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J Biol Chem. 2008 August 1; 283(31): 21462–21468. doi: 10.1074/jbc.M803150200. | PMCID: PMC2490774 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. The Hereditary Hemochromatosis Protein, HFE, Inhibits Iron Uptake via
Down-regulation of Zip14 in HepG2
Cells *Junwei Gao,‡ Ningning Zhao,§ Mitchell D. Knutson,§ and Caroline A. Enns‡1 ‡Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon 97239 and the §Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611 Received April 24, 2008; Revised June 2, 2008. Lack of functional hereditary hemochromatosis protein, HFE, causes iron
overload predominantly in hepatocytes, the major site of HFE expression in the
liver. In this study, we investigated the role of HFE in the regulation of
both transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI) uptake
in HepG2 cells, a human hepatoma cell line. Expression of HFE decreased both
TBI and NTBI uptake. It also resulted in a decrease in the protein levels of
Zip14 with no evident change in the mRNA level of Zip14. Zip14 (Slc39a14) is a
metal transporter that mediates NTBI into cells (Liuzzi, J. P., Aydemir, F.,
Nam, H., Knutson, M. D., and Cousins, R. J. (2006) Proc. Natl. Acad. Sci.
U. S. A. 103, 13612–13617). Knockdown of Zip14 with siRNA abolished
the effect of HFE on NTBI uptake. To determine if HFE had a similar effect on
Zip14 in another cell line, HeLa cells expressing HFE under the
tetracycline-repressible promoter were transfected with Zip14. As in HepG2
cells, HFE expression inhibited NTBI uptake by ~50% and decreased Zip14
protein levels. Further analysis of protein turnover indicated that the
half-life of Zip14 is lower in cells that express HFE. These results suggest
that HFE decreases the stability of Zip14 and therefore reduces the iron
loading in HepG2 cells. Iron absorbed from the intestine is transported directly to the liver, a
key organ involved in iron homeostasis. Hepatocytes, making up about 80% of
the liver in volume, play an important role in maintaining iron homeostasis
and iron sensing. They take up both transferrin-bound iron
(TBI) 2 and
non-transferrin-bound iron (NTBI). NTBI uptake requires both reduction by
ferric reductase and transport by a ferrous transporter. Steap3 is the
candidate reductase in the liver
( 1,
2), and DMT1 (divalent metal
transporter 1) and Zip14 (Zrt- and Irt-like protein 14) are candidate
transporters. DMT1 was the first iron transporter identified and is
ubiquitously expressed ( 3,
4). Zip14, a member of the
SLC39A metal ion transporter family, initially identified as zinc transporter
( 5,
6), was recently reported to be
abundantly expressed in hepatocytes and involved in NTBI uptake
( 7). Patients with hereditary
hemochromatosis have significant levels of NTBI in their serum
( 8,
9). Mutation of a single base pair in the hereditary hemochromatosis gene
( HFE) causes iron overload in the liver, heart, pancreas, and
parathyroid and pituitary glands, leading to multiorgan dysfunction
( 10,
11). This mutation results in
a substitution of tyrosine for cysteine in the hereditary hemochromatosis
protein, HFE, which disrupts a disulfide bond required for proper folding,
preventing it from binding to β 2-microglobulin and trafficking
to the cell surface ( 12). The
importance of functional HFE in iron metabolism is also supported by the
evidence that hepatocytes from Hfe-/- knock-out mice can
take up more NTBI ( 9) and have
an 8-fold higher iron accumulation in the liver than wild-type mice
( 13). Several mechanisms have been proposed by which HFE regulates iron
metabolism. HFE competes with transferrin (Tf) for binding to TfR1
(transferrin receptor 1), lowering iron uptake into cells
( 14– 16).
Alternately, Tf binding to TfR1 releases HFE to bind to TfR2 in hepatocytes to
increase hepcidin transcription
( 17). Hepcidin is a hormone
secreted by the liver that negatively regulates dietary iron uptake by the
intestine. HFE can also lower NTBI uptake in isolated primary mouse
hepatocytes ( 9) and in Chinese
hamster ovary cells lacking endogenous TfR1
( 18). Thus, the mechanism by
which HFE regulates iron metabolism still remains elusive. In the present study, the regulation of both TBI and NTBI uptake by HFE was
studied in HepG2 cells, a human hepatoma cell line. We found that expression
of HFE decreased both TBI and NTBI uptake. Expression of HFE resulted in a
decrease in the protein level of Zip14 with no evident change in the level of
Zip14 mRNA. Knockdown of Zip14 with siRNA abolished the effect of HFE on NTBI
uptake. HeLa cells had no detectable Zip14 protein. When HeLa cells expressing
HFE were transfected with Zip14, NTBI uptake was decreased by comparison with
the corresponding controls. HFE was also found to reduce NTBI uptake by
~50% and to decrease Zip14 protein level. These results suggest that HFE
controls the stability of Zip14, which consequently influences the iron
loading of hepatocytes. Cell Culture and Transfection—HepG2 cells were maintained in
minimum essential medium Eagle (Invitrogen) supplemented with 1.0
mm sodium pyruvate, 0.1 mm nonessential amino acids
(Invitrogen), and 10% fetal bovine serum. Cells (2 × 106)
were transfected with 5 μg of pcDNA3.1/HFE-FLAG (HepG2/HFE) or pcDNA3.1
(HepG2/Con) using Nucleofector kit V (Amaxa Biosystems, Gaithersburg, MD)
according to the manufacturer's instructions and plated into a
78-cm2 dish. Stable cell clones were obtained by selection with 800
μg/ml G418. The HeLa/tTA-HFE-FLAG cell line, expressing FLAG epitope-tagged HFE under
control of the tetracycline-responsive promoter
( 19), was maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum, 400 μg/ml G418, and 300 ng/ml puromycin with or without 2 μg/ml
doxycyline (dox). HeLa/tTA-HFE-FLAG cells (70–80% confluent) in
78-cm 2 dishes were transiently transfected using 100 μl of
Lipofectamine (Invitrogen) and 10 μg of pCMV/Zip14-myc encoding mouse Zip14
tagged with the Myc epitope at the C terminus. Two days later, cells were
split into a 12-well plate in the presence or absence of 2 μg/μl dox for
24 h. Iron Treatment of Cells—Fe-NTA was freshly prepared by
mixing 500 m m FeCl 3 and 530 m m NTA at a
volume ratio of 1:3.77 ( 18).
HepG2 cells and HepG2/HFE cells were seeded in a 6-well plate at a
concentration of 2 × 10 5 cells/well. After 2 days, cells were
treated with 100 μ m Fe-NTA for 24 h and harvested. 55Fe-NTA Uptake—Cells grown in a 6-well plate
were washed twice and equilibrated in DMEM supplemented with 20 m m
Hepes (pH 7.4) at 37 °C, 5% CO 2 for 15 min. The medium was
replaced with 1 ml of 100 n m 55Fe-NTA in DMEM
supplemented with 20 m m Hepes (pH 7.4) and 2 mg/ml ovalbumin. After
5, 15, 30, or 60 min at 37 °C, 5% CO 2, cells were washed twice
with 2 ml of 5 m m EDTA, phosphate-buffered saline at 4 °C and
solubilized with 1 ml of solubilization detergent (0.1% Triton X-100, 0.1%
NaOH). Lysates were mixed with 6 ml of UniverSol scintillation fluid (CN
Chemical Co., Costa Mesa, CA), and the radioactivity was counted for 10 min in
a scintillation counter ( 20,
21). 55Fe-Tf Uptake— 55Fe-Tf uptake was
conducted as previously described
( 21). Briefly, cells grown in
a 6-well plate were washed twice and equilibrated in DMEM with 20
m m Hepes (pH 7.4) for 15 min at 37 °C, 5% CO 2. Wash
medium was replaced with 1 ml of uptake medium containing 100 n m
55Fe-Tf in DMEM supplemented with 20 m m Hepes (pH 7.4)
and 2 mg/ml ovalbumin, pH 7.4. After 1 h, cells were placed on ice, and
externally bound 55Fe-Tf was stripped with an acidic buffer (0.2
n acetic acid, 500 m m NaCl, 1 m m
FeCl 3) for 3 min. Cells were solubilized in 1 ml of solubilization
detergent (0.1% Triton X-100, 0.1% NaOH). Lysates were mixed with 6 ml of
UniverSol scintillation fluid, and the radioactivity was counted for 10 min in
a scintillation counter ( 20,
21). 55Fe-NTA Efflux—Cells grown in a 6-well plate
were washed twice and equilibrated in DMEM with 20 m m Hepes (pH
7.4) for 15 min at 37 °C, 5% CO 2. The medium was replaced with
1 ml of DMEM containing 100 n m 55Fe-NTA, 20
m m Hepes (pH 7.4), and 2 mg/ml ovalbumin. After 3 h, cells were
immediately washed twice at room temperature with 2 ml of 5 m m EDTA
in phosphate-buffered saline (pH 7.4) to remove nonspecific 55Fe
from the cell surface, and 1 ml of efflux medium (DMEM, 20 m m Hepes
(pH 7.4), 1 m m Fe-NTA) was added to the cells, and they were
incubated at 37 °C for 0, 5, 15, 30, or 60 min. The plates were quickly
washed twice with 2 ml of 5 m m EDTA in phosphate-buffered saline to
remove any extracellular 55Fe, and the cells were solubilized in 1
ml of solubilization detergent (0.1% Triton X-100, 0.1% NaOH). Lysates were
mixed with 6 ml of UniverSol scintillation fluid, and the radioactivity was
counted for 10 min in a scintillation counter
( 20,
21). Real Time Quantitative Reverse Transcription (qRT)-PCR—Total
RNA was isolated from cells using the RNeasy RNA isolation kit (Qiagen,
Valencia, CA) and treated with DNase (Roche Applied Science) to remove any
contaminating genomic DNA. cDNA was synthesized using oligo(dT) primers and
Superscript II reverse transcriptase according to the manufacturer's
instructions. Primers specific for human HFE, TfR1, DMT1 (for both
iron-responsive element and non-iron-responsive element DMT1 forms), Zip14,
and GAPDH were designed using the Primer Express software package (PE
Biosystems, Foster City, CA)
( 20). The primer sequences are
listed in Table 1. The qRT-PCR
was carried out in triplicate for each sample in at least three independent
experiments using a SYBR Green detection system on an ABI PRISM 7900 machine
(Applied Biosystems, Foster City, CA)
( 20,
22). The reaction volume was
15 μl. Forty cycles of PCR amplification were denatured at 95 °C for 15
s, were annealed at 55 °C for 30 s, and were extended at 72 °C for 30
s. PCR products were detected by measuring the increase of fluorescence from
the binding of SYBR Green to double-stranded DNA. Melting curve experiments
previously established that the fluorescent signal for each amplicon was
derived from the products only and not from primer dimers. All primer sets
used in these studies were validated against the reference primers (GAPDH) to
ensure that they amplified equally across the range of template
concentrations. | TABLE 1List of primers used for qRT-PCR |
Knockdown of Zip14 Using siRNA—Lipofectamine RNAiMAX
transfection reagent (Invitrogen) was used to transfect siRNA specific for
human Zip14 (Dharmacon, Lafayette, CO) or negative control siRNA into cells at
a final concentration of 10 n m following the manufacturer's
instructions ( 7,
23). Briefly, 2 μl of
Lipofectamine RNAiMAX and 12 pmol of RNAi duplex were mixed in 200 μl of
Opti-MEM medium and added into each well of a 12-well plate. After incubation
at room temperature for 15 min, ~2 × 10 5 cells in 1 ml of
minimum essential medium Eagle supplemented with 1.0 m m sodium
pyruvate, 0.1 m m nonessential amino acids, and 10% fetal bovine
serum were added to each well. Three days later, Zip14 mRNA levels were
detected using qRT-PCR, and protein levels were detected using immunoblots to
determine the efficiency of knockdown. Immunoblot—Cells were washed with cold phosphate-buffered
saline twice and lysed on ice in NET-Triton buffer (150 m m NaCl, 5
m m EDTA, 10 m m Tris, 1% Triton X-100, pH 7.4) with
1× Complete Mini Protease Inhibitor Mixture (Roche Applied Science) and
1 m m phenylmethylsulfonyl fluoride. The cell lysate was centrifuged
at 16,000 × g for 5 min, and the supernatant was kept. Protein
concentrations of the cell extracts were measured using the BCA Protein Assay
(Pierce). The cell extracts were reduced and denatured with Laemmli buffer
( 24) for 5 min at 95 °C
and subjected to SDS-PAGE on 12% gels. Protein was transferred to
nitrocellulose. Immunoblot analysis was carried out using rabbit anti-ferritin
(1:4,000; DAKO, Carpinteria, CA), M2 anti-FLAG (1:10,000; Sigma), mouse
anti-Myc (1:5,000; Invitrogen), rabbit anti-Zip14 (1:2,000), and mouse
anti-actin (1:10,000; Sigma) followed by goat anti-rabbit or anti-mouse
secondary antibodies conjugated to horseradish peroxidase (1:10,000; Chemicon,
Temecula, CA). Bands were detected by enhanced chemiluminescence (SuperSignal
West-Pico; Pierce). To quantify the amount of Zip14 and actin on the blot,
Alexa 680 goat anti-rabbit (1:5,000; Molecular Probes, Carlsbad, CA) and IRDye
800 donkey anti-mouse (1:5,000; Rockland Immunochemicals, Gilbertsville, PA)
fluorescent secondary antibodies were used, respectively. The intensity of the
band was quantified by fluorescence imaging (Odyssey Infrared Imaging System;
Li-Cor, Lincoln, NE). HFE Decreases Intracellular Iron Uptake in HepG2 Cells—HepG2
cells were used to investigate the role of HFE in iron uptake. Intracellular
iron status was assessed initially by measuring levels of ferritin, an iron
storage protein. When the intracellular iron concentration is low,
iron-regulatory proteins 1 and 2 (IRP1 and -2) bind to a conserved
iron-responsive element located in the 5′-untranslated regions of
ferritin mRNAs to inhibit its translation. Conversely, increased iron
concentration leads to decreased binding of IRP1 and -2 to the iron-responsive
element, resulting in increased ferritin synthesis
( 25– 27).
We found that HFE expression in HepG2 cells decreased ferritin levels
(), indicating
lower cellular iron concentrations. Reduced ferritin levels may result from a
decrease in iron uptake and/or an increase in iron export. Iron uptake into
cells is controlled by Tf and non-Tf-mediated pathways. Cells loaded with
Fe-NTA (NTBI) showed increased ferritin levels, and cells expressing HFE
showed decreased iron loading both with and without Fe-NTA treatment
(). To test how
much NTBI uptake was inhibited by HFE expression, a direct assay of iron
uptake using 55Fe-NTA was employed. HFE expression inhibited NTBI
uptake by about 42% in HepG2 cells after 60 min
().
Furthermore, HFE expression decreased the TBI uptake through Tf-mediated iron
transport pathway by ~48% in HepG2 cells
(), indicating
that the inhibition of cellular iron loading by HFE could be due to a decrease
of both TBI and NTBI uptake. Iron efflux measurements were made to examine the
possibility that the lower basal intracellular iron levels in HFE-HepG2 cells
could result from an increase in iron export. HFE had no effect on iron export
in HepG2 cells (). These results suggest that HFE decreases
intracellular iron through inhibition of NTBI and TBI uptake rather than iron
export. HFE Expression Increases TfR1 but Has No Effect on Zip14 or DMT1 mRNA
Levels in HepG2 Cells—TfR1, DMT1, and Zip14 are iron transporters
important in iron uptake. TfR1 mediates TBI uptake. DMT1 and Zip14 mediate
NTBI uptake. The mRNA levels of TfR1, DMT1, and Zip14 were measured by qRT-PCR
to investigate whether the inhibitory effect of HFE on iron uptake in HepG2
cells was caused by the change in expression of these mRNAs. HFE expression
increased TfR1 mRNA levels in HepG2 cells
(). These results are
consistent with increased stability of TfR1 mRNA under low iron conditions
( 28). Thus, increased TfR1
expression in HFE-expressing HepG2 cells can be attributed to lower
intracellular iron (). DMT1 and Zip14 mRNA levels were not affected by HFE
(). Notably, the relative
level of Zip14 mRNA was high in HepG2 cells. Knockdown of Zip14 Abolishes the Inhibitory Effect of HFE on NTBI
Uptake in HepG2 Cells—Since the level of Zip14 mRNA was high in
HepG2 cells and HFE expression inhibited NTBI uptake, we used siRNA knockdown
of Zip14 in HepG2 cells to determine whether NTBI uptake was mediated by Zip14
and whether the inhibitory effect of HFE on NTBI uptake was mediated through
Zip14. Knockdown of Zip14 resulted in a 71% decrease in mRNA level detected by
qRT-PCR ().
Immunoblot analysis of cells treated with Zip14 siRNA indicated that the level
of Zip14 protein was undetectable in HepG2 cells transfected with Zip14 siRNA
compared with control cells (). In addition, knockdown of Zip14 decreased NTBI
uptake by about 52% (), implying that Zip14 is responsible for approximately
half of the NTBI uptake in HepG2 cells. HFE expression decreased endogenous Zip14 protein levels
() and
inhibited NTBI uptake in HepG2 cells. This inhibition of HFE on NTBI uptake
was abolished by Zip14 knockdown (). These results suggest that in HepG2 cells, the
inhibitory effect of HFE on NTBI uptake is mainly mediated by the
down-regulation of endogenous Zip14 protein. Expression of HFE Promotes Zip14 Degradation in HepG2
Cells—The observation that HFE expression reduced Zip14 protein
while not changing the level of Zip14 mRNA suggested that HFE might affect the
stability of the Zip14 protein. We measured Zip14 half-life by immunoblot
using cycloheximide to inhibit protein synthesis in control and HFE-expressing
HepG2 cells. The half-life of Zip14 decreased from 11.0 h in control cells to
7.5 h in HFE-expressing HepG2 cells (), indicating that HFE expression
promotes Zip14 degradation in HepG2 cells. HFE Inhibits Zip14-mediated NTBI Uptake in HeLa Cells Transfected with
Zip14—Since HFE has no detectable effect on NTBI uptake in HeLa
cells and HT29 cells ( 20,
21), we wanted to test whether
transfection of HeLa cells expressing HFE with Zip14 would alter NTBI uptake.
First, the endogenous mRNA levels of DMT1 and Zip14 were measured in HeLa and
HepG2 cells. At the mRNA level, HeLa cells expressed approximately the same
amount of DMT1 as HepG2 cells but less Zip14 than HepG2 cells normalized to
the GAPDH of each cell line (). No Zip14 protein could be detected in HeLa cells by
immunoblot (data not shown). The difference in the endogenous expression of
Zip14 could be the reason why HFE inhibited NTBI uptake in HepG2 cells but had
no effect in HeLa cells. Low levels of endogenous Zip14 expression makes HeLa
cells a perfect tool to investigate the effect of HFE on exogenous
Zip14-mediated NTBI uptake by transfection with Zip14. In HeLa/tTA-HFE-FLAG
cells expressing FLAG epitope-tagged HFE under control of the tetracycline
responsive promoter, induction of HFE expression had no effect on NTBI uptake
(), consistent
with previous observations
( 21). Zip14 expression
significantly increased NTBI uptake in HeLa cells. The increase was partially
inhibited by induction of HFE expression
(). Induction
of HFE expression also significantly decreased Zip14 protein level in
HeLa/tTA-HFE-FLAG cells (). Thus, in HeLa cells expressing Zip14, Zip14
increases NTBI uptake, and HFE expression inhibits Zip14-mediated NTBI uptake
through the down-regulation of Zip14 at the protein level. HFE appears to function at several different levels in the liver. Genetic
evidence shows that it plays a role in the regulation of hepcidin in
hepatocytes ( 29,
30). In the latter study, the
authors speculated that HFE might increase the transcription of hepcidin
through its interaction with TfR2. Earlier studies on the function of HFE
demonstrated that HFE also decreases iron uptake in some cell types and lowers
iron efflux in other cell types (reviewed in Ref.
31). HepG2 cells possess many
of the key features of hepatocytes, including the ability to polarize and
secrete hepatocyte-specific proteins, such as albumin, transferrin, and
hepcidin ( 32). Because of
their similarity to hepatocytes, we used HepG2 cells to observe the effect of
HFE on cellular iron homeostasis. Stable transfection of HFE in HepG2 cells
decreased ferritin levels and lowered levels of iron uptake. Notably, HFE
expression not only reduced TBI uptake but also NTBI uptake, which is
different from the observation in HeLa cells, where HFE reduced only TBI
uptake, leaving NTBI uptake unaffected
( 21). DMT1 and Zip14 are iron
transporters for NTBI ( 3,
7,
33). Expression of HFE in
HepG2 cells resulted in a lower amount of Zip14 protein. This was partially
due to a decreased stability of Zip14 rather than changes in the mRNA levels.
The insensitivity of iron uptake to HFE expression after Zip14 knockdown by
siRNA implies that HFE has a direct effect on Zip14-mediated iron transport.
These results suggest that in HepG2 cells, Zip14 is involved in NTBI uptake,
and the reduction in NTBI uptake by HFE expression may be mediated through
Zip14. Zip14 ( SLA39A14) is a member of the SLC39A metal ion transporter
family, which was initially characterized as a zinc transporter. A recent
study indicated that NTBI uptake increases in HEK 293 cells and Sf9 insect
cells transfected with Zip14 and decreases in AML12 mouse hepatocytes when
Zip14 is knocked down by siRNA
( 7). Moreover, Zip14 is
expressed at the plasma membrane of hepatocytes
( 34) and can efficiently
transport iron at pH 7.4 ( 7),
the normal pH at the plasma membrane surface of hepatocytes. In contrast,
DMT1, the first iron transporter to be identified, transports iron optimally
at pH 5.5 ( 35) and is readily
detected in endosomes but not on the plasma membrane
( 36). These observations
suggest that Zip14, rather than DMT1, plays the predominant role in NTBI
uptake by hepatocytes. The variation in phenotypes of patients with the HFE mutation has led to
the hypothesis that there are HFE modifiers, including iron importers
( 37– 39).
Interestingly, the survival of Slc11a2-/- mice that lack
DMT1 is significantly improved when HFE alleles were inactivated. This finding
suggests that in Slc11a2-/- mice, lacking HFE might lead
to the up-regulation of another iron importer
( 40). Zip14 expression in
duodenum tissue is higher in Hfe-/- mice than that of
control mice by microarray analysis
( 41). In that case, the mRNA
levels for Zip14 increase. No such change was noted in the mRNA levels of
Zip14 in the liver sample, consistent with our results. Given the findings in
the present study, Zip14 is a potential candidate for an HFE modifier involved
in TBI and NTBI uptake in HepG2 cells. To investigate the mechanism by which HFE lowers NTBI in HepG2 cells, we
found that knockdown of Zip14 abolished the inhibitory effect of HFE on NTBI
uptake. This suggested that the reduction of NTBI uptake by HFE expression was
mediated through Zip14 in HepG2 cells. We further observed the effect of HFE
on Zip14 expression in HepG2 cells by immunoblot and qRT-PCR. HFE
significantly reduces Zip14 protein with no change in the level of Zip14 mRNA.
We also found that HFE reduced Zip14 half-life from 11.0 h to 7.5 h in HepG2
cells. These results imply that HFE lowers Zip14-mediated NTBI uptake by
decreasing Zip14 stability. In HeLa/tTA-HFE-FLAG cells, inducing HFE
expression by withdrawal of dox does not affect NTBI uptake
( 21). Transfection of this
cell line with Zip14 increased NTBI uptake 7-fold over untransfected cells.
This increase in NTBI uptake was inhibited by HFE expression. Analysis of
Zip14 expression by immunoblot demonstrated that HFE expression reduces Zip14
protein level in HeLa/tTA-HFE-FLAG cells transfected with Zip14. These
observations were consistent with the results obtained in HepG2 cells, which
endogenously express Zip14. The evidence that HFE reduced both TBI uptake and NTBI in HepG2 cells
raises the question of whether both TBI uptake and NTBI share a common pathway
in HepG2 cells ( 9). HFE reduces
NTBI uptake in Chinese hamster ovary cells lacking endogenous TfR1 and TBI
uptake in Chinese hamster ovary cells transfected with TfR1
( 18). A single divalent iron
transporter could explain the shared pathway for TBI and NTBI uptake, which
functions in the endosome for TBI uptake and on cell membrane for NTBI uptake.
DMT1 overexpression in hepatoma (HLA) cells does not change TfR1-dependent
iron uptake ( 36). These
results suggest that Zip14, which can be down-regulated by HFE, mediates TBI
and NTBI uptake in HepG2 cells. Future studies on how HFE increases the
turnover of Zip14, the localization and the pH dependence of Zip14, and a
direct association of HFE and Zip14 will be necessary to test these
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