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Data from small clinical trials in the USA and India suggest zinc supplementation reduces infection in adolescents and adults with sickle cell anemia (SCA), but no studies of zinc supplementation for infection prevention have been conducted in young children with SCA living in Africa, who have higher infection rates. We conducted a randomized double-blind placebo-controlled trial to assess the effectiveness of zinc supplementation for prevention of severe or invasive infections in Ugandan children 1.00-4.99 years with SCA. Of the 252 enrolled participants, 124 were assigned zinc (10 mg) and 126 assigned placebo once daily for 12 months. The primary outcome was incidence of protocol-defined severe or invasive infections. Infection incidence did not differ between treatment arms (282 vs. 270 severe or invasive infections per 100 person-years, respectively, incidence rate ratio of 1.04 [95% confidence interval (CI), 0.81, 1.32, p=0.78]), adjusting for duration on hydroxyurea. There was also no difference between treatment arms in incidence of serious adverse events or SCA-related events. Children receiving zinc had increased serum levels after 12-months, but at study exit, 41% remained zinc deficient (

Anemia is the most prevalent nutrition-related disorder worldwide. Zinc is an essential trace element for various biological processes in the body, and zinc deficiency has been associated with anemia in humans. However, the molecular mechanisms by which zinc availability alters red blood cell development remain uncertain. The present study identifies the essentiality of zinc during erythroid development, particularly for normal heme biosynthesis. G1E-ER4 mouse cells were used as an in vitro model of terminal erythroid differentiation, which featured elevated cellular zinc content by development. Restriction of zinc import compromised the rate of heme and α-globin production and, thus, the hemoglobinization of the erythroid progenitors. Heme is synthesized by the incorporation of iron into protoporphyrin. The lower heme production under zinc restriction was not due to changes in iron but was attributable to less porphyrin synthesis. The requirement of adequate zinc for erythroid heme metabolism was confirmed in another erythropoietic cell model, MEL-DS19. Additionally, we found that a conventional marker of iron deficiency anemia, the ZnPP-to-heme ratio, responded to zinc restriction differently from iron deficiency. Collectively, our findings define zinc as an essential nutrient integral to erythroid heme biosynthesis and, thus, a potential therapeutic target for treating anemia and other erythrocyte-related disorders.

Summary Objective Osteoarthritis (OA) and rheumatoid arthritis (RA) are both debilitating diseases that cause significant morbidity and disability globally. This study aims to investigate the causal effects of varying blood levels of five minerals -- iron, zinc, copper, calcium, and magnesium, on OA and RA. Design We performed two-sample Mendelian randomization (MR) analyses to assess the associations of five circulating minerals with OA and RA. Single nucleotide polymorphisms (SNPs) serving as genetic instruments for the circulating mineral levels were selected from large genome-wide association studies of European-descent individuals. The associations of these SNPs with OA and RA were evaluated in UK Biobank participants. Multiple sensitivity analyses were applied to detect and correct for the presence of pleiotropy. Results Genetically determined copper and zinc status were associated with OA, but not with RA. Per standard deviation (SD) increment in copper increases the risk of OA (OR = 1.07, 95% CI: 1.02–1.13) and one of its subtypes, localized OA (OR = 1.08, 95% CI: 1.03–1.15). Per SD increment in zinc is positively associated with risks of OA (OR = 1.07, 95% CI: 1.01–1.13), generalized OA (OR = 1.18, 95% CI: 1.05–1.31), and unspecified OA (OR = 1.21, 95% CI: 1.11–1.31). Additionally, per SD increment in calcium decreases the risk of localized OA (OR = 0.83, 95% CI: 0.69–0.98). Conclusions Genetically high zinc and copper status were positively associated with OA, but not with RA. Given the modifiable nature of circulating mineral status, these findings warrant further investigation for OA prevention strategies.

Gestating sows (n = 267) were used to determine if timing of feeding elevated dietary zinc in a commercial setting would reduce pre-weaning mortality of piglets. Sows from 3 consecutive weekly farrowing groups were assigned randomly within parity to 1 of 3 treatments consisting of: 1) Control: a corn-soybean meal diet containing 125 ppm supplemental Zn supplied by zinc hydroxychloride; 2) Breed-to-Farrow (B-to-F): as Control + 141 ppm supplemental Zn as ZnSO4 (266 ppm total supplemental Zn) fed from 5 d after breeding to farrowing; and 3) Day 110-to-Farrow (D110-to-F): as Control diet fed from breeding to farrowing + 2,715 ppm supplemental Zn as ZnSO4 (2,840 ppm total supplemental Zn) starting on d 110 of gestation until farrowing. The statistical model considered fixed effects of treatment, farrowing group, and the random effect of their interaction. Dietary treatments did not influence number of total pigs born or pigs weaned per litter (Table). No differences were observed for any piglet growth criteria evaluated across treatments (P > 0.05). For B-to-F sows, there was an increase in percent of pigs born alive compared with Control and D110-to-F sows (P = 0.002). Percentage of stillborn pigs tended to decrease for sows assigned to the B-to-F treatment (P = 0.052) compared with Control or D110-to-F sows. Feeding elevated zinc throughout gestation increased (P = 0.015) percentage of low-birth-weight pigs born alive compared with Control sows. Mortality of low-birth-weight piglets did not differ among treatments (P = 0.592). However, a trend for decreasing post-natal mortality (P = 0.065) of normal birth weight pigs was observed for pigs born to sows fed elevated zinc. The incidence of low-birth-weight pigs was higher (P < 0.001) for sows fed the D110-to-F treatment compared with Control sows. Overall, feeding elevated zinc to sows throughout gestation increased the proportion of pigs born alive and marginally increased survival of normal birth weight pigs.

OBJECTIVES: Zinc is an essential nutrient for diverse biological processes in the body. Cellular zinc homeostasis is established through differential expressions of the transmembrane zinc transporter proteins, ZnTs and ZIPs. The aims of the current studies were to elucidate the roles of cellular zinc in erythrocyte maturation, and to determine the zinc transporters essential to erythroid zinc homeostasis. METHODS: G1E-ER4 mouse cells were employed as an in vitro study model of terminal erythroid differentiation. A cell-impermeable zinc chelator, diethylenetriamine pentaacetate (DTPA), was used to limit extracellular zinc availability. For gene silencing, gene-specific siRNAs were introduced to cells via Nucleofection. Functional impacts of zinc and gene deficiency were assessed via ICP-MS-based metal quantitation, heme assays, and gene expression assays using RNA-seq, qPCR, and western analyses. RESULTS: G1E-ER4 cells featured a 1.7-fold increase in total cellular zinc contents after 48 h of differentiation. Restriction of zinc import by 50 µM of DTPA led to less red coloration and lower increases in mean corpuscular hemoglobin contents by development. The heme deficiency by DTPA was fully restored by the addition of equimolar zinc, and was not due to changes in cellular iron contents. Zinc-deficient G1E-ER4 cells differentiated with normal Alas2 and Hbb-b1 transcript responses, but less Alad and alpha-globin expressions. Among the 24 zinc transporter genes, Zip10 produced the most prominent response to zinc restriction in differentiating erythroid cells. ZIP10-deficient G1E-ER4 cells were less efficient than control cells in hemoglobin production under zinc restriction. ZIP10 deficiency alone had no effects on the molecular indices of red cell hemoglobinization. CONCLUSIONS: Our studies characterize zinc as a nutrient essential to normal erythroid maturation and heme synthesis. Moreover, we have identified a compensatory role of ZIP10 for erythroid zinc homeostasis during zinc restriction. Thus, poor zinc status and ZIP10 mutations might serve as potential risk factors and thus new therapeutic targets for anemia and other erythrocyte-related disorders. FUNDING SOURCES: Supported by CFANS Graduate Fellowship to JK, and the Allen Foundation, Inc. and USDA NIFA Hatch Funds to M-SR.

Developing red blood cells exhibit multiple, redundant systems for regulating and coordinating the uptake of iron, the synthesis of heme, and the formation of hemoglobin during terminal differentiation. We recently described the roles of poly rC-binding protein (PCBP1) and nuclear coactivator 4 (NCOA4) in mediating the flux of iron through ferritin in developing erythroid cells, with PCBP1, an iron chaperone, delivering iron to ferritin and NCOA4, an autophagic cargo receptor, directing ferritin to the lysosome for degradation and iron release. Ferritin iron flux is critical, as mice lacking these factors develop microcytic anemia. Here we report that these processes are regulated by cellular iron levels in a murine model of ex vivo terminal differentiation. PCBP1 delivers iron to ferritin via a direct protein-protein interaction. This interaction is developmentally regulated, enhanced by iron deprivation, and inhibited by iron excess, both in developing cells and in vitro. NCOA4 activity also exhibited developmental regulation and regulation by cellular iron levels. Excess iron uptake during differentiation triggered lysosomal degradation of NCOA4, which was dependent on the E3 ubiquitin ligase HERC2. Thus, developing red blood cells express a series of proteins that both mediate and regulate the flux of iron to the mitochondria.

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells.

Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

OBJECTIVES: To disentangle the shared and unique causal clinical effects of five circulating minerals – iron, zinc, copper, calcium, and magnesium. METHODS: Genetic instruments for circulating minerals were curated from existing genome-wide association studies. Candidate clinical outcomes were identified with a phenome-wide association study (PheWAS) between these genetic instruments and 853 phenotypes in 310,999 individuals from the UK Biobank. Two-sample Mendelian Randomization (MR) analyses were performed to evaluate the causal associations between genetically predicted circulating mineral levels and candidate clinical outcomes. Multiple sensitivity tests were applied to detect and correct for the presence of horizontal pleiotropy. RESULTS: Iron and copper were found to share the most clinical outcomes. Genetically predicted higher blood levels of iron and copper are associated with lower risks of iron deficiency anemia and lipid metabolism disorders, including hyperlipidemia and hypercholesterolemia. Consistently, they are also associated with lower blood levels of total cholesterol and low-density lipoprotein cholesterol. On the other hand, osteoarthrosis (OA) and its subcategories are associated with the most minerals, including zinc, copper, and calcium. Genetically predicted higher blood levels of zinc and copper are associated with increased risk of OA, while calcium exhibits protective effects. Another five shared clinical effects were identified. For instance, zinc and magnesium are associated with a higher risk of bacterial infection; calcium and magnesium increase the risk of renal colic; iron increases while calcium decreases the risk of varicose veins. Unique clinical effects of each blood mineral were also pinpointed, such as iron enhancing the risk of glossitis and acquired foot deformities. CONCLUSIONS: Our comparative PheWAS-MR study of five circulating minerals comprehensively characterized their shared and unique clinical effects, highlighting the causal roles of specific minerals in hyperlipidemia and OA. Our findings emphasize managing blood minerals, probably through dietary adjustments, for disease prevention. FUNDING SOURCES: United States Department of Agriculture; National Institute of Food and Agriculture Hatch Funds; Allen Foundation Inc.; University of Georgia Research Foundation.

Blood levels of iron and copper, even within their normal ranges, have been associated with a wide range of clinical outcomes. The available epidemiological evidence for these associations is often inconsistent and suffers from confounding and reverse causation. This study aims to examine the causal clinical effects of blood iron and copper with Mendelian randomization (MR) analyses. Genetic instruments for the blood levels of iron and copper were curated from existing genome-wide association studies. Candidate clinical outcomes were identified based on a phenome-wide association study (PheWAS) between these genetic instruments and a wide range of phenotypes in 310,999 unrelated individuals of European ancestry from the UK Biobank. All signals passing stringent correction for multiple testing were followed by MR analyses, with replication in independent data sources where possible. We found that genetically predicted higher blood levels of iron and copper are both associated with lower risks of iron deficiency anemia (odds ratio (OR) = 0.75, 95% confidence interval (CI): 0.67&ndash, 0.85, p = 1.90 &times, 10&minus, 6 for iron, OR = 0.88, 95% CI: 0.78&ndash, 0.98, p = 0.032 for copper), lipid metabolism disorders, and its two subcategories, hyperlipidemia (OR = 0.90, 95% CI: 0.85&ndash, 0.96, p = 6.44 &times, 4, OR = 0.92, 95% CI: 0.87&ndash, 0.98, p = 5.51 &times, 3) and hypercholesterolemia (OR = 0.90, 95% CI: 0.84&ndash, 0.95, p = 5.34 &times, OR = 0.93, 95% CI: 0.89&ndash, 0.99, p = 0.022). Consistently, they are also associated with lower blood levels of total cholesterol and low-density lipoprotein cholesterol. Multiple sensitivity tests were applied to assess the presence of pleiotropy and the robustness of causal estimates. Regardless of the approaches, consistent evidence was obtained. Moreover, the unique clinical effects of each blood mineral were identified. Notably, genetically predicated higher blood iron is associated with an enhanced risk of varicose veins (OR = 1.28, 95% CI: 1.15&ndash, 1.42, p = 4.34 &times, 6), while blood copper is positively associated with the risk of osteoarthrosis (OR = 1.07, 95% CI: 1.02&ndash, 1.13, p = 0.010). Sex-stratified MR analysis further revealed some degree of sex differences in their clinical effects. Our comparative PheWAS-MR study of iron and copper comprehensively characterized their shared and unique clinical effects, highlighting their potential causal roles in hyperlipidemia and hypercholesterolemia. Given the modifiable nature of blood mineral status and the potential for clinical intervention, these findings warrant further investigation.

OBJECTIVES: Iron recycled from erythrophagocytosis by macrophages serves as a primary source of systemic iron. NCOA4 mediates ferritin turnover via ferritinophagy. Yet, whether NCOA4 is important in macrophages or erythrophagocytosis-mediated iron recycling remains unclear, and thus was assessed in vitro. METHODS: J774 cells were employed as an in vitro model of macrophages. Iron studies involved treatments of ferric ammonium citrate (FAC) or an iron chelator, deferoxamine (Dfo). To recapitulate systemic iron recycling and overload, cells were treated with opsonized erythrocytes and minihepcidin, respectively. NCOA4 knock-down was achieved by siRNA transfection. Iron gene responses were measured by qPCR and western analyses, and viable cell counts were colorimetrically determined by CCK8 assays as functional outcomes. RESULTS: NCOA4 protein abundance was inversely related to iron availability and ferritin in macrophages. Loss of NCOA4 resulted in impaired ferritin turnover, and led to a reduction in viable cells when combined with iron deficiency. By erythrophagocytosis, a peak in ferritin abundance was observed at 12 h with a subsequent decrease at 24 h. This loss in ferritin was NCOA4-dependent. Minihepcidin caused accumulation of ferritin, along with a repression of NCOA4 in both control and erythrocyte-laden macrophages. Hepcidin activity had no effect on ferritin when NCOA4 was depleted. CONCLUSIONS: NCOA4 mediates the release of ferritin iron during cellular iron restriction and iron recycling by macrophages. Moreover, our studies suggest that macrophage NCOA4 is integral to systemic iron homeostasis by responding to the iron regulatory hormone, hepcidin. Thus, NCOA4 and ferritinophagy may potentially serve as therapeutic targets for treatments of iron disorders and anemia of chronic disease. FUNDING SOURCES: Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.

OBJECTIVES: Iron is essential for proper cell function and development. However, mishandled iron may lead to ferroptotic cell death. NCOA4 manages the cellular labile iron pool by controlling the release of ferritin iron via ferritinophagy. The present studies examined the capacity of hippocampal cells in handling iron fluctuation, with particular focus on NCOA4 and ferritin. METHODS: HT22 mouse hippocampal cells were treated with ferric ammonium citrate (FAC) and deferoxamine (Dfo) to produce cellular iron overload and deprivation, respectively. Ferroptosis was determined by measures of ferrostatin-1 effects and Ptgs2 mRNA. For ferritinophagy studies, Ncoa4 was silenced by siRNA transfections. Functional impacts of impaired ferritinophagy were assessed via CCK-8 cell viability assays and western and qPCR analyses of iron-related genes. RESULTS: HT22 cells were highly susceptible to cellular iron overload. FAC-treated cells featured acute morphological changes, decreased viability, and elevated Ptgs2 mRNA abundance. Iron effects were prevented by ferrostatin-1, indicating ferroptosis by cellular iron overload. Dfo alone had minimal impact on cell morphology and viability. NCOA4 protein, but not mRNA, levels were acutely upregulated by Dfo treatments. Ferritin turnover by iron deficiency was impaired in NCOA4-depleted cells, presumably due to impaired ferritinophagy. Moreover, HT22 cells became sensitive to iron deficiency by loss of NCOA4. CONCLUSIONS: Our studies demonstrate iron can induce ferroptosis in neuronal cells. We also identify NCOA4-mediated ferritinophagy as an integral process for neuronal cell survival during iron deficiency. Further investigation using multi-omics approaches are in progress to determine the mechanisms by which NCOA4 depletion leads to cell death when extracellular iron supply is limited. FUNDING SOURCES: Supported by the NIFA, USDA, Hatch project under MIN-18–118 and intramural support to M-S.R.

Background: Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool. Objective: The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans. Design: A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins. Results: Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet. Conclusions: The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT01221129","term_id":"NCT01221129"}}NCT01221129.

Implementation of zinc interventions for subjects suspected of being zinc-deficient is a global need, but is limited due to the absence of reliable biomarkers. To discover molecular signatures of human zinc deficiency, a combination of transcriptome, cytokine, and microRNA analyses was applied to a dietary zinc depletion/repletion protocol with young male human subjects. Concomitant with a decrease in serum zinc concentration, changes in buccal and blood gene transcripts related to zinc homeostasis occurred with zinc depletion. Microarray analyses of whole blood RNA revealed zinc-responsive genes, particularly, those associated with cell cycle regulation and immunity. Responses of potential signature genes of dietary zinc depletion were further assessed by quantitative real-time PCR. The diagnostic properties of specific serum microRNAs for dietary zinc deficiency were identified by acute responses to zinc depletion, which were reversible by subsequent zinc repletion. Depression of immune-stimulated TNFα secretion by blood cells was observed after low zinc consumption and may serve as a functional biomarker. Our findings introduce numerous novel candidate biomarkers for dietary zinc status assessment using a variety of contemporary technologies and which identify changes that occur prior to or with greater sensitivity than the serum zinc concentration which represents the current zinc status assessment marker. In addition, the results of gene network analysis reveal potential clinical outcomes attributable to suboptimal zinc intake including immune function defects and predisposition to cancer. These demonstrate through a controlled depletion/repletion dietary protocol that the illusive zinc biomarker(s) can be identified and applied to assessment and intervention strategies.

Iron is an essential nutrient required by every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mono- and dinuclear iron centers) are involved in virtually every major metabolic process in the cell. Iron deficiency continues to be the most common nutritional deficiency in the world, while iron overload is a feature of an increasing number of human diseases, including genetic disorders such as hereditary hemochromatosis, thalassemias, and Friedreich ataxia, as well as chronic inflammatory diseases of the liver, such as hepatitis C and steatohepatitis. The biochemical mechanisms by which iron causes toxicity are not completely known. Previously, we identified Poly rC-binding protein 1 (Pcbp1) as a protein that directly binds and delivers iron to ferritin, the major iron storage protein in mammalian cells. Pcbp1 and its paralog Pcbp2 are multifunctional adaptors that bind cellular RNA, DNA, proteins, and iron, altering the fate of their binding partners. Ferritin is a large polypeptide, containing 24 subunits of H- and L-chains assembled into a hollow sphere. Reduced (ferrous) iron enters the sphere through pores formed between the subunits and is oxidized on the interior surface to form nanocrystals of ferric oxyhydroxides. Pcbp1 is the first example of an iron chaperone- a protein that specifically binds iron ions and delivers them to target proteins, such as ferritin, through direct protein-protein interactions. In cultured cells, Pcbp1 is also required for efficient iron delivery to non-heme enzymes in addition to ferritin. These enzymes include the mononuclear iron-containing prolyl hydroxylases that regulate HIF and the dinuclear iron-containing deoxyhypusine hydroxylase, which is required for the modification of lysine to hypusine. In the adult human, 70% of total body iron is present in circulating erythrocytes, which are produced by the bone marrow at a rate of 2 million reticulocytes per second. We examined the roles of Pcbp1 and Ncoa4 in this extraordinary flux of iron through the erythron. Ncoa4 is the cargo receptor that recruits ferritin into the autophagosome for degradation in the lysosome. Using an in vitro model of erythroid differentiation, we showed that depletion of Pcbp1 or Ncoa4 impeded trafficking of iron through ferritin, which impaired the synthesis of heme and hemoglobin. Mice with tamoxifen-induced Pcbp1 deficiency exhibited a microcytic anemia typical of iron deficiency, with activation of compensatory erythropoiesis. The role of ferritin in erythropoiesis has been controversial, but our studies indicate that iron flux through ferritin is an obligatory process in the early stages of erythroid terminal differentiation. Our in vitro studies of erythrocytes revealed that the interactions of Pcbp1 and Ncoa4 with ferritin changed during differentiation, with maximal binding of Pcbp1 at early stages and Ncoa4 at late stages. These binding activities are regulated by cellular iron in mechanistically distinct ways. Ongoing studies of Pcbp1 deficiency in other murine tissues suggest the importance of iron chaperones in maintaining the bioavailable pool of iron in mammalian cells. Disclosures No relevant conflicts of interest to declare.

Zinc is essential for normal erythroid cell functions and therefore intracellular zinc homeostasis during erythroid differentiation is tightly regulated. However, a characterization of zinc transporters in erythrocytes has not been conducted. The membrane fraction of mature mouse RBC was screened for zinc transporter expression using western analysis as a first step in the characterization process. ZnT1, Zip8, and Zip10 were detected among the 12 transporter proteins tested. We examined expression of these zinc transporters during erythropoietin (EPO)-induced differentiation of splenic erythroid progenitor cells into reticulocytes. Both Zip8 and Zip10 mRNA increased by 2-6 h after addition of EPO to the cells. In contrast, maximal RNA levels for the zinc transporter ZnT1 and erythroid delta-aminolevulinic acid synthase were only produced by 24 h after EPO. We confirmed these changes in transcript abundance by western analysis. Dietary zinc status influences zinc-dependent functions of RBC. To determine whether the identified zinc transporters respond to dietary zinc status, mice were fed a zinc-deficient or control diet. Incorporation of (65)Zn into erythrocytes in vitro was significantly increased in cells from the zinc-deficient mice. Western analysis and densitometry revealed that erythrocyte Zip10 was upregulated and ZnT1 was downregulated in the zinc-depleted mice. Zip8 was not affected by restricted zinc intake. Collectively, these data suggest that the zinc transporters ZnT1, Zip8, and Zip10 are important for zinc homeostasis in erythrocytes and that ZnT1 and Zip10 respond to the dietary zinc supply.

Eukaryotic cells contain hundreds of proteins that require iron cofactors for activity. These iron enzymes are located in essentially every subcellular compartment; thus, iron cofactors must travel to every compartment in the cell. Iron cofactors exist in three basic forms: Heme, iron–sulfur clusters, and simple iron ions (also called non-heme iron). Iron ions taken up by the cell initially enter a kinetically labile, exchangeable pool that is referred to as the labile iron pool. The majority of the iron in this pool is delivered to mitochondria, where it is incorporated into heme and iron–sulfur clusters, as well as non-heme iron enzymes. These cofactors must then be distributed to nascent proteins in the mitochondria, cytosol, and membrane-bound organelles. Emerging evidence suggests that specific systems exist for the distribution of iron cofactors within the cell. These systems include membrane transporters, protein chaperones, specialized carriers, and small molecules. This review focuses on the distribution of iron ions in the cytosol and will highlight differences between the iron distribution systems of simple eukaryotes and mammalian cells.

Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn(2+) and non-transferrin-bound Fe(2+) in vitro. Using a Zip14(-/-) mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/-) mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/-) mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/-) phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14(-/-) mice and their phenotype shows defects in glucose homeostasis.

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.

In general human cell models, Poly(rC)-binding proteins (Pcbp) mediate the delivery and integration of iron into the iron storage protein. Conversely, nuclear receptor coactivator 4 (Ncoa4) initiates the liberation of ferritin iron by localizing the protein to autophagosomes. The erythron is the major consumer of body iron. Whether these regulators of ferritin iron are functional in developing red cells remains unclear, and thus was investigated. Both Pcbp1 and Ncoa4 were readily detectable in immunoprecipitates of erythroid ferritin. The peak of Pcbp1-binding to ferritin preceded the stage of maximal Ncoa4-ferritin interaction, which coincided with the induction of β-globin, during erythroid maturation. Both Pcbp1 and Ncoa4 deficiency resulted in lower iron incorporation into heme and hemoglobin by differentiation, without compromised Alas2 gene expression. In Pcbp1-deficent cells, less iron loading into ferritin accompanied the erythropoietic defects. Ncoa4-lacking cells exhibited a massive accumulation of iron in ferritin. No apparent interaction between Pcbp2 and ferritin was detectable in erythroid cells. However, Pcbp2 depletion affected ferritin mineralization in a manner opposite to Pcbp1 deficiency. This was associated with an increase in Pcbp1-ferritin complex formation detected by coimmunoprecipitation. Supplemental iron led to decreases in the amount of both Pcbp1 and Ncoa4 interacting with ferritin. The iron-responsiveness of Ncoa4's ferritin-binding activity was primarily due to post-transcriptional changes in protein abundance. The down-regulation of Ncoa4 protein by high iron required intact lysosome activity, and the expression of an E3 ubiquitin protein ligase, Herc2. Collectively, the present studies provide evidence of the metallochaperone activity of Pcbp1 for ferritin mineralization, and the involvement of Ncoa4 in the mobilization process of stored iron in erythroid cells. Furthermore, the impaired hemoglobinization by insufficiency of these genes identifies ferritin as an essential source of iron for heme production during terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Iron is an essential nutrient for every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mononuclear and diiron centers) are involved in virtually every major metabolic process in the cell. Hundreds of iron, zinc, copper, and manganese proteins are expressed in human cells, yet little is known about the mechanisms by which these metalloproteins acquire their native metal ligands and avoid mis-metallation. Significant advances have been made in understanding the delivery of iron to iron-dependent enzymes in the cytosol. Poly(rC)-binding proteins (PCBPs) are multifunctional adaptors that mediate interactions between single-stranded nucleic acids, iron cofactors, and other proteins, affecting the fate and activity of the components of these interactions. PCBP1 is an iron-binding protein that delivers iron to ferritin in human cells via a direct protein-protein interaction and can be described as an iron chaperone. PCBP2, a human paralog of PCBP1, is also involved in the delivery of iron to ferritin, both in yeast cells and in human cells, suggesting that PCBP1 and PCBP2 work together in iron delivery. PCBP1 and PCBP2 can also deliver iron to the two major families of non-heme iron enzymes: the mononuclear and dinuclear iron-dependent oxygenases. The prolyl hydroxylases (PHDs) are mononuclear iron enzymes that regulate the degradation of hypoxia inducible factor 1 (HIF1). Misregulation of the HIF transcription factors leads to the development of a variety of cancers in humans. Cells depend on the iron chaperones PCBP1 and PCBP2 to maintain iron in the enzymatic active site of PHDs and the related enzyme, asparagyl hydroxylase and to maintain proper regulation of HIF1a, especially under conditions of iron limitation. Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme that is required for the posttranslational modification of a single lysine residue on eukaryotic initiation factor 5A (eIF5A). EIF5A and the conversion of this conserved lysine to hypusine are essential in all eukaryotes, as it enables the translation of peptides containing polyproline sequences. We found that cells depleted of PCBP1 or PCBP2 exhibited reduced activity of DOHH, which was due to a loss of iron in the active site of the enzyme. Thus, PCBPs are basic components of a cytosolic iron delivery system that serves both of the major classes of non-heme iron enzymes in the cytosol. Recent work has indicated that a second type of iron delivery system in the cytosol is mediated by a monothiol glutaredoxin, Glrx3, which, in vitro, can bind and transfer iron-sulfur clusters to recipient apo-iron-sulfur proteins. We have determined that PCBP1 directly interacts with Glrx3-containing complexes and can affect the coordination of iron-sulfur clusters by Glrx3. The huge flux of iron through the developing erythroid cell represents unique challenges for the utilization of cellular iron. We have examined the role of PCBPs as iron chaperones in terminal erythroid differentiation. The role of ferritin in erythroid cell maturation is controversial, but our data indicate that ferritin, PCBPs and NCOA4 are critical factors in erythrocyte development. The flux of iron through ferritin via the lysosome appears to be critical for the transfer of iron to mitochondria for heme synthesis. Disclosures No relevant conflicts of interest to declare.