1 Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University; 2 Department of Neurology, Kennedy-Krieger Institute; and 3 Department of Neurology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller Vmax for iron uptake compared with control cell lines. 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, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.
divalent metal transporter 1; transport; intestine
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INTRODUCTION |
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DIVALENT METAL TRANSPORTER (DMT1), also known as Nramp2 (13) (natural resistance-associated macrophage protein) or Dct1 (divalent cation transporter), is a proton-coupled metal-ion transporter that is ubiquitously expressed in mammals (14) and absorbs ferrous iron in the proximal duodenum (8). DMT1 is one component of an emergent functional model of iron transport in the intestine, to which has recently been added the iron export protein ferroportin1/IREG1/MTP1 (10) and the ferric reductase Dcytb (20).
Intestinal absorption of metals probably involves three steps, apical uptake, cell traversal, and basolateral exit or transfer, and DMT1 has been shown to mediate the first step in this pathway, at least for iron. Dietary iron is taken up through DMT1 possibly in concert with the ferric reductase Dcytb (20), traversing the cell through some as yet unknown mechanism, and is currently thought to be exported through ferroportin1/IREG1/MTP1, using the multicopper oxidase hephaestin (10), to circulating transferrin. In frog oocytes, high levels of DMT1 expression resulted in a preference for iron transport, but data were provided suggesting that DMT1 also mediates the transport of manganese, nickel, zinc, copper, cadmium, and lead (14). DMT1 is therefore an interesting candidate for the study of essential and toxic metal absorption from the diet, especially because expression in the proximal duodenum, the site of metal transport, has been shown to be dramatically increased by dietary iron deficiency (8).
The role of iron and iron deficiency has been a consistent theme in the absorption of the toxic metals lead and cadmium. Dietary iron deficiency increases the mucosal uptake of both iron and cadmium in rats, whereas lead transfer, but not uptake, is increased under the same conditions (12). In humans, epidemiological evidence on the effect of iron status on body lead can be contradictory, reporting positive (7) or no association (27), whereas for cadmium there seems to be more uniform animal and human evidence relating iron deficiency and cadmium absorption (12). Furthermore, hereditary hemochromatosis, a disease state in which there is unrestrained uptake of iron, can also be associated with increased cadmium (3) and lead body burdens (6), albeit at low levels. This evident complex nature of lead and cadmium absorption warrants further study, particularly at the molecular level, to clarify both pathways of transport and the role of iron.
The Caco-2 cell line is a human intestinal cell-line that has been used widely as a representative model of mammalian intestinal absorbing cells (21). When confluent, the cells differentiate to form a distinct apical and basolateral surface, the former of which is enriched in DMT1(15, 30), along with other markers of differentiation (9, 19). These cells have become useful tools for the study of uptake and transport of nutrients (21, 24), including iron (30) and cadmium (17). The involvement of DMT1 in metal transport has been, until now, based on overexpression of DMT1 by transfection (32) and control of expression using iron treatment (30). Although these approaches represent useful strategies, overexpression can lead to abnormally high expression of the transporter that does not reflect physiological events. Therefore, to study the involvement of endogenous DMT1 in metal transport, we inhibited the expression of DMT1 by means of a U1/ribozyme.
In this study, we demonstrated a saturable, pH-dependent mechanism of iron, lead, and cadmium transport in wild-type Caco-2 cells. We then used a U1/ribozyme to knockdown DMT1 mRNA levels, the functional consequence of which was reduced uptake of iron and cadmium, but not lead, over a range of pH values from 5.5 to 7.4. These result show that lead does not enter Caco-2 cells through DMT1, although the transport pathway seems to be hydrogen coupled.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD). Cells were cultured at 37°C in a humidified atmosphere of 95% air-5% CO2 by using Eagle's minimum essential medium with Earle's salts and L-glutamine (Invitrogen, Carlsbad, CA), supplemented with 20% fetal bovine serum, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/l sodium bicarbonate (all from Invitrogen) as recommended by ATCC. Confluent 100-mm plates were split 1:4 by using 0.25% (wt/vol) trypsin-0.03% (wt/vol) EDTA (Invitrogen), and the medium was changed every 3 days. For uptake experiments, the cells were grown for 11-14 days postconfluence on 24-well or 100-mM plates, depending on the sensitivity of the assay.
Construct preparation.
The pU1 vector, described elsewhere, was derived from pZEoSV
(Invitrogen), which has been modified to contain a U1 snRNA expression cassette and has been used successfully to knockdown the expression of
other genes (1, 22). The framework of the construct,
U1snRNA (Fig. 1, inset), is an
essential component of the nuclear spliceosome complex and is abundant
and stable in the mammalian nucleus, and the U1snRNA promoter is potent
and constitutively active in mammalian cells. The unusual
trimethylguanosine 5' cap and Sm proteins are thought to signal
transport of the U1snRNA back into the nucleus, thus improving the
efficiency of the knockdown. The plasmid contains unique
EcoR1 and Spe1 restriction sites for insertion of
a synthesized oligonucleotide/hammerhead ribozyme complex (Fig.
1A) that binds to and cleaves target RNA immediately 3' of a
GUC consensus sequence (16). Specificity is conferred by
the two flanking oligonucleotides that bind to their complimentary RNA
sequences (Fig. 1)
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Transfection and screening. Caco-2 cells were transfected with pU1/DMT1 or pU1 (lacking the ribozyme/antisense) only as control by using Fugene transfection reagent (Roche Molecular Biochemistry) according to the manufacturer's instructions. Transfected cells were selected in the presence of 100 µg/ml zeocin (Invitrogen), and clonal cell lines were established. Clones were maintained in medium containing zeocin and screened for DMT1 knockdown by Northern analysis.
Northern analysis. Total RNA was isolated using RNeasy according to the manufacturer's instructions, and Northern analysis was carried out with modifications to a previous procedure (23, 25). Briefly, 10 µg of total RNA were fractionated on a 1% agarose gel by electrophoresis and transferred to Nytran membrane (Schleicher & Schuell, Dassel, Germany). Hybridization, using full-length DMT1 cDNA (from M. Garrick, State University of New York at Buffalo) that was [32P]dCTP-labeled using a random priming kit (Amersham, Amersham, UK), was carried out for 18 h at 42°C. Membranes were washed in 1× SSC/0.1% SDS once for 5 min and then twice for 60 min, followed by exposure to high-performance chemiluminescence film with quantification by densitometry. Blots were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (1.9 kb) to control for RNA loading, and relative transfer rates and results were normalized to GAPDH mRNA levels. A similar procedure has been used to detect both forms of DMT1 mRNA in Madin-Darby canine kidney cells (23).
Uptake assays. Caco-2 cells were used for assays at 11-14 days post confluence. Uptake buffer consisted of 140 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2 and 900 mg (0.09%) of glucose. For pH assays, 10 mM HEPES (pH 7.0 or 7.4), 10 mM PIPES (pH 6.5), or 10 mM MES (pH 5.5) was added to the buffer and the pH was adjusted with HCl or NaOH. Before uptake assays, medium was removed and cells were washed briefly with uptake buffer pH 7.4. To make an 55Fe dosing solution, 10 mM sodium ascorbate was added to a 1:4 Fe-NTA (FeCl3: nitrilotriacetic acid) solution, and 1 ml of this was radiolabeled with 10 µl of a 10 mCi/ml 55FeCl3 (NEN) solution to give a 1 mM 55Fe[Fe-NTA] solution. Fe-NTA solutions maintain iron solubility (31), and ascorbate maintains iron as Fe2+. To initiate dosing, we added aliquots of this 1 mM 55Fe[Fe-NTA] solution to cells bathed in uptake buffer and then incubated cells at 37°C for specific uptake or 4°C for nonspecific uptake. To terminate uptake, we placed cells on ice and replaced the uptake buffer with an ice-cold wash solution consisting of 10 mM HEPES, 1 mM NTA, and 150 mM NaCl to remove nonspecifically bound metal. This was repeated three times for 5 min each. Cells were lysed with 200 mM NaOH for several hours, followed by neutralization with 200 mM HCl and scintillation counting. For 109Cd uptake, aliquots of a 1:15,000 dilution of 109CdCl2 (specific activity 3.893 mCi/ml; NEN) were added to cells in uptake buffer, followed by incubation. With lead transport, aliquots of a 1 mM 1:5 Pb:citrate [Pb(NO3)2:sodium citrate] solution were added to cells in uptake buffer, followed by incubation. Citrate ions maintain the solubility of lead in solution and have been previously used for lead uptake experiments using atomic absorption spectroscopy (28). Uptake was terminated for lead and cadmium by placing the cells on ice and replacing the uptake buffer with ice-cold wash buffer containing 1 mM EDTA instead of NTA, followed by washing. For lead analysis, cells were lysed in matrix modifier solution consisting of 0.2% HNO3, 0.2% ammonium dihydrogen orthophosphate, and 0.1% Triton X-100, followed by graphite furnace atomic absorption spectrometry as previously described (4).
Statistics. All experiments were repeated at least twice. Data are means ± SE of three replicates. Nonlinear regression was fitted to a Michaelis-Menten equation using GraphPad Prism version 2. Two-away ANOVA was carried out using STATA statistical software.
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RESULTS |
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Iron and lead uptake by wild-type Caco-2 cells is saturable and pH
dependent.
We first examined uptake of iron and lead in wild-type cells to
establish a benchmark for functional assessment of the knockdown model.
In wild-type Caco-2 cells, iron uptake was saturable (Fig. 2A), indicating a limited
number of carriers mediating uptake, and at least twofold greater at pH
5.5 than that at pH 7.4, depending on the concentration. For lead, over
the same concentration range as iron, uptake was also saturable and
increased up to twofold with decreasing pH (Fig. 2B). When
the data for both metals were fitted to a Michaelis-Menten equation,
the estimated Km values were 2.9 ± 0.4 µM for iron and 2.5 ± 0.6 µM for lead, whereas the
Vmax was 257 ± 12 fmol · µg
protein1 · min
1 for
iron but 80 ± 6 fmol · µg
protein
1 · min
1 for
lead, which is over threefold less than that for iron.
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Iron uptake is affected by changes in divalent iron concentration
and the presence of other cations.
Acid-stimulated (pH 5.5 vs. 7.4) iron uptake was strongly inhibited by
500 µM iron, manganese, and cadmium (Fig.
3) but not by zinc or lead. This finding
demonstrated that acid-stimulated uptake of iron, a property of
DMT1, was abrogated by the presence of divalent ions of iron,
manganese, and cadmium but was not affected by lead or zinc.
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Effect of DMT1 knockdown on uptake of iron.
Two clonal knockdown DMT1 cell lines (KD1 and KD2) and two control cell
lines (Con1 and Con2) were established from wild-type Caco-2 cells as
described in MATERIALS AND METHODS. KD1 and KD2 exhibited
much lower levels of DMT1 mRNA than Con1 and Con2 (Fig. 4A). The
KD1 and KD2 cell lines also displayed lower levels of iron uptake
compared with the Con1 and Con2 cell lines (Fig. 4, B and
C). Saturable uptake decreased by 50% in the KD1 cell line compared with the Con1 cell line, with a decrease in
Vmax from 776.4 to 267.4 fmol · µg
protein1 · min
1.
Saturable uptake also declined in the KD2 cell line compared with the
Con2 cell line, with a decrease in Vmax from 558 to 319 fmol · µg
protein
1 · min
1. When
the four cells lines were compared at the substrate concentration of 20 µM, the KD cell lines exhibited significantly lower levels of iron
uptake than the controls. The control clones were not different from
each other, and the knockdown clones were not different from each other
(Fig. 4D). Differences in the kinetics of the control clones
and the wild-type Caco-2 cells were observed (Fig. 2A) for
reasons that are presently unknown.
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Effect of pH on the uptake of iron.
The pH dependence of iron uptake was compared in the KD1 and Con1 cell
lines. The KD1 cell line displayed an ~50% decrease in the uptake of
iron that was consistent across a pH range from 5.5 to 7.4 compared
with that in the Con2 cell line (Fig. 5). Uptake was inversely proportional to pH, with the highest uptake at pH
5.5.
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Effect of DMT1 knockdown on uptake of cadmium and lead.
We also examined the effect of diminished expression of DMT1 mRNA on
uptake of toxic metals in the KD1 and Con1 cell lines. An ~50%
decrease in the uptake of 1 µM cadmium across a pH range was observed
(Fig. 6A). When cells
were exposed to 10 µM lead, uptake increased with decreasing pH up to
that at pH 5.5, as would be expected in cells expressing DMT1, but
there was no consistent difference between the knockdown and control
cell lines (Fig. 6B), suggesting that DMT1 is not a major
pathway by which lead enters these cells. This is consistent with the
data in Fig. 3, showing that lead does not inhibit the uptake of iron
in wild-type Caco-2 cells whereas cadmium strongly inhibits iron
uptake.
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DISCUSSION |
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Previous studies have shown that DMT1 mediates the transport of iron in Caco-2 cells (30), making it a suitable model for the study of essential and toxic metal acquisition in humans. In our experiments, uptake of iron depended on hydrogen ion concentration and was saturable, properties associated with DMT1.
Our studies provide further evidence for the involvement of DMT1 in the transport of iron and cadmium. Knockdown clonal DMT1 Caco-2 cell lines were established displaying lower levels of DMT1 mRNA compared with control cell lines. The functional consequence of the knockdown was a reduction in the Vmax for iron, indicating a decrease in the number of transporters, as well as reduced uptake of iron across a pH range from 5.5 to 7.4. We also found a decrease in the transport of cadmium across a pH range from 5.5 to 7.4 and competition between cadmium and iron in uptake. Our studies corroborate previous studies suggesting that DMT1 mediates the transport of Cd (11, 29). The competition by cadmium during iron transport would decrease the amount of iron absorbed during digestion. Lower levels of body iron would likely result in increase in DMT1 expression in the intestine (2) because of the IRE located at the 3'-end of DMT1 mRNA. Hence, the competition between cadmium and iron provides a molecular mechanism to explain both the process of iron deficiency induced by chronic exposure to cadmium (12) and the increased absorption of dietary cadmium shown in humans who are iron deficient (26).
In wild-type cells, the effect of pH and concentration was similar for both lead and iron, providing evidence that lead could be transported by DMT1. However, lead did not inhibit iron uptake or vice versa (data not shown). Furthermore, the knockdown DMT1 Caco-2 cell line displayed levels of lead uptake similar to those of the control cell line. Collectively, our evidence demonstrates that DMT1 is not the major pathway by which lead enters Caco-2 cells, even though the properties are consistent with hydrogen-coupled transport. Similar to lead, zinc does not inhibit transport of iron and does not appear to be transported by DMT1 in Caco-2 cells (30). In contrast, transport of zinc in intestinal brush-border membrane vesicles from rats exhibited properties similar to a DMT1-mediated mechanism (18). Furthermore, in one of the original studies describing DMT1, a broad range of metals, including zinc and lead, were transported in frog oocytes expressing DMT1 (14). Finally, increased transport of lead was observed in yeast and human fibroblasts overexpressing DMT1 (5). To reconcile these disparate observations, we suggest that high levels of DMT1 are needed for the DMT1-mediated transport of lead and zinc. Caco-2 cells, rather, express low levels of DMT1 but express other transporters in sufficient amounts to transport lead and zinc. Unfortunately, the identity of the transporters mediating the uptake of lead in Caco-2 cells is unknown.
Previous studies had shown that iron uptake in DMT1-transfected COS cells reaches a pH optimum at 6.75 (32), but our work consistently demonstrated that metal uptake increases with increasing hydrogen ions up to pH 5.5, in agreement with findings of other workers (13, 30). These differences might be explained by the different cell lines expressing DMT1. Interestingly, the KD DMT1 cell line displayed a diminished capacity to transport iron and cadmium across the pH range, rather than only at an acidic pH. Thus DMT1 appears to transport iron at physiological pH, albeit at lower amounts, suggesting the possible involvement of DMT1 in the transport of iron in cell types other than intestinal cells, where the extracellular fluid is at a physiological pH.
In summary, our results provide evidence for the direct involvement of DMT1 in Caco-2 cells for the uptake of iron and cadmium. Our results explain why iron deficiency is a risk factor for cadmium poisoning. Interestingly, the transport of lead was not mediated by DMT1 in Caco-2 cells, suggesting the presence of a different transporter.
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ACKNOWLEDGEMENTS |
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We thank Pamela D. Jones for help in manuscript preparation.
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FOOTNOTES |
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This work was funded by National Institute of Environmental Health Sciences Grants ES-03819 (Center Grant), R01-ES-08785 (J. Bressler), and P01-ES-08131 (G. Goldstein) and by a Center for Alternatives to Animal Testing-American Society for the Prevention of Cruelty to Animals Lasker Fellowship (D. Bannon).
Address for reprint requests and other correspondence: J. P. Bressler, Kennedy Krieger Research Institute, 707 N. Broadway, Baltimore, MD 21205 (E-mail: Bressler{at}kennedykrieger.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published August 22, 2002;10.1152/ajpcell.00184.2002
Received 22 April 2002; accepted in final form 19 August 2002.
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