Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C

Luisa Olivi1, Jeanne Sisk1, and Joseph Bressler1,2

1 Department of Neurology, Kennedy Krieger Research Institute, and 2 Department of Environmental Health Sciences, School of Public Health and Hygiene, Johns Hopkins University, Baltimore, Maryland 21205


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.

divalent metal transporter 1; cadmium; Madin-Darby canine kidney cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CADMIUM (Cd) is a highly toxic metal that can be found in food and water in contaminated areas. Cd is absorbed by the gastrointestinal tract and is distributed quickly to the kidney and liver. Because Cd is nonessential, it most likely utilizes other metal transporters to gain entry into cells. For example, Cd is taken up by PC-12 cells (15) and neurons (32) through voltage-dependent Ca channels. Moreover, evidence from studies on nutrition and on metal transport suggests that Fe transporters may also mediate the uptake of Cd. For example, dietary Cd interferes with Fe absorption (8), and Fe supplementation reduces Cd uptake (26). Fe was also shown to block the uptake of Cd in kidney epithelial cells (9), and Cd interferes with the transport of Fe in human intestinal cells (29).

Several Fe transporters have been described. In mammals, the best-studied mechanism of Fe uptake is the process of transferrin receptor-mediated endocytosis (13, 24). Another mode of uptake that does not involve transferrin uptake of Fe is divalent metal transporter 1 (DMT1, also referred to as Nramp2 or divalent-cation transporter), which was identified in rat intestine by using a frog oocyte expression cloning system (12). DMT1 was shown to mediate the uptake of a number of different heavy metals. At the same time that DMT1 was identified in rat intestine, mutations in the murine DMT1 were implicated as the cause of microcytic anemia in the mk/mk mouse (7) and as the cause of Fe deficiency in the anemic Belgrade rat (6). DMT1 appears to transport Fe but not Zn in the Caco-2 cell intestinal cell line (29), and it has been proposed as the primary transporter for Fe absorption in the intestine. DMT1 is similar to the oligopeptide transporter that has been described in kidney and intestine (10) because it cotransports H+ and requires a cell membrane potential. In the rat, one form of DMT1 mRNA with a molecular mass of 4.5 kDa is found in the intestine, whereas two forms, 3.5 and 4.5 kDa, are found in the brain (33), kidney, and thymus (12).

In the present study, we compared the transport of Cd to the transport of Fe in Madin-Darby canine kidney (MDCK) cells, which have been used as a model to study kidney epithelial cells. We also studied the role of protein kinase C in regulating transport of Cd because other studies have shown the influence of protein kinase C on Fe homeostasis (1, 18, 25). We found that the uptake of Cd displayed properties that were similar to those of DMT1-mediated uptake of Fe. For example, uptake of Cd was attenuated by decreasing the cell membrane potential, increased in cells bathed in a buffer at low pH, and blocked by Fe. Similarly, uptake of Fe was decreased by reducing the cell membrane potential and was blocked by Cd. Uptake of Fe and Cd was also increased in a cell line that overexpresses DMT1. Activation of protein kinase C resulted in an increase in the uptake of Fe and Cd and an increase DMT1 mRNA. Our data suggest that DMT1 mediates the uptake of Cd and is regulated by protein kinase C.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. MDCK cells were obtained from American Type Culture Collection (ATCC). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and Lipofectamine were obtained from Life Technologies. 109Cd (2-6 mCi/mg), 55Fe (3 mCi/mg), and [3H]tetraphenylphosphonium (TPP+; 30 Ci/mmol) were purchased from NEN; [alpha -32P]dCTP (3,000 Ci/mmol) was from Amersham. Nitran paper was purchased from Schleicher and Schuell. RNeasy kit and random priming kits were purchased from Qiagen and Boehringer Mannheim, respectively. Nonidet P-40 was purchased from Calbiochem, and RNasin was obtained from Promega. Phorbol 12,13-dibutyrate (PDBu), dithiothreitol, diethylpyrocarbonate, and all other chemicals for preparing buffers were of reagent grade and obtained from Sigma. The pZeoSV was purchased from Invitrogen, and the DMT1 expression vector was a gift from M. Garick (State University of New York at Buffalo).

Cell culture. MDCK cells were maintained in 100-mm petri dishes at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum. After 3 to 4 days in culture, cells were dislodged with 0.2% trypsin and plated at a 1:5 dilution.

To construct a cell line overexpressing DMT1, HEK-293 human kidney fibroblasts were obtained from ATCC and grown in DMEM containing 10% fetal bovine serum. The cells were transfected with two plasmids at 6 µg/100-mm plate; one plasmid (a gift) was a DMT1 expression vector that was constructed by subcloning the 1.7-kb open reading frame of DMT1 (Fe response element-containing form of DMT1) into the EcoRI site of pMT2 (6). A cell line transfected with only pZeoSV served as the control. Lipofectamine was used to transfect cells according to the instructions provided by Life Technologies for HEK-293cells. Clones were selected for resistance to 100 µg/ml of Zeocin and expanded. Cells were dislodged with trypsin after reaching confluence and plated at a 1:5 dilution. Expression of DMT1 was confirmed by Northern analysis.

Uptake of metals. MDCK cells were plated in 12- or 24-well plates and washed three times with a balanced salt solution (1 mM glucose, 1 mM CaCl2, 0.5 mM MgCl2, 145 mM NaCl, 7 mM KCl, and 25 mM HEPES, pH 7.4). To measure the effect of pH on Cd uptake, 25 mM 2-(N-morpholino)ethanesulfonic acid replaced HEPES at pH 5.5. 109CdCl2 was added to a final concentration of 80 nM to each well, and the cells were incubated for 20 min (unless otherwise indicated) at 37°C. Cells were washed with ice-cold PBS made with 1 mM EDTA, and the radioactivity was extracted with 0.5% SDS in water and measured by liquid scintillation spectroscopy.

The procedure to prepare a buffer containing Fe was adapted from Teichmann and Stremmel (30). 55FeCl3 in 0.5 M HCl was added to a fourfold excess of nitrilotriacetic acid (NTA) and diluted in Hanks' balanced salts (HBSS). The pH was adjusted to pH 6.0, and the uptake of Fe was measured by incubating cells at 37°C and 4°C with 2.3 µM Fe and 9.2 µM NTA in HBSS at pH 6.0 containing 20 µM ascorbic acid. Specific uptake was determined by subtracting total uptake (determined at 37°C) from nonspecific uptake (4°C). Radioactivity was determined as described for the experiments on the uptake of Cd. Unlabeled solutions of Fe in HCl were diluted in HBSS and adjusted to pH 6.0. Ascorbic acid was added to obtain a 1:10 molar ratio (Fe:ascorbate).

Cell membrane potential. Cell monolayers were washed with HBSS and incubated with 5 µM [3H]TPP+ at 1 µCi/ml for 40 min. Monolayers were washed with ice-cold PBS made with 1 mM TPP+. The radioactivity was extracted from the monolayers with 0.5% SDS in water and measured by liquid scintillation spectroscopy. The cell membrane potential was altered by incubating cells in isotonic HBSS made with 75 mM KCl replacing NaCl.

Northern blot analysis for DMT1. Total RNA was isolated using the RNeasy kit according to the manufacturer's instructions. A 15-µg fraction of each sample was denatured with glyoxal/dimethyl sulfoxide, subjected to electrophoresis through a 1.0% agarose gel, and transferred directly to Nitran membranes in 3 M NaCl/0.3 M sodium citrate (17). The RNA was fixed to the membrane and hybridized with cDNA probes. A cDNA probe for DMT1 was prepared from a 1.7-kb fragment cut with EcoRI from the pMT2 plasmid and labeled with [alpha -32P]dCTP by random priming. After the membranes were stripped by being heated for 10 min at 85°C in a 0.1% solution of SDS in 1× saline sodium citrate (SSC), the membranes were reprobed with a cDNA fragment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Radioactivity was quantified by autoradiography and densitometry and expressed as a DMT1:GAPDH ratio.

Nuclear run-on assays. These assays were performed according to published procedures with some modification (2, 27). Nuclei were prepared from two 150-cm2 flasks of cells by scraping the cells into lysis buffer [10 mM Tris · HCl, 10 mM NaCl, 3 mM MgCl2, and 0.5% (vol/vol) Nonidet P-40]. Isolated nuclei were resuspended in glycerol storage buffer [50 mM Tris · HCl, pH 8.3, 40% (vol/vol) glycerol, 5 mM MgCl2, and 0.1 mM EDTA] and stored at -70°C after being frozen in liquid nitrogen. Nuclear run-on transcription assays were carried out on thawed nuclei at 24°C for 20 min by addition of 70 µl of 2× reaction buffer [10 mM Tris · HCl, pH 8.0, 5 mM MgCl2, 0.3 mM KCl, 1 mM ATP, 1 mM GTP, 1 mM CTP, 27 µl [alpha -32P]UTP (270 µCi, 3,000 Ci/mmol), and 3 µl of RNasin ribonuclease inhibitor]. The reaction was stopped by treating the nuclei with guanidine thiocyanate/N-lauroylsarcosine and extracting the RNA with water-saturated phenol and chloroform/isoamyl alcohol (49:1). The RNA was precipitated with ethanol in the presence of glycogen/yeast tRNA and resuspended in hybridization buffer (5× sodium chloride-sodium phosphate-EDTA, 0.5% SDS, 50% deionized formamide, and 1× Denhardt's solution, pH 7.6). The template cDNA was immobilized on Nitran paper, prehybridized at 42° for 4 h, and added to tubes containing [32P]RNA. Hybridization was allowed to proceed at 42°C for 36 h. The paper was washed three times for 20 min at 50°C with 2× SSC/0.1% SDS and twice with 0.1× SSC and 0.1% SDS for 30 min at 65°C. The paper was dried, exposed to X-ray film, and processed for autoradiography.

Statistical analysis. The data were analyzed by either Student's t-test or by a two-way ANOVA followed by Fisher's protected least significant differences test post hoc by using StatView software. P < 0.05 is considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uptake of Cd was dependent on time and temperature. The uptake of 109Cd was dependent on temperature; uptake was higher at 37°C than at 22°C, whereas little uptake was observed at 4°C (Fig. 1). Uptake of 109Cd occurred quickly at 37°C, but never reached saturation. Indeed, accumulation continued even after 12 h (data not shown). Finally, very little 109Cd leaked from the cell (data not shown).


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Fig. 1.   Effect of temperature and time on the uptake of cadmium (Cd). Madin-Darby canine kidney (MDCK) cells were washed and equilibrated for 30 min at the appropriate temperature in a balanced salt solution (BSS) as described in MATERIALS AND METHODS. Eighty nanomolars 109CdCl2 (100 nCi) was then added to each well. After incubation with 109CdCl2 for different lengths of time, cells were washed 3 times with ice-cold PBS/1 mM EDTA. Radioactivity was determined in cell lysates by liquid scintillation spectroscopy. Each data point is the mean ± SE from triplicate wells. Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Cd uptake is higher in MDCK cells incubated with a buffer at an acidic pH. Because DMT1 requires extracellular H+, the effect of pH on the uptake of Cd was examined. Cells were incubated for different lengths of time with Cd in a balanced salt solution at pH 5.5 and 7.4. The uptake of Cd was higher at several time points in cells that were bathed in the acidic buffer (Fig. 2), which is within the range of the pH of urine and likely similar to the pH in the distal tubule.


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Fig. 2.   Effect of pH on Cd uptake. MDCK cells were incubated in BSS made with 2-(N-morpholino)ethanesulfonic acid, pH 5.5, or made with HEPES, pH 7.4, and 80 nM 109CdCl2 (100 nCi) was added at the indicated times. Radioactivity was determined as described in Fig. 1. Each data point is the mean ± SE from triplicate wells. *Mean values for pH 5.5 and pH 7.4 were significantly different from each other (P < 0.05, ANOVA followed by Fisher's protected least significant differences test). Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Cell membrane potential affects the uptake of Fe and Cd. Membrane potential has been shown to affect the uptake of Fe by DMT1 (12, 29). To reduce cell membrane potential, cells were bathed in a buffer enriched in K+ for 60 min. The buffer reduced the accumulation of TPP+ (Fig. 3A), which depends on the cell membrane potential for uptake (20). The buffer also resulted in reduced uptake of Cd (Fig. 3B) and Fe (Fig. 3C), indicating that cell membrane potential affects the uptake of these metals.


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Fig. 3.   Effect of membrane potential on uptake of Fe and Cd. Cells were incubated in isotonic Hanks' balanced salts made with 75 mM KCl at pH 5.5 for a total of 60 min. A: to measure permeability to tetraphenylphosphonium (TPP+), cells were incubated with 5 µM [3H]TPP+ at 1 µCi/ml for the remaining 30 min and then washed with ice-cold PBS containing 1 mM TPP+. B: 109Cd uptake was measured in the remaining 20 min as described in Fig. 1. C: 55Fe uptake was measured by incubating cells at 37°C and 4°C for the remaining 20 min. Cells were washed, and radioactivity was measured as described in MATERIALS AND METHODS. Each data point is the mean ± SE from triplicate wells. *Mean values for control and high potassium in each plot were significantly different from each other (P < 0.05, Student's t-test). Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Transport of Cd is blocked by Fe and the transport of Fe is blocked by Cd. The uptake of 109Cd was inhibited by micromolar concentrations of Fe (Fig. 4A), and uptake of 55Fe was inhibited by micromolar concentrations of Cd (Fig. 4B). It is possible that the amount of Fe needed to inhibit uptake of Cd was greater than the amount of Cd needed to inhibit uptake of Fe because of nonspecific binding of Fe. Almost 20% of total uptake of Fe was nonspecific (data not shown), whereas <5% of the uptake of Cd was nonspecific (Fig. 1). Cu also inhibited uptake of 55Fe and 109Cd, but Zn and Mn did not (data not shown).


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Fig. 4.   Effect of Fe on Cd uptake and Cd on Fe uptake. Different concentrations of unlabeled Fe were added to 109Cd, and different concentrations of Cd were added to 55Fe. Fe was prepared as described in MATERIALS AND METHODS. Uptake of Cd (A) and Fe (B) was measured after 20 min at pH 5.5. Each data point is the mean ± SE from triplicate wells. Results are from an individual experiment and are similar to data obtained from 3 other experiments. *Mean values for 10-1,000 µM Fe were significantly different from 0.3 and 3 µM Fe (A), and 0.1-100 µM Cd was significantly different from 0.03 µM Cd (B) (P < 0.05, ANOVA followed by Fisher's protected least significant differences test).

Overexpression of DMT1 increases uptake of 109Cd and 55Fe. For a more direct determination of the role of DMT1 in Cd uptake, a cell line that overexpresses DMT1 was constructed by transfecting HEK-293 cells with a DMT1 expression vector and a Zeocin resistance vector. Overexpression of DMT1 resulted in an increase in the time-dependent uptake of 109Cd (Fig. 5A) and 55Fe (Fig. 5B) compared with the control cell line, which was transfected only with the Zeocin resistance vector. In the Northern analysis, two bands of DMT1 mRNA were found in the cell line overexpressing DMT1 (Fig. 5C). The band with the higher mass represents the endogenous DMT1 mRNA expressed by the HEK-293 cells, and the lower band represents the mRNA from the DMT1 expression vector, which is missing much of the 3'-untranslated region (UTR). The lower band is missing from the control cell line.


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Fig. 5.   Uptake of Cd and Fe in cells overexpressing divalent metal transporter 1 (DMT1). A control cell line and one that overexpressed DMT1 were constructed as described in MATERIALS AND METHODS. Uptake of Cd (A) and Fe (B) at pH 5.5 was measured at the indicated times. Northern analysis of the DMT1 cell line probed with DMT1 cDNA is shown (C). For Northern analysis, total RNA was isolated from cells, and a 15-µg fraction was denatured with glyoxal/dimethyl sulfoxide, subjected to electrophoresis through a 1.0% agarose gel, and transferred directly to Nitran membranes in 3 M NaCl/0.3 M sodium citrate. The RNA was fixed to the membrane and hybridized with cDNA probes that were labeled with [32P]dCTP by random priming. After being stripped by heating, the membranes were reprobed with a labeled cDNA fragment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each data point is the mean ± SE from triplicate wells. Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Activation of protein kinase C increases uptake of 109Cd and 55Fe. The involvement of the protein kinase C signaling pathway in the regulation of the uptake of Cd and Fe was examined. An overnight treatment with the protein kinase C activator PDBu at 300 nM increased the uptake of 109Cd in MDCK cells. The saturation curve is shown in Fig. 6. The apparent maximal velocity in treated and untreated cells was 44.8 ± 5.4 pmol/mg of protein per minute and 20 ± .8 ± 3.3 pmol/mg of protein per minute, respectively (Table 1). Significant differences in the apparent Michaelis-Menten constant were not observed. Protein kinase C activation by PDBu also resulted in an increase in the time-dependent uptake of 55Fe (Fig. 7).


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Fig. 6.   MDCK cells were treated with or without 300 nM phorbol 12,13-dibutyrate (PDBu) for ~16 h and then assayed for uptake as described in Fig. 1, except that uptake was determined as a function of concentration of Cd. The values for the apparent Michaelis-Menten constant and maximal velocity (V) were determined by nonlinear regression analysis and are shown in Table 1. Results are from an individual experiment and are similar to data obtained from 2 other experiments. [S], saturation.


                              
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Table 1.   Estimation of constants for nonlinear regression model



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Fig. 7.   Effect of protein kinase C activation on Fe uptake. MDCK cells were treated for 16 h with 300 µM PDBu. 55Fe uptake was measured at different lengths of time at pH 5.5 as described in MATERIALS AND METHODS. Each data point is the mean ± SE from triplicate wells. *Mean values were significantly different from each other (P < 0.05, ANOVA followed by Fisher's protected least significant differences test). Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Protein kinase C activation increases expression of DMT1 mRNA. If the increase in Cd and Fe uptake was due to DMT1 after activation of protein kinase C, then treatment with PDBu should increase expression of DMT1 mRNA. MDCK cells were found to express two species of DMT1 mRNA with different molecular masses. Overnight treatment with PDBu resulted in increases in both species of DMT1 mRNA (Fig. 8).


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Fig. 8.   Levels of DMT1 mRNA in MDCK cells after protein kinase C activation. Total RNA was isolated from MDCK cells after treatment with 300 µM PDBu for 16 h or left untreated and subjected to Northern analysis as described in Fig. 5 and in MATERIALS AND METHODS. A: autoradiogram. B: plot of the radioactivity quantified by densitometry expressed as optical density (O.D.) in arbitrary units. Each data point is the mean ± SE from duplicate plates. *Mean values of controls of were significantly different from PDBu treated for each species of mRNA (P < 0.05, ANOVA followed by Fisher's protected least significant differences test). Small refers to the lower band and large to the upper band. Results are from an individual experiment and are similar to data obtained from 4 other experiments.

Protein kinase C activation does not increase newly transcribed DMT1 mRNA. The increase in DMT1 mRNA after protein kinase C activation may be due to an increase in newly transcribed mRNA or an increase in DMT1 mRNA stability, or both. Therefore, nascent transcript levels of DMT1 mRNA were measured in a nuclear run-on assay in control cells and in cells treated with PDBu for 4 or 16 h. The results obtained from these two different incubation times were almost identical: there was no observed increase in newly transcribed DMT1 mRNA at 4 h (data not shown) or 16 h (Fig. 9).


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Fig. 9.   Nuclear run-on transcription assays for the DMT1 mRNA gene in MDCK cells treated with PDBu. Nuclei from untreated cells and from cells treated for 16 h with 300 nM PDBu were prepared as described in MATERIALS AND METHODS. Nuclear run-on transcription assays were carried out at 24°C for 20 min by adding reaction buffer and RNasin ribonuclease inhibitor to the nuclei. The reaction was stopped by treating the nuclei with guanidine thiocyanate/N-lauroylsarcosine and extracting the RNA with water-saturated phenol and chloroform/isoamyl alcohol (49:1). The RNA was precipitated and hybridized to template cDNA that was immobilized on Nitran paper. After prehybridization at 42°C for 4 h and hybridization at 42°C for 36 h, the paper was washed, dried, and exposed to X-ray film. Each data point is the mean ± SE from triplicate plates. The relative intensities of the nascent mRNA signals for DMT1 in untreated and treated cultures are 1,496 ± 85 and 1,302 ± 13, respectively, and for GAPDH are 1,302 ± 13 and 905 ± 35, respectively. Results are from an individual experiment and are similar to data obtained from 2 other experiments.

Protein kinase C activation increases stability of DMT1 mRNA. Because no change in newly transcribed DMT1 mRNA was observed, the increase observed in the Northern analysis may have been due to an increase in mRNA stability. We examined the effect of protein kinase C activation on stability of the smaller species of DMT1 mRNA by measuring levels of DMT1 mRNA at different times in cells in which transcription was inhibited by actinomycin D at time 0. The level of GAPDH mRNA, a housekeeping gene, was also measured at each time to control for overall effects of actinomycin D on transcription. The data are expressed as a percentage, which is the amount of DMT1 mRNA at a time interval divided by the amount of DMT1 mRNA at time 0 multiplied by 100. We found that the decline in DMT1 mRNA was slower in MDCK cells treated with PDBu compared with controls, indicating an increase in stability (Fig. 10). We did not examine the stability of the larger species of DMT1 mRNA because the effect of PDBu was too small.


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Fig. 10.   Effect of PDBu on the degradation of DMT1 mRNA. MDCK cells were either treated for 16 h with 300 µM PDBu or left untreated. The cells were then incubated with 5 µg/ml of actinomycin D, and at subsequent time intervals, total RNA was isolated and levels of the smaller DMT1 and GAPDH mRNA were measured by Northern blotting as described in Fig. 8. The percentage of DMT1 mRNA was determined by dividing the ratio (DMT1 mRNA:GAPDH mRNA) at the time interval by the ratio at time 0 multiplied by 100. Each data point is the mean ± SE from duplicate plates. Bands representing DMT1 and GAPDH mRNA from one plate of each duplicate are shown. Results are from an individual experiment and are similar to data obtained from 3 other experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of these studies was to determine the involvement of Fe transporters in the uptake of Cd in kidney epithelial cells. Fe transporters have been implicated in Cd uptake in several studies demonstrating an interaction between these metals during transport. For example, Cd competes with Fe for uptake in erythroleukemic cells and intestinal cells, whereas Fe was shown to block Cd transport in kidney epithelial cells (9).

The transport of Cd and of Fe in MDCK cells displayed similar properties, suggesting that Fe transporters do mediate the uptake of Cd in MDCK cells. Indeed, results from three different experiments indicated that DMT1 appears to mediate Cd uptake. First, a decrease in the cell membrane potential resulted in a decrease in the uptake of Fe and Cd. Similarly, Fe transport mediated by DMT1 in frog oocytes and in Caco-2 cells was also affected by altering the cell membrane potential. DMT1 is similar to the electrogenic H+-coupled oligopeptide cotransporter found in intestine and kidney (5, 21). Second, Cd uptake was higher in cells bathed in a buffer at pH 5.5 than 7.4. Because DMT1 requires H+ to transport metals, Cd uptake should be higher in cells bathed in a buffer at low pH. Third, overexpression of DMT1 in mammalian cells resulted in increased uptake of Cd and Fe. Another property of Fe uptake by DMT1 is acidification of intracellular pH (pHi) (29). However, because Cd has been shown to interfere with mechanisms by which pHi is regulated (14, 16), changes in pHi may not necessarily have been a property of Cd uptake. In any event, the biochemical properties of Cd uptake are consistent with DMT1-mediated transport. Similarly, overexpression of DMT1 was recently shown to mediate the uptake of Cd in Chinese hamster ovary cells (23). The uptake of Cd by DMT1 may explain why exposure to Cd is associated with a reduction in Fe uptake. Because DMT1 is most likely the major pathway by which Fe is transported in the intestine, Cd in the diet would block the transport of Fe and thus decrease the amount of Fe that is absorbed.

An overnight treatment with the protein kinase C activator PDBu increased uptake of Cd and Fe as well as expression of the both species of DMT1 mRNA. The increase in expression of DMT1 mRNA was likely due to a decrease in the rate of degradation of DMT1 mRNA, since there was no evidence of an increase in transcription of DMT1 mRNA in the nuclear run-on assay. One possible mechanism for stabilizing mRNA is through the interaction of RNA-binding proteins that recognize specific consensus sequences at the 3'-UTR (4). For example, transferrin mRNA is stabilized through the interaction between the iron response element (IRE), located on the 3'-UTR, and an iron response protein (IRP) (3, 22). Protein kinase C has been shown to phosphorylate the IRP, resulting in increased binding between the IRP and the IRE. This mechanism may explain why activation of protein kinase C increased the level of the larger DMT1 species but does not explain the effects on the smaller one (12), which is missing the IRE. Alternatively, other binding proteins recognizing sequence motifs in the 3-'UTR have been described and may participate in stabilizing DMT1 mRNA. A sequence motif with a high content of adenines and uridines has long been recognized in cytokines and immediate early gene mRNAs, but it is not apparent on the 3.5-kb mRNA in the rat. Nonetheless, new motifs have been shown on mRNAs for lactate dehydrogenase (28, 31), angiotensin II (19), and cytochrome P-450 monooxygenases (11). The identification of proteins that bind to the 3'-UTR will be necessary to identify the motifs on the DMT1 mRNA.

In summary, evidence has been provided suggesting the involvement of DMT1 in the uptake of Cd in kidney epithelial cells. We also found that activation of protein kinase C increased expression of DMT1, probably by increasing the stability of the mRNA. Our results suggest that hormones and growth factors that activate protein kinase C have the potential to increase the uptake of Fe and Cd transport by increasing expression of DMT1.


    ACKNOWLEDGEMENTS

We thank Angela T. Williams for assistance in the preparation of this article.


    FOOTNOTES

This work was supported by National Institute of Environmental Health Sciences Grants RO1-ES-07980 (to J. Bressler) and ES-03819.

Address for reprint requests and other correspondence: J. Bressler, 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.

Received 4 December 2000; accepted in final form 2 April 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(3):C793-C800
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