From the Department of Cell and Developmental
Biology, Oregon Health Sciences University,
Portland, Oregon 97201-3098 and ¶ Progenitor Inc.,
Menlo Park, California 94025
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ABSTRACT |
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HFE is the protein product of the gene mutated in
the autosomal recessive disease hereditary hemochromatosis (Feder,
J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A.,
Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C.,
Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E.,
Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B.,
Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana,
L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna,
D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996)
Nat. Genet. 13, 399-408). At the cell surface, HFE
complexes with transferrin receptor (TfR), increasing the dissociation
constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998)
J. Biol. Chem. 273, 22068-22074; Feder, J. N.,
Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A.,
Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A
95, 1472-1477). HFE does not remain at the cell surface, but traffics
with TfR to Tf-positive internal compartments (Gross et
al., 1998). Using a HeLa cell line in which the expression of HFE
is controlled by tetracycline, we show that the expression of HFE
reduces 55Fe uptake from Tf by 33% but does not affect the
endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to
reduce cellular acquisition of iron from Tf within endocytic
compartments. HFE specifically reduces iron uptake from Tf, as
non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not
altered. These results explain the decreased ferritin levels seen in
our HeLa cell system and demonstrate the specific control of HFE over
the Tf-mediated pathway of iron uptake. These results also have
implications for the understanding of cellular iron homeostasis in
organs such as the liver, pancreas, heart, and spleen that are iron
loaded in hereditary hemochromatotic individuals lacking functional HFE.
HFE is the protein product of the gene mutated in the autosomal
recessive disease hereditary hemochromatosis
(1), which was first cloned in 1996 (1).
It is therefore a relatively new member of the growing group of
proteins involved in iron metabolism. HFE is remarkable in that it is a
nonclassical major histocompatibility complex class I type molecule, a
characteristic that prevented its role in iron homeostasis from being
recognized immediately. Over 80% of hereditary hemochromatosis
patients have the same mutation in the Although the gene encoding HFE was discovered by genetic mapping of
individuals with a disease of iron overload, the function of the
protein in iron metabolism has not been immediately appreciated. The
region of the HFE molecule normally associated with peptide binding and
cell surface presentation in major histocompatibility complex class I
molecules is too narrow to bind peptides. HFE does not appear to bind
iron either (5). HFE does, however, associate with the transferrin
receptor (TfR),1 a well
characterized member of the iron metabolic pathway. The TfR is a type
II transmembrane protein that binds the serum protein, diferric
transferrin (Tf), at the cell surface. The Tf-TfR complex is
constitutively taken into the cell through clathrin-mediated endocytosis. At the low pH of the endosome, a conformational change in
the TfR facilitates iron release from Tf (6-9). Apotransferrin (apo-Tf) remains bound to TfR until the complex cycles back to the cell
surface. At the neutral pH of the extracellular environment, apo-Tf is
quickly replaced by diferric Tf and is free to bind more iron (for
further discussion, see reviews in Refs. 10 and 11).
The association of HFE with the TfR has been demonstrated in tissues
and in cell culture (12, 13). Wild type HFE has been shown to associate
with TfR in the placenta (13) and to have the same immunohistochemical
staining pattern as TfR in the crypt cells of the intestine (14). In
cultured cells, HFE traffics with TfR between the cell surface and
Tf-positive perinuclear compartments (15). The association between HFE
and TfR has been shown to lower the affinity of TfR for its ligand
10-fold (5, 12, 15). Other nonclassical major histocompatibility
complex class I molecules have been shown to affect ligand affinity for other receptors, such as the ability of the HLA H-2 protein to associate with the insulin receptor and reduce all insulin binding sites to low affinity sites (16-20) and the ability of anti-HLA antibodies to reduce epidermal growth factor binding to its receptor (21, 22).
The physiological importance of the interaction between HFE and TfR is
not immediately clear. Diferric Tf is present at concentrations of
approximately 5 µM in the blood (23). The dissociation
constant of Tf for the TfR-HFE complex is approximately 11 nM. Thus, the binding of diferric Tf to its receptor is
saturated in the presence of HFE. These results emphasize the putative
role of HFE as a regulator of iron homeostasis that acts through the
TfR cycle rather than having a role as a transporter or iron-binding protein.
To investigate its influence on cellular iron metabolism, the ability
of HFE to specifically regulate the Tf-mediated pathway of iron uptake
was tested. We found that HFE does not alter the cycling kinetics of
the TfR. Rather, HFE reduces the amount of iron assimilated from Tf by
~33% without affecting non-Tf-mediated uptake of Fe-NTA. This
finding gives more insight into the role of HFE as an iron regulator
through the Tf-mediated pathway. Additionally, this focuses attention
on the role of Tf-iron stores as a critical part of the "sensing
machinery" that regulates dietary iron uptake and the kinetics of
metabolic iron recycling.
Cell Lines
The fWTHFE/tTA HeLa cell line expressing FLAG epitope-tagged HFE
(fHFE) under the tetracycline-responsive promoter has been previously
described (15). Cells were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum, 400 µg/ml
G418 (Geneticin, Calbiochem), 200 ng/ml puromycin, and with (HFE Iodination
Human holotransferrin (Intergen, Co.) was labeled with
Na[125I] (NEN Life Science Products) using
lactoperoxidase as described previously (24).
125I-Tf Uptake Protocol
The rate of 125I-Tf uptake was determined as
described previously (24) with the following modifications. Uptakes
were performed on subconfluent HFE 125I-Tf Efflux Protocol
The rate of Tf efflux was determined as described previously by
McGraw and Maxfield (25) with the following modifications. Monolayers
of subconfluent HFE Efflux--
Medium was removed by pipetting, and cells were
washed one time with 1 ml of final wash, which was pooled and counted
in gamma counter (Packard, CobraII Auto-Gamma).
Surface--
Cells were incubated in 1 ml of acid wash (0.2 N acetic acid, 0.5 M NaCl) for 3 min at 4 °C
and then washed 1 time with 1 ml of final wash, which was pooled and counted.
Internal--
Cells were stripped and washed as per surface
samples and then solubilized in 2 ml of solubilization detergent and counted.
TfR Distribution
The relative TfR distribution between cell surface (external)
and internal compartments was determined as described previously (25,
26) with the following modifications. Subconfluent HFE 55Fe Loading of Human Apotransferrin
55FeCl3 (NEN Life Science Products) was
complexed to nitrilotriacetic acid in a 1:200 ratio (Fe:NTA). Then, the
55Fe-NTA was incubated with Tf in a 2:1 ratio for 1 h
in carbonate buffer (10 mM NaHCO3, 250 mM Tris-HCl). 55Fe-transferrin was separated
from free 55Fe on a 20-ml G-50 Sephadex (Sigma) column. The
resulting transferrin was 82% saturated with iron.
55Fe-Tf Uptake Protocol
55Fe uptake from transferrin was determined
essentially as described above for 125I-Tf uptake with the
following modifications. Uptakes were performed on subconfluent HFE 55Fe-NTA Uptake Protocol
55Fe-NTA uptake procedures were modified from a
previously described protocol by Inman and Wessling-Resnick (27). A
55Fe-NTA solution was made by the addition of
55FeCl3 to NTA buffer (80 µM NTA,
20 mM HEPES-Tris) for a final concentration of 20 µM. Subconfluent fWTHFE/tTA HeLa cells grown in 35-mm
dishes were washed two times with 2 ml of McCoy's 5A-20 mM
HEPES at 37 °C. Wash medium was replaced with 1 ml of specific (200 nM 55Fe-NTA in wash medium) or nonspecific
(specific medium with 1 mM Fe-NTA) medium. After 2, 5, 10, 15, or 30 min of uptake, cells were placed on ice, and 3 ml of 4 °C
quench buffer (1 mM Fe-NTA, 25 mM HEPES, 150 mM NaCl) was added for 20 min. Finally, the cells were
washed three times with 2 ml of 150 mM NaCl at 4 °C,
solubilized, and counted as for 55Fe-Tf uptake protocol
described above.
Iron Treatments
HFE Western Immunodetection
Cell extracts from 2 × 105 cells were diluted
with 4× Laemmli buffer (28) to 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and subjected to
electrophoresis on 12% SDS-polyacrylamide gels under reducing
conditions. The proteins were transferred to nitrocellulose. Immunoblot
analysis was performed using sheep anti-human transferrin receptor
serum (described previously (24); 1:10,000 dilution), and sheep
anti-human ferritin antibody (The Binding Site, Ltd., 1:100 dilution)
followed by swine anti-goat secondary antibody conjugated to
horseradish peroxidase (Boehringer Mannheim). Chemiluminescence
(SuperSignal, Pierce) was performed per the manufacturer's directions.
fHFE Does Not Alter Tf Endocytosis--
The influence of HFE on
TfR cycling kinetics was measured to determine how HFE regulates iron
levels in the cell. In previous studies, a cell line expressing fHFE
under the control of the tetracycline repressible system was
established (15). Induction of HFE expression by the withdrawal of
tetracycline from the medium results in decreased ferritin (Ft) levels.
One mechanism by which HFE could lower the amount of iron taken up into
cells would be by decreasing the rate of endocytosis of the TfR. HFE
co-traffics with TfR between the cell surface and Tf positive
perinuclear compartments (15) and therefore might have some influence
on TfR endocytic kinetics.
The rate of 125I-Tf uptake was measured to test whether or
not HFE alters the endocytosis of TfR and therefore the amount of Tf-mediated iron uptake in fWTHFE/tTA HeLa cells expressing fHFE (fWTHFE/tTA HeLa cells). Uptake experiments were performed in the
presence of 50 nM 125I-Tf. The association of
HFE with the TfR decreases the affinity of the TfR for Tf, resulting in
an increase in the Kd from 1.2 to 11 nM
(15). At 50 nM Tf, essentially all the TfRs are occupied
with Tf, even in the presence of HFE. Cells were incubated with
125I-Tf for 2, 4, 6, or 8 min at 37 °C with 5%
CO2. The surface was stripped of bound Tf, and the amount
of 125I-Tf inside the cell was counted. Both HFE fHFE Does Not Alter TfR Exocytosis--
The amount of iron taken
up into cells is a function of the kinetics of TfR cycling that depends
on both the endocytic and exocytic rates. Because fHFE had no effect on
TfR endocytosis, the rate of 125I-Tf release was measured
to test whether fHFE alters the exocytic rate of TfR in HFE TfR Distribution Is Not Changed in fHFE-expressing
Cells--
Because the uptake and efflux kinetics of Tf did not change
with the expression of fHFE, the steady state distribution of external
and internal TfRs should also remain the same between cells expressing
and not expressing fHFE. To test this prediction, steady state external
and internal pools of 125I-Tf were measured in control
cells (HFE fHFE Reduces Iron Uptake from Tf But Does Not Alter Non-Tf-mediated
Iron Uptake--
We wanted to determine whether HFE affected the
amount of Tf-mediated or non-Tf-mediated iron uptake because no effect
on the cycling kinetics of the TfR was detected. HFE
To determine whether the influences of HFE are restricted to the
Tf-mediated pathway of iron uptake or if HFE additionally affects the
non Tf-mediated iron uptake pathway, HFE Ft Levels Are Responsive to Iron Treatment in fWTHFE/tTA HeLa
Cells--
The response of HFE
The response of cells to iron depletion and iron loading was measured
in HFE
TfR levels also changed, as shown in Fig. 6, as predicted by the known
influence of IRPs. TfR levels were slightly elevated in fHFE-expressing
cells versus HFE
The sensitivity of the cellular IRP response to iron was also tested in
the presence and absence of HFE. Even at Tf concentrations below
saturation of the TfR and HFE/TfR complex, such as 5 nM, Ft
levels were greater for both HFE The mechanism by which HFE controls iron homeostasis is not yet
known. We have previously reported that HFE specifically reduces Ft
levels in HeLa cells expressing fHFE, showing a direct relationship between HFE expression and cellular iron homeostasis (15). HFE traffics
with TfR to Tf-positive perinuclear vesicles, which are one site of
cellular iron absorption (15). Regulated Tf and non-Tf-mediated iron
uptake pathways have both been described for the HeLa tissue culture
system (32-34) (for further discussion, see review in Ref. 35). This
study shows that HFE specifically decreases the Tf-mediated pathway of
iron uptake.
fHFE has no effect on the kinetics of TfR cycling in cells. If HFE were
to slightly decrease the endocytic rate or increase the exocytic rate
of TfR, less Tf and therefore less iron would be taken up per unit of
time. At concentrations of Tf that saturate Tf binding to the HFE/TfR
complex (50-100 nM), no difference in the uptake or
release of 125I-Tf from HFE Although HFE does not affect TfR cycling kinetics, it does regulate
cellular iron homeostasis through the Tf-mediated iron uptake pathway.
At saturating concentrations of 55Fe-loaded Tf (100 nM), 33% less iron was taken up by fWTHFE/tTA HeLa cells
expressing fHFE. Because saturating amounts of Tf were used in these
experiments, the ability of HFE to lower the TfR affinity for Tf is not
the mechanism responsible for the observed decrease in cellular iron.
This experiment does not differentiate between release of iron from
transferrin and transport across the endosomal membrane to the
cytoplasm. HFE might effect either of these steps in Tf-mediated iron
transport. Because Ft levels are also reduced for HFE+ fWTHFE/tTA HeLa
cells in culture, these results suggest that acquisition of iron from
fetal bovine Tf in tissue culture medium takes place through the TfR.
Despite the larger dissociation constant of fetal bovine Tf for the
human TfR, iron is still removed from the bovine Tf but the uptake is reduced in HFE+ cells.
fWTHFE/tTA HeLa cells were incubated in the presence of 200 nM 55Fe-NTA to confirm that HFE specifically
regulates Tf-mediated iron uptake. Iron presented in this form bypasses
the Tf-TfR pathway and enters the cell directly through a transporter
(27, 36). Candidate transporters are SFT and DCT1/Nramp2 although
other, currently unidentified, cell surface iron transporters may exist as well (34, 37). No significant difference was seen in the amount of
non-Tf bound iron taken up by HFE+ cells as compared with HFE Western blots were used to determine whether cells expressing HFE can
respond to bioavailable iron by increasing their Ft levels. Under
control tissue culture conditions, HFE These studies demonstrate that in a nonpolarized cell line, fHFE
reduces the amount of iron taken up from the Tf-mediated iron uptake
pathway. Several factors are involved in iron uptake from the endosome,
any one of which could be affected by HFE. One characteristic of the
endosome is its relatively low pH (between 5.5 and 6.5, depending on
the cell type (9)). This low pH is responsible for a conformational
change in both Tf and the TfR that facilitates the release of iron
(6-9). HFE might regulate the lumenal pH of the endosome, preventing
efficient removal of iron from Tf. In conflict with this hypothesis is
evidence that suggests increases in endosomal pH slows recycling of TfR
(38). We do not see any perturbations of TfR recycling, suggesting that the endosomal pH is close to normal.
We favor the hypothesis that HFE prevents the pH-induced conformation
of the TfR that potentiates the release of iron from Tf. Aisen and
co-workers (6-8) and Sipe and Murphy (9) showed the importance of the
TfR-induced conformational change in Tf for the release of iron in the
endosome. At pH 6.0, the association of Tf with the TfR approximately
doubles the rate of loss of iron from Tf (39). If this change was
prevented, then less iron would be taken up into cells. One caveat of
this hypothesis is the observation that soluble TfR does not detectably
bind to soluble HFE at pH 6.0 (5). For this mechanism to be responsible
for the lower uptake of iron into cells, either HFE does interact with
the TfR at the slightly higher pH of many endosomes or the membrane
bound forms of the TfR and HFE interact with each other at endosomal pHs.
Alternatively, HFE might regulate the function of one of the known
endosomal iron transporters, such as Nramp2/DCT1 or SFT (40, 41),
thereby reducing iron transport across the endosomal membrane. HFE
might simply use its association with TfR for endocytosis. Once in the
endosome, HFE might complex with transmembrane proteins responsible for
transporting, iron into the cytoplasm. HFE could modulate the
transporters affinity for iron or its kinetics of iron transport across
the endosomal membrane. Thus, HFE may be involved in maintaining
homeostatic equilibrium of iron through its regulation of such a
transporter. By modulating transporter activity in Tf-positive
endosomes, HFE could facilitate appropriate flux of Tf-derived iron
across the intestinal enterocyte. These three hypothesis need to be tested.
With the addition of the present findings from the fWTHFE/tTA HeLa
system, we propose a new model for the role of HFE in iron regulation
(Fig. 8). HFE imposes an additional
regulatory step for iron uptake in cells expressing HFE. Iron
responsive element-mediated iron regulation does not appear to be
sufficient to regulate iron homeostasis because organisms lacking HFE
or Tf but having functional IRPs eventually succumb to iron overload.
We propose that HFE limits iron uptake from Tf by reducing the amount
of iron released from Tf. Without HFE (as is the case in hereditary
hemochromatosis patients), organs normally expressing HFE, such as the
lives, clear more iron from circulating Tf, contributing to hepatic
iron overload.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 domain of HFE
that prevents its heterodimerization with
2
microglobulin (2). Consistent with the classification of HFE as an
major histocompatibility complex class I type molecule, this C282Y
mutation has been shown to prevent cell surface expression and,
presumably, function of the protein (2, 3). The generation of the HFE
knockout mouse supports the finding that the C282Y mutation is, indeed,
a loss of function mutation because the phenotype of the knockout mouse
parallels the manifestation of hereditary hemochromatosis in humans
(4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) or
without (HFE+) 2 µg/ml tetracycline.
and HFE+ fWTHFE/tTA HeLa cultures
(~1 × 106 cells) washed two times with 2 ml of
DMEM-20 mM HEPES, pH 7.4, and preincubated in the same
medium for 15 min at 37 °C with 5% CO2. At the 0 min
mark, 1 ml of specific (DMEM-20 mM HEPES, 2 mg/ml
ovalbumin, 50 nM 125I-Tf) or nonspecific
(specific medium with 1 mg/ml cold Tf) medium was added to the
appropriate cells. Cells were incubated at 37 °C with 5%
CO2 for 2, 4, 6, or 8 min. Externally bound Tf was stripped
with an acidic buffer (0.5 N acetic acid, 0.5 M
NaCl) for 3 min at 4 °C. Then, the cells were washed four times with 2 ml of 4 °C final wash buffer (150 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, pH 7.4) before
addition of solubilization detergent (0.1% Triton X-100, 0.1% NaOH)
and counting in a gamma counter (Packard, CobraII Auto-Gamma). Surface
TfR numbers were determined by counting the amount of
125I-Tf bound after incubation with 50 nM
125I-Tf for 90 min on ice at 4 °C. Following the 90-min
incubation, the medium was removed, and cells were washed four times
with 2 ml of 4 °C final wash buffer before solubilization and counting.
and HFE+ fWTHFE/tTA HeLa cells grown in 35-mm
plates were washed three times with 2 ml of DMEM-20 mM
HEPES and then preincubated in the same medium for 15 min at 37 °C
and 5% CO2. The medium was removed, and cells were
incubated for 2 h with specific (50 nM
125I-Tf in DMEM-20 mM HEPES and 2 mg/ml
ovalbumin) or nonspecific (specific medium with 1 mg/ml unlabeled Tf)
medium at 37 °C and 5% CO2. Cells were washed with 2 ml
of 37 °C mild acid buffer (500 mM NaCl, 50 mM MES, pH 5.0) for 2 min and then washed two times with 2 ml of 37 °C final wash buffer with 100 µM
desferoxamine (desferal; Ciba-Geigy Ltd.; Basel, Switzerland) and 3 µg/ml Tf to prevent loading and rebinding of apo-125I-Tf.
1 ml of 37 °C medium (DMEM-20 mM HEPES, 2 mg/ml
ovalbumin) with 3 µg/ml Tf and 100 µM desferoxamine was
added at the 0 min mark, and then at each of the appropriate time
points, plates were placed on ice and the appropriate efflux, surface
and internal samples were collected as follows.
and HFE+
fWTHFE/tTA HeLa cells grown in 35-mm plates were either incubated for
90 min with 100 nM 125I-Tf at 37 °C with 5%
CO2 for total and internal measurements or with 100 nM 125I-Tf at 4 °C for external measurements
in 1 ml of specific (100 nM 125I-Tf in DMEM-20
mM HEPES) or nonspecific (specific medium with 1 mg/ml
unlabeled Tf) medium at 4 °C. At the end of the incubation time,
cells were placed on ice. Total and external measurements were
determined by washing four times with 2 ml of final wash at 4 °C and
then solubilizing and counting. The amount of internalized 125I-Tf was determined by stripping the surface for 3 min
with acid wash, washing four times with 2 ml of final wash, and then
solubilizing and counting in gamma counter (Packard, CobraII
Auto-Gamma).
and HFE+ fWTHFE/tTA HeLa cells grown in 35-mm dishes. Specific medium
contained 100 nM 55Fe-Tf and 2 mg/ml ovalbumin
in wash medium (McCoy's 5A with 20 mM HEPES). Nonspecific
medium was the same as specific with the addition of 1 mg/ml unlabeled
Tf. After 45, 90, 135, or 225 min of uptake, cells were placed on ice,
and externally bound Tf was stripped with an acidic buffer (0.2 N acetic acid, 500 mM NaCl, 1 mM
FeCl3) for 3 min at 4 °C. Cells were solubilized in 1 ml of the same solubilization detergent described above. Lysates were
mixed with 6 ml UniverSol (ICN) and counted for 10 min in a
scintillation counter (Beckman LS 6000SC) with a window of 0-350 nm.
TfR numbers were determined as described for the 125I-Tf
uptake protocol above.
and HFE+ fWTHFE/tTA HeLa cells were seeded at ~1.5 × 105 cells per 35-mm dish. After 2 days, cells were
treated with either 50 µM desferoxamine, 100 nM human holotransferrin (Intergen Co.), or 100 µM Fe-NTA overnight. Cells were lysed in NET-Triton (150 mM NaCl, 5 mM EDTA, and 10 mM Tris
(pH 7.4) with 1% Triton X-100) after at least 12 h of treatment.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HFE+
cells took up approximately 0.20 Tf/surface TfR/min (Fig.
1). The same rates of internalization
were measured in cells treated with 100 nM 125I-Tf, confirming that the binding of Tf to its receptor
was rapid and saturating (data not shown). fHFE did not significantly
alter the amount of 125I-Tf uptake per TfR in fWTHFE/tTA
cells and therefore did not affect the kinetics of TfR endocytosis.
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Fig. 1.
fHFE does not significantly alter Tf
endocytosis. 125I-Tf uptake was measured in 7.2 × 105 HFE (
, dashed line) or 7.0 × 105 HFE+ (
, solid line) fWTHFE/tTA HeLa
cells. The rates of 125I-Tf uptake/surface receptor/min for
HFE
and HFE+ were 0.198 (r2 = 0.901) and 0.213 (r2 = 0.925), respectively. Linear regression
was determined in the Cricket Graph program. These results are
representative of three experiments performed with quadruplicate data
points without significant variation between experiments.
and HFE+
fWTHFE/tTA HeLa cells. Cells were loaded with 125I-Tf for
2 h at 37 °C with 5% CO2 to saturate internal and
external TfR with Tf. Then, the cell surface was stripped of Tf, and
fresh medium was added to the cells. The Tf released from internal
cellular compartments was collected and counted at 2, 4, 6, and 8 min
time points. HFE
and HFE+ fWTHFE/tTA HeLa cells did not differ
significantly in their rate of TfR exocytosis (Fig.
2).
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Fig. 2.
fHFE does not significantly alter Tf
exocytosis. 125I-Tf release was measured in 9.3 × 105 HFE (
, dashed line) or 7.7 × 105 HFE+ (
, solid line) fWTHFE/tTA HeLa
cells. The rates of 125I-Tf release as a percentage of the
total cell associated/125I-Tf/min for HFE
and HFE+ were 0.052 (r2 = 0.916) and 0.047 (r2 = 0.929), respectively. Linear regression
was determined in the Cricket Graph program. These results are
representative of three experiments performed with quadruplicate
data points without significant variation between experiments.
) and in cells expressing fHFE (HFE+). fHFE expression did
not significantly alter the relative surface and internal distributions
of TfR (Fig. 3). Approximately 20% of
cellular TfR was found at the cell surface, whereas 80% was found in
internal vesicles. The distribution of TfR between the cell surface and
internal compartments was similar to that reported previously in other
cell lines expressing similar amounts of TfR (29-31). Combined with
the uptake and efflux data reported above, these findings support the
conclusion that HFE has no effect on TfR cycling kinetics even though
HFE associates and co-traffics with the TfR (15).
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Fig. 3.
fHFE does not significantly alter TfR
distribution. TfR distribution was measured in 7.6 × 105 HFE (light gray) or 6.1 × 105 HFE+ (dark gray) fWTHFE/tTA HeLa cells. The
percentage of receptors on the cell surface or in internal compartments
did not differ significantly between the two cell types. Both express
approximately 20% of TfR on the cell surface and 80% in intracellular
vesicles. These results are representative of three experiments
performed with quadruplicate data points without significant
variation between experiments.
and HFE+
fWTHFE/tTA HeLa cells were incubated in the presence of 100 nM 55Fe-loaded Tf for up to 240 min, and the
rate of 55Fe uptake from Tf for HFE
and HFE+ cells was
determined. Control cells took up 55Fe from Tf at a rate of
0.291 pmol of Fe/106 cells/min, whereas cells expressing
fHFE took up 55Fe from Tf at a rate of 0.193 pmol of
Fe/106 cells/min (Fig. 4).
These data show that HFE-expressing cells take up approximately 33%
less iron from Tf than control cells that do not express fHFE.
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Fig. 4.
fHFE significantly reduces iron uptake from
Tf. 55Fe uptake from Tf was measured in 6.8 × 105 HFE (
, dashed line) or 5.6 × 105 HFE+ (
, solid line) fWTHFE/tTA HeLa
cells. The rate of 55Fe uptake/min/106 HFE
and HFE+ cells from Tf was 0.291 (r2 = 0.939)
and 0.193 (r2 = 0.923), respectively; a 33%
reduction specific to HFE expression. Linear regression was determined
in the Cricket Graph program. These results are representative of three
experiments performed with quadruplicate data points without
significant variation between experiments.
and HFE+ fWTHFE/tTA HeLa
cells were incubated in the presence of 200 nM 55Fe-NTA for up to 30 min. No significant difference was
seen in iron uptake between HFE
and HFE+ cells (Fig.
5). Combined, these results support the
conclusion that the regulation of iron uptake by HFE is specific to the
Tf-mediated iron uptake pathway. The effect of HFE on Tf-mediated iron
uptake is therefore a direct effect on the Tf-derived iron pathway
rather than an alteration of all cellular iron metabolism.
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Fig. 5.
fHFE does not significantly alter
non-Tf-mediated iron uptake. 55Fe uptake from
55Fe-NTA was measured in 1.5 × 106 HFE
(
, dashed line) or 1.8 × 106 HFE+ (
,
solid line) fWTHFE/tTA HeLa cells. The rates of
55Fe uptake in pmol/million cells/min for HFE
and HFE+
were 0.275 (r2 = 0.964) and 0.255 (r2 = 0.975), respectively. Linear regression
was determined in the Cricket Graph program. These results are
representative of three experiments performed with quadruplicate
data points without significant variation between experiments.
and HFE+ fWTHFE/tTA HeLa cells to
iron loading or iron chelation was observed by detection of TfR and Ft
levels on Western blots. These experiments were performed to test
whether HFE affects the ability of the cells to compensate for changing
iron levels in the medium as predicted by the known influences of iron
regulatory proteins (IRPs) on Ft and TfR expression. IRPs are the most
extensively characterized mode of regulating intracellular iron
concentrations. In the presence of low intracellular iron, IRPs bind to
iron responsive elements in the mRNA of iron-regulated proteins.
This interaction either stabilizes the mRNA by binding to the
3'-end of the transcript (as is the case for TfR mRNA), or blocks
translation by binding to the 5'-end of the transcript, close to the
site of translation initiation (as is the case for Ft mRNA). Thus,
at low intracellular iron levels, the cell increases iron uptake by
increasing TfR levels. It decreases iron storage by lowering Ft levels.
When intracellular iron concentrations are high enough, the IRP no
longer binds the iron responsive element. This acts to reduce iron
uptake from Tf by reducing TfR levels and increasing Ft levels.
and HFE+ fWTHFE/tTA HeLa cells grown for 2 days in untreated
growth medium and treated for at least 12 h with growth medium
supplemented with 50 µM desferoxamine to chelate iron,
human Tf, or Fe-NTA. Under control conditions, cells expressing fHFE
had no detectable Ft over background, whereas those that do not express
HFE showed significant amounts of Ft (Fig.
6). HFE
and HFE+ cells showed increased
Ft expression following treatment with 100 nM human Tf.
HFE
cells increased Ft levels only about 2-fold upon inspection of
the Western blot. HFE+ cells had slightly less Ft than HFE
cells for
the same treatment condition, but this was a significant increase over
the undetectable Ft levels in HFE+ cells without Tf treatment. In the
same way, both HFE
and HFE+ cells showed increased Ft expression
following treatment with 100 µM Fe-NTA.
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Fig. 6.
Ft levels are responsive to iron treatment in
fWTHFE/tTA HeLa cells. Lysates of 2 × 105 HFE
or HFE+ fWTHFE/tTA HeLa cells were run on a 12% denaturing
polyacrylamide gel under reducing conditions. Proteins were transferred
to nitrocellulose and detected with sheep anti-human transferrin
receptor (1:10,000) and sheep anti-ferritin (1:100) antibodies and
swine anti-goat horseradish peroxidase-conjugated secondary antibody
(1:10,000). ~94- and 19/21-kDa bands (by chemiluminescent detection)
represent TfR and Ft, respectively. The increased TfR expression and
decreased Ft expression in fHFE-expressing cells is consistent with
previously reported results. Both cell types increased Ft levels in the
presence of an iron source and increased TfR levels in the presence of
the iron chelator desferoxamine. No Ft was detectable in HFE+ cells in
the presence of 50 µM desferoxamine. These results are
representative of four experiments without significant variation
between experiments.
cells in keeping with their iron-depleted
status. Both HFE
and HFE+ cells treated with desferoxamine showed
increased TfR levels above that in untreated cells. For both HFE
and
HFE+ cells, TfR levels slightly decreased upon treatment with human Tf
or Fe-NTA as compared with untreated cells. These results demonstrate
that the outcome of the iron-responsive mechanisms used by the cell are
not perturbed by HFE expression. Rather, HFE imposes an additional but
separate homeostatic mechanism.
and HFE+ cells than without Tf
treatment (Fig. 7). However, due to the
difference in the Tf binding affinity and the reduced iron uptake from
Tf, HFE+ cells expressed consistently smaller amounts of Ft than HFE
cells under the same Tf concentrations below 50 nM. Ft
levels were also increased in HFE
and HFE+ cells when treated with
Fe-NTA at concentrations as low as 50 nM. Ft levels for
HFE+ cells remained lower than HFE
cells due to their initially
depressed intracellular iron levels. The observation that the cells
responded to Tf-derived iron at much lower concentrations than
NTA-derived iron is consistent with the association constants and the
kinetics of the protein components of those systems. Detection limits
of the enhanced chemiluminescence and x-ray film prevent quantitative
conclusions from these data; however, the qualitative results provide
strong evidence that HFE does not prevent the IRP-dependent
regulation of Ft and TfR expression. Rather, HFE alters the "set
point" of intracellular iron levels in a Tf-mediated manner.
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Fig. 7.
Sensitivity of fWTHFE/tTA HeLa cells to iron
treatment. Lysates of 4 × 105 HFE or HFE+
fWTHFE/tTA HeLa cells treated for 12 h with the indicated amounts
of Tf (A) or Fe-NTA (B) were run on a 12%
denaturing polyacrylamide gel under reducing conditions. Proteins were
transferred to nitrocellulose and detected with sheep anti-ferritin
(1:100) antibodies and swine anti-goat horseradish
peroxidase-conjugated secondary antibody (1:10,000). Sensitivity to Tf-
and NTA-derived iron sources is within the concentration range
consistent with the association constants of the TfR and iron transport
proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and HFE+ fWTHFE/tTA HeLa
cells was detected. These results were confirmed by measuring the
steady state distribution of TfRs. No redistribution of TfRs was
detected between the cell surface and internal compartments when fHFE
is expressed at steady state.
cells,
suggesting that HFE acts specifically through the Tf/TfR-mediated
pathway of iron uptake.
cells have more Ft than HFE+
cells. Cells that do not express fHFE are capable of depleting Ft
levels under low iron conditions, such as in the presence of the iron
chelator desferoxamine. Both HFE
and HFE+ fWTHFE/tTA HeLa cells
responded to iron loading via the Tf-mediated and non-Tf-mediated
pathways by increasing intracellular Ft levels and decreasing TfR
levels. These results emphasize that cells expressing HFE still
regulate Ft levels under high and low iron conditions, presumably
through the translational regulation of Ft by the IRPs. HFE has simply
changed the total amount of iron that is stored within the cell.
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Fig. 8.
Model of HFE action on cellular iron
homeostasis. Diferric Tf is present at a concentration of
approximately 5 µM in the blood (43) and is therefore
capable of saturating all TfRs, even in the presence of HFE
(arrow 1A). HFE traffics with Tf and TfR to the endosome
(arrow 2A). Once the endosome has acidified, iron is
released from transferrin and transported through an endosomal iron
transporter, such as Nramp2/DCT1 or SFT (arrow 3A). In the
presence of HFE, TfR does not potentiate the full release of iron from
Tf, leaving some iron bound to Tf. Tf then cycles out of the endosome
and back to the cell surface, resulting in release of ferric rather
than apo-Tf (arrow 4A). TfR that is not associated with HFE
(as is the case in HH patients) also binds diferric Tf preferentially
at the cell surface (arrow 1B). TfR internalization is
identical to that of the TfR/HFE complex (arrow 2B). Once
the endosome has acidified, TfR potentiates the release of iron from
Tf, which is transported across the endosomal membrane (arrow
3B). More iron is released from Tf that is bound to TfR alone than
is released from Tf bound to the HFE TfR complex. Tf then cycles out
of the endosome and back to the cell surface, where apo-Tf is released
(arrow 4B).
We have shown previously that HFE co-traffics with TfR from the time of
its initial synthesis to its cycling through Tf-positive endosomes
(15). HFE may traffic with TfR from the basolateral plasma membrane to
endocytic vesicles of the enterocytes. This would be consistent with
the perinuclear immunohistochemical staining of HFE (14) and TfR
localization (42) in the cells of the intestinal crypts. HFE in the
enterocyte endosome, where iron derived from apical dietary uptake and
iron derived from bodily Tf stores meet, may play a key role in the
regulation of iron transport out of the enterocyte into the blood. The
mechanisms for such events remain unresolved.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 40608.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.
§ Supported by the Training Program in Molecular Hematology, T32-HL00781, NHLBI, National Institutes of Health.
To whom correspondence should be addressed: Dept. of Cell and
Developmental Biology, L215, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-5845; Fax: 503-494-4253; E-mail: ennsca{at}ohsu.edu.
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ABBREVIATIONS |
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The abbreviations used are: Tf, transferrin; TfR, Tf receptor; fHFE, FLAG epitope-tagged HFE; Ft, ferritin; IRP, iron regulatory protein; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; tTA, tetracycline transactivatable; DMEM, Dulbecco's modified Eagle's medium.
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