes in IRP1 con®guration and are not generally due to changes in protein levels. However, under certain conditions when iron enters a cell, IRP1 levels may be substantially reduced due to degradation, though this is very controversial [19,40,41]. A recent report showed that iron chelators and heme synthesis inhibitors (see Fig. 1) blocked iron induced IRP1 degradation, but not hemeinduced IRP1 degradation [19]. One report has shown that IRP1 is dimerised and then degraded by heme [40], although another report demonstrated that eects on IRP1 are by dierent forms of iron [41]. Indeed, an earlier work demonstrated that translational control of mRNAs coding for H- and L-ferritin was regulated by chelateable intracellular iron pools [42]. The mechanism involved in the postulated hemeinduced degradation of IRP1 is unknown [19]. The pool of IRP1 targetted for degradation is unclear, but may comprise the subset of proteins that lack an iron±sulphur cluster [19]. Clearly, further research is required in this controversial area. 3.2. Hormone (erythropoietin, TRH, T3) and kinase induced modulation Ligands, other than iron, have recently been shown to alter IRP1 binding activity. Protein sequence pro®les indicate that Ser 138 and Ser 711 are potential PKC phosphorylation sites in IRP1. Activation of cellular PKCs with phorbol12 13-myristate acetate (PMA) results in phosphorylation of IRP1 and an increase in binding to IRE [17,34]. Thus, activation of various signalling pathways that converge on PKC may have the potential to modify the phosphorylation sta- 1146 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 tus and binding activity of IRP1. Indeed, we have con®rmed that PKC contributes to basal levels of IRP1 binding activity. Interestingly, mitogen activated protein kinases (MAPKs) contribute to maintaining basal IRP1 binding levels. Furthermore, we have recently demonstrated in pituitary cells that basal IRP1 binding to a ferritin IRE is regulated by T3 [18] and have subsequently found that TRH also has an eect. This IRP-regulation in the pituitary is accompanied by multiple kinase and phosphatase activation. This suggests that multiple hormones have the capacity to regulate IRP1 RNA-binding by activation of various signalling pathways (Fig. 1). Interestingly, RNA bound IRP1 is less susceptible to phosphorylation by PKC than nonRNA bound IRP1 [17,34]. Phosphorylation of Ser 138 in IRP1 may serve as a means of partitioning the protein between its two opposing functions by selectively aecting the ability of residues 116±151 (RNA-binding) and residues 125 and 126 (essential for aconitase function) to support the separate functions of IRP1. It is hypothesised that phosphorylation may block or inhibit cluster assembly, inhibiting reversion of the apoprotein to the haloprotein. This may also alter the set point at which iron aects IRP1 function [17]. Erythropoietin is a kidney produced glycoprotein that enhances red blood cell mass by stimulating proliferation and dierentiation of near mature red blood progenitor cells. Erythropoietin has recently been shown to increase the binding activity of IRP1 to TfR mRNA speci®cally in erythroid cells [15], leading to an increase in iron uptake via an increase in TfR number due to an increase in TfR mRNA stability. Binding of erythropoietin to its cell surface receptor sets in motion a cascade of kinase activation, including MAPKs and PKCs, which are most likely responsible for modulating IRP1 phosphorylation and RNA-binding activity [43]. 3.3. Nitric oxide (NO) induced modulation An interesting connection between NO and the IRP has been established [44±46]. NO is a powerful vascular dilator, and much interest surrounds the possible role of iron in regulating vascular tone. The observation some years ago that NO could modulate IRP-binding suggested a plausible connection between the two. NO induces a slow increase in IRP1 binding activity resulting in decreased ferritin and increased transferrin synthesis in hepatic cells [44]. NO activates IRP1 via a cycloheximide-insensitive posttranslational mechanism [45]. Although the precise mechanisms are unclear, NO may regulate IRP1-binding through either its ability to disassemble Fe±S clusters, or, as most recent evidence suggests, to act as a cytoplasmic iron-chelator thereby limiting intracellular iron availability [45]. The eect is readily detected in adjacent cells due to NO diusion [47] and is present in all cell types and tissues studied to date [44,46]. The eect is particularly evident in in¯ammatory conditions in which bacterial endotoxin and cytokines (IL-1b, IL-6, TNF-a, INF-g ), induce nitric oxide synthase isoform-2 (NOS-2) production, leading to an increase in NO production. 3.4. Oxidative stress, phosphatases, hypoxia and reoxygenation induced modulation Oxidative stress and the regulation of iron metabolism are tightly coupled in bacteria. It is therefore not surprising to ®nd that oxidative stress (by H2O2) induces a rapid (<60 min) increase in IRP1 binding activity in vertebrates, unlike the slow response (>12 h) observed with NO [13,46]. There is a corresponding alteration in ferritin (decrease) and transferrin receptor (increase) synthesis. This eect is due to posttranslational modi®cation of IRP1 and is okadaic acid sensitive [48]. The current hypothesis suggests phosphatases are involved in the signalling mechanism, being recruited by H2O2, and acting through a plasma-membrane associated H2O2 sensor [13]. Interestingly, a recent study [14] has shown that hypoxia leads to the inactivation of IRP1 binding in hepatoma cells, without any changes in IRP1 protein levels. This eect was reversible on subsequent exposure of the cells to oxygen. The mechanism governing this IRP1 modulation is unknown but is thought to be a phosphatase and/or oxygen radical- A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 dependent [14] posttranslational event, mimicking the mechanism involved in the H2O2-induced modulation of IRP1. 4. Regulation of IRP2 binding activity 4.1. Iron induced modulation The discovery of a second vertebrate IRP, IRP2, led to the postulate that two IRPs are essential for high ®delity control of iron homeostasis in cells that are exposed to multiple internal and external stimuli [47,49±51]. As a consequence, there has been a resurgence of interest in understanding the mechanisms controlling the regulation of IRP2 in mammalian cells in the last few years. Indeed, recent studies have demonstrated the important role it plays in regulating iron homeostasis. Most signi®cantly, IRP2 was shown to be resposible for maintaining normal cellular function and iron homeostasis in Ba/F3 prolymphocytes which lack IRP1 [39]. IRP2, unlike IRP1, does not form an iron±sulphur cluster in the putative cleft, although the location of the 3 cysteines (C512, C578, C581) is conserved when compared with IRP1. Furthermore, IRP2 does not exhibit any aconitase activity, due to the lack of conservation of certain amino acids that play a crucial role in aconitase enzymatic function [47,49±51]. As mentioned earlier, another major dierence between IPR2 and IRP1 is the 73 amino acid insertion in IRP2 that is involved in binding iron and is the target for a cytoplasmic protease that mediates IRP2 degradation [52±54]. Variations in iron concentration has little eect on IRP2 transcription. However, IRP2 protein is degraded eciently by the proteosome in iron-replete cells [52±54], whilst its degradation rate is decreased in iron-deplete cells [52±54]. IRP2 undergoes an iron-dependent oxidative modi®cation in iron-replete cells that increases degradation by the proteosome by a ubiquitinmediated pathway [52±54]. Mutations of the cysteine residues, C168 and C201, within the unique 73 amino acid region eliminates the ability of this sequence to mediate iron-dependent 1147 degradation [52]. It is hypothesised that these cysteines are involved in the ligation of an iron± sulphur cluster, and so act as an iron sensor [52]. Free iron is not necessary for the ubiquitination steps once the protein has been oxidised [53]. The degradation process may also be induced by heme levels, similar to IRP1 degradation, rather than a direct eect of iron [19]. It is now clear that the two cysteine residues, C168 and C201, within the unique 73 amino acid region of IRP2 that are predicted to be especially redox active [17,39], are indispensable for iron induced degradation. 4.2. Hormone (erythropoietin, TRH, T3) and kinase induced modulation Similar to IRP1, the RNA-binding activity of IRP2 can be upregulated by phosphorylation via activation of PKCs [17,34,39]. This eect does not require a change in IRP2 protein level [17,34,39]. Interestingly, the putative PKC phosphorylation sites of IRP2 dier to those of IRP1 [17,39]. In addition, putative PKC and MAPK phosphorylation sites have been found in the unique 73 amino acid region of IRP2. Phosphorylation of speci®c serine and threonine residues in this region may aect the redox properties of the nearby cysteine residues that contribute to the redox and iron regulation of IRP2 [39]. Somewhat unexpectedly, the basal level of phosphorylation of IRP2 is higher than IRP1 in HL-60 cells [39]. The observation that IRP2 binding may be regulated by phosphorylation without changes in IRP2 protein levels provides a second level of regulation. IRP2 binding activity increases in erythropoietin stimulated cells [15], and this is most likely due to changes in phosphorylation status of the IRP induced by activation of PKCs and MAPKs. In our studies to investigate the regulation of gene expression in the pituitary, we recently found that IRP2 predominates (see Fig. 4A). The binding activity of IRP2 is regulated by several dierent stimuli. These include TRH and T3 which appear to modulate the activation of PKCs, MAPKs, and phosphatases, resulting in a change in phosphorylation of IRP2, without changes in IRP2 1148 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 protein levels (data not shown). These data and those of Schalinske et al. [34] support the concept that IRP2 may have an important eect on iron homeostasis in speci®c cells and may predominate over IRP1. 4.3. Nitric oxide (NO) induced modulation The eect of NO on IRP2 RNA-binding activity remains controversial. The binding activity of IRP2 is increased, as with IRP1, after NO stimulation [45], though this appears to be cell speci®c [12]. One hypothesis suggests that NO limits the availabilty of cytoplasmic iron, thereby reducing binding of iron to the 73 amino acid insertion and oxidation of IRP2, as well as degradation by the proteasome [54]. In those cells where NO increases IRP2 binding activity, it is slow (>12 h) and appears to be due to an increase in de novo IRP2 synthesis [45]. A major drawback in all of these studies to date relates to the use of cells in which IRP2 is not the predominant isoform. Thus, the eect may be dierent in cells, such as the pituitary, and remains to be investigated. 4.4. Oxidative stress, phosphatases, hypoxia and reoxygenation induced modulation Despite extensive studies, H2O2 does not appear to modify IRP2 binding activity [1,13,45,47]. This is consistent with work demonstrating no eect of hypoxia and reoxygenation on IRP2 binding activity [10]. Again these studies have suered from using cells with concentrations of IRP1 greater than IRP2. However, these data are consistent with other stimuli such as phosphatases and kinases being the major modulators of IRP2 binding action. In sum, it is evident that a more detailed study of the mechanisms of IRP2 modulation are required to fully understand the regulation of RNA-binding of this important protein. However, our data and that of others demonstrate that IRP2 can be regulated by many stimuli and that the regulation of IRP2 expression is complex and diers signi®cantly from that of IRP1. These dierences between IRP1 and IRP2 regulation provide a far greater level of complexity for controlling intracellular iron homeostasis than previously envisaged. How each is dierentially regulated, and the contribution of each in speci®c cell types is the focus of current work in this area. 5. Invertebrate IRP1-isoforms Interestingly, IRP1-isoforms, but no IRP2-isoforms, have recently been identi®ed in D.melanogaster and C.elegans[7]. One IRP was found in C.elegans and two IRPs, IRP1A and IRP1B (86% identical to each other), in D.melanogaster. IRP1A has been localised to position 94C1-8 and IRP1B to position 86B3-6 on the right arm of chromosome 3 and both show 67% homology to IRP1. Human IRP1 has been localised to human chromosome 9 [55]. The invertebrate IRPs do not contain an IRP2-like amino acid insertion, but the aconitase active site residues are of IRP1-type [7]. It is postulated that the D.melaonogaster and vertebrate IRPs resulted from an IRP gene duplication event, and that this may have occurred prior to the diversion of invertebrates and vertebrates [7]. In contrast to vertebrates, in which insertion of a 73 amino acid coding exon produces IRP2, evolutionary pressures may have been such that further changes to Drosophila IRPs were not essential. Intriguingly, IRP1A and IRP1B are not dierentially expressed in speci®c organs, unlike mammalian IRP1 and IRP2 [7]. However, the fact that invertebrates evolved a second IRP, albeit another IRP1 derivative, reinforces the notion that two IRPs are essential for high ®delity control of iron homeostasis in cells exposed to multiple internal and external stimuli. It is apparent from all the above that the IRP/ IRE interactions play a pivotal role in controlling iron homeostasis in invertebrates and vertebrates via regulation of ferritin gene expression. However, cis-elements, other than IREs, have recently been identi®ed that have a major impact on ferritin gene expression. A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 6. Non-IRP/IRE regulation of ferritin translation Ð the acute box There has long been a clinical association between serum iron levels, serum ferritin levels, intracellular ferritin levels and cytokine induced in¯ammation [56]. Indeed, elevated serum ferritin levels have been used as a diagnostic for in¯ammation and disease. IL-1 is the major in¯ammatory cytokine that lowers blood serum iron levels [57], and an increase in ferritin synthesis immediately precedes a lowering in serum iron levels [58]. The elevated ferritin levels lead to a lowering of serum iron levels through iron sequestration. IL-1 induces an increase in hepatic ferritin synthesis by increasing ferritin mRNA translation in hepatic cells [59]. This IL-1 induced response increases serum ferritin levels due to tissue export of ferritin to the bloodstream [28]. This increase in tissue and serum ferritin levels also occurs in hereditary cataract hyperferritinemia syndrome, in which an IRE mutation in the L-ferritin gene reduces IRP binding and leads to uncontrolled L-ferritin synthesis [28,60]. Remarkably, a 20 nt acute box cis-element downstream of the IRE in the 5 '-UTR of H- and L-ferritin mRNAs was recently identi®ed (Figs. 1 and 2) which mediates this IL-1 eect on ferritin translation in both hepatic and endothelial cells [61,24]. The importance of the acute box as a major regulator of ferritin synthesis was established in experiments in which mutations of the 5'-UTR sequence resulted in an eightfold reduction in the level of ferritin synthesis [24]. The mutations may change the steric con®guration of the 5'-UTR and reduce the mRNA anity to the translational pre-initiation complex. Recent investigations have found a trans-acting RNA-binding protein in hepatic cells that binds to the acute box cis-element in H- and L-ferritin mRNAs (J.T. Rogers, unpublished results), and mutations in the acute box may alter binding anity of this RNA-binding protein. Our current hypothesis is that during in¯ammation ferritin translation is primarily regulated by IL-1 modulation of binding of this RBP to the ferritin mRNA 5'-UTR acute box. The acute box also plays an important role in the regulation of ferritin synthesis in pitu- 1149 itary lactotroph cells stimulated with TRH and T3 in which there is reduced IRP/IRE binding, but a decrease in ferritin synthesis (A.M. Thomson, J.T. Rogers and P.J. Leedman, unpublished results). Thus, evidence is evolving to indicate that the posttranscriptional regulation of ferritin levels involves at least two cis-acting mRNA elements and two sets of RBPs, which may act together or independently and which may be controlled by multiple stimuli. 7. Concluding remarks Vertebrates and invertebrates have devised elaborate and exquisitely controlled systems to regulate iron homeostasis. The IRPs, which are conserved across species, play a central role as bifunctional regulators of ferritin mRNA translational eciency and TfR mRNA stability. Acting in concert, these IRP/IRE interactions are a superb example of how cells have crafted a master control system based on a single high anity RNA±protein interaction that has great complexity, due to the expanding set of compounds that regulate the interaction. The discovery of reciprocal and coordinate regulation of IRP/IRE interactions and establishing their role in modulating iron homeostasis represent major discoveries in the ®eld. At the molecular level, the interplay between IRPs and IREs of ferritin and transferrin receptor mRNA have formed the basis of much of our understanding of RNA±protein interactions and posttranscriptional regulation of genes. De®ning the mechanisms controlling ferritin translation represents a major advance in understanding translational initiation. Elucidation of the regulation of TfR mRNA stability has provided great insights into the regulation of mRNA turnover. The discovery of a second IRP has presented many challenges to de®ne the role of each in what appears to be a cellspeci®c set of regulatory events. It has also provided the framework for extremely ®ne tuning of intracellular iron homeostasis through dierential expression of the isoforms. The ubiquitination and subsequent degradation by the proteasome of oxidised IRP2 has added to the knowledge of 1150 A.M. Thomson et al. / The International Journal of Biochemistry & Cell Biology 31 (1999) 1139±1152 cellular control of oxidatively damaged cytoplasmic proteins. Appreciation of the role of the acute box in regulating ferritin synthesis has emphasised that posttranscriptional regulation of mRNAs is likely to involve the interaction of multiple RNA-binding proteins and their cognate cis-acting mRNA sequences. The last decade has seen an explosion of information in the biology of the IRPs. 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