The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.     

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.

     

Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro

MDCK cells expressing an inducible duodenal cytochrome b-green fluorescent protein (Dcytb-EGFP) fusion construct were used to investigate the function of Dcytb. The Dcytb-EGFP protein was targeted correctly to the plasma membrane, and cells displayed increased ferric and cupric reductase activities, which were greatly reduced in the presence of doxycycline. The data suggests that Dcytb plays a physiological role in both iron and copper uptake, through divalent metal transporter 1 (DMT1) and copper transporter 1, respectively. In support of this hypothesis, we show that 59Fe uptake was significantly enhanced in Dcytb-EGFP expressing MDCK cells which endogenously express DMT1.     

Effect of DMT1 knockdown on iron,cadmium, and lead uptake in Caco-2 cells

DMT1 (divalent metal transporter 1) is ahydrogen-coupled divalent metal transporter with a substrate preferencefor iron, although the protein when expressed in frog oocytestransports a broad range of metals, including the toxic metals cadmiumand lead. Wild-type Caco-2 cells displayed saturable transport of leadand iron that was stimulated by acid. Cadmium and manganese inhibitedtransport of iron, but zinc and lead did not. The involvement of DMT1in the transport of toxic metals was examined by establishing clonalDMT1 knockdown and control Caco-2 cell lines. Knockdown cell linesdisplayed much lower levels of DMT1 mRNA and a smaller V_max for iron uptake compared with control celllines. One clone was further characterized and found to display an~50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, butuptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters thesecells through an independent hydrogen-driven mechanism.

     

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.     

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.     

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.     

Molecular evidence for the role of a ferric reductase in iron transport

Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brush-border membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/DMT1 (divalent metal-transporter 1). The protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.     

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.     

Ferroportin 1 and hephaestin expression in BeWo cell line with different iron treatment

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase.     

Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess

We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 muM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.     

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.     

Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.     

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.     

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.     

Colocalization of ferroportin-1 with hephaestin on the basolateral membrane of human intestinal absorptive cells

An iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) are predicted to be expressed on the basolateral membrane of the enterocyte and involved in the processes of iron export across the basolateral membrane of the enterocyte. However, it is not clear where these proteins are exactly located in the intestinal absorptive cell. We examined cellular localization of FPN-1 and Heph in the intestinal absorptive cells using the fully differentiated Caco-2 cells. Confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane and they are associated with the transferrin receptor (TfR) in fully differentiated Caco-2 cells grown on microporous membrane inserts. However, Heph protein was not detected in the crypt cell-like proliferating Caco-2 cell. In stably transfected human intestinal absorptive cells expressing human FPN-1 modified by the addition of GFP at the C-terminus, we show that FPN-1-GFP is located on the basolateral membrane and it is associated with Heph suggesting the possibility that FPN-1 might associate and interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell.