High Demand For Iron Research Articles

Low Cytosolic Non-Heme Iron Levels in Erythroid Cells Prevent IRP2-Mediated Ferritin Upregulation during Differentiation.

Erythroid cells are the major consumers of iron in the human body. Differentiating erythroid cells shuttle the metal with very high efficiency towards mitochondria for the formation of heme. To satisfy their high iron needs, developing red blood cells (RBC) have to sustain high expression of transferrin receptor 1 (TfR) despite increasing cellular iron concentration. Moreover, synthesis of ferritin must not be activated by incoming iron, since this would represent a counterproductive storage during the phase of high iron demand. Recently we have demonstrated that during terminal differentiation primary erythroid cells satisfy their exceptionally high requirements for iron by switching to a mode where the post-transcriptional, iron-dependent regulatory system, formed by iron responsive proteins (IRP1 and IRP2) and iron responsive elements (IREs), seems to sense a low-iron state. This occurs despite a massive net increase of iron import into the cell (Schranzhofer et al., Blood 107:4159, 2006). To examine the hypothesis that erythroid cells have low non-heme iron levels in their cytosol, we experimentally increased the cytosolic iron pool by either inhibiting heme biosynthesis or overloading cells with iron. Both block of heme synthesis by either succinylacetone or isonicotinic acid hydrazide (INH) or administration of ferric ammonium citrate, resulted in a clear increase in ferritin levels. This increase was directly proportional to the increase in the cellular concentration of non-heme-iron. Moreover, the effect of INH, the inhibitor of 5-aminolevulinic acid (ALA) synthase, could be reversed by the addition of ALA. Strikingly, increases in ferritin expression upon perturbation of cellular iron homeostasis strongly correlated with the loss of IRE-binding activity of IRP2 but not IRP1, as determined by mobility shift assays. This suggests that IRP2 is the major regulator of ferritin expression in erythroid cells. To further elaborate on this observation, we cultured primary erythroblasts derived from IRP1−/− and IRP2−/− mice (kindly provided by Drs. M. Hentze and B. Galy). In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization. Moreover, only IRP2−/− cells showed a significant increase in ferritin expression, whereas developing RBC lacking IRP1 had levels of ferritin protein equal to wild type cells. We conclude that in erythroid cells efficient shuttling of incoming iron towards mitochondria and its prompt use for heme formation is important to keep the cytosol in an iron-deprived state and consequently ferritin protein levels low. This translational repression seems to be mainly achieved by IRP2. Together with the observation that surface expression of TfR was reduced in IRP2−/− erythroblasts during self renewal but not during terminal differentiation, our results suggest that not only down-regulation of TfR, but also up-regulation of ferritin may be a major factor for the anemic phenotype observed in IRP2−/− mice.

Two Isoforms of a Divalent Metal Transporter (DMT1) in Schistosoma mansoni Suggest a Surface-associated Pathway for Iron Absorption in Schistosomes

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.

Ferritin as an Iron Concentrator and Chelator Target

Ferritin has a broad role to play in new strategies for managing Cooley's anemia and the thalassemias. Serum ferritin iron content is relegated to reporting on tissue iron concentrations. Recently, a new property of ferritin was discovered: gated pores, which are highly conserved in ferritins of humans down to bacteria, and control iron flow to chelators. The pore gates can be selectively opened to increase chelator access by mutation, temperature, and physiological concentrations of urea. In another recent observation, the iron in ferritin from seeds such as soybeans has been shown to be readily available to tissues with high demand for iron, such as red blood cells, but slower to be mobilized in other tissues, compared to nonheme iron salts, presumably through a controlled iron gating mechanism. Because the iron pore gating property of ferritin is more thoroughly investigated, and the knowledge that much of the iron to be chelated in the thalassemias is from a solid iron mineral inside the ferritin protein nanocage, a new role of ferritin in regulating cellular iron homeostasis is established. Two new areas, based on recent knowledge of the molecular properties of ferritin, are (1) exploration of food ferritin as a potentially safer form of dietary nonheme iron, and (2) development of chelators targeted to ferritin protein pores that control chelator access.

The Prevalence of Anemia in a WIC Population: A Comparison by Homeless Experience

Families with children make up 38% of the homeless population in the United States, an 11% increase from 1985 to 1996, and children account for 27% of homeless Americans (1). Iron deficiency may be one of the many negative sequelae of homelessness (2,3). Inadequate iron intake and the resulting iron deficiency and anemia contribute to cognitive deficits and poor developmental out comes in children, the consequences of which may remain even after iron status is restored to normal (4–6). Children under the age of 2 years are particularly vulnerable to iron deficiency due to high growth velocity and resulting high demand for iron (7). The primary objective of this study was to determine whether anemia was more prevalent in homeless children participating in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) when compared with never-homeless children in the WIC and children in the WIC with a previous experience of homelessness.

In Vitro Fusion of Reticulocyte Endocytic Vesicles with Liposomes

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Influence of iron on plutonium absorption by the adult and neonatal rat

To determine how iron affects plutonium absorption, adult rats were gavaged with 238Pu nitrate (pH 2) after they had been fed an iron-deficient diet or treated with iron supplements. Neonatal rats born to dams on an iron-deficient diet were also gavaged with 238Pu. An iron-deficient diet resulted in enhanced 238Pu absorption both in the adults and in neonates born to iron-deficient dams. Ferric iron increased 238Pu absorption 12-fold in adult rats; injected iron-dextran reduced that increase; gavaged ferrous iron reduced 238Pu absorption to one-third of the control value. Rat neonates absorbed 30 to 40 times as much 238Pu as adults; absorption was lowered in groups that received iron supplements: Iron-dextran caused a 50% reduction; ferric iron, 95%; and ferrous iron, >95%. The results demonstrate an effect of the oxidation state of iron on plutonium absorption in adult rats different from that observed in suckling rats. The results suggest that the high rate of 238Pu absorption by neonatal animals is due not only to the permeability of their intestines but also to their high demand for iron.