Regulation Of Cellular Iron Metabolism Research Articles

Dysregulation of the sensory and regulatory pathways controlling cellular iron metabolism in unilateral obstructive nephropathy.

Chronic kidney disease involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in Irp1+/+ and Irp1-/- mice, we asked if iron regulatory proteins (IRPs), the central regulators of cellular iron metabolism and suppressors of EPO production, contribute to the etiology of anemia in kidney failure. We identified a significant reduction in IRP protein level and RNA binding activity that associates with a loss of the iron uptake protein transferrin receptor 1 (TfR1), increased expression of the iron storage protein subunits H- and L-ferritin, and a low but overall variable level of stainable iron in the obstructed kidney. This reduction in IRP RNA binding activity and ferritin RNA levels suggests the concomitant rise in ferritin expression and iron content in kidney failure is IRP dependent. In contrast, the reduction in the Epo mRNA level in the obstructed kidney was not rescued by genetic ablation of IRP1, suggesting disruption of normal hypoxia-inducible factor (HIF)-2α regulation. Furthermore, reduced expression of some HIF-α target genes in UUO occurred in the face of increased expression of HIF-α proteins and prolyl hydroxylases 2 and 1, the latter of which is not known to be HIF-α mediated. Our results suggest that the IRP system drives changes in cellular iron metabolism that are associated with kidney failure in UUO but that the impact of IRPs on EPO production is overridden by disrupted hypoxia signaling.NEW & NOTEWORTHY This study demonstrates that iron metabolism and hypoxia signaling are dysregulated in unilateral obstructive nephropathy. Expression of iron regulatory proteins (IRPs), central regulators of cellular iron metabolism, and the iron uptake (transferrin receptor 1) and storage (ferritins) proteins they target is strongly altered. This suggests a role of IRPs in previously observed changes in iron metabolism in progressive renal disease. Hypoxia signaling is disrupted and appeared to dominate the action of IRP1 in controlling erythropoietin expression.

HDAC inhibition induces EMT and alterations in cellular iron homeostasis to augment ferroptosis sensitivity in SW13 cells

Epithelial-to-mesenchymal transition (EMT) is an essential mechanism for development and wound healing, but in cancer it also mediates the progression and spread of aggressive tumors while increasing therapeutic resistance. Adoption of a mesenchymal state is also associated with increased iron uptake, but the relationship between EMT and the key regulators of cellular iron metabolism remains undefined. In this regard, the human adrenal cortical carcinoma SW13 cell line represents an invaluable research model as HDAC inhibitor treatment can convert them from an epithelial-like (SW13-) cell type to a mesenchymal-like (SW13+) subtype. In this study we establish SW13 cells as a model for exploring the link between iron and EMT. Increased iron accumulation following HDAC inhibitor mediated EMT is associated with decreased expression of the iron export protein ferroportin, enhanced ROS production, and reduced expression of antioxidant response genes. As availability of redox active iron and loss of lipid peroxide repair capacity are hallmarks of ferroptosis, a form of iron-mediated cell death, we next examined whether HDAC inhibitor treatment could augment ferroptosis sensitivity. Indeed, HDAC inhibitor treatment synergistically increased cell death following induction of ferroptosis. The exact mechanisms by which HDAC inhibition facilitates cell death following ferroptosis induction requires further study. As several HDAC inhibitors are already in use clinically for the treatment of certain cancer types, the findings from these studies have immediate implications for improving iron-targeted chemotherapeutic strategies.

Ascorbate and Tumor Cell Iron Metabolism: The Evolving Story and Its Link to Pathology.

Significance: Vitamin C or ascorbate (Asc) is a water-soluble vitamin and an antioxidant that is involved in many crucial biological functions. Asc's ability to reduce metals makes it an essential enzyme cofactor. Recent Advances: The ability of Asc to act as a reductant also plays an important part in its overall role in iron metabolism, where Asc induces both nontransferrin-bound iron and transferrin-bound iron uptake at physiological concentrations (∼50 μM). Moreover, Asc has emerged to play an important role in multiple diseases and its effects at pharmacological doses could be important for their treatment. Critical Issues: Asc's role as a regulator of cellular iron metabolism, along with its cytotoxic effects and different roles at pharmacological concentrations, makes it a candidate as an anticancer agent. Ever since the controversy regarding the studies from the Mayo Clinic was finally explained, there has been a renewed interest in using Asc as a therapeutic approach toward cancer due to its minimal side effects. Numerous studies have been able to demonstrate the anticancer activity of Asc through selective oxidative stress toward cancer cells via H2O2 generation at pharmacological concentrations. Studies have demonstrated that Asc's cytotoxic mechanism at concentrations (>1 mM) has been associated with decreased cellular iron uptake. Future Directions: Recent studies have also suggested other mechanisms, such as Asc's effects on autophagy, polyamine metabolism, and the cell cycle. Clearly, more has yet to be discovered about Asc's mechanism of action to facilitate safe and effective treatment options for cancer and other diseases.

342-LB: Iron Regulatory Protein 2 Regulates Proinsulin Production in Pancreatic Beta Cells through Iron-Dependent Cdkal1-Catalyzed tRNA Modification

Iron regulatory protein 2 (Irp2) is a key regulator of cellular iron metabolism in vertebrates. Irp2 controls the post-transcriptional expression of the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin. Here we show that Irp2-/-mice develop diabetes characterized by glucose intolerance and impaired glucose-stimulated insulin secretion from pancreatic β-cells. Impaired glucose-stimulated insulin secretion in Irp2-/-β-cells is not due to insulin resistance but rather to a reduction in insulin stores caused by impaired proinsulin processing. Loss of Irp2 reduces TfR1 expression and iron content, and increases ferritin expression and iron sequestration causing cellular iron deficiency that impairs mitochondrial Fe-S cluster biosynthesis. This in turn reduces the function of Cdk5-regulated subunit-associated protein 1 like (Cdkal1), a 4Fe-4S cluster enzyme that catalyzes the 2-methythio (ms) modification of t6A37 of tRNALys(UUU)to ms2t6A37, which is critical for efficient translation of lysine codons in proinsulin. Irp2-deficient β cells show reduced ms2t6A37 levels and lysine incorporation into proinsulin. Increasing cellular iron content in Irp2-deficient β cells normalizes mitochondrial function, ms2t6A37 levels, proinsulin and insulin content, and insulin secretion. These study reveals a previously unidentified link between insulin processing and cellular iron homeostasis, and show that Irp2 is required for normal β cell function and whole body glucose homeostasis. Disclosure M.C. Ferreira dos Santos: None. M. Rajan: None. E. Leibold: None. Funding American Diabetes Association (1-18-PMF-020 to M.C.F.D.S.); National Institute of Diabetes and Digestive and Kidney Diseases

Paradoxical Increase of Insulin Sensitivity Despite Blunted Adipose Tissue Browning in Genetic Ablation of IRP1 (OR09-08-19)

Abstract Objectives Iron regulatory protein 1 (IRP1) plays a key regulator of cellular iron metabolism, systemic oxygen sensing, and erythropoiesis. Deletion of IRP1 leads to profound HIF2a-dependent abnormalities in erythropoiesis and iron metabolism. Previously, we demonstrated that modulation of adipose tissue iron metabolism is necessary for adipose tissue browning. However, the role of IRP1 in adipose tissue browning and its metabolic consequences are uncertain. This study aimed to investigate the role of IRP1 in regulating adipose tissue browning in a mouse model of genetic ablation of IPR1 (IRP1−/−). Methods The IRP1−/− mice and wildtype (WT) controls were kept either at room (25°C) or cold (6°C) temperature for 7 days. Adipose tissue browning was evaluated by UCP1 expression and prevalence of beige-like structure in inguinal fat. Thermogenic heat release captured by infrared camera and core body temperature was measured by a rectal thermometer. The modulation of iron metabolism was assessed by serum levels of ferritin, hematocrit, and erythropoietin levels by ELISA. To investigate the role of IRP1 on energy metabolism, IRP1−/− and WT controls were fed a high-fat diet (45%) for 14 weeks. Insulin sensitivity was determined by glucose and insulin tolerance test and HOMA-IR score. [3H]-2-deoxyglucose (DOG) was injected to determine the distribution of 3H-radioactivity was quantified. Results IRP1−/− mice dramatically increased serum levels of erythropoietin but decreased hepcidin. IRP1−/− developed polycythemia and reticulocytosis, which was not affected by cold exposure. IRP1−/− were completely blunted in cold-induced browning in the inguinal fat showing no changes in UCP1 and adipocyte morphology. Unexpectedly, IRP1−/− showed higher core body temperature and heat release than control independent of UCP1 expression. Chronic intake of HF diet paradoxically increased the insulin sensitivity regardless of obesity. 2-DOG distribution was significantly increased in red blood cells, suggesting that red blood cell-dependent energy expenditure significantly contributed to rapid glucose disposal. Conclusions Disruption of IRP1 blunted adipose tissue browning. The paradoxical rise in insulin sensitivity in IRP1−/− is likely due to red blood cells-mediated energy expenditure. Funding Sources None.

Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

AbstractIn macrophages, cellular iron metabolism status is tightly integrated with macrophage phenotype and associated with mitochondrial function. However, how molecular events regulate mitochondrial activity to integrate regulation of iron metabolism and macrophage phenotype remains unclear. Here, we explored the important role of the actin-regulatory protein glia maturation factor-γ (GMFG) in the regulation of cellular iron metabolism and macrophage phenotype. We found that GMFG was downregulated in murine macrophages by exposure to iron and hydrogen peroxide. GMFG knockdown altered the expression of iron metabolism proteins and increased iron levels in murine macrophages and concomitantly promoted their polarization toward an anti-inflammatory M2 phenotype. GMFG-knockdown macrophages exhibited moderately increased levels of mitochondrial reactive oxygen species (mtROS), which were accompanied by decreased expression of some mitochondrial respiration chain components, including the iron-sulfur cluster assembly scaffold protein ISCU as well as the antioxidant enzymes SOD1 and SOD2. Importantly, treatment of GMFG-knockdown macrophages with the antioxidant N-acetylcysteine reversed the altered expression of iron metabolism proteins and significantly inhibited the enhanced gene expression of M2 macrophage markers, suggesting that mtROS is mechanistically linked to cellular iron metabolism and macrophage phenotype. Finally, GMFG interacted with the mitochondrial membrane ATPase ATAD3A, suggesting that GMFG knockdown–induced mtROS production might be attributed to alteration of mitochondrial function in macrophages. Our findings suggest that GMFG is an important regulator in cellular iron metabolism and macrophage phenotype and could be a novel therapeutic target for modulating macrophage function in immune and metabolic disorders.

Iron Regulatory Protein 1 Sustains Mitochondrial Iron Loading and Function in Frataxin Deficiency

Mitochondrial iron accumulation is a hallmark of diseases associated with impaired iron-sulfur cluster (Fe-S) biogenesis, such as Friedreich ataxia linked to frataxin (FXN) deficiency. The pathophysiological relevance of the mitochondrial iron loading and the underlying mechanisms are unknown. Using a mouse model of hepatic FXN deficiency in combination with mice deficient for iron regulatory protein 1 (IRP1), a key regulator of cellular iron metabolism, we show that IRP1 activation in conditions of Fe-S deficiency increases the available cytosolic labile iron pool. Surprisingly, our data indicate that IRP1 activation sustains mitochondrial iron supply and function rather than driving detrimental iron overload. Mitochondrial iron accumulation is shown to depend on mitochondrial dysfunction and heme-dependent upregulation of the mitochondrial iron importer mitoferrin-2. Our results uncover an unexpected protective role of IRP1 in pathological conditions associated with altered Fe-S metabolism.

The IRP/IRE system in vivo: insights from mouse models.

Iron regulatory proteins 1 and 2 (IRP1 and IRP2) post-transcriptionally control the expression of several mRNAs encoding proteins of iron, oxygen and energy metabolism. The mechanism involves their binding to iron responsive elements (IREs) in the untranslated regions of target mRNAs, thereby controlling mRNA translation or stability. Whereas IRP2 functions solely as an RNA-binding protein, IRP1 operates as either an RNA-binding protein or a cytosolic aconitase. Early experiments in cultured cells established a crucial role of IRPs in regulation of cellular iron metabolism. More recently, studies in mouse models with global or localized Irp1 and/or Irp2 deficiencies uncovered new physiological functions of IRPs in the context of systemic iron homeostasis. Thus, IRP1 emerged as a key regulator of erythropoiesis and iron absorption by controlling hypoxia inducible factor 2α (HIF2α) mRNA translation, while IRP2 appears to dominate the control of iron uptake and heme biosynthesis in erythroid progenitor cells by regulating the expression of transferrin receptor 1 (TfR1) and 5-aminolevulinic acid synthase 2 (ALAS2) mRNAs, respectively. Targeted disruption of either Irp1 or Irp2 in mice is associated with distinct phenotypic abnormalities. Thus, Irp1−/− mice develop polycythemia and pulmonary hypertension, while Irp2−/− mice present with microcytic anemia, iron overload in the intestine and the liver, and neurologic defects. Combined disruption of both Irp1 and Irp2 is incombatible with life and leads to early embryonic lethality. Mice with intestinal- or liver-specific disruption of both Irps are viable at birth but die later on due to malabsorption or liver failure, respectively. Adult mice lacking both Irps in the intestine exhibit a profound defect in dietary iron absorption due to a “mucosal block” that is caused by the de-repression of ferritin mRNA translation. Herein, we discuss the physiological function of the IRE/IRP regulatory system.

Regulation of cellular iron metabolism and its implications in lung cancer progression.

Iron is essential for life and is involved in numerous metabolic processes including cell growth and proliferation. However, excess iron in the body raises the risk of developing cancer due to its capacity to engage in redox cycling and free radical production. Therefore, iron can contribute to both carcinogenesis and tumor growth. Both epidemiologic and laboratory studies have demonstrated that the effects of iron overload are associated with the tumorigenesis of lung cancer and growth of lung cancer cells. In particular, the discovery of hepcidin and several iron transporters in the past decade may warrant reconsideration of the role of iron in carcinogenesis and tumor cell proliferation in lung cancer. Pathways of iron uptake, storage, efflux, and regulation are all disturbed in cancer, suggesting that reprogramming of iron metabolism is a critical aspect of tumor cell survival. Although these pathways in lung cancer have been identified and extensively studied, many issues on the metabolic processes of iron in lung cancer cells have not been addressed. Targeting metabolic pathways of iron may provide new tools for lung cancer prognosis and therapy.

Mammalian Iron Homeostasis in Health and Disease: Uptake, Storage, Transport, and Molecular Mechanisms of Action

Iron is a crucial factor for life. However, it also has the potential to cause the formation of noxious free radicals. These double-edged sword characteristics demand a tight regulation of cellular iron metabolism. In this review, we discuss the various pathways of cellular iron uptake, cellular iron storage, and transport. Recent advances in understanding the reduction and uptake of non-transferrin-bound iron are discussed. We also discuss the recent progress in the understanding of transcriptional and translational regulation by iron. Furthermore, we discuss recent advances in the understanding of the regulation of cellular and systemic iron homeostasis and several key diseases resulting from iron deficiency and overload. We also discuss the knockout mice available for studying iron metabolism and the related human conditions.

Iron Regulatory Protein 1 Outcompetes Iron Regulatory Protein 2 in Regulating Cellular Iron Homeostasis in Response to Nitric Oxide

In mammals, iron regulatory proteins (IRPs) 1 and 2 posttranscriptionally regulate expression of genes involved in iron metabolism, including transferrin receptor 1, the ferritin (Ft) H and L subunits, and ferroportin by binding mRNA motifs called iron responsive elements (IREs). IRP1 is a bifunctional protein that mostly exists in a non-IRE-binding, [4Fe-4S] cluster aconitase form, whereas IRP2, which does not assemble an Fe-S cluster, spontaneously binds IREs. Although both IRPs fulfill a trans-regulatory function, only mice lacking IRP2 misregulate iron metabolism. NO stimulates the IRE-binding activity of IRP1 by targeting its Fe-S cluster. IRP2 has also been reported to sense NO, but the intrinsic function of IRP1 and IRP2 in NO-mediated regulation of cellular iron metabolism is controversial. In this study, we exposed bone marrow macrophages from Irp1(-/-) and Irp2(-/-) mice to NO and showed that the generated apo-IRP1 was entirely responsible for the posttranscriptional regulation of transferrin receptor 1, H-Ft, L-Ft, and ferroportin. The powerful action of NO on IRP1 also remedies the defects of iron storage found in IRP2-null bone marrow macrophages by efficiently reducing Ft overexpression. We also found that NO-dependent IRP1 activation, resulting in increased iron uptake and reduced iron sequestration and export, maintains enough intracellular iron to fuel the Fe-S cluster biosynthetic pathway for efficient restoration of the citric acid cycle aconitase in mitochondria. Thus, IRP1 is the dominant sensor and transducer of NO for posttranscriptional regulation of iron metabolism and participates in Fe-S cluster repair after exposure to NO.

Cellular Iron Depletion Stimulates the JNK and p38 MAPK Signaling Transduction Pathways, Dissociation of ASK1-Thioredoxin, and Activation of ASK1

The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion.

Regulation of cellular iron metabolism

Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages. The binding of iron-laden transferrin to the cell-surface transferrin receptor 1 results in endocytosis and uptake of the metal cargo. Internalized iron is transported to mitochondria for the synthesis of haem or iron–sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. Iron metabolism is controlled at different levels and by diverse mechanisms. The present review summarizes basic concepts of iron transport, use and storage and focuses on the IRE (iron-responsive element)/IRP (iron-regulatory protein) system, a well known post-transcriptional regulatory circuit that not only maintains iron homoeostasis in various cell types, but also contributes to systemic iron balance.

Effect of hypoxia on the expression of iron regulatory proteins 1 and the mechanisms involved

Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Iron regulatory proteins (IRPs, IRP1 and IRP2) are master regulators of cellular iron metabolism. Hypoxia triggers a broad range of gene responses that are primarily mediated by hypoxia-inducible factor-1 (HIF-1). In this study, we have shown that hypoxia could not only upregulate the expression of hypoxia inducible factor-1 but also downregulate the expression of IRP1. However, the molecular mechanisms that govern the IRP1 response to hypoxia are not known. Herein we suggested that HIF/HRE system was an essential link between IRP1 and hypoxia. The HRE of IRP1 5'-regulation regions could combine with HIF-1 in vitro. Dual-luciferase reporter assay showed that IRP1 was directly downregulated by HIF/HRE system.

Iron-dependent degradation of IRP2 requires its C-terminal region and IRP structural integrity

BackgroundIron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism, undergoes iron-dependent degradation via the ubiquitin-proteasome pathway. A stretch of 73 amino acids within the N-terminal domain 1 of the protein was reported to function as an iron sensor. However, mutants lacking this fragment remain sensitive to degradation in iron-replete cells.ResultsTo identify elements within IRP2 involved in the control of its stability, we undertook a systematic mutagenesis approach. Truncated versions of IRP2 were expressed in H1299 cells and analyzed for their response to iron. Deletion mutants lacking the entire C-terminal domain 4 (amino acids 719–963) of IRP2 remained stable following iron treatments. Moreover, the replacement of domain 4 of IRP1 with the corresponding region of IRP2 sensitized the chimerical IRP11–3/IRP24 protein to iron-dependent degradation, while the reverse manipulation gave rise to a stable chimerical IRP21–3/IRP14 protein. The deletion of just 26 or 34 C-terminal amino acids stabilized IRP2 against iron. However, the fusion of C-terminal IRP2 fragments to luciferase failed to sensitize the indicator protein for degradation in iron-loaded cells.ConclusionOur data suggest that the C-terminus of IRP2 contains elements that are necessary but not sufficient for iron-dependent degradation. The functionality of these elements depends upon the overall IRP structure.

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.

The Regulation of Cellular Iron Metabolism

While iron is an essential trace element required by nearly all living organisms, deficiencies or excesses can lead to pathological conditions such as iron deficiency anemia or hemochromatosis, respectively. A decade has passed since the discovery of the hemochromatosis gene, HFE, and our understanding of hereditary hemochromatosis (HH) and iron metabolism in health and a variety of diseases has progressed considerably. Although HFE-related hemochromatosis is the most widespread, other forms of HH have subsequently been identified. These forms are not attributed to mutations in the HFE gene but rather to mutations in genes involved in the transport, storage, and regulation of iron. This review is an overview of cellular iron metabolism and regulation, describing the function of key proteins involved in these processes, with particular emphasis on the liver's role in iron homeostasis, as it is the main target of iron deposition in pathological iron overload. Current knowledge on their roles in maintaining iron homeostasis and how their dysregulation leads to the pathogenesis of HH are discussed.

NADPH oxidase inhibitor diphenyliodonium abolishes lipopolysaccharide‐induced down‐regulation of transferrin receptor expression in N2a and BV‐2 cells

The activation of cellular inflammatory response is tightly linked to induced production of reactive oxygen species (ROS) and nitric oxide (NO), which in turn have been identified as important regulators of cellular iron metabolism. In the present study, we have used the microglia cell line BV-2 and the neuroblastoma cell line N2a to study the regulatory effects of the microbial agent lipopolysaccharide (LPS) on the expression of the transferrin receptor (TfR) and ferritin in cell lines with different characteristics. The receptor mainly responsible for LPS recognition is the Toll-like receptor 4 (TLR4) that triggers a variety of intracellular signalling cascades leading to the induction of transcription of target genes involved in the innate immune response. Among the pathways to be activated is the MAPK cascade leading to the activation of nuclear factor-kappaB that induces transcription of a variety of genes, e.g., inducible nitric oxide synthase (iNOS). The TLR4-mediated LPS response also induces the production of ROS through a mechanism(s) suggested to involve the activation of NADPH oxidase(s). This study shows that exposure of BV-2 and N2a cells to LPS results in decreased TfR protein levels and increased H-ferritin mRNA levels. The LPS down-regulatory effect on TfR protein expression is abolished by the NADPH oxidase inhibitor diphenyliodonium (DPI) but is not affected by the free radical scavenger N-acetyl-L-cysteine (NAC) or the iNOS inhibitor aminoguanidine (AG). The increased H-ferritin mRNA levels in response to LPS are not affected by DPI, NAC, or AG.

Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2)

AbstractIron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1. (Blood. 2005;106: 2580-2589)

What have we learnt from CDNA microarray gene expression studies about the role of iron in MPTP induced neurodegeneration and Parkinson's disease?

There have been numerous hypotheses concerning the etiology and mechanism of dorsal raphe dopaminergic neurodegeneration in Parkinson's disease and its animal models, MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and 6-hydroxydopamine. The advent of cDNA microarray gene expression where expression of thousands of genes can be globally assessed has indicated that mechanism of neurodegeneration by MPTP is a complex cascade of vicious circles. One of these is the alteration of genes associated with iron metabolism, a transitional metal closely associated with inducing the formation of reactive oxygen species and inducing oxidative stress. cDNA gene expression analyses support the established hypothesis of oxidative induced neurodegeneration involving iron deposition in substantia nigra pars compacta (SNPC) parkinsonian brains. The regulation of cellular iron metabolism has been further enhanced by the recent discovery of two iron regulatory proteins, IRP1 and IRP2 which control the level of iron with in the cell. When the cellular level of iron increases IRP2 is degraded by ubiquitination and no further iron accumulates. The reverse occurs when the level of iron is low within the cell. Knock-out IRP1 and IRP2 mice have shown that in latter mice brain iron accumulation precedes the neurodegeneration, ataxia and bradykinesia observed in these animals. Indeed MPTP treatment, which results in iron accumulation in SNCP, abolishes IRP2 with the concomitant increase in alpha-synuclein. Iron chelators such as R-apomorphine and EGCG, which protect against MPTP neurotoxicity, prevent the loss of IRP2 and the increase in alpha-synuclein. The presence of iron together with alpha-synuclein in SNPC may be detrimental for dopaminergic neurons. Since, iron has been shown to cause aggregation of alpha-synuclein to a neurotoxic agent. The use of iron chelators penetrating the blood brain barrier as neuroprotective drugs has been envisaged.