Enhanced Iron Uptake Research Articles

Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia.

Novel Ferroxidases in Mammalian Intestinal Epithelial Cells

Hephaestin (HEPH) is an intestinal ferroxidase (FOX) that has been implicated as essential for intestinal iron absorption. HEPH oxidizes ferrous iron exported by ferroportin 1 (FPN1), to allow binding of ferric iron to transferrin in the blood. We have previously discovered: 1) the existence of a soluble, intracellular form of HEPH, and 2) that additional novel FOXs in mammalian enterocytes. The purpose of the current study was to test the hypothesis that novel FOXs exist in enterocytes that compensate for the lack of HEPH. A HEPH knockdown (KD) Caco-2 cell line was developed by stable transfection of HEPH-specific or negative control, plasmid-derived shRNAs. 59Fe flux was measured in fully-differentiated Caco-2 cells grown on collagen-coated, bicameral cell culture inserts. HEPH KD enhanced iron uptake (2-fold) under control conditions and enhanced iron efflux (>6-fold) under iron-deficient conditions. Increased FPN1 expression in HEPH KD cells may provide a mechanistic explanation for enhanced iron efflux. FOX activity was then assessed using a spectrophotometric transferrin-coupled assay. Consistent with enhanced iron efflux, HEPH KD and iron deprivation increased cytosolic and membrane FOX activity. Next, intestine-specific HEPH knockout (KO) and wild type (WT) male and female mice were fed control or iron-deficient diets for 5-6 weeks. Intestinal iron absorption was subsequently tested by oral gavage of 59Fe. Adult HEPH KO mice had normal hemoglobin levels and absorbed iron as efficiently as WT littermates. In conclusion, HEPH is not required for efficient iron transport and novel FOXs exist in mammalian enterocytes that compensate for the lack of HEPH.

ROLE OF IRON IN PLANT GROWTH AND METABOLISM

Iron is an essential micronutrient for almost all living organisms because of it plays critical role in metabolic processes such as DNA synthesis, respiration, and photosynthesis. Further, many metabolic pathways are activated by iron, and it is a prosthetic group constituent of many enzymes. An imbalance between the solubility of iron in soil and the demand for iron by the plant are the primary causes of iron chlorosis. Although abundant in most well-aerated soils, the biological activity of iron is low because it primarily forms highly insoluble ferric compounds at neutral pH levels. Iron plays a significant role in various physiological and biochemical pathways in plants. It serves as a component of many vital enzymes such as cytochromes of the electron transport chain, and it is thus required for a wide range of biological functions. In plants, iron is involved in the synthesis of chlorophyll, and it is essential for the maintenance of chloroplast structure and function. There are seven transgenic approaches and combinations, which can be used to increase the concentration of iron in rice seeds. The first approach involves enhancing iron accumulation in rice seeds by expressing the ferritin gene under the control of endosperm-specific promoters. The second approach is to increase iron concentrations in rice through overexpression of the nicotianamine synthase gene (NAS). Nicotianamine, which is a chelator of metal cations, such as Fe+2 and zinc (Zn+2), is biosynthesized from methionine via S-adenosyl methionine synthase. The third approach is to increase iron concentrations in rice and to enhance iron influx to seeds by expressing the Fe+2- nicotianamine transporter gene OsYSL2. The fourth approach to iron biofortification involves enhancing iron uptake and translocation by introducing genes responsible for biosynthesis of mugineic acid family phytosiderophores (MAs). The fifth approach to enhance iron uptake from soil is the over expression of the OsIRT1 or OsYSL15 iron transporter genes. The sixth approach to enhanced iron uptake and translocation is overexpression of the iron homeostasis-related transcription factor OsIRO2. OsIRO2 is responsible for the regulation of key genes involved in MAs-related iron uptake. The seventh approach to enhanced iron translocation from flag leaves to seeds utilizes the knockdown of the vacuolar iron transporter gene OsVIT1 or OsVIT2. The present review discusses iron toxicity in plants with regard to plant growth and metabolism, metal interaction, iron-acquisition mechanisms, biofortification of iron, plant-iron homeostasis, gene function in crop improvement, and micronutrient interactions.

Possible involvement of iron-induced oxidative insults in neurodegeneration

Involvement of iron in the development of neurodegenerative disorders has long been suggested, and iron that cannot be stored properly is suggested to induce iron toxicity. To enhance iron uptake and suppress iron storage in neurons, we generated transgenic (Tg) mice expressing iron regulatory protein 2 (IRP2), a major regulator of iron metabolism, in a neuron-specific manner. Although very subtle, IRP2 was expressed in all regions of brain examined. In the Tg mice, mitochondrial oxidative insults were observed including generation of 4-hydroxynonenal modified proteins, which appeared to be removed by a mitochondrial quality control protein Parkin. Inter-crossing of the Tg mice to Parkin knockout mice perturbed the integrity of neurons in the substantia nigra and provoked motor symptoms. These results suggest that a subtle, but chronic increase in IRP2 induces mitochondrial oxidative insults and accelerates neurodegeneration in a mouse model of Parkinson's disease. Thus, the IRP2 Tg may be a useful tool to probe the roles of iron-induced mitochondrial damages in neurodegeraration research.

Amla ( Phyllanthus emblica L.) Enhances Iron Dialysability and Uptake in In vitro Models

Phyllanthus emblica L. (Indian gooseberry or amla ) is a well-known dietary supplement ( Rasayana ) in Ayurveda used in the management of iron deficiency anaemia ( Pandu ). Amla is said to act by regulating the 'metabolic fire' ( agni ), which is important for proper digestion and absorption of nutrients. In the present study standard cell-free and cell-based models that are employed in biomedical sciences to study digestion and bioavailability of nutrients were used to examine the influence of amla fruit juice on iron dialysability and uptake. Amla juice contained 0.35% ascorbic acid (AA), 0.33% tannins (gallic acid equivalent), 0.13% gallic acid, 0.58% total organic acid and 0.002% iron on a w/w basis. Amla juice exhibited a dose response to iron dialysability with an optimum at 1 : 0.25 molar ratio of Fe : amla juice (AA equivalent) in the cell-free digestion model and 1 : 0.5 in both Caco-2 and HepG2 cell lines. Amla juice increased the dialysable iron three times more than the FeSO 4 alone control in the cell-free digestion model. Iron uptake in Caco-2 and HepG2 cell lines increased 17.18 and 18.71 times more than the control respectively, in the presence of amla juice. AA, a known Fe bioavailability enhancer, at the same molar ratios showed an enhancement only by 1.45 times in the cell-free model and 13.01 and 12.48 times in the Caco-2 and HepG2 models respectively. As a dietary supplement that enhances iron dialysability and uptake, amla fruits can be explored further as a low-cost intervention in the management of iron deficiency anaemia.

Hydroxyl radicals cause fluctuation in intracellular ferrous ion levels upon light exposure during photoreceptor cell death

Iron accumulation is a potential pathogenic event often seen in age-related macular degeneration (AMD) patients. In this study, we focused on the relationship between AMD pathology and concentrations of ferrous ion, which is a highly reactive oxygen generator in biological systems. Murine cone-cells-derived 661W cells were exposed to white florescence light at 2500 lx for 1, 3, 6, or 12 h. Levels of ferrous ions, reactive oxygen species (ROS), and hydroxyl radicals were detected by RhoNox-1, a novel fluorescent probe for the selective detection of ferrous ion, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), and 3′-p-(aminophenyl) fluorescein, respectively. Reduced glutathione, total iron levels and photoreceptor cell death were also measured. Two genes related to iron metabolism, transferrin receptor 1 (TfR1) and H ferritin (HFt), were quantified by RT-PCR. The effects of ferrous ion on cell death and hydroxyl radical production were determined by treatment with a ferrous ion chelating agent, 2,2′-bipyridyl. We found that the ferrous ion level decreased with light exposure in the short time frame, whereas it was upregulated during a 6-h light exposure. Total iron, ROS, cell death rate, and expression of TfR and HFt genes were significantly increased in a time-dependent manner in 661W cells exposed to light. Chelation with 2,2′-bipyridyl reduced the level of hydroxyl radicals and protected against light-induced cell death. These results suggest that light exposure decreases ferrous ion levels and enhances iron uptake in photoreceptor cells. Ferrous ion may be involved in light-induced photoreceptor cell death through production of hydroxyl radicals.

Silencing the Menkes Copper-Transporting ATPase (Atp7a) Gene in Rat Intestinal Epithelial (IEC-6) Cells Increases Iron Flux via Transcriptional Induction of Ferroportin 1 (Fpn1)1–3

The Menkes copper-transporting ATPase (Atp7a) gene is induced in rat duodenum during iron deficiency, consistent with copper accumulation in the intestinal mucosa and liver. To test the hypothesis that ATP7A influences intestinal iron metabolism, the Atp7a gene was silenced in rat intestinal epithelial (IEC-6) cells using short hairpin RNA (shRNA) technology. Perturbations in intracellular copper homeostasis were noted in knockdown cells, consistent with the dual roles of ATP7A in pumping copper into the trans-Golgi (for cuproenzyme synthesis) and exporting copper from cells. Intracellular iron concentrations were unaffected by Atp7a knockdown. Unexpectedly, however, vectorial iron (59Fe) transport increased (∼33%) in knockdown cells grown in bicameral inserts and increased further (∼70%) by iron deprivation (compared with negative control shRNA-transfected cells). Additional experiments were designed to elucidate the molecular mechanism of increased transepithelial iron flux. Enhanced iron uptake by knockdown cells was associated with increased expression of a ferrireductase (duodenal cytochrome b) and activity of a cell-surface ferrireductase. Increased iron efflux from knockdown cells was likely mediated via transcriptional activation of the ferroportin 1 gene (by an unknown mechanism). Moreover, Atp7a knockdown significantly attenuated expression of an iron oxidase [hephaestin (HEPH); by ∼80%] and membrane ferroxidase activity (by ∼50%). Cytosolic ferroxidase activity, however, was retained in knockdown cells (75% of control cells), perhaps compensating for diminished HEPH activity. This investigation has thus documented alterations in iron homeostasis associated with Atp7a knockdown in enterocyte-like cells. Alterations in copper transport, trafficking, or distribution may underlie the increase in transepithelial iron flux noted when ATP7A activity is diminished.

SKB1/PRMT5‐mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana

Histone modifications play critical roles in the perception of environmental cues by plants. Here, we report that Shk1 binding protein 1 (SKB1/AtPRMT5), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is involved in iron homeostasis in Arabidopsis. The SKB1 lesion mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency than the wild type. The expression of SKB1 was not affected by iron, but the level of H4R3sme2 mediated by SKB1 was related to iron status in plants. We showed by chromatin immunoprecipitation (ChIP) and genome-wide ChIP-seq that SKB1 associated with the chromatin of the Ib subgroup bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101), and symmetrically dimethylated histone H4R3. The quantity of SKB1 that associated with chromatin of the Ib subgroup bHLH genes and the level of H4R3sme2 corresponded to the iron status of plants (higher with increased iron supply and lower when iron was removed). We conclude that SKB1-mediated H4R3sme2 regulates iron homeostasis in Arabidopsis in the context of increasing or decreasing expression of Ib subgroup bHLH genes. Iron deficiency may cause an increase in the disassociation of SKB1 from chromatin of the bHLH genes and a decrease in the level of H4R3sme2, thereby elevating their transcription and enhancing iron uptake. Our findings provide new insight into the molecular mechanisms of iron homeostasis in strategy I plants.

Enhancing iron density and uptake in grain and straw of aerobic rice through mulching and rhizo-foliar fertilization of iron

A field experiment was carried out during kharif seasons (June to October) of 2008 to 2009 at New Delhi, to study the Fe density and uptake in aerobic rice as influenced by mulching and iron nutrition. The treatments comprising four methods of rice production viz. bare soil (no mulch), wheat straw mulch (5 t ha-1), Sesbania aculeata mulch and transplanted rice (paddy control) in main plots and five treatments of Fe fertilization, that is, control, FeSO4 50 kg ha-1, FeSO4 100 kg ha-1, FeSO4 50 kg ha-1 + 2 foliar sprays of 2.0% FeSO4 and 3 foliar sprays of 2.0% FeSO4 at maximum tillering, pre-flowering and flowering in sub-plots, replicated thrice in a split plot design. Transplanted rice (Paddy control) was found to be superior for improving Fe concentration and their uptake in grain and straw of rice compared with other treatments. Application of mulch increased Fe concentration and uptake in grain and straw in aerobic rice compared to no mulch aerobic rice treatment. Application of 3 foliar sprays of 2.0% Fe SO4 recorded higher Fe concentration and with significant improvement in uptake in grain as well as in straw of aerobic rice. Fe application increased the Fe concentration 5.4 to 19.9% and 5.8 to 13.3% in grain and straw of aerobic rice. Besides that, methods of rice production increased the Fe concentration 5 to 11% and 4.1 to 6.3% over bare soil (no mulch) in grain and straw, respectively. Transplanted rice produced more yield compared to all aerobic rice treatments. But application of wheat straw/Sesbania mulch recorded significant higher yields compared to no mulch aerobic rice treatment. Application of Fe fertilizer increased the grain and straw yield 4 to 8.2% and 2.7 to 6.7% over control, respectively. Our study showed that, growing of aerobic rice with Sesbania mulch and Fe fertilization produced higher grain and straw yield of aerobic rice with sufficient Fe nutrition. Key words: Aerobic rice, Fe concentration, iron nutrition, Sesbania mulch, wheat straw mulch.

Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

Ovarian Carcinoma: Quantitative Biexponential MR Imaging Relaxometry Reveals the Dynamic Recruitment of Ferritin-expressing Fibroblasts to the Angiogenic Rim of Tumors

To quantitatively monitor the dynamic perivascular recruitment of ferritin heavy chain (FHC)-overexpressing fibroblasts to ovarian carcinoma xenografts by using R2 mapping and biexponential magnetic resonance (MR) relaxometry. In vivo studies of female mice were approved by the institutional animal care and use committee. In vitro analysis included MR-based R2 relaxation measurements of monkey kidney cell line (CV1) fibroblasts that overexpress FHC, followed by inductively coupled plasma mass spectrometry to assess cellular iron content. For in vivo analysis, CV1-FHC fibroblasts were either mixed with fluorescent human ovarian carcinoma cells before subcutaneous implantation (coinjection) or injected intraperitoneally 4 days after the cancer cells were injected (remote recruitment). Dynamic changes in tumor R2 were used to derive CV1-FHC cell fraction in both models. In coinjection tumors, dynamic contrast material-enhanced MR imaging was used to measure tumor fractional blood volume. Whole-body fluorescence imaging and immunohistochemical staining were performed to validate MR results. One-way repeated measures analysis of variance was used to assess MR and fluorescence imaging results and tumor volume, and one-way analysis of variance was used to assess spectrometric results, fractional blood volume, and immunohistochemical evaluation. CV1-FHC fibroblasts (vs CV1 fibroblasts) showed enhanced iron uptake (1.8 mmol ± 0.5 × 10(-8) vs 0.9 mmol ± 0.5 × 10(-8); P < .05), retention (1.6 mmol ± 0.5 × 10(-8) vs 0.5 mmol ± 0.5 × 10(-8), P < .05), and cell density-dependent R2 contrast. R2 mapping in vivo revealed preferential recruitment of CV1-FHC cells to the tumor rim in both models. Measurement of fractional blood volume was similar in all tumors (2.6 AU ± 0.5 × 10(-3) for CV1, 2.3 AU ± 0.3 × 10(-3) for CV1-FHC, 2.9 ± 0.3 × 10(-3) for CV1-FHC-ferric citrate). Dynamic changes in CV1-FHC cell fraction determined at MR relaxometry in both models were confirmed at immunohistochemical analysis. FHC overexpression, when combined with R2 mapping and MR relaxometry, enabled in vivo detection of the dynamic recruitment of exogenously administered fibroblasts to the vasculature of solid tumors.

Identification of bean polyphenols that inhibit and enhance iron uptake by Caco‐2 cells

Polyphenolic compounds present in bean seed coats are known to be inhibitors of Fe bioavailability; however, the specific polyphenols in bean seed coats that inhibit Fe bioavailability have not been identified. In addition, the antioxidative properties of some polyphenols may actually promote Fe absorption. The objective of this study was to identify the major polyphenols that affect Fe bioavailability in black beans. Extracts of white beans and black beans were evaluated for effect on Fe uptake using a Caco‐2 cell model. LC/MS was then used to identify compounds present in active extracts. As expected, Iron uptake was inhibited by extracts from black bean seed coats relative to that of white beans. Examination of the effect of individual polyphenols present in black bean seed coats showed varying dose related Fe uptake responses, ranging from inhibition to stimulation. Myricetin, quercetin and quercetin 3‐glucoside inhibited iron uptake; ferulic acid and salicylic acid had no effect; and caffeic acid, epicatechin, gallic acid and kaempferol stimulated iron uptake in Caco‐2 cells. The results clearly indicate that not all polyphenols inhibit iron uptake and that some are promoters. Targeting these compounds in breeding and or processing of beans could yield improvements of Fe bioavailability. Funded by USDA‐ARS.

Regulation of erythropoiesis by hypoxia-inducible factors

A classic physiologic response to systemic hypoxia is the increase in red blood cell production. Hypoxia-inducible factors (HIFs) orchestrate this response by inducing cell-type specific gene expression changes that result in increased erythropoietin (EPO) production in kidney and liver, in enhanced iron uptake and utilization and in adjustments of the bone marrow microenvironment that facilitate erythroid progenitor maturation and proliferation. In particular HIF-2 has emerged as the transcription factor that regulates EPO synthesis in the kidney and liver and plays a critical role in the regulation of intestinal iron uptake. Its key function in the hypoxic regulation of erythropoiesis is underscored by genetic studies in human populations that live at high-altitude and by mutational analysis of patients with familial erythrocytosis. This review provides a perspective on recent insights into HIF-controlled erythropoiesis and iron metabolism, and examines cell types that have EPO-producing capability. Furthermore, the review summarizes clinical syndromes associated with mutations in the O2-sensing pathway and the genetic changes that occur in high altitude natives. The therapeutic potential of pharmacologic HIF activation for the treatment of anemia is discussed.

Angiotensin II promotes iron accumulation and depresses PGI2and NO synthesis in endothelial cells: effects of losartan and propranolol analogs

Angiotensin may promote endothelial dysfunction through iron accumulation. To research this, bovine endothelial cells (ECs) were incubated with iron (30 µmol·L⁻¹) with or without angiotensin II (100 nmol·L⁻¹). After incubation for 6 h, it was observed that the addition of angiotensin enhanced EC iron accumulation by 5.1-fold compared with a 1.8-fold increase for cells incubated with iron only. This enhanced iron uptake was attenuated by losartan (100 nmol·L⁻¹), d-propranolol (10 µmol·L⁻¹), 4-HO-propranolol (5 µmol·L⁻¹), and methylamine, but not by vitamin E or atenolol. After 6 h of incubation, angiotensin plus iron provoked intracellular oxidant formation (2'7'-dichlorofluorescein diacetate (DCF-DA) fluorescence) and elevated oxidized glutathione; significant loss of cell viability occurred at 48 h. Stimulated prostacyclin release decreased by 38% (6 h) and NO synthesis was reduced by 41% (24 h). Both oxidative events and functional impairment were substantially attenuated by losartan or d-propranolol. It is concluded that angiotensin promoted non-transferrin-bound iron uptake via AT-1 receptor activation, leading to EC oxidative functional impairment. The protective effects of d-propranolol and 4-HO-propranolol may be related to their lysosomotropic properties.

Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation.

BackgroundBiogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient.ResultsThe combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes.ConclusionsBeneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.

An Anti-Apoptotic Molecule, Anamorsin, Functions in Both Iron-Sulfur Protein Assembly and Cellular Iron Homeostasis

Abstract 2106 Introduction:Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells (J.Exp.Med. 199:581–592, 2004). AM has no structural homology to any known anti-apoptosis molecules such as Bcl-2 and IAP family members. AM-deficient (AM−/−) mice are embryonic lethal at late gestation. Its embryos are anemic and the size of embryos is very small. It is thought that AM plays a crucial role in hematopoiesis, however the precise biological mechanisms of AM remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569-5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the cellular iron regulation. Methods and Results:At first, in order to analyze molecular and cellular events, we established cell lines from wild-type or AM-deficient embryonic fetal liver (14.5dpc) by using SV40 large T antigen. We isolated 5 wild-type cell lines (AM WTs) and 2 AM-deficient cell lines (AM KOs) respectively. Next, we compared the cell growth and apoptosis in both cell lines and found that the growth rate of AM KOs were slightly lower than that of AM WTs although these cell lines were immortalized. AM KOs showed more significant apoptosis induced by oxidative stress; the percents of Annexin V positive fraction were 12 ± 4 and 36 ± 6 in AM WTs and AM KOs respectively under the condition of 0.1mM H2O2 for 16hr. In addition to oxidative stress, AM KOs were more sensitive to UV irradiation. These differences were cancelled by transduction of AM-expression retrovirus vector in AM KOs.It was reported that Dre2 functions in cytosolic Fe/S protein biogenesis. We examined whether AM might be involved in the maturation of cytosolic Fe/S proteins. Iron regulatory protein 1 (IRP1) is a well-known cytosolic Fe/S protein with dual functions; in the presence of an [4Fe-4S] cluster it functions as a cytosolic aconitase, while IRP1 binds to mRNA stem-loop structures called iron responsive elements (IREs) and confer the mRNA stability when the [4Fe-4S] cluster is missing. In the iron-deficient cells, IRP1 binds to IREs located at the mRNA of iron transferrin receptor (TfR), ferritin and other iron metabolism transcripts, thereby enhancing iron uptake. In this way, it is thought that IRP1 plays important roles in iron homeostasis. We therefore compared the aconitase activity and IRE binding activities of IRP1 between AM WTs and AM KOs and found that AM deficiency resulted in the decrease of cytosolic aconitase activity (approximately 30% compared to AM WTs). In contrast to cytosolic aconitase activity, the mitochondrial aconitase activity showed little change regardless of AM deficiency. In order to analyze whether AM deficiency might increase IRE binding activity of IRP1, cytoplasmic extracts of AM WTs and AM KOs were compared by RNA precipitation assay. In AM KOs, the expression level of IRP1 decreased approximately one third compared to AM WTs. However, the binding activity of IRP1 to biotin-labeled IRE increased in the extract of AM KOs approximately three-fold in comparison to AM WTs. These differences was cancelled by transduction of AM-expression retrovirus vector to AM KOs. All these findings demonstrated the involvement of AM in the maturation of the cytosolic Fe/S protein, IRP1.Furthermore, we examined the expression of TfR, which is known to be modulated by IRP1-mediated posttranscriptional regulation. In the presence of iron chelator, desferrioxamine, the expression of TfR in AM WTs was markedly elevated. On the other hand, in AM KOs, the expression of TfR was hardly elevated. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration due to the decreased function of IRP1. Conclusion:Our current findings indicate that AM is essential for cytosolic Fe/S cluster biogenesis and iron homeostasis. Now the influence of the AM-mediated iron homeostasis on hematopoiesis is under investigation. Disclosures:No relevant conflicts of interest to declare.

Pathogenic implications of iron accumulation in multiple sclerosis

Iron, an essential element used for a multitude of biochemical reactions, abnormally accumulates in the CNS of patients with multiple sclerosis (MS). The mechanisms of abnormal iron deposition in MS are not fully understood, nor do we know whether these deposits have adverse consequences, that is, contribute to pathogenesis. With some exceptions, excess levels of iron are represented concomitantly in multiple deep gray matter structures often with bilateral representation, whereas in white matter, pathological iron deposits are usually located at sites of inflammation that are associated with veins. These distinct spatial patterns suggest disparate mechanisms of iron accumulation between these regions. Iron has been postulated to promote disease activity in MS by various means: (i) iron can amplify the activated state of microglia resulting in the increased production of proinflammatory mediators; (ii) excess intracellular iron deposits could promote mitochondria dysfunction; and (iii) improperly managed iron could catalyze the production of damaging reactive oxygen species (ROS). The pathological consequences of abnormal iron deposits may be dependent on the affected brain region and/or accumulation process. Here, we review putative mechanisms of enhanced iron uptake in MS and address the likely roles of iron in the pathogenesis of this disease.

Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats

Diquat toxicity causes iron-mediated oxidative stress; however, it remains unclear how diquat affects iron metabolism. Here, we examined the effect of diquat-induced oxidative stress on iron metabolism in male Fischer-344 rats, with particular focus on gene expression. Hepatic nonheme iron content was unchanged until 20h after diquat treatment. Hepatic free iron levels increased markedly in the early stages following treatment and remained elevated for at least 6h, resulting in severe hepatotoxicity, until returning to control levels at 20h. The level of hepatic ferritin, especially the H-subunit, increased 20h after diquat treatment due to elevated hepatic ferritin-H mRNA expression. These results indicate that early elevated levels of free iron in the liver of diquat-treated rats cause hepatotoxicity, and that this free iron is subsequently sequestered by ferritin synthesized under conditions of oxidative stress, thus limiting the pro-oxidant challenge of iron. The plasma iron concentration decreased at 6 and 20h after diquat treatment, whereas the level of plasma interleukin-6 increased markedly at 3h and remained high until 20h. In the liver of diquat-treated rats, expression of hepcidin mRNA was markedly upregulated at 3 and 6h, whereas ferroportin mRNA expression was downregulated slightly at 20h. Transferrin receptor 1 mRNA expression was significantly upregulated at 3, 6, and 20h. These results indicate that inhibition of iron release from iron-storage tissues, through stimulation of the interleukin-6-hepcidin-ferroportin axis, and enhanced iron uptake into hepatocytes, mediated by transferrin receptor 1, cause hypoferremia.

The importance of reductive mechanisms for intestinal uptake of iron from ferric maltol and ferric nitrilotriacetic acid (NTA)

Intestinal iron absorption is thought to proceed with iron mainly in the ferrous form, yet the novel iron complex, ferric maltol is an effective oral preparation. Although possessing a high oil: water partition coefficient, ferric maltol does not diffuse across the intestine but donates its iron to the endogenous uptake system. Reduction of the ferric iron in the gut lumen appears to precede iron uptake both from ferric maltol and from ferric nitrilotriacetic acid (NTA) which is a non-penetrating iron ligand. Uptake of radiolabelled iron (59Fe) into isolated fragments of rat small intestine was inhibited by the ferrous chelator, bathophenanthroline sulphonate (BPS) and enhanced at low concentrations by the reducing agent ascorbic acid. Spectrophotometric evidence was obtained that ferrous ions are generated from these ferric complexes in the presence of ascorbic acid and other reducing agents. The rate of ferrous ion formation was independent of ferric maltol concentration at low ascorbic acid levels and decreased with increasing ferric maltol concentration at higher levels of ascorbate. Maltol has a high affinity for ferric ions and may delay reduction at higher concentrations. By contrast, a higher rate of ferrous ion generation was seen with ferric NTA and this increased with iron ligand concentration. Washings from the intestinal lumen also brought about ferrous ion formation from these ferric ligands. Gel filtration revealed these reducing factors to be of low molecular weight. The washings, however, interfered with 59Fe uptake into the isolated fragments, but when reducing fractions only from the filtered washings were used, enhanced iron uptake was seen.(ABSTRACT TRUNCATED AT 250 WORDS)