Heme oxygenase 1 overexpression increases iron fluxes in caco-2 cells

Heme oxygenase-1 is a microsomal enzyme that, when induced by stress, protects the cells from oxidative injury. Heme oxygenase-1 participates in the cleavage of the heme ring producing biliverdin, CO and ferrous Fe. The released Fe becomes part of intracellular Fe pool and can be stored in ferritin or released by an iron exporter. The mechanism by which heme enters cells is not completely understood, although it had been suggested that it might be internalized by an endocytosis process. In this study, we expressed a full-length Heme oxygenase-1 cDNA in Caco-2 cells and measured intracellular iron content, heme-iron uptake and transport and immunolocalization of heme oxygenase-1 in these cells. We found that heme oxygenasc-1 expressing cells showed increased apical heme iron uptake and transepithelial transport when compared to control cells. These results suggested that heme oxygenase-1 mediates heme iron influx and efflux in intestinal cells.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

Molecular mechanisms of iron homeostasis

Iron metabolism in mammals requires a complex and tightly regulated molecular network. The classical view of iron metabolism has been challenged over the past ten years by the discovery of several new proteins, mostly Fe (II) iron transporters, enzymes with ferro-oxydase (hephaestin or ceruloplasmin) or ferri-reductase (Dcytb) activity or regulatory proteins like HFE and hepcidin. Furthermore, a new transferrin receptor has been identified, mostly expressed in the liver, and the ability of the megalin-cubilin complex to internalise the urinary Fe (III)-transferrin complex in renal tubular cells has been highlighted. Intestinal iron absorption by mature duodenal enterocytes requires Fe (III) iron reduction by Dcytb and Fe (II) iron transport through apical membranes by the iron transporter Nramp2/DMT1. This is followed by iron transfer to the baso-lateral side, export by ferroportin and oxidation into Fe (III) by hephaestin prior to binding to plasma transferrin. Macrophages play also an important role in iron delivery to plasma transferrin through phagocytosis of senescent red blood cell, heme catabolism and recycling of iron. Iron egress from macrophages is probably also mediated by ferroportin and patients with heterozygous ferroportin mutations develop progressive iron overload in liver macrophages. Iron homeostasis at the level of the organism is based on a tight control of intestinal iron absorption and efficient recycling of iron by macrophages. Signalling between iron stores in the liver and both duodenal enterocytes and macrophages is mediated by hepcidin, a circulating peptide synthesized by the liver and secreted into the plasma. Hepcidin expression is stimulated in response to iron overload or inflammation, and down regulated by anemia and hypoxia. Hepcidin deficiency leads to iron overload and hepcidin overexpression to anemia. Hepcidin synthesis in response to iron overload seems to be controlled by the HFE molecule. Patients with hereditary hemochromatosis due to HFE mutation have impaired hepcidin synthesis and forced expression of an hepcidin transgene in HFE deficient mice prevents iron overload. These results open new therapeutic perspectives, especially with the possibility to use hepcidin or antagonists for the treatment of iron overload disorders.     

Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently.

Iron is required for the growth of Saccharomyces cerevisiae. High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron. We demonstrate that S. cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter. This transporter is highly selective for Fe(II). Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport. Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II). Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities. In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not. When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels. These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.     

Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells

The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.     

Molecular mechanism of intestinal iron absorption

Iron is an essential trace metal in the human diet because of its role in a number of metabolic processes including oxygen transport. In the diet, iron is present in two fundamental forms, heme and non-heme iron. This article presents a brief overview of the molecular mechanisms of intestinal iron absorption and its regulation. While many proteins that orchestrate iron transport pathway have been identified, a number of key factors that control the regulation of iron absorption still remain to be elucidated. This review also summarizes new and emerging information about iron metabolic regulators that coordinate regulation of intestinal iron absorption.     

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7-8 days in media supplemented with 1 microM CuCl(2) had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.     

A novel mammalian iron-regulated protein involved in intracellular iron metabolism

We have isolated and characterized a novel iron-regulated gene that is homologous to the divalent metal transporter 1 family of metal transporters. This gene, termed metal transporter protein (mtp1), is expressed in tissues involved in body iron homeostasis including the developing and mature reticuloendothelial system, the duodenum, and the pregnant uterus. MTP1 is also expressed in muscle and central nervous system cells in the embryo. At the subcellular level, MTP1 is localized to the basolateral membrane of the duodenal epithelial cell and a cytoplasmic compartment of reticuloendothelial system cells. Overexpression of MTP1 in tissue culture cells results in intracellular iron depletion. In the adult mouse, MTP1 expression in the liver and duodenum are reciprocally regulated. Iron deficiency induces MTP1 expression in the duodenum but down-regulates expression in the liver. These data indicate that MTP1 is an iron-regulated membrane-spanning protein that is involved in intracellular iron metabolism.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Iron Imports. V. Transport of iron through the intestinal epithelium

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

The new role of poly (rC)-binding proteins as iron transport chaperones: Proteins that could couple with inter-organelle interactions to safely traffic iron

BackgroundIntracellular iron transport is mediated by iron chaperone proteins known as the poly(rC)-binding proteins (PCBPs), which were originally identified as RNA/DNA-binding molecules.Scope of reviewPCBPs assume a role as not only as cytosolic iron carriers, but also as regulators of iron transport and recycling. PCBP1 is involved in the iron storage pathway that involves ferritin, while PCBP2 is involved in processes that include: iron transfer from the iron importer, divalent metal ion transporter 1; iron export mediated by ferroportin-1; and heme degradation via heme oxygenase 1.Major conclusionsBoth PCBP1 and PCBP2 possess iron-binding activity and form hetero/homo dimer complexes. These iron chaperones have a subset of non-redundant functions and regulate iron metabolism independently.General significanceThis intracellular iron chaperone system mediated by PCBPs provide a transport “gateway” of ferrous iron that may potentially link with dynamic, inter-organelle interactions to safely traffic intracellular iron.     

Transferrin as a donor of iron to mitochondria. Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

Structural biology of heme binding in the Staphylococcus aureus Isd system

Iron is an absolute requirement for nearly all organisms, but most bacterial pathogens are faced with extreme iron-restriction within their host environments. To overcome iron limitation pathogens have evolved precise mechanisms to steal iron from host supplies. Staphylococcus aureus employs the iron-responsive surface determinant (Isd) system as its primary heme-iron uptake pathway. Hemoglobin or hemoglobin-haptoglobin complexes are bound by Near iron-Transport (NEAT) domains within cell surface anchored proteins IsdB or IsdH. Heme is stripped from the host proteins and transferred between NEAT domains through IsdA and IsdC to the membrane transporter IsdEF for internalization. Once internalized, heme can be degraded by IsdG or IsdI, thereby liberating iron for the organism. Most components of the Isd system have been structurally characterized to provide insight into the mechanisms of heme binding and transport. This review summarizes recent research on the Isd system with a focus on the structural biology of heme recognition.     

The intestinal heme transporter revealed

The iron-containing porphyrin heme provides a rich source of dietary iron for mammals. The fact that animals can derive iron from heme implies the existence of a transporter that would transport heme from the gut lumen into intestinal epithelial cells. In this issue of Cell, Shayeghi, McKie, and co-workers (Shayeghi et al., 2005) now describe a heme transporter that is expressed in the apical region of epithelial cells in the mouse duodenum. Their identification of heme carrier protein 1 (HCP1) provides a major missing piece in our understanding of iron uptake and mammalian nutrition.     

A Golgi-localized MATE transporter mediates iron homoeostasis under osmotic stress in Arabidopsis

Iron is an essential micronutrient that acts as a cofactor in a wide variety of pivotal metabolic processes, such as the electron transport chain of respiration, photosynthesis and redox reactions, in plants. However, its overload exceeding the cellular capacity of iron binding and storage is potentially toxic to plant cells by causing oxidative stress and cell death. Consequently, plants have developed versatile mechanisms to maintain iron homoeostasis. Organismal iron content is tightly regulated at the steps of uptake, translocation and compartmentalization. Whereas iron uptake is fairly well understood at the cellular and organismal levels, intracellular and intercellular transport is only poorly understood. In the present study, we show that a MATE (multidrug and toxic compound extrusion) transporter, designated BCD1 (BUSH-AND-CHLOROTIC-DWARF 1), contributes to iron homoeostasis during stress responses and senescence in Arabidopsis. The BCD1 gene is induced by excessive iron, but repressed by iron deficiency. It is also induced by cellular and tissue damage occurring under osmotic stress. The activation-tagged mutant bcd1-1D exhibits leaf chlorosis, a typical symptom of iron deficiency. The chlorotic lesion of the mutant was partially recovered by iron feeding. Whereas the bcd1-1D mutant accumulated a lower amount of iron, the iron level was elevated in the knockout mutant bcd1-1. The BCD1 protein is localized to the Golgi complex. We propose that the BCD1 transporter plays a role in sustaining iron homoeostasis by reallocating excess iron released from stress-induced cellular damage.