Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml^–1, with a mean value of 479±286 (SD) ng ml^–1. Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml^–1 in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml^–1. There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112±0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.     

The many faces of the octahedral ferritin protein

Iron is an essential trace nutrient required for the active sites of many enzymes, electron transfer and oxygen transport proteins. In contrast, to its important biological roles, iron is a catalyst for reactive oxygen species (ROS). Organisms must acquire iron but must protect against oxidative damage. Biology has evolved siderophores, hormones, membrane transporters, and iron transport and storage proteins to acquire sufficient iron but maintain iron levels at safe concentrations that prevent iron from catalyzing the formation of ROS. Ferritin is an important hub for iron metabolism because it sequesters iron during times of iron excess and releases iron during iron paucity. Ferritin is expressed in response to oxidative stress and is secreted into the extracellular matrix and into the serum. The iron sequestering ability of ferritin is believed to be the source of the anti-oxidant properties of ferritin. In fact, ferritin has been used as a biomarker for disease because it is synthesized in response to oxidative damage and inflammation. The function of serum ferritin is poorly understood, however serum ferritin concentrations seem to correlate with total iron stores. Under certain conditions, ferritin is also associated with pro-oxidant activity. The source of this switch from anti-oxidant to pro-oxidant has not been established but may be associated with unregulated iron release from ferritin. Recent reports demonstrate that ferritin is involved in other aspects of biology such as cell activation, development, immunity and angiogenesis. This review examines ferritin expression and secretion in correlation with anti-oxidant activity and with respect to these new functions. In addition, conditions that lead to pro-oxidant conditions are considered.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

Red blood cells upregulate cytoprotective proteins and the labile iron pool in dividing human T cells despite a reduction in oxidative stress

We have recently reported that red blood cells (RBC) promote T cell growth and survival by inhibiting activation-induced T cell death. In the present study, we have examined parameters of oxidative stress and intracellular iron in activated T cells and correlated these data with the expression of ferritin, heme oxygenase-1 (HO-1), and the transferrin receptor CD71. T cells growing in the presence of RBC had reduced levels of reactive oxygen species (ROS) and oxidatively modified proteins, suggesting that RBC efficiently counteracted ROS production on the activated T cells. Flow cytometry and immunodetection demonstrated that T cells dividing in the presence of RBC had increased levels of intracellular ferritin rich in L-subunits and HO-1 along with a downmodulation in CD71 expression. Finally, using the fluorescent iron indicator calcein and flow cytometry analysis, we were able to show that a relative amount of the labile iron pool (LIP) was upregulated in T cells growing in the presence of RBC. These findings are consistent with a typical response to iron overload. However, neither heme compounds nor ferric iron reproduced the levels of expansion and survival of T cells induced by intact RBC. Altogether, these data suggest that RBC inhibit apoptosis of activated T cells by a combination of ROS scavenging and upregulation of cytoprotective proteins such as ferritin and HO-1, which may counteract a possible toxic effect of the increased intracellular free iron.     

Ferritin as a source of iron for oxidative damage.

The generation of deleterious activated oxygen species capable of damaging DNA, lipids, and proteins requires a catalyst such as iron. Once released, ferritin iron is capable of catalyzing these reactions. Thus, agents that promote iron release may lead to increased oxidative damage. The superoxide anion formed enzymatically, radiolytically, via metal-catalyzed oxidations, or by redox cycling xenobiotics reductively mobilizes ferritin iron and promotes oxidative damage. In addition, a growing list of compounds capable of undergoing single electron oxidation/reduction reactions exemplified by paraquat, adriamycin, and alloxan have been reported to release iron from ferritin. Because the rapid removal of iron from ferritin requires reduction of the iron core, it is not surprising that the reduction potential of a compound is a primary factor that determines whether a compound will mobilize ferritin iron. The reduction potential does not, however, predict the rate of iron release. Therefore, ferritin-dependent oxidative damage may be involved in the pathogenesis of diseases where increased superoxide formation occurs and the toxicity of chemicals that increase superoxide production or have an adequate reduction potential to mobilize ferritin iron.     

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.     

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.     

Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis

Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, over-accumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage.     

Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.     

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.     

Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells

Iron is essential for life in almost all organisms and, in mammals, is absorbed through the villus cells of the duodenum. Using a human colonic carcinoma cell line that has many duodenal characteristics, HT29, we show that genes involved in intestinal iron transport are endogenously expressed. When stably transfected to express the hereditary hemochromatosis protein HFE these cells have increased ferritin levels. We demonstrate that this is not due to an effect on the transferrin (TF)-mediated iron uptake pathway but rather due to inhibition of iron efflux from the cell. The effect of HFE was independent of its interaction with TF receptor 1 as indicated by similar results using both the wild type HFE and the W81A mutant that binds TF receptor 1 with greatly reduced affinity. HFE expression did not affect the mRNA levels of most of the genes involved in iron absorption that were tested; however, it did correspond to a decrease in hephaestin message levels. These results point to a role for HFE in inhibition of iron efflux in HT29 cells. This is a distinct role from that in HeLa and human embryonic kidney 293 cells where HFE has been shown to inhibit TF-mediated iron uptake resulting in decreased ferritin levels. Such a distinction suggests a multifunctional role for HFE that is dependent upon expression levels of proteins involved in iron transport.     

Iron chelation in the biological activity of curcumin

Curcumin is among the more successful chemopreventive compounds investigated in recent years, and is currently in human trials to prevent cancer. The mechanism of action of curcumin is complex and likely multifactorial. We have made the unexpected observation that curcumin strikingly modulates proteins of iron metabolism in cells and in tissues, suggesting that curcumin has properties of an iron chelator. Curcumin increased mRNA levels of ferritin and GSTalpha in cultured liver cells. Unexpectedly, however, although levels of GSTalpha protein increased in parallel with mRNA levels in response to curcumin, levels of ferritin protein declined. Since iron chelators repress ferritin translation, we considered that curcumin may act as an iron chelator. To test this hypothesis, we measured the effect of curcumin on transferrin receptor 1, a protein stabilized under conditions of iron limitation, as well as the ability of curcumin to activate iron regulatory proteins (IRPs). Both transferrin receptor 1 and activated IRP, indicators of iron depletion, increased in response to curcumin. Consistent with the hypothesis that curcumin acts as an iron chelator, mice that were fed diets supplemented with curcumin exhibited a decline in levels of ferritin protein in the liver. These results suggest that iron chelation may be an additional mode of action of curcumin.     

Iron homeostasis is dysregulated,but the iron-hepcidin axis is functional,in chronic liver disease

BackgroundPerturbations in iron homeostasis have been reported to be associated with irreversible liver injury in chronic liver disease (CLD). However, it is not clear whether liver dysfunction per se underlies such dysregulation or whether other factors also contribute to it. This study attempted to examine the issues involved.MethodsPatients diagnosed to have chronic liver disease (n = 63), who underwent a medically-indicated upper gastrointestinal endoscopy, were the subjects of this study. Patients with dyspepsia, who underwent such a procedure, and were found to have no endoscopic abnormalities, were used as control subjects (n = 49). Duodenal mucosal samples were obtained to study mRNA and protein levels of duodenal proteins involved in iron absorption. A blood sample was also obtained for estimation of hematological, iron-related, inflammatory and liver function-related parameters.ResultsPatients with CLD had impaired liver function, anemia of inflammation and lower serum levels of hepcidin than control subjects. Gene (mRNA) expression levels of duodenal ferroportin and duodenal cytochrome b (proteins involved in iron absorption) were decreased, while that of divalent metal transporter–1 (DMT-1) was unchanged. Protein expression of DMT-1 was, however, decreased while that of ferroportin was unchanged. In the CLD group, serum hepcidin was predicted independently by serum ferritin and hemoglobin, but not by C-reactive protein (a marker of inflammation). CLD patients with serum ferritin greater than 300 μg/dL had significantly greater liver dysfunction (as indicated by significantly higher serum concentrations of bilirubin, AST and ALT, and MELD scores), higher serum concentrations of CRP and hepcidin, and higher ferroportin protein expression, than those with serum ferritin ≤ 300 μg/dL.ConclusionsIn patients with CLD, anemia of inflammation and low serum hepcidin levels were found to paradoxically co-exist. Expression of duodenal proteins involved in iron absorption were either decreased or unaltered in these patients. The hepcidin response to higher body iron levels and/or inflammation appeared to be functional in these patients, despite the presence of liver disease.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

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.     

Ferritin accumulation under iron scarcity in Drosophila iron cells

Ferritins are highly stable, multi-subunit protein complexes with iron-binding capacities that reach 4500 iron atoms per ferritin molecule. The strict dependence of cellular physiology on an adequate supply of iron cofactors has likely been a key driving force in the evolution of ferritins as iron storage molecules. The insect intestine has long been known to contain cells that are responsive to dietary iron levels and a specialized group of “iron cells” that always accumulate iron-loaded ferritin, even when no supplementary iron is added to the diet. Here, we further characterize ferritin localization in Drosophila melanogaster larvae raised under iron-enriched and iron-depleted conditions. High dietary iron intake results in ferritin accumulation in the anterior midgut, but also in garland (wreath) cells and in pericardial cells, which together filter the circulating hemolymph. Ferritin is also abundant in the brain, where levels remain unaltered following dietary iron chelation, a treatment that depletes ferritin from the aforementioned tissues. We attribute the stability of ferritin levels in the brain to the function of the blood-brain barrier that may shield this organ from systemic iron fluctuations. Most intriguingly, our dietary manipulations demonstrably iron-depleted the iron cells without a concomitant reduction in their production of ferritin. Therefore, insect iron cells may constitute an exception from the evolutionary norm with respect to iron-dependent ferritin regulation. It will be of interest to decipher both the physiological purpose served and the mechanism employed to untie ferritin regulation from cellular iron levels in this cell type.     

Ferritin levels in microglia depend upon activation: modulation by reactive oxygen species

Iron is one of the trace elements playing a key role in the normal cellular metabolism. Since an excess of free iron is catalyzing the Fenton reaction, most of the intracellular iron is sequestered in the iron storage protein ferritin. The binding of iron into ferritin is well described for physiological conditions, however, under certain pathophysiological situations, the efficiency of this process is unknown. In the brain, microglial cells are among others the cell population most importantly responsible for the maintenance of the extracellular environment. These cells might undergo activation, and little is known about the expression of ferritin during activation of microglial cells. Therefore, we tested the microglial model cell line RAW264.7 for the expression of ferritin after LPS activation. A significant decrease in the levels of the ferritin H-chain during activation and a significant increase in the early recovery phase were found. We were able to demonstrate that reactive oxygen species are responsible for a suppression of the H-chain of ferritin, whereas iNOS expression and NO synthesis are counteracting the reactive oxygen species effect. The balance of reactive oxygen species and NO production are, therefore, determining expression levels of the ferritin H-chain during activation of microglial cells.