Iron metabolism in the reticuloendothelial system

Comprised mainly of monocytes and tissue macrophages, the reticuloendothelial system (RES) plays two major roles in iron metabolism: it recycles iron from senescent red blood cells and it serves as a large storage depot for excess iron. Although iron recycling by the RES represents the largest pathway of iron efflux in the body, the precise mechanisms involved have remained elusive. However, studies characterizing the function and regulation of Nramp1, DMT1, HFE, FPN1, CD163, and hepcidin are rapidly expanding our knowledge of the molecular aspects of RE iron handling. This review summarizes fundamental physiological and biochemical aspects of iron metabolism in the RES and focuses on how recent studies have advanced our understanding of these areas. Also discussed are novel insights into the molecular mechanisms contributing to the abnormal RE iron metabolism characteristic of hereditary hemochromatosis and the anemia of chronic disease.     

The roles of iron in health and disease

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

Iron homeostasis and tumorigenesis: molecular mechanisms and therapeutic opportunities

Excess iron is tightly associated with tumorigenesis in multiple human cancer types through a variety of mechanisms including catalyzing the formation of mutagenic hydroxyl radicals, regulating DNA replication, repair and cell cycle progression, affecting signal transduction in cancer cells, and acting as an essential nutrient for proliferating tumor cells. Thus, multiple therapeutic strategies based on iron deprivation have been developed in cancer therapy. During the past few years, our understanding of genetic association and molecular mechanisms between iron and tumorigenesis has expanded enormously. In this review, we briefly summarize iron homeostasis in mammals, and discuss recent progresses in understanding the aberrant iron metabolism in numerous cancer types, with a focus on studies revealing altered signal transduction in cancer cells.     

Regulation of SLC11A3 by inflammation

Acute and chronic inflammatory states are characterized by changes in body iron metabolism. These changes include a drop in serum iron, an increase in the rate of plasma iron disappearance, a decline in the rate of plasma iron turnover, reticuloendothelial system (RES) cell iron sequestration and a decline in intestinal iron absorption. This response is elicited by a variety of metabolic conditions and acute bacterial infections, especially gram-negative bacteria, and by experimental mediators of inflammation such as endotoxin and turpentine. These changes in iron metabolism contribute to the development of the anemia of chronic diseases. SLC11A3 (aka MTP1, ferroportin 1, IREG1) is a metal transporter that exports iron from the cytosol of cells and was initially identified as the duodenal epithelial basolateral iron transporter. Recent identification of a MTP1 mutation leading to hemochromatosis in man adds further weight to the hypothesis that MTP1 is involved in iron homeostasis. RES cells are responsible for the recycling of iron from the breakdown of heme from senescent erythrocytes and MTP1 has been hypothesized to be the key iron exporter in these cells. Supporting this hypothesis is the observation that MTP1 is expressed in the RES macrophages of the spleen, Kupffer cells, bone marrow and lymph node histiocytes, mesangial cells, brain microglial cells. In a mouse (C57/Bl6) model of lipopolysaccharide (LPS) induced acute inflammation, MTP1 expression in the cells of the RES is regulated by acute inflammation. Immunohistochemical staining of tissues, using an anti-MTP1 antibody, of mice given parenteral injections of LPS demonstrated down-regulation of MTP1 expression in the RES cells of the spleen and liver and also in the duodenal epithelial cells compared to control animals. Western blotting of total liver and spleen lysates confirmed the decline in MTP1 protein expression induced by LPS. In addition, RT-PCR analysis showed that LPS treatment also resulted in a decline in MTP1 mRNA in spleen, liver and duodenum compared to controls. One clue to the molecular signaling mechanism for MTP1 down-regulation by LPS comes from the study of the C3H/HeJ mouse, which lacks a functional LPS receptor, toll-like receptor 4 (TLR4). C3H/HeJ mice are resistant to the toxic and hypoferraemic effects of LPS. Similarly, a down-regulation of MTP1 in response to LPS in the C3H/HeJ mice was not observed. This finding indicates that the down-regulation of MTP1 by LPS requires signaling through TLR4. Despite resistance to LPS, treatment of C3H/HeJ mice with turpentine, an inducer of sterile inflammation, for a period of 24 hours resulted in down-regulation of MTP1 expression in the spleen. These data indicate that LPS mediated down-regulation of MTP1 requires a functional TLR4, but that there are non-TLR4 dependent mechanisms for the down-regulation of MTP1 by inflammatory stimuli. In vitro treatment of mouse adherent splenocytes with 5 ug ml of LPS also resulted in down-regulation of MTP1 mRNA. This in vitro down-regulation was not abrogated by co-treatment of cells with pyrrolidinedithiocarbamate (PDTC), a well-characterized inhibitor of NF-KB activation or anti-tumor necrosis factor-a antibodies. In addition, in vitro treatment of mouse splenocytes with recombinant TNF- did not result in down-regulation of MTP1 mRNA. The lack of antagonism between LPS and PDTC and the lack of an effect of TNF- in vitro indicates that NF-B activation may not be required for MTP1 mRNA down-regulation. This inflammation-mediated down-regulation of MTP1 expression in the RES may be a component responsible for iron sequestration in the RES in both acute and chronic inflammatory states.     

Insect iron binding proteins: insights from the genomes

Sequencing of the genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, and Bombyx mori provided an opportunity to examine the diversity and organization of genes encoding insect transferrins (Tsf) and ferritins. Information obtained from the genomes significantly advances our knowledge of these major players in insect iron metabolism and complements the results of molecular studies on their temporal, spatial, and inducible expression pattern and regulatory mechanisms conducted in diverse insect species. Analysis of genes encoding new members of the Tsf family and non-secreted ferritin subunits allows making preliminary hypotheses about their possible functions and opens possibilities to study lesser-known aspects of insect iron homeostasis. Proteomic and gene expression studies that followed the whole genome sequencing quickly contribute to defining or better understanding of the important and diverse biological roles of Tsf and ferritin, particularly their involvement in insect's defenses against oxidative stress and infection.     

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.     

A large number of the human microRNAs target lentiviruses, retroviruses, and endogenous retroviruses

Retroelements (including transposons, retrotransposons, retroviruses, and lentiviruses) make up a significant portion of eukaryotic genomes. Given their ability to mutate genes these mobile elements always present a threat to the integrity of the host genomes. Recent studies have revealed complex molecular mechanisms that silence the mutagenic ability of these RE as well strategically express the pieces of the incorporated RE that are utilized to silence human endogenous retroviruses (HERVs) or invading exogenous retroviruses (IERV). We have hypothesized that small endogenous RNA originally evolved to quell “foreign” IERV-genes and subsequently emerged into elaborate silencing systems that include RNA interference, miRNA-based gene regulation and other gene silencing mechanisms. Here, we present evidence that the replication of complex RE are most likely silenced or regulated by homologous miRNA that are found as a part of the cellular repertoire. We analyzed Homo sapiens miRNAs for possible target genetic sequences in selected HERVs and IERV found in humans and other large primates. We identified several miRNAs that have >80% sequence homology with human HERVs; -L, -W, and -K, and IERV like SIVcpz, HTLV-1, and HTLV-2. We found an inverse correlation between the numbers and relative degree of homology of miRNAs to the relative replication capacity of a specific RE. Therefore, larger numbers of miRNAs with greater degree of homology are found against the least active RE and the least numbers of miRNAs with smaller degree of homology are found against the most active RE (i.e. HERV-K). Implications of these observations in RE disease and therapy are discussed.     

The ferroportin-ceruloplasmin system and the mammalian iron homeostasis machine: regulatory pathways and the role of lactoferrin

In the last 20 years, several new genes and proteins involved in iron metabolism in eukaryotes, particularly related to pathological states both in animal models and in humans have been identified, and we are now starting to unveil at the molecular level the mechanisms of iron absorption, the regulation of iron transport and the homeostatic balancing processes. In this review, we will briefly outline the general scheme of iron metabolism in humans and then focus our attention on the cellular iron export system formed by the permease ferroportin and the ferroxidase ceruloplasmin. We will finally summarize data on the role of the iron binding protein lactoferrin on the regulation of the ferroportin/ceruloplasmin couple and of other proteins involved in iron homeostasis in inflamed human macrophages.     

Chemistry and biology of eukaryotic iron metabolism

With rare exceptions, virtually all studied organisms from Archaea to man are dependent on iron for survival. Despite the ubiquitous distribution and abundance of iron in the biosphere, iron-dependent life must contend with the paradoxical hazards of iron deficiency and iron overload, each with its serious or fatal consequences. Homeostatic mechanisms regulating the absorption, transport, storage and mobilization of cellular iron are therefore of critical importance in iron metabolism, and a rich biology and chemistry underlie all of these mechanisms. A coherent understanding of that biology and chemistry is now rapidly emerging. In this review we will emphasize discoveries of the past decade, which have brought a revolution to the understanding of the molecular events in iron metabolism. Of central importance has been the discovery of new proteins carrying out functions previously suspected but not understood or, more interestingly, unsuspected and surprising. Parallel discoveries have delineated regulatory mechanisms controlling the expression of proteins long known--the transferrin receptor and ferritin--as well as proteins new to the scene of iron metabolism and its homeostatic control. These proteins include the iron regulatory proteins (IRPs 1 and 2), a variety of ferrireductases in yeast an mammalian cells, membrane transporters (DMT1 and ferroportin 1), a multicopper ferroxidase involved in iron export from cells (hephaestin), and regulators of mitochondrial iron balance (frataxin and MFT). Experimental models, making use of organisms from yeast through the zebrafish to rodents have asserted their power in elucidating normal iron metabolism, as well as its genetic disorders and their underlying molecular defects. Iron absorption, previously poorly understood, is now a fruitful subject for research and well on its way to detailed elucidation. The long-sought hemochromatosis gene has been found, and active research is underway to determine how its aberrant functioning results in disease that is easily controlled but lethal when untreated. A surprising connection between iron metabolism and Friedreich's ataxia has been uncovered. It is no exaggeration to say that the new understanding of iron metabolism in health and disease has been explosive, and that what is past is likely to be prologue to what is ahead.     

Control of intracellular heme levels: Heme transporters and heme oxygenases

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology.     

Effects of hypoxic preconditioning on the expression of iron influx and efflux proteins in primary neuron culture

The mechanisms of neuroprotection induced by hypoxic preconditioning (HP) and the effects of HP on iron metabolism proteins in the brain have not been fully elucidated. Based on the accumulated information, we hypothesized that HP would be able to affect the expression of iron metabolism proteins in the brain and that the changes in the expression of these proteins induced by HP might be partly associated with the HP-induced neuroprotection. Here, we demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (Fpn1) proteins and thus increase transferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release, and also a progressive increase in cellular iron content in the cultured neurons. We concluded that HP has the ability to speed iron transport rate and proposed that the increase in iron transport rate and cellular iron in neurons might be one of the mechanisms involved in neuroprotection in the HP neurons. We also demonstrated that Fpn1 expression was significantly affected by HIF-1alpha, implying that the gene encoding this iron efflux protein is hypoxia-inducible.     

Time course of molecular responses of human skeletal muscle to acute bouts of resistance exercise.

Resistance exercise (RE) training, designed to induce hypertrophy, strives for optimal activation of anabolic and myogenic mechanisms to increase myofiber size. Clearly, activation of these mechanisms must precede skeletal muscle growth. Most mechanistic studies of RE have involved analysis of outcome variables after many training sessions. This study measured molecular level responses to RE on a scale of hours to establish a time course for the activation of myogenic mechanisms. Muscle biopsy samples were collected from nine subjects before and after acute bouts of RE. The response to a single bout was assessed at 12 and 24 h postexercise. Further samples were obtained 24 and 72 h after a second exercise bout. RE was induced by neuromuscular electrical stimulation to generate maximal isometric contractions in the muscle of interest. A single RE bout resulted in increased levels of mRNA for IGF binding protein-4 (84%), MyoD (83%), myogenin (approximately 3-fold), cyclin D1 (50%), and p21-Waf1 (16-fold), and a transient decrease in IGF-I mRNA (46%). A temporally conserved, significant correlation between myogenin and p21 mRNA was observed (r = 0.70, P < or = 0.02). The mRNAs for mechano-growth factor, IGF binding protein-5, and the IGF-I receptor were unchanged by RE. Total skeletal muscle RNA was increased 72 h after the second serial bout of RE. These results indicate that molecular adaptations of skeletal muscle to loading respond in a very short time. This approach should provide insights on the mechanisms that modulate adaptation to RE and may be useful in evaluating RE training protocol variables with high temporal resolution.     

Helicobacter pylori-related iron deficiency anemia: a review

Several clinical reports have demonstrated that Helicobacter pylori gastric infection has emerged as a new cause of refractory iron deficiency anemia, unresponsive to iron therapy, and not attributable to usual causes such as intestinal losses or poor intake, malabsorption or diversion of iron in the reticulo-endothelial system. Although the interaction between infection and iron metabolism is now well consolidated, our understanding of the pathogenetic mechanism underlying the anemia is still wanting. Microbiological and ferrokinetic studies seem to suggest that Helicobacter pylori infected antrum could act as a sequestering focus for serum iron by means of outer membrane receptors of the bacterium, that in vitro are able to capture and utilize for growth iron from human lactoferrin. The proposed hypothesis does not answer why this complication is such a rare disease outcome in a common human infection but it may be used as a template for further controlled studies to determine the mechanisms of this atypical, medically important putative sequelae of H. pylori infection.     

Bioenergetic challenges of microbial iron metabolisms

Before cyanobacteria invented oxygenic photosynthesis and O(2) and H(2)O began to cycle between respiration and photosynthesis, redox cycles between other elements were used to sustain microbial metabolism on a global scale. Today these cycles continue to occur in more specialized niches. In this review we focus on the bioenergetic aspects of one of these cycles - the iron cycle - because iron presents unique and fascinating challenges for cells that use it for energy. Although iron is an important nutrient for nearly all life forms, we restrict our discussion to energy-yielding pathways that use ferrous iron [Fe(II)] as an electron donor or ferric iron [Fe(III)] as an electron acceptor. We briefly review general concepts in bioenergetics, focusing on what is known about the mechanisms of electron transfer in Fe(II)-oxidizing and Fe(III)-reducing bacteria, and highlight aspects of their bioenergetic pathways that are poorly understood.