Non-transferrin-bound Iron Uptake Research Articles

Plasma non-transferrin-bound iron uptake by the small intestine leads to intestinal injury and intestinal flora dysbiosis in an iron overload mouse model and Caco-2 cells.

Iron overload often occurs during blood transfusion and iron supplementation, resulting in the presence of non-transferrin-bound iron (NTBI) in host plasma and damage to multiple organs, but effects on the intestine have rarely been reported. In this study, an iron overload mouse model with plasma NTBI was established by intraperitoneal injection of iron dextran. We found that plasma NTBI damaged intestinal morphology, caused intestinal oxidative stress injury and reactive oxygen species (ROS) accumulation, and induced intestinal epithelial cell apoptosis. In addition, plasma NTBI increased the relative abundance of Ileibacterium and Desulfovibrio in the cecum, while the relative abundance of Faecalibaculum and Romboutsia was reduced. Ileibacterium may be a potential microbial biomarker of plasma NTBI. Based on the function prediction analysis, plasma NTBI led to the weakening of intestinal microbiota function, significantly reducing the function of the extracellular structure. Further investigation into the mechanism of injury showed that iron absorption in the small intestine significantly increased in the iron group. Caco-2 cell monolayers were used as a model of the intestinal epithelium to study the mechanism of iron transport. By adding ferric ammonium citrate (FAC, plasma NTBI in physiological form) to the basolateral side, the apparent permeability coefficient (Papp) values from the basolateral to the apical side were greater than 3×10-6 cm s-1. Intracellular ferritin level and apical iron concentration significantly increased, and SLC39A8 (ZIP8) and SLC39A14 (ZIP14) were highly expressed in the FAC group. Short hairpin RNA (shRNA) was used to knock down ZIP8 and ZIP14 in Caco-2 cells. Transfection with ZIP14-specific shRNA decreased intracellular ferritin level and inhibited iron uptake. These results revealed that plasma NTBI may cause intestinal injury and intestinal flora dysbiosis due to the uptake of plasma NTBI from the basolateral side into the small intestine, which is probably mediated by ZIP14.

Functional role of endothelial transferrin receptor 1 in iron sensing and homeostasis.

Systemic iron homeostasis is regulated by the hepatic hormone hepcidin to balance meeting iron requirements while limiting toxicity from iron excess. Iron-mediated induction of bone morphogenetic protein (BMP) 6 is a central mechanism for regulating hepcidin production. Liver endothelial cells (LECs) are the main source of endogenous BMP6, but how they sense iron to modulate BMP6 transcription and thereby hepcidin is uncertain. Here, we investigate the role of endothelial cell transferrin receptor 1 (TFR1) in iron uptake, BMP6 regulation, and systemic iron homeostasis using primary LEC cultures and endothelial Tfrc (encoding TFR1) knockout mice. We show that intracellular iron regulates Bmp6 expression in a cell-autonomous manner, and TFR1 mediates iron uptake and Bmp6 expression by holo-transferrin in primary LEC cultures. In addition, endothelial Tfrc knockout mice exhibit altered iron homeostasis compared with littermate controls when fed a limited iron diet, as evidenced by increased liver iron and inappropriately low Bmp6 and hepcidin expression relative to liver iron. However, endothelial Tfrc knockout mice have a similar iron phenotype compared to littermate controls when fed an iron-rich standard diet. Finally, ferritin and non-transferrin bound iron (NTBI) are additional sources of iron that mediate Bmp6 induction in primary LEC cultures via TFR1-independent mechanisms. Together, our data demonstrate a minor functional role for endothelial cell TFR1 in iron uptake, BMP6 regulation, and hepatocyte hepcidin regulation under iron limiting conditions, and suggest that ferritin and/or NTBI uptake by other transporters have a dominant role when iron availability is high.

DMT1 Protects Macrophages from Salmonella Infection by Controlling Cellular Iron Turnover and Lipocalin 2 Expression.

Macrophages are at the center of innate pathogen control and iron recycling. Divalent metal transporter 1 (DMT1) is essential for the uptake of non-transferrin-bound iron (NTBI) into macrophages and for the transfer of transferrin-bound iron from the endosome to the cytoplasm. As the control of cellular iron trafficking is central for the control of infection with siderophilic pathogens such as Salmonella Typhimurium, a Gram-negative bacterium residing within the phagosome of macrophages, we examined the potential role of DMT1 for infection control. Bone marrow derived macrophages lacking DMT1 (DMT1fl/flLysMCre(+)) present with reduced NTBI uptake and reduced levels of the iron storage protein ferritin, the iron exporter ferroportin and, surprisingly, of the iron uptake protein transferrin receptor. Further, DMT1-deficient macrophages have an impaired control of Salmonella Typhimurium infection, paralleled by reduced levels of the peptide lipocalin-2 (LCN2). LCN2 exerts anti-bacterial activity upon binding of microbial siderophores but also facilitates systemic and cellular hypoferremia. Remarkably, nifedipine, a pharmacological DMT1 activator, stimulates LCN2 expression in RAW264.7 macrophages, confirming its DMT1-dependent regulation. In addition, the absence of DMT1 increases the availability of iron for Salmonella upon infection and leads to increased bacterial proliferation and persistence within macrophages. Accordingly, mice harboring a macrophage-selective DMT1 disruption demonstrate reduced survival following Salmonella infection. This study highlights the importance of DMT1 in nutritional immunity and the significance of iron delivery for the control of infection with siderophilic bacteria.

Verapamil downregulates iron uptake and upregulates divalent metal transporter 1 expression in H9C2 cardiomyocytes.

Belgrade rats have a defect in divalent metal transport 1 (DMT1) with a reduced heart iron, indicating that DMT1 plays a physiological role in non-transferrin-bound iron (NTBI) uptake by cardiomyocytes. However, L-type voltage-dependent Ca2+ channel (LVDCC) blockers were recently demonstrated to significantly reduce NTBI uptake by cardiomyocytes, implying that LVDCC plays a dominant role in NTBI uptake by cardiomyocytes under iron-overloaded conditions. These findings led us to hypothesize that the LVDCC blocker-induced reduction in NTBI uptake might result not only from the inhibition of LVDCC-mediated NTBI uptake but also from the suppression of DMT1-mediated NTBI uptake. We therefore investigated the effects of the LVDCC blocker verapamil on NTBI uptake as well as DMT1 expression in H9C2 cells by the measurement of radio-labeled 55 Fe(II), reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis. We demonstrated that verapamil induced a significant reduction in NTBI uptake by H9C2 cells but also unexpectedly a remarkable increase rather than decrease in the expression of DMT1 mRNA and protein in H9C2 cells. Our findings imply that the verapamil-induced reduction in NTBI uptake by H9C2 cells is not associated with DMT1 and also indicate that verapamil stimulates rather than inhibits DMT1 expression and DMT1-mediated iron uptake by heart cells.

Plasma Non-transferrin-Bound Iron Could Enter into Mice Duodenum and Negatively Affect Duodenal Defense Response to Virus and Immune Responses.

Plasma non-transferrin-bound iron (NTBI) exists when the plasma iron content exceeds the carrying capacity of transferrin and can be quickly cleared by the liver, pancreas, and other organs. However, whether it could enter the small intestine and its effects still remain unclear. Herein, these issues were explored. Mice were intravenously administrated of ferric citrate (treatment) or citrate acid (control) 10min after the saturation of the transferrin. Two hours later, hepatic, duodenal, and jejunal iron content and distribution were measured and duodenal transcriptome sequencing was performed. Significant increase of duodenal and hepatic iron content was detected, indicating that plasma NTBI could be absorbed by the duodenum as well as the liver. A total of 103 differentially expressed genes were identified in the duodenum of mice in the treatment group compared to the control group. Gene Ontology (GO) functional analysis of these genes showed that they were mainly involved in defense response to virus and immune response. The results of Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) analysis revealed that there were major changes in the hematopoietic cell lineage and some virus infection pathways between the two groups. Determination of 7 cytokines in the duodenum were further conducted, which demonstrated that the anti-inflammatory factors interferon (IL)-4 and IL-10 in the duodenum were significantly decreased after NTBI uptake. Our findings revealed that NTBI in plasma can enter the duodenum, which would change the duodenal hematopoietic cell lineage and have a negative impact on defense response to the virus and immune responses.

The (Bio)Chemistry of Non-Transferrin-Bound Iron.

In healthy individuals, virtually all blood plasma iron is bound by transferrin. However, in several diseases and clinical conditions, hazardous non-transferrin-bound iron (NTBI) species occur. NTBI represents a potentially toxic iron form, being a direct cause of oxidative stress in the circulating compartment and tissue iron loading. The accumulation of these species can cause cellular damage in several organs, namely, the liver, spleen, and heart. Despite its pathophysiological relevance, the chemical nature of NTBI remains elusive. This has precluded its use as a clinical biochemical marker and the development of targeted therapies. Herein, we make a critical assessment of the current knowledge of NTBI speciation. The currently accepted hypotheses suggest that NTBI is mostly iron bound to citric acid and iron bound to serum albumin, but the chemistry of this system remains fuzzy. We explore the complex chemistry of iron complexation by citric acid and its implications towards NTBI reactivity. Further, the ability of albumin to bind iron is revised and the role of protein post-translational modifications on iron binding is discussed. The characterization of the NTBI species structure may be the starting point for the development of a standardized analytical assay, the better understanding of these species’ reactivity or the identification of NTBI uptake mechanisms by different cell types, and finally, to the development of new therapies.

Metal-Ion Transporter SLC39A14 Is Required for Cardiac Iron Loading in the Hjv Mouse Model of Iron Overload

Although iron overload-related cardiomyopathy is a leading cause of morbidity and mortality in iron-overload disorders (e.g., thalassemia major and hemochromatosis), the molecular mechanisms that mediate cardiac iron uptake and accumulation are incompletely understood. Previous studies using Slc39a14 knockout mice have revealed that SLC39A14 is required for the uptake of non-transferrin-bound iron (NTBI) by the liver and pancreas and is essential for iron loading of hepatocytes and pancreatic acinar cells. To investigate the requirement for SLC39A14 in cardiac iron accumulation, we generated cardiomyocyte-specific Slc39a14 knockout (Slc39a14 hrt/hrt) mice and crossed them with iron-loading hemojuvelin (Hjv) knockout mice to generate Hjv -/-;Slc39a14 hrt/hrt animals. At 12 and 24 weeks of age, cardiac nonheme iron levels were ~340% higher in Hjv -/- mice than in controls. By contrast, cardiac nonheme iron levels in Hjv -/-;Slc39a14 hrt/hrt mice at these ages were only ~60% higher than those than in controls, and ~65% less than those in Hjv -/- mice. Moreover, cardiac nonheme iron levels in Hjv -/-;Slc39a14 +/hrt (heterozygous conditional Slc39a14 knockout) mice were between those of Hjv -/- and Hjv -/-;Slc39a14 hrt/hrt mice, suggesting a gene-dosage effect of Slc39a14 on cardiac iron accumulation. A role for voltage-dependent calcium channels in mediating the uptake of NTBI into cardiomyocytes has been proposed based on observations of the effects of L-type calcium-channel blockers on iron uptake and accumulation in vitro and in vivo. We considered the possibility that these observations could be explained if SLC39A14 were reactive with calcium-channel blockers. To test this hypothesis, we examined the effects of blockers on the activity of SLC39A14 by using radiotracer assays in RNA-injected Xenopus oocytes expressing mouse SLC39A14. We found that 100 µM amlodipine (Amld), nifedipine, and nicardipine each afforded modest inhibition of SLC39A14-mediated 55Fe 2+. Inhibition of iron transport by Amld was dose-dependent, EC 50 = 167 µM ± (SEM) 30 µM. Our findings implicate SLC39A14 in mediating cardiomyocyte NTBI uptake in the mouse and raise doubts about the relative importance of calcium channels as a mechanism by which NTBI gains entry to the heart. DisclosuresNo relevant conflicts of interest to declare.

Non-Transferrin-Bound Iron Serves As Iron Source for Aspergillus Fumigatus Growth

The appearance of non-transferrin-bound iron (NTBI) as a consequence of saturated transferrin (Tf) levels in patients undergoing bone marrow transplantation is multifactorial. Chemotherapy-induced tissue damage on the one hand side, causing iron release from apoptotic cells, and chemotherapy-induced bone marrow suppression leading to diminished iron consumption for erythropoiesis on the other hand side as well as chronic transfusion therapy for anemia are among the major contributing factors for iron overload. The existence of NTBI is known to be associated with therapy-related complications, including infections. Nowadays invasive aspergillosis is, in spite of antifungal prophylaxis, still a serious problem with mortality rates ranging up to 70%-90%. The possible interaction of invasive aspergillosis and iron metabolism has been reported.In this study we specifically assessed the role of NTBI on the in vitro growth of Aspergillus fumigatus (A. fumigatus ; ATCC46645) in the presence of plasma. Therefore the growth of A. fumigatus in cultures containing 10% human plasma and various NTBI levels was explored. This assay revealed that the presence of NTBI, but not high Tf-saturation without NTBI, led to a considerable growth advantage. These findings suppose that A. fumigatus growth depends on the presence of NTBI rather than total iron availability. Correspondingly, addition of sufficient amounts of human apo-Tf to eliminate NTBI prevented A. fumigatus growth. These results clearly show that NTBI only exists and promotes A. fumigatus growth if Tf is saturated and disproves the widely believed notion that that Aspergillus utilizes iron from Tf. In contrast to apo-Tf, iron chelators (e.g. deferiprone or deferoxamine) tested in our in vitro system stimulated, rather than inhibited A. fumigatus growth, despite elimination of NTBI.To better understand the underlying molecular mechanisms, different A. fumigatus mutant strains with defects in iron acquisition systems are under investigation. Preliminary data indicate that the uptake of NTBI is not solely dependent on siderophore-mediated iron uptake.In conclusion our findings clearly indicate that NTBI is relevant for fungal growth, at least in vitro in the presence of plasma. Further ongoing experiments aim to clarify whether different clinical A. fumigatus isolates differ in their ability to utilize Tf- bound iron and NTBI. In addition, plasma samples from patients, who had undergone bone marrow transplantation, with already known levels of NTBI, should help to further corroborate the relevance of NTBI for fungal growth in vivo . DisclosuresPlatzbecker:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding.

Genome-Wide CRISPR/Cas9-Mediated Loss-of-Function Screening in Human Erythroleukemic Cells Identifies Novel Cellular Components Involved in Iron and Heme Trafficking

To meet the high demand for iron during erythropoiesis, erythroid precursors utilize efficient yet tightly regulated mechanisms of iron trafficking. Transferrin-bound iron (TBI), the major cellular iron source for erythroid cells under physiological conditions, is taken up through the transferrin receptor (TfR) by endocytosis. Non-transferrin bound iron (NTBI), which exists in the circulation under conditions of iron overload, is also utilized by erythroid cells and its uncontrolled uptake by erythroid precursors presumably results in ineffective erythropoiesis (IE). The majority of iron acquired by erythroid cells is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. In this study, we employed a functional approach to identify cellular components involved in TBI and NTBI uptake and heme trafficking in human erythroid cells. We performed genome-wide loss-of-function screens using the CRISPR-Cas9 system in K562 erythroleukemic cells, which can utilize either TBI or NTBI as an essential iron source for proliferation and can be differentiated into erythroid cells by hemin. In addition to genes that are known to be involved in the studied processes, our approach revealed novel candidates whose roles in iron uptake, heme trafficking and erythroid differentiation were validated using targeted confirmatory approaches. Our screen on iron uptake identified 25 genes potentially involved in TBI acquisition in addition to 18 NTBI uptake candidate genes. The transferrin receptor (TfR1) was one of the top TBI uptake candidates indicating that our approach effectively revealed mechanistically relevant components in the process. Unsurprisingly, several components of the endocytic pathway were shown potentially indispensable for transferrin acquisition. We identified a role for the vacuolar-type H+ - ATPase (V-ATPase) assembly factor CCDC115 in TBI uptake and validated this role by generating individual CCDC115 deficient K562 cells. Our study on heme trafficking revealed several candidate genes relevant to cellular heme uptake, heme-induced differentiation and heme export. Pathway analysis indicates a major role for clathrin-mediated endocytosis and vesicle acidification in heme acquisition by K562 cells. Interestingly, we demonstrated that CCDC115, which was also identified as a significant heme uptake candidate, is indispensable for heme-induced hemoglobinization. We further identified components involved in heme detoxification including two ATP-binding cassette (ABC) transporters that could play a critical unrevealed role in mitochondrial and cellular heme export. Our work demonstrates the strength of functional genomics in studying the various mechanisms affecting erythropoiesis. DisclosuresFraenkel:Sanofi: Employment, Other: Sanofi had no role in funding or influencing this study.

Iron uptake by ZIP8 and ZIP14 in human proximal tubular epithelial cells

In patients with iron overload disorders, increasing number of reports of renal dysfunction and renal iron deposition support an association between increased iron exposure and renal injury. In systemic iron overload, elevated circulating levels of transferrin-bound (TBI) and non-transferrin-bound iron (NTBI) are filtered to the renal proximal tubules, where they may cause injury. However, the mechanisms of tubular iron handling remain elusive. To unravel molecular renal proximal tubular NTBI and TBI handling, human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were incubated with 55Fe as NTBI and fluorescently labeled holo-transferrin as TBI. Ferrous iron importers ZIP8 and ZIP14 were localized in the ciPTEC plasma membrane. Whereas silencing of either ZIP8 or ZIP14 alone did not affect 55Fe uptake, combined silencing significantly reduced 55Fe uptake compared to control (p < 0.05). Furthermore, transferrin receptor 1 (TfR1) and ZIP14, but not ZIP8, colocalized with early endosome antigen 1 (EEA1). TfR1 and ZIP14 also colocalized with uptake of fluorescently labeled transferrin. Furthermore, ZIP14 silencing decreased 55Fe uptake after 55Fe-Transferrin exposure (p < 0.05), suggesting ZIP14 could be involved in early endosomal transport of TBI-derived iron into the cytosol. Our data suggest that human proximal tubular epithelial cells take up TBI and NTBI, where ZIP8 and ZIP14 are both involved in NTBI uptake, but ZIP14, not ZIP8, mediates TBI-derived iron uptake. This knowledge provides more insights in the mechanisms of renal iron handling and suggests that ZIP8 and ZIP14 could be potential targets for limiting renal iron reabsorption and enhancing urinary iron excretion in systemic iron overload disorders.

New Perspectives on Iron Uptake in Eukaryotes.

All eukaryotic organisms require iron to function. Malfunctions within iron homeostasis have a range of physiological consequences, and can lead to the development of pathological conditions that can result in an excess of non-transferrin bound iron (NTBI). Despite extensive understanding of iron homeostasis, the links between the “macroscopic” transport of iron across biological barriers (cellular membranes) and the chemistry of redox changes that drive these processes still needs elucidating. This review draws conclusions from the current literature, and describes some of the underlying biophysical and biochemical processes that occur in iron homeostasis. By first taking a broad view of iron uptake within the gut and subsequent delivery to tissues, in addition to describing the transferrin and non-transferrin mediated components of these processes, we provide a base of knowledge from which we further explore NTBI uptake. We provide concise up-to-date information of the transplasma electron transport systems (tPMETSs) involved within NTBI uptake, and highlight how these systems are not only involved within NTBI uptake for detoxification but also may play a role within the reduction of metabolic stress through regeneration of intracellular NAD(P)H/NAD(P)+ levels. Furthermore, we illuminate the thermodynamics that governs iron transport, namely the redox potential cascade and electrochemical behavior of key components of the electron transport systems that facilitate the movement of electrons across the plasma membrane to the extracellular compartment. We also take account of kinetic changes that occur to transport iron into the cell, namely membrane dipole change and their consequent effects within membrane structure that act to facilitate transport of ions.

Non-Transferrin-Mediated Iron Delivery

In iron overload conditions such as thalassemia major and hereditary hemochromatosis, the iron-carrying capacity of plasma transferrin is exceeded, giving rise to non-transferrin-bound iron (NTBI). NTBI is taken up preferentially by the liver, and to a lesser extent, the kidney, pancreas, and heart. How NTBI is taken up by various tissues has been elusive. We recently demonstrated that the plasma membrane metal-ion transporter SLC39A14 (ZIP14) mediates NTBI uptake and iron loading of the liver and pancreas, but not the kidney, heart or most other tissues¹ Given that the heart is particularly susceptible to iron-related toxicity, we are currently investigating the contribution of other iron transporters to iron loading of this organ. Possible alternative cardiac iron importers include L-type and T-type calcium channels, divalent metal transporter 1 (DMT1), and SLC39A8 (ZIP8). To examine the role of DMT1 and ZIP8 in cardiac iron metabolism, we generated mice with cardiomyocyte-specific disruption of DMT1 (Dmt1heart/heart) or ZIP8 (Zip8heart/heart). The mice were then crossed with hemojuvelin knockout (Hjv-/-) mice, a model of juvenile hemochromatosis characterized by high circulating levels of NTBI. Dmt1heart/heart mice were found to have cardiac non-heme iron concentrations that were 30% lower (P<0.01) than those of wild-type littermate controls at 6 weeks of age. Interestingly, however, double mutant Hjv-/-; Dmt1heart/heart mice accumulated more cardiac non-heme iron (3.9X control) than did single-mutant Hjv-/- mice (2.3X control) at 6 weeks of age. Cardiac-specific disruption of Zip8 did not affect cardiac non-heme iron concentrations under basal conditions or when mice were crossed with Hjv-/- mice. Collectively, these data indicate that DMT1 and ZIP8 are dispensable for iron loading of the heart in a mouse model of hemochromatosis. Our data additionally suggest that DMT1 may play a role in normal cardiac iron metabolism.Reference:Jenkitkasemwong S, Wang C, Coffey R, et al. SLC39A14 is required for the development of hepatocellular iron overload in murine models of hereditary hemochromatosis.Cell Metabolism.2015; 22(1):138-150. DisclosuresNo relevant conflicts of interest to declare.

The plasma membrane metal-ion transporter ZIP14 contributes to nontransferrin-bound iron uptake by human β-cells.

The relationship between iron and β-cell dysfunction has long been recognized as individuals with iron overload display an increased incidence of diabetes. This link is usually attributed to the accumulation of excess iron in β-cells leading to cellular damage and impaired function. Yet, the molecular mechanism(s) by which human β-cells take up iron has not been determined. In the present study, we assessed the contribution of the metal-ion transporters ZRT/IRT-like protein 14 and 8 (ZIP14 and ZIP8) and divalent metal-ion transporter-1 (DMT1) to iron uptake by human β-cells. Iron was provided to the cells as nontransferrin-bound iron (NTBI), which appears in the plasma during iron overload and is a major contributor to tissue iron loading. We found that overexpression of ZIP14 and ZIP8, but not DMT1, resulted in increased NTBI uptake by βlox5 cells, a human β-cell line. Conversely, siRNA-mediated knockdown of ZIP14, but not ZIP8, resulted in 50% lower NTBI uptake in βlox5 cells. In primary human islets, knockdown of ZIP14 also reduced NTBI uptake by 50%. Immunofluorescence analysis of islets from human pancreatic sections localized ZIP14 and DMT1 nearly exclusively to β-cells. Studies in primary human islets suggest that ZIP14 protein levels do not vary with iron status or treatment with IL-1β. Collectively, these observations identify ZIP14 as a major contributor to NTBI uptake by β-cells and suggest differential regulation of ZIP14 in primary human islets compared with other cell types such as hepatocytes.

Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis

Nearly all forms of hereditary hemochromatosis are characterized by pathological iron accumulation in the liver, pancreas, and heart. These tissues preferentially load iron because they take up non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. Yet, how tissues take up NTBI is largely unknown. We report that ablation of Slc39a14, the gene coding for solute carrier SLC39A14 (also called ZIP14), in mice markedly reduced the uptake of plasma NTBI by the liver and pancreas. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14(-/-) mice with Hfe(-/-) and Hfe2(-/-) mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The data suggest that inhibition of SLC39A14 may mitigate hepatic and pancreatic iron loading and associated pathologies in iron overload disorders.

Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma and processes, implying a crucial role in iron accumulation from NTBI and transferrin-bound iron (TBI) by hippocampal neurons.

Effect of Glutamate on Brain Iron Metabolism and the Regulation Mechanism

Glutamate is an excitatory transmitter and can induce neurotoxicity, it can also increase the iron concentrations in the brain, but little is known about the detailed molecular regulation mechanism of iron metabolism by Glu. Based on our previous data, iron metabolism related proteins might be associated with the increase of brain iron contents induced by neurotransmitter. To investigate the issues, the iron contents, non-transferrin-bound iron (NTBI) uptake and the expression of iron uptake and iron release proteins were firstly examined in vivo and in vitro with iron histochemistry, inductively coupled plasma mass spectroscopy (ICP-MS), 55Fe radioactive liquid scintillation counting and western blot methods. Data showed that glutamate induced the increase of total iron contents, storage iron contents and NTBI uptake activity. Moreover, only divalent metal transporter 1, one of iron uptake proteins, was increased in rat brain and PC12 cells treated with glutamate. Further investigations revealed that nuclear factor ÐoB (NF-ÐoB) and protein kinase C (PKC) were involved in the regulation of DMT1 in PC12 cells treated with glutamate. These findings demonstrate that glutamate increases iron contents in the brain through increased NTBI, and that DMT1 is the key molecule underlying regulation of iron metabolism by glutamate, Furthermore, NF-ÐoB and PKC play important roles in the regulatory pathway of DMT1 expression by glutamate. Thus, it implicates that inhibiting the expression of DMT1 and disruption of its regulation pathway might be effective strategies in attenuating glutamate neurotoxicity through decreased iron contents.

Angiotensin II Inhibits Iron Uptake and Release in Cultured Neurons

Based on the well-confirmed roles of angiotensin II (ANGII) in iron transport of peripheral organs and cells, the causative link of excess brain iron with and the involvement of ANGII in neurodegenerative disorders, we speculated that ANGII might also have an effect on expression of iron transport proteins in the brain. In the present study, we investigated effects of ANGII on iron uptake and release using the radio-isotope methods as well as expression of cell iron transport proteins by Western blot analysis in cultured neurons. Our findings demonstrated for the first time that ANGII significantly reduced transferrin-bound iron and non-transferrin bound iron uptake and iron release as well as expression of two major iron uptake proteins transferrin receptor 1 and divalent metal transporter 1 and the key iron exporter ferroportin 1 in cultured neurons. The findings suggested that endogenous ANGII might have a physiological significance in brain iron metabolism.

Physiological implications of NTBI uptake by T lymphocytes

In iron overload disorders a significant fraction of the total iron circulates in the plasma as low molecular weight complexes not bound to transferrin, known as non-transferrin-bound iron (NTBI). By catalyzing the formation of free radicals, NTBI accumulation results in oxidative stress and cellular damage, being a major cause of organ toxicity. NTBI is rapidly and preferentially cleared from circulation by the liver and the myocardium, the main disease targets in iron overload conditions. We have recently demonstrated that human peripheral blood T lymphocytes take up NTBI in vitro, with a pattern that resembles that of hepatocytes. Since T lymphocytes constitute a numerically important component of the circulating cell pool, these findings support a putative role for this cell type in the systemic protection against iron toxicity. Here we tested the hypothesis that the circulating peripheral blood T lymphocyte pool constitutes an important storage compartment for NTBI and is thus a modifier of NTBI deposition in target organs. First we show that NTBI uptake by human T lymphocytes increases the expression of the iron-storage protein ferritin and of the iron exporter ferroportin via an IRE-dependent mechanism. NTBI retention by T lymphocytes is shown to be critically controlled by the hepcidin-mediated modulation of ferroportin both in vitro and in vivo. Finally, the protective effect of T lymphocytes was tested by analyzing the patterns of iron accumulation in the T lymphocyte-deficient mouse model Foxn1nu before and after reconstitution with T lymphocytes by adoptive transfer. The results confirmed a significant increase of liver and pancreas iron accumulation in T lymphocyte-deficient mice. NTBI accumulation in the liver and spleen was prevented by reconstitution with syngeneic T lymphocytes. Altogether, our results demonstrate that T lymphocytes are important components of a circulating “NTBI storage compartment” and show its physiological relevance as a modifier of tissue iron overload.

Non-Transferrin-Bound Iron (NTBI) Uptake by T Lymphocytes: Evidence for the Selective Acquisition of Oligomeric Ferric Citrate Species

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.