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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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; [
-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 [
-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 [
-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.
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RESULTS |
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.
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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.
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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.
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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).
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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.
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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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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.
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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.
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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.
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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.
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 |
DISCUSSION |
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.
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ACKNOWLEDGEMENTS |
We thank Angela T. Williams for assistance in the preparation of
this article.
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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
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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.
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