An oxidative stress-mediated positive-feedback iron uptake loop in neuronal cells

Intracellular reactive iron is a source of free radicals and a possible cause of cell damage. In this study, we analyzed the changes in iron homeostasis generated by iron accumulation in neuroblastoma (N2A) cells and hippocampal neurons. Increasing concentrations of iron in the culture medium elicited increasing amounts of intracellular iron and of the reactive iron pool. The cells had both IRP1 and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium increased from 1 to 40 microm, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron increased up to 20 microm, and then, unexpectedly, increased. IRP1 activity at iron concentrations above 20 microm was functional as it correlated with increased (55) Fe uptake. The increase in IRP1 activity was mediated by oxidative-stress as it was largely abolished by N-acetyl-L-cysteine. Culturing cells with iron resulted in proteins and DNA modifications. In summary, iron uptake by N2A cells and hippocampus neurons did not shut off at high iron concentrations in the culture media. As a consequence, iron accumulated and generated oxidative damage. This behavior is probably a consequence of the paradoxical activation of IRP1 at high iron concentrations, a condition that may underlie some processes associated with neuronal degeneration and death.     

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.     

HOIL-1 is not required for iron-mediated IRP2 degradation in HEK293 cells

Iron regulatory protein 2 (IRP2) binds to iron-responsive elements (IREs) to regulate the translation and stability of mRNAs encoding several proteins involved in mammalian iron homeostasis. Increases in cellular iron stimulate the polyubiquitylation and proteasomal degradation of IRP2. One study has suggested that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) binds to a unique 73-amino acid (aa) domain in IRP2 in an iron-dependent manner to regulate IRP2 polyubiquitylation and degradation. Other studies have questioned the role of the 73-aa domain in iron-dependent IRP2 degradation. We investigated the potential role of HOIL-1 in the iron-mediated degradation of IRP2 in human embryonic kidney 293 (HEK293) cells. We found that transiently expressed HOIL-1 and IRP2 interact via the 73-aa domain, but this interaction is not iron-dependent, nor does it enhance the rate of IRP2 degradation by iron. In addition, stable expression of HOIL-1 does not alter the iron-dependent degradation or RNA-binding activity of endogenous IRP2. Reduction of endogenous HOIL-1 by siRNA has no affect on the iron-mediated degradation of endogenous IRP2. These data demonstrate that HOIL-1 is not required for iron-dependent degradation of IRP2 in HEK293 cells, and suggest that a HOIL-1 independent mechanism is used for IRP2 degradation in most cell types.     

Iron-activated iron uptake: A positive feedback loop mediated by iron regulatory protein 1

The love-hate relationship between iron and living matter has generated mechanisms to maintain iron concentration in a narrow range, above and below which deleterious effects occur. At the cellular level, iron homeostasis is accomplished by the activity of the IRP proteins, which, under conditions of iron depletion, up-regulate the expression of the iron acquisition proteins TfR and DMT1. It has been shown that hydrogen peroxide activates IRP1 and that this activation mediates a potentially harmful increase in cell iron uptake. Here we show that IRP1 activity is also induced by iron-mediated oxidative stress. When cells were incubated with up to 20 M of iron, a typical decrease in IRP1 and IRP2 activity was observed. Interestingly, when iron was further increased to 40 or 80 M, IRP1 was reactivated in three of the four different cell lines tested, i.e., Caco-2 cells, N2A cells and HepG2 cells. In the fourth cell line (K562) IRP1 activity did not increase, but neither did it decrease. This response to iron was largely abrogated when the antioxidant N-acetyl cysteine was added along with iron to the culture medium. Thus, the effect of iron was mediated by oxidative stress. Increases in IRP1 activity were accompanied by increases in cell iron uptake, an indication that the activated IRP1 was functional in the activation of iron uptake. Hence, this iron-induced iron uptake feedback loop results in the increase of intracellular iron and increased oxidative stress.     

Multiple,conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.     

Crystal structure of human iron regulatory protein 1 as cytosolic aconitase

Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data.     

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.     

Iron accumulation and iron-regulatory protein activity in human hepatoma (HepG2) cells

Iron may populate distinct hepatocellular iron pools that differentially regulate expression of proteins such as ferritin and transferrin receptor (TfR) through iron-regulatory mRNA-binding proteins (IRPs), and may additionally regulate uptake and accumulation of non-transferrin-bound iron (NTBI). We examined iron-regulatory protein (IRP) binding activity and ferritin/TfR expression in human hepatoma (HepG2) cells exposed to iron at different levels for different periods. Several iron-dependent RNA-binding activities were identified, but only IRP increased with beta-mercaptoethanol. With exposures between 0 and 20 microg/ml iron, decreases in IRP binding accompanied large changes in TfR and ferritin expression, while chelation of residual iron with deferoxamine (DFO) caused a large increase in IRP binding with little additional effect on TfR or ferritin expression. Cellular iron content increased beyond 4 days of exposure to iron at 20 microg/ml, when IRP binding, TfR, and ferritin had all reached stable levels. However, iron content of the cells plateaued by 7 days, or decreased with 24 h exposure to very high concentrations (>50 microg/ml) of iron. These results indicate that iron-replete HepG2 cells exhibit a narrow range of maximal responsiveness of the IRP-regulatory mechanism, whose functional response is blunted both by excessive iron exposure and by removal of iron from a chelatable pool. HepG2 cells are able to limit iron accumulation upon higher or prolonged exposure to NTBI, apparently independent of the IRP mechanism.