In vivo and in vitro modulation of the mRNA-binding activity of iron-regulatory factor. Tissue distribution and effects of cell proliferation, iron levels and redox state.

The mRNA-binding protein, iron-regulatory factor (IRF) has a central role in iron metabolism. It coordinately increases transferrin-receptor mRNA stability and inhibits translation of ferritin and erythroid delta-aminolevulinate synthase mRNA by binding to specific mRNA structures, the iron-responsive elements (IRE). In gel-retardation assays, IRF had a broad tissue distribution, showing activity in cytosolic extracts from 12 mouse organs tested. In all these extracts, IRF could be further activated in vitro by 2-mercaptoethanol. In cultured mouse 3T6 fibroblasts, growth stimulation after low serum arrest increased IRF activity 10-fold, mainly through activation of existing inactive IRF. No change was observed during progression of 3T6 cells through the cell cycle. IRF activation by iron chelators has been postulated to result in the reduction of an intramolecular sulfhydryl group. In a search for redox conditions that regulate IRE binding of IRF, we studied several compounds in vitro or in vivo. Hemin, known to inactivate IRF in vivo, showed a similar, reversible effect in vitro, presumably by oxidizing IRF. However, this did not appear to be relevant for the mode of IRF regulation in vivo. Addition of protoporphyrin IX to intact cells induced IRF activity almost to the same extent as desferrioxamine. This effect was inhibited by iron salts, indicating that IRF is activated in vivo through depletion of a chelatable iron pool. In vitro activation by reductants other than 2-mercaptoethanol suggested some selectivity in their access to relevant sulfhydryl groups, but did not reveal which natural redox-sensitive compound might regulate IRF in vivo. However, in cultured cells, inactivation of free IRF by the sulfhydryl-specific oxidizing agent diamide was much more rapidly reversed than inactivation by iron salts. This indicates the direct involvement of a cellular reductant in setting IRF activity and suggests a rate-limiting IRF conformation that is reached only in the presence of iron, but not after diamide oxidation.     

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.     

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.     

Effect of iron and retinoic acid on the control of transferrin receptor and ferritin in the human promonocytic cell line U937.

The effect of changes in iron availability and induction of differentiation on transferrin receptor expression and ferritin levels has been examined in the promonocytic cell line U937. Addition of iron (as 200 micrograms/ml saturated transferrin) or retinoic acid (1 microM) both caused approx. 70% reduction in the average number of surface transferrin receptors, while the iron chelator desferrioxamine caused an 84% increase. Comparable changes also occurred in the levels of transferrin receptor mRNA. Neither iron nor retinoic acid significantly altered the half-life of transferrin receptor mRNA in the presence of actinomycin D (approx. 75 min) but a 10-fold increase in stability occurred in the presence of desferrioxamine. Iron and retinoic acid both caused an increase in intracellular ferritin levels (approx. 4-and 3-fold, respectively), while desferrioxamine reduced ferritin levels by approx. two-thirds. The effect of iron and retinoic acid added together did not differ greatly from that of each agent alone. None of the treatments greatly affected levels of L-ferritin mRNA. Virtually no H-ferritin mRNA was detected in U937 cells. These results show that changes in ferritin and transferrin receptor caused by treatment with retinoic acid are similar to those induced by excess iron, and suggest that changes in these proteins during cell differentiation are due to redistribution of intracellular iron into the regulatory pool(s), rather than to iron-independent mechanisms.     

Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.     

Role of HIF-1 in iron regulation: potential therapeutic strategy for neurodegenerative disorders

A disruption in optimal iron levels within different brain regions has been demonstrated in several neurodegenerative disorders. Although iron is an essential element that is required for many processes in the human body, an excess can lead to the generation of free radicals that can damage cells. Iron levels are therefore stringently regulated within cells by a host of regulatory proteins that keep iron levels in check. The iron regulatory proteins (IRPs) have the ability to sense and control the level of intracellular iron by binding to iron responsive elements (IREs) of several genes encoding key proteins such as the transferrin receptor (TfR) and ferritin. Concurrently, the hypoxia-inducible factor (HIF) has also been shown in previous studies to regulate intracellular iron by binding to HIF-responsive elements (HREs) that are located within the genes of iron-related proteins such as TfR and heme oxygenase-1 (HO-1). This review will focus on the interactions between the IRP/IRE and HIF/HRE systems and how cells utilize these intricate networks to regulate intracellular iron levels. Additionally, since iron chelation has been suggested to be a therapeutic treatment for disorders such as Parkinson's and Alzheimer's disease, understanding the exact mechanisms by which iron acts to cause disease and how the brain would be impacted by iron chelation could potentially give us novel insights into new therapies directed towards preventing or slowing neuronal cell loss associated with these disorders.     

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.     

A ferritin-responsive internal ribosome entry site regulates folate metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.     

Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)

Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.     

Secretion of ferritin by iron-laden macrophages and influence of lipoproteins

Increasing evidence supports a role of cellular iron in the initiation and development of atherosclerosis. We and others reported earlier that iron-laden macrophages are associated with LDL oxidation, angiogenesis, nitric oxide production and apoptosis in atherosclerotic processes. Here we have further studied perturbed iron metabolism in macrophages, their interaction with lipoproteins and the origin of iron accumulation in human atheroma. In both early and advanced human atheroma lesions, hemoglobin and ferritin accumulation correlated with the macrophage-rich areas. Iron uptake into macrophages, via transferrin receptors or scavenger receptor-mediated erythrophagocytosis, increased cellular iron and accelerated ferritin synthesis at both mRNA and protein levels. The binding activity of iron regulatory proteins was enhanced by desferrioxamine (DFO) and decreased by hemin and iron compounds. Iron-laden macrophages exocytosed both iron and ferritin into the culture medium. Exposure to oxidized low-density lipoprotein (oxLDL, >or=50 microg/mL) resulted in <20% apoptosis of iron-laden human macrophages, but cells remained impermeable after a 24 h period and an increased excretion of ferritin could be observed by immunostaining techniques. Exposure to high-density lipoprotein (HDL) significantly decreased ferritin excretion from these cells. We conclude: (i) erythrophagocytosis and hemoglobin catabolism by macrophages contribute to ferritin accumulation in human atherosclerotic lesions and; (ii) iron uptake into macrophages leads to increased synthesis and secretion of ferritin; (iii) oxidized LDL and HDL have different effects on these processes.     

Secretion of Ferritin by Iron-laden Macrophages and Influence of Lipoproteins

Increasing evidence supports a role of cellular iron in the initiation and development of atherosclerosis. We and others reported earlier that iron-laden macrophages are associated with LDL oxidation, angiogenesis, nitric oxide production and apoptosis in atherosclerotic processes. Here we have further studied perturbed iron metabolism in macrophages, their interaction with lipoproteins and the origin of iron accumulation in human atheroma. In both early and advanced human atheroma lesions, hemoglobin and ferritin accumulation correlated with the macrophage-rich areas. Iron uptake into macrophages, via transferrin receptors or scavenger receptor-mediated erythrophagocytosis, increased cellular iron and accelerated ferritin synthesis at both mRNA and protein levels. The binding activity of iron regulatory proteins was enhanced by desferrioxamine (DFO) and decreased by hemin and iron compounds. Iron-laden macrophages exocytosed both iron and ferritin into the culture medium. Exposure to oxidized low-density lipoprotein (oxLDL, ≥50?μg/mL) resulted in <20% apoptosis of iron-laden human macrophages, but cells remained impermeable after a 24?h period and an increased excretion of ferritin could be observed by immunostaining techniques. Exposure to high-density lipoprotein (HDL) significantly decreased ferritin excretion from these cells. We conclude: (i) erythrophagocytosis and hemoglobin catabolism by macrophages contribute to ferritin accumulation in human atherosclerotic lesions and; (ii) iron uptake into macrophages leads to increased synthesis and secretion of ferritin; (iii) oxidized LDL and HDL have different effects on these processes.