The cellular mechanisms of body iron homeostasis

Cells tightly regulate iron levels through the activity of iron regulatory proteins (IRPs) that bind to RNA motifs called iron responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Similarly, body iron homeostasis is maintained through the control of intestinal iron absorption. Intestinal epithelia cells sense body iron through the basolateral endocytosis of plasma transferrin. Transferrin endocytosis results in enterocytes whose iron content will depend on the iron saturation of plasma transferrin. Cell iron levels, in turn, inversely correlate with intestinal iron absorption. In this study, we examined the relationship between the regulation of intestinal iron absorption and the regulation of intracellular iron levels by Caco-2 cells. We asserted that IRP activity closely correlates with apical iron uptake and transepithelial iron transport. Moreover, overexpression of IRE resulted in a very low labile or reactive iron pool and increased apical to basolateral iron flux. These results show that iron absorption is primarily regulated by the size of the labile iron pool, which in turn is regulated by the IRE/IRP system.     

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.     

Evolution of the iron-responsive element

An RNA hairpin structure referred to as the iron-responsive element (IRE) and iron regulatory proteins (IRPs) are key players in the control of iron metabolism in animal cells. They regulate translation initiation or mRNA stability, and the IRE is found in a variety of mRNAs, such as those encoding ferritin, transferrin receptor (Tfr), erythroid aminolevulinic acid synthase (eALAS), mitochondrial aconitase (mACO), ferroportin, and divalent metal transporter 1 (DMT1). We have studied the evolution of the IRE by considering all mRNAs previously known to be associated with this structure and by computationally examining its occurrence in a large variety of eukaryotic organisms. More than 100 novel sequences together with approximately 50 IREs that were previously reported resulted in a comprehensive view of the phylogenetic distribution of this element. A comparison of the different mRNAs shows that the IREs of eALAS and mACO are found in chordates, those of ferroportin and Tfr1 are found in vertebrates, and the IRE of DMT1 is confined to mammals. In contrast, the IRE of ferritin occurs in a majority of metazoa including lower metazoa such as sponges and Nematostella (sea anemone). These findings suggest that the ferritin IRE represents the ancestral version of this type of translational control and that during the evolution of higher animals the IRE structure was adopted by other genes. On the basis of primary sequence comparison between different organisms, we suggest that some of these IREs developed by "convergent evolution" through stepwise changes in sequence, rather than by recombination events.     

Accommodating variety in iron-responsive elements: Crystal structure of transferrin receptor 1 B IRE bound to iron regulatory protein 1

Iron responsive elements (IREs) are short stem-loop structures found in several mRNAs encoding proteins involved in cellular iron metabolism. Iron regulatory proteins (IRPs) control iron homeostasis through differential binding to the IREs, accommodating any sequence or structural variations that the IREs may present. Here we report the structure of IRP1 in complex with transferrin receptor 1 B (TfR B) IRE, and compare it to the complex with ferritin H (Ftn H) IRE. The two IREs are bound to IRP1 through nearly identical protein-RNA contacts, although their stem conformations are significantly different. These results support the view that binding of different IREs with IRP1 depends both on protein and RNA conformational plasticity, adapting to RNA variation while retaining conserved protein-RNA contacts.     

Detection of intracellular iron by its regulatory effect

Intracellular iron regulates gene expression by inhibiting the interaction of iron regulatory proteins (IRPs) with RNA motifs called iron-responsive elements (IREs). To assay this interaction in living cells we have developed two fluorescent IRE-based reporters that rapidly, reversibly, and specifically respond to changes in cellular iron status as well as signaling that modifies IRP activity. The reporters were also sufficiently sensitive to distinguish apo- from holotransferrin in the medium, to detect the effect of modifiers of the transferrin pathway such as HFE, and to detect the donation or chelation of iron by siderophores bound to the lipocalin neutrophil gelatinase-associated lipocalin (Ngal). In addition, alternative configurations of the IRE motif either enhanced or repressed fluorescence, permitting a ratio analysis of the iron-dependent response. These characteristics make it possible to visualize iron-IRP-IRE interactions in vivo. iron regulatory proteins; iron-responsive element; labile iron pool; transferrin; HFE; neutrophil gelatinase-associated lipocalin; siderophore     

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.     

Excess capacity of the iron regulatory protein system

Iron regulatory proteins (IRP1 and IRP2) are master regulators of cellular iron metabolism. IRPs bind to iron-responsive elements (IREs) present in the untranslated regions of mRNAs encoding proteins of iron storage, uptake, transport, and export. Because simultaneous knockout of IRP1 and IRP2 is embryonically lethal, it has not been possible to use dual knockouts to explore the consequences of loss of both IRP1 and IRP2 in mammalian cells. In this report, we describe the use of small interfering RNA to assess the relative contributions of IRP1 and IRP2 in epithelial cells. Stable cell lines were created in which either IRP1, IRP2, or both were knocked down. Knockdown of IRP1 decreased IRE binding activity but did not affect ferritin H and transferrin receptor 1 (TfR1) expression, whereas knockdown of IRP2 marginally affected IRE binding activity but caused an increase in ferritin H and a decrease in TfR1. Knockdown of both IRPs resulted in a greater reduction of IRE binding activity and more severe perturbation of ferritin H and TfR1 expression compared with single IRP knockdown. Even though the knockdown of IRP-1, IRP-2, or both was efficient, resulting in nondetectable protein and under 5% of wild type levels of mRNA, all stable knockdowns retained an ability to modulate ferritin H and TfR1 appropriately in response to iron challenge. However, further knockdown of IRPs accomplished by transient transfection of small interfering RNA in stable knockdown cells completely abolished the response of ferritin H and TfR1 to iron challenge, demonstrating an extensive excess capacity of the IRP system.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains.

Iron regulatory proteins (IRPs) are cytoplasmic mRNA binding proteins involved in intracellular regulation of iron homeostasis. IRPs regulate expression of ferritin and transferrin receptor at the mRNA level by interacting with a conserved RNA structure termed the iron-responsive element (IRE). This concordant regulation of transferrin receptors and ferritin is designed so a cell can obtain iron when it is needed, and sequester iron when it is in excess. However, we have reported that iron accumulates in the brain in Alzheimer's disease without a concomitant increase in ferritin. An increase in iron without proper sequestration can increase the vulnerability of cells to oxidative stress. Oxidative stress is a component of many neurological diseases including Alzheimer's. We hypothesized that alterations in the IRP/IRE interaction could be the site at which iron mismanagement occurs in the Alzheimer's brains. In this report we demonstrate that in normal human brain extracts, the IRP is detected as a double IRE/IRP complex by RNA band shift assay, but in 2 of 6 Alzheimer's brain (AD) extracts examined a single IRE/IRP complex was obtained. Furthermore, the mobility of the single IRE/IRP complex in Alzheimer's brain extracts is decreased relative to the double IRE/IRP complex. Western blot and RNA band super shift assay demonstrate that IRP1 is involved in the formation of the single IRE/IRP complex. In vitro analyses suggest that the stability of the doublet complex and single AD complex are different. The single complex from the AD brain are more stable. A more stable IRE/IRP complex in the AD brain could increase stability of the transferrin receptor mRNA and inhibit ferritin synthesis. At the cellular level, the outcome of this alteration in the molecular regulatory mechanism would be increased iron accumulation without an increase in ferritin; identical to the observation we reported in AD brains. The appearance of the single IRE/IRP complex in Alzheimer's brain extracts is associated with relatively high endogenous ribonuclease activity. We propose that elevated RNase activity is one mechanism by which the iron regulatory system becomes dysfunctional.     

Regulation of the 75-kDa subunit of mitochondrial complex I by iron

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.