Abnormal body iron distribution and erythropoiesis in a novel mouse model with inducible gain of iron regulatory protein (IRP)-1 function.

Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1) using Cre/Lox technology. Systemic activation of the IRP1 transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1 activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson's disease or Friedreich's ataxia.

Rat duodenal IRP1 activity and iron absorption in iron deficiency and after HO perfusion.

BACKGROUND: Iron regulatory protein 1 (IRP1), a post-transcriptional regulator of iron metabolism, is activated in the duodenum of iron-deficient animals, which is associated with increased iron absorption. In cell cultures IRP1 was also activated by iron-independent signals, such as H(2)O(2). Here we investigate whether luminal perfusion of rat duodenum with H(2)O(2) activates duodenal IRP1 and modulates duodenal iron absorption. METHODS: Duodena from iron-adequate Sprague-Dawley rats were luminally perfused with H(2)O(2). Iron regulatory protein-1 activity was determined in duodenal mucosa or in villus and crypt preparations by an electrophoretic mobility shift assay. Duodenal (59)Fe absorption was measured in isolated, perfused duodenal segments ex vivo and in ligated loops in vivo. (59)Fe uptake from the blood side was assessed after i.v. injection of (59)Fe-nitrilotriacetic acid. RESULTS: Similar to iron deficiency, the perfusion with 0-50 mM of H(2)O(2) increases duodenal IRP1 activity along the entire crypt villus-axis in a dose-dependent manner. After H(2)O(2) treatment, IRP1 remains activated for 12-24 h in the tips and for 72 h in the crypts. In iron-deficiency, IRP activation correlates with increased (59)Fe absorption. However, the H(2)O(2) treatment fails to stimulate any increase in (59)Fe uptake, without promoting damage of mucosal architecture or impairing glucose and water transport. CONCLUSION: Duodenal (59)Fe uptake is not affected by the H(2)O(2)-mediated activation of IRP1.

Iron-dependent regulation of the divalent metal ion transporter.

The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.

Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.

Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.

Fertility facts: male and female germ cell development requires translational control by CPEB.

In the August issue of Developmental Cell, Tay and Richter examine the consequences of eliminating CPEB function in mice. Their studies reveal an important role for this translational regulator at the pachytene stage of germ cell differentiation.

A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation.

Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.

HFE downregulates iron uptake from transferrin and induces iron-regulatory protein activity in stably transfected cells.

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular "labile iron pool." The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

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.

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.

Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress.

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

Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements.

Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.

Activation of iron regulatory protein-1 by oxidative stress in vitro.

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-gammaS and GTP-gammaS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.

Iron-regulatory protein-1 (IRP-1) is highly conserved in two invertebrate species--characterization of IRP-1 homologues in Drosophila melanogaster and Caenorhabditis elegans.

Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.

Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.

Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N-terminal part, and binds eIF3 and eIF4A through the central and C-terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap-independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G-mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.

Binary specification of nonsense codons by splicing and cytoplasmic translation.

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.

A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose-phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.

Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intracellular oxidative stress.

We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kühn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 microM H2O2 suffice for complete IRP-1 activation within 60 min when H2O2 is generated extracellularly at steady-state. By contrast, rapid cellular H2O2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4-triazole (to diminish H2O2 degradation); in parallel, 2',7'-dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H2O2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H2O2, but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H2O2. IRP-1 is thus activated both by extra- and intracellular generation of ROIs. While extracellular H2O2 rapidly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activation. IRP-1 thus represents a novel example of an H2O2-regulated protein that responds differentially to alterations of extra- and intracellular H2O2 levels. Our data also suggest that a direct attack on the 4Fe-4S cluster of IRP-1 by H2O2 (or an H2O2-derived reactive species) represents an unlikely explanation for IRP-1 activation by oxidative stress.

Pathways for the regulation of macrophage iron metabolism by the anti-inflammatory cytokines IL-4 and IL-13.

Macrophage effector functions are influenced by their iron status and by shifts in the balance between type 1 Th1 and Th2 cells. To elucidate the influence of the Th2 cytokines IL-4 and IL-13 on macrophage iron metabolism, we investigated activated primary mouse macrophages and the murine macrophage cell line J774. Stimulation of J774 cells and primary macrophages with IFN-gamma/LPS activates the RNA binding affinities of iron regulatory protein-1 (IRP-1) and IRP-2 for iron-responsive elements, leading to translational repression of the iron storage protein ferritin. Activation of IRP-1 and IRP-2 is caused by increased formation of nitric oxide (NO) via stimulation of the inducible NO synthase by IFN-gamma/LPS. Treatment of macrophages with IL-4 and/or IL-13 before stimulation with IFN-gamma/LPS suppresses NO formation and IRP activation, with concomitantly enhanced ferritin synthesis despite a small reduction in ferritin heavy chain mRNA levels. The mRNA levels for the membrane receptor for iron uptake, transferrin receptor (TfR), decrease following stimulation with IFN-gamma/LPS, although IRP-mediated stabilization of the TfR mRNA would have been expected. This as yet unidentified proximal inhibitory signal by IFN-gamma/LPS is antagonized by IL-4 and/or IL-13, which leads to increased TfR mRNA expression in an IRP-independent manner. Thus, IL-4 and IL-13 regulate the iron metabolism of activated macrophages by at least two different pathways: first, by opposing NO-mediated IRP activation, thereby increasing ferritin translation; and second, by an IRP-independent augmentation of TfR mRNA expression. We suggest that IL-4 and IL-13 may enhance iron uptake and storage in activated macrophages and thereby contribute to down-regulation of macrophage effector functions.