Iron,Copper, and Zinc Transport: Inhibition of Divalent Metal Transporter 1 (DMT1) and Human Copper Transporter 1 (hCTR1) by shRNA

Iron (Fe), copper (Cu), and zinc (Zn) fulfill various essential biological functions and are vital for all living organisms. They play important roles in oxygen transport, cell growth and differentiation, neurotransmitter synthesis, myelination, and synaptic transmission. Because of their role in many critical functions, they are commonly used in food fortification and supplementation strategies globally. To determine the involvement of divalent metal transporter 1 (DMT1) and human copper transporter 1 (hCTR1) on Fe, Cu, and Zn uptake, Caco-2 cells were transfected with four different shRNA plasmids to selectively inhibit DMT1 or hCTR1 transporter expression. Fe and Cu uptake and total Zn content measurements were performed in shRNA-DMT1 and shRNA-hCTR1 cells. Both shRNA-DMT1 and shRNA-hCTR1 cells had lower apical Fe uptake (a decrease of 51% and 41%, respectively), Cu uptake (a decrease of 25.8% and 38.5%, respectively), and Zn content (a decrease of 23.1% and 22.7%, respectively) compared to control cells. These results confirm that DMT1 is involved in active transport of Fe, Cu, and Zn although Zn showed a different relative capacity. These results also show that hCTR1 is able to transport Fe and Zn.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Dietary iron induces rapid changes in rat intestinal divalent metal transporter expression

The divalent metal transporter (DMT1, also known as NRAMP2 or DCT1) is the likely target for regulation of intestinal iron absorption by iron stores. We investigated changes in intestinal DMT1 expression after a bolus of dietary iron in iron-deficient Belgrade rats homozygous for the DMT1 G185R mutation (b/b) and phenotypically normal heterozygous littermates (+/b). Immunofluorescent staining with anti-DMT1 antisera showed that DMT1 was located in the brush-border membrane. Duodenal DMT1 mRNA and protein levels were six- and twofold higher, respectively, in b/b rats than in +/b rats. At 1.5 h after dietary iron intake in +/b and b/b rats, DMT1 was internalized into cytoplasmic vesicles. At 1.5 and 3 h after iron intake in +/b and b/b rats, there was a rapid decrease of DMT1 mRNA and a transient increase of DMT1 protein. The decrease of DMT1 mRNA was specific, because ferritin mRNA was unchanged. After iron intake, an increase in ferritin protein and decrease in iron-regulatory protein binding activity occurred, reflecting elevated intracellular iron pools. Thus intestinal DMT1 rapidly responds to dietary iron in both +/b and b/b rats. The internalization of DMT1 may be an acute regulatory mechanism to limit iron uptake. In addition, the results suggest that in the Belgrade rat DMT1 with the G185R mutation is not an absolute block to iron.     

Uptake of lead and iron by divalent metal transporter 1 in yeast and mammalian cells

Although the divalent metal transporter (DMT1) was suggested to transport a wide range of metals in Xenopus oocytes, recent studies in other models have provided contrasting results. Here, we provide direct evidence demonstrating that DMT1 expressed in yeast mutants defective for high affinity iron transport facilitates the transport of iron with an 'apparent K(m)' of approximately 1.2 microM, and transport of lead with an 'apparent K(m)' of approximately 1.8 microM. DMT1-dependent lead transport was H(+)-dependent and was inhibited by iron. Human embryonic kidney fibroblasts (HEK293 cells) overexpressing DMT1 also showed a higher uptake of lead than HEK293 cells without overexpressing DMT1. These results show that DMT1 transports lead and iron with similar affinity in a yeast model suggesting that DMT1 is a transporter for lead.     

Vesicular transport of fe and interaction with other metal ions in polarized Caco2 cell monolayers

Two aspects of the mechanisms by which iron is absorbed by the intestine were studied in the Caco2 cell model, using 59Fe(II)-ascorbate. Data showing the importance of vesicular processes and cycling of apotransferrin (apoTf) to uptake and overall transport of Caco2 cell monolayers (or basolateral 59Fe release) were obtained by comparing effects of: a) adding apoTf to the basal chamber; b) adding vesicular transport inhibitors; or c) cooling to 4 degrees C. These showed that apoTf may be involved in as much as half of Fe transfer across the basolateral membrane, and that vesicular processes may also play a role in non-apoTf-dependent Fe transport. Studies were initiated to examine potential interactions of other metal ions with Fe(II) via DMT1. Kinetic data showed a single, saturable process for uptake of Fe(II) that was pH dependent and had a Km of 7 microM. An excess of Mn(II) and Cu(I) over Fe(II) of 200: 1 (microM: microM) in 1 mM ascorbate markedly inhibited Fe uptake. The kinetics were not competitive. Km increased and Vmax decreased. We conclude that vesicular transport, involving endo- and exocytosis at both ends of the enterocyte, is a fundamental aspect of intestinal iron absorption and that DMT1 may function as a transporter not just for divalent but also for monovalent metal ions.     

DMT1, a physiologically relevant apical Cu1+ transporter of intestinal cells

Despite important advancesin the understanding of copper secretion and excretion, the molecularcomponents of intestinal copper absorption remain a mystery. DMT1, alsoknown as Nramp2 and DCT1, is the transporter responsible for intestinaliron uptake. Electrophysiological evidence suggests that DMT1 can alsobe a copper transporter. Thus we examined the potential role of DMT1 asa copper transporter in intestinal Caco-2 cells. Treatment of cellswith a DMT1 antisense oligonucleotide resulted in 80 and 48%inhibition of iron and copper uptake, respectively. Cells incorporatedconsiderable amounts of copper as Cu¹⁺, whereasCu²⁺ transport was about 10-fold lower. Cu¹⁺inhibited apical Fe²⁺ transport. Fe²⁺, but notFe³⁺, effectively inhibited Cu¹⁺ uptake. Theiron content of the cells influenced both copper and iron uptake. Cellswith low iron content transported fourfold more iron and threefold morecopper than cells with high iron content. These results demonstratethat DMT1 is a physiologically relevant Cu¹⁺ transporter inintestinal cells, indicating that intestinal absorption of copper andiron are intertwined.

     

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.     

Functional properties of transfected human DMT1 iron transporter

Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mk mouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 microM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

Interleukin-6 and Lipopolysaccharide Modulate Hepcidin mRNA Expression by Hepg2 Cells

Iron homeostasis is controlled by hepcidin (Hpc) as well as other ways. Hpc expression is regulated by iron (Fe) storage and by inflammation, but the joint effect of both stimuli remains unclear. We studied the modulatory role of inflammatory agents (IL6 and LPS) over Hpc and DMT1 mRNA expression in HepG2 cells preloaded with Fe. HepG2 cells were preloaded with different Fe concentrations (holo-Tf or Fe-NTA) and then incubated with IL6 or LPS. We measured intracellular Fe levels by AAS with graphite furnace, transferrin receptor (TfR) by ELISA and mRNA relative abundance of Hpc and DMT1 by qRT-PCR. The maximum effect on Fe uptake was observed in cells incubated with 30?ng/ml IL6 (p?<?0.01) and 500?ng/ml LPS (p?<?0.05). In HepG2 cells preloaded with holo-Tf or Fe-NTA and challenged with IL6 and LPS, we observed a decreased: (a) Hpc mRNA relative abundance (two-way ANOVA: p?<?0.05 and p?<?0.001, respectively), (b) DMT1 mRNA relative abundance and TfR1 protein levels (two-way ANOVA: p?<?0.001), and (c) intracellular Fe concentration (two-way ANOVA: p?<?0.001 and p?<?0.01, respectively) compared to control cells incubated only with Fe (holo-Tf or Fe-NTA). Our results support the idea that Fe storage and inflammation act together to regulate Fe homeostasis and suggest a negative regulation in this hepatic cellular model to prevent excessive increases in Hpc.     

Manganese uptake and release by cultured human hepatocarcinoma (Hep-G2) cells

The liver is the primary organ involved in manganese (Mn) homeostasis. The human hepato-carcinoma cell line, Hep-G2, shows many liver specific functions. Consequently, Hep-G2 cells were investigated as a possible model of hepatic metabolism of Mn. Initial experiments showed that the concentration of Mn in the diet, or culture medium, similarly affected the retention of Mn by isolated rat hepatocytes and Hep-G2 cells. Manganese uptake by Hep-G2 cells suggested that uptake was followed by release from the cell. Uptake was saturable and half-maximal at 2.0 μmol Mn/L, and was inhibited by iodoacetate, vanadate, cold, and bepridil. The cations Fe²⁺, Cu²⁺, Ni²⁺, Cd²⁺, and Zn²⁺ decreased Mn uptake. Uptake was dependent on Calcium (Ca) concentration in a manner that resembled saturation kinetics. Cells that were pulsed with⁵⁴Mn and then placed into nonradioactive medium quickly released a large portion of their internalized Mn. Release of internalized Mn could be inhibited by low temperature, nocodozole, quinacrine and sodium azide. These data show that Hep-G2 cells are a potentially good model of hepatic Mn metabolism. Mn is taken up by a facilitated process that may be related to Ca uptake. Release apparently is an active, controlled process, that may involve microtubules and lysosomes. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Stereoselectivity of the reduced folate carrier in Caco-2 cells

Stereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco-2 cells using methotrexate (l-amethopterin or l-MTX) and its antipode (d-amethopterin or d-MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a K(m) value of approximately 0.6 microM. The Eadie-Hofstee plot of the RFC1-mediated FA uptake revealed a single component for FA uptake into Caco-2 cells, demonstrating that only RFC1 is involved in FA uptake. l-MTX inhibited FA uptake in a competitive manner with a K(i) value of approximately 2 microM, similar to the K(m) value of l-MTX. d-MTX also competitively inhibited FA uptake with a K(i) value being approximately 120 microM, indicating that the affinity of d-MTX is ca. 60-fold less than that of l-MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans.     

Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276–1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, β-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and β-2 microglobulin (β2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE–β2m to the apical membrane. The amount of HFE–β2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or β2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE–β2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.