From the Department of Cell Biology, The Lerner
Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the § Department of Biological, Geological and
Environmental Sciences, Cleveland State University,
Cleveland, Ohio 44115
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ABSTRACT |
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The balance required to maintain appropriate
cellular and tissue iron levels has led to the evolution of multiple
mechanisms to precisely regulate iron uptake from transferrin and low
molecular weight iron chelates. A role for ceruloplasmin (Cp) in
vertebrate iron metabolism is suggested by its potent ferroxidase
activity catalyzing conversion of Fe2+ to
Fe3+, by identification of yeast copper oxidases homologous
to Cp that facilitate high affinity iron uptake, and by studies of
"aceruloplasminemic" patients who have extensive iron deposits in
multiple tissues. We have recently shown that Cp increases iron uptake
by cultured HepG2 cells. In this report, we investigated the mechanism
by which Cp stimulates cellular iron uptake. Cp stimulated the rate of
non-transferrin 55Fe uptake by iron-deficient K562 cells by
2-3-fold, using a transferrin receptor-independent pathway. Induction
of Cp-stimulated iron uptake by iron deficiency was blocked by
actinomycin D and cycloheximide, consistent with a transcriptionally
induced or regulated transporter. Cp-stimulated iron uptake was
completely blocked by unlabeled Fe3+ and by other trivalent
cations including Al3+, Ga3+, and
Cr3+, but not by divalent cations. These results indicate
that Cp utilizes a trivalent cation-specific transporter. Cp
ferroxidase activity was required for iron uptake as shown by the
ineffectiveness of two ferroxidase-deficient Cp preparations,
copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in
which iron reduction and subsequent re-oxidation by Cp are essential
for an iron uptake pathway with high ion specificity.
Iron is important in many biological processes because it is an
ideal O2 carrier and because it can function as a
protein-bound redox element. Iron deficiency is common worldwide and in
infants can cause severe neurological deficit (1). In contrast, iron in
excess of cellular needs is toxic and dietary overload or hereditary hemochromatosis (HH)1 leads
to tissue iron deposition and injury (2, 3). Alterations of iron pools
have been implicated in diverse human diseases, including
neurodegenerative disease, aging, atherosclerosis, cancer, and
microbial infection (4). The precise balance required to maintain
appropriate intracellular and tissue iron levels has led to the
evolution of several independent cellular iron transport pathways.
The transferrin-mediated iron uptake pathway has been extensively
studied and is an important model for understanding the recycling,
receptor-mediated endocytosis pathway. In this pathway, diferric
transferrin is endocytosed by a cell surface transferrin receptor and
iron is delivered to the cell by an endosomal pathway accompanied by
recycling of transferrin and its receptor (5). The physiological
significance of the transferrin receptor is suggested by its presence
in virtually all cultured cells and by the fact that
hypotransferrinemic animals develop severe hemochromatosis (6). The
central importance of the transferrin pathway is shown by recent
studies of HH, the most common inherited disease in people of northern
European descent. Positional cloning studies of Feder et al.
(7) identified HFE as a candidate gene for HH, and two HFE mutations
account for about 80-85% of all HH. The normal HFE gene product, by
binding to the transferrin receptor and lowering its affinity for
transferrin, down-regulates intestinal iron absorption (8). Mutant HFE
does not bind the transferrin receptor and lacks this suppressor activity.
Transferrin-independent cellular iron uptake has been described in many
cultured cells including HepG2 (9) and K562 (10) cells. The function of
the transferrin-independent system is unknown, but Kaplan et
al. (11) have proposed that it clears potentially toxic, low
molecular weight iron chelates (e.g. non-transferrin bound
iron). Such chelates accumulate to a very large extent in HH patient
plasma (12). Transferrin-independent uptake mechanisms are not well
understood; however, two putative transporters, DCT1 and SFT, have been
recently identified in mammalian cells (13, 14). A cell surface
ferrireductase is also active and may be required for
transferrin-independent iron uptake (15, 16).
Important insights into eukaryotic non-transferrin iron metabolism have
come from recent yeast genetic studies. Two iron transporters (or
"permeases"), Ftr1p from Saccharomyces cerevisiae (17)
and the homologous Fip1p from Schizosaccharomyces pombe
(18), facilitate high affinity iron uptake by iron-deficient yeast. A
surface ferrireductase, Fre1p (or its homologue Fre2p), is also
necessary for yeast iron uptake (19). A requirement for copper in iron
uptake has led to the cloning of genes encoding copper-containing
proteins, Fet3p in S. cerevisiae (20-22) and its homologue
Fio1p in S. pombe (18), that are required for iron uptake.
These proteins are membrane-spanning, multicopper oxidases which
interact at the cell membrane with the transporters (17). Fet3p (and
Fio1p) has limited sequence homology to human ceruloplasmin (Cp), and
like Cp is a copper protein with a ferroxidase activity that catalyzes
conversion of ferrous to ferric ion.
The similarities between Fet3p and Cp suggest a possible role for the
latter in vertebrate cell iron uptake. We have shown recently that Cp
increases 55Fe uptake by iron-deficient HepG2 cells by a
transferrin receptor-independent pathway (23). Furthermore, iron
deprivation transcriptionally induces Cp synthesis by HepG2 cells (23),
and Fet3p synthesis in yeast (20, 21). Thus maintenance of iron
homeostasis in liver (and possibly other tissues) during periods of
iron deficiency may be an important physiological function of Cp. The
recent observation that "aceruloplasminemia" patients with Cp gene
defects have massive iron deposits in liver, pancreas, brain, retina,
and other tissues indicates an important role for Cp in iron
homeostasis in vivo (22, 24, 25).
In this report we investigate the molecular mechanism by which Cp
increases iron uptake by erythroleukemic K562 cells. Our results show
that Cp-stimulated iron uptake requires Cp ferroxidase activity and
utilizes a novel, trivalent cation-specific transport pathway.
Materials--
Purified human Cp was obtained from Calbiochem
(La Jolla, CA). Cp homogeneity was verified by an absorbance ratio (610 nm/280 nm) higher than 0.045, and by SDS-PAGE and Coomassie Blue
staining. Densitometric analysis showed that about 80-85% was present
as the intact, 132-kDa form of Cp. 55FeCl3 (20 mCi/mg), 59FeCl3 (19.22 mCi/mg), and
51CrCl3 (789 mCi/mg) were from NEN Life Science
Products Inc. (Boston, MA). Rabbit anti-human apotransferrin antibody
was from Accurate (Westbury, NY), anti-human transferrin receptor mouse
monoclonal antibody (H68.4) was from Zymed Laboratories
Inc. (South San Francisco, CA), and rabbit anti-human ferritin
IgG was from Boehringer-Mannheim. Nitrocellulose filters were from
Millipore (Bedford, MA). Ammonium tetrathiomolybdate was from
Sigma-Aldrich (Milwaukee, WI), and human apotransferrin, horse spleen
apoferritin, reduced glutathione (GSH), cycloheximide,
nitrilotriacetic acid (NTA), bathophenanthroline disulfonic acid, and
other assay reagents were from Sigma.
Measurement of 55Fe Uptake by K562 Cells--
Human
erythroleukemic K562 cells (ATCC, Rockville, MA) were grown in
iron-free RPMI 1640 medium supplemented with 10% fetal bovine serum.
The cells were made iron-deficient by resuspension in serum-free RPMI
1640 medium containing bathophenanthroline disulfonate (100 µM) for 4-8 h. The cells were washed twice with phosphate-buffered saline and once with RPMI 1640 medium, and then
resuspended in the same medium. A solution of 55Fe-NTA was
prepared by mixing 55FeCl3 (15-20
µM) with a 4-fold molar excess of NTA in colorless RPMI
1640 medium containing 1 mM ascorbate (26). To measure iron
uptake, 5 × 106 K562 cells were incubated with
55Fe-NTA (1.4 µM) in 0.5 ml of RPMI 1640 medium for 20-30 min at 25 °C. An aliquot (0.1 ml) was removed and
applied to a nitrocellulose filter subjected to vacuum. The filter was
washed twice with 3 ml of 150 mM NaCl containing 10 µM EDTA (to remove surface-bound iron) and
cell-associated radioactivity was measured by liquid scintillation
counting. The amount of 55Fe bound after a "zero time"
incubation (less than 15 s) was subtracted from experimental
measurements to correct for nonspecific binding.
Measurement of 59Fe Uptake and Incorporation into K562
Cell Protein--
K562 cells, made iron-sufficient and iron-deficient
as above, were incubated with 59Fe-NTA (1.4 µM) in the presence or absence of Cp (30 µg/ml) for 20 min at 25 °C. The cells were washed two times by centrifugation at
16,000 × g, and then lysed in 20 mM HEPES,
pH 8.0, 2 mM MgCl2, 1% aprotinin, and 200 µg/ml leupeptin by repeated passage through a 27-gauge needle at
0 °C. Unbroken cells, nuclei, and debris were removed by
centrifugation at 2000 × g for 5 min. Membrane fragments were separated from the cytosol-containing fraction by
centrifugation at 16,000 × g for 10 min at 4 °C.
Radioactivity in the intact cells, and membrane and cytosol fractions
was measured by Preparation of Apoceruloplasmin and Reconstitution of
Holoceruloplasmin--
Apoceruloplasmin (apoCp) was prepared by
removal of Cp copper by complexing with cyanide under reducing
conditions as described by Musci et al. (27). Cp was
dialyzed at 4 °C against 100 mM sodium acetate buffer
(pH 5.9) containing 10 mM ascorbate under anaerobic
conditions achieved by continuous bubbling with N2 gas. Dialysis was continued until the "blue" coppers were reduced as shown by complete decolorization of the sample. Potassium cyanide (50 mM) was added and the dialysis continued for 5 h. The
copper-cyanide complex and excess cyanide were removed by dialysis
against 100 mM sodium acetate containing 1 mM
cysteine, and then by an overnight dialysis against buffer containing
50 mM MOPS, 150 mM potassium chloride (pH 7.0).
Inactivation was verified by an "in-gel" ferroxidase assay (see below).
Holoceruloplasmin was reconstituted from apoCp as described by Musci
et al. (27). A Cu1+-glutathione complex was
prepared immediately before use (28) by addition of copper sulfate (0.1 mM) to GSH (0.3 mM) in 0.1 M
phosphate buffer (pH 7.0). A 7-fold molar excess of the
Cu1+-GSH complex was added to apoCp in 50 mM
MOPS, 150 mM potassium chloride, 5 mM magnesium
chloride (pH 7.0) for 4 h. Unbound copper was removed by overnight
dialysis against phosphate-buffered saline.
Inhibition of Cp Ferroxidase Activity by Thiomolybdate--
Cp
(7.61 µM) was incubated for 20 min with ammonium
tetrathiomolybdate (1.52 mM) in acetate buffer (320 mM, pH 6.0) as described by Chidambaram et al.
(29). Excess thiomolybdate was removed by dialysis overnight against
the same buffer, then against phosphate-buffered saline. Removal of
unreacted thiomolybdate was monitored by monitoring optical density at
470 nm.
Measurement of Cp Ferroxidase Activity--
Cp ferroxidase
activity was measured by a modification of the method of Schen and
Rabinovitz (30). In brief, Cp or modified Cp was subjected to
electrophoresis on a 1% agarose gel (Seakem Gold, FMC, Rockland, ME)
under nondenaturing conditions. After transfer to an Immobilon-P
membrane, the blot was incubated with 0.08% ferrous sulfate in acetate
buffer (pH 5.7), for 30 min at 37 °C, followed by incubation with
1% potassium ferrocyanide in 0.1 N HCl. Ferric ion (formed
by Cp-catalyzed oxidation of ferrous ion) was detected by the rapid
formation of a "Prussian blue" band which was stable for at least
24 h.
Characterization of Cp-stimulated Iron Uptake by K562
Cells--
We have previously shown that Cp stimulates high affinity
iron uptake by iron-deficient HepG2 cells (23). For a more detailed analysis of the mechanism of Cp-stimulated iron uptake, we used human
erythroleukemic K562 cells since the transferrin-dependent and -independent uptake pathways are well characterized (10). Unlike
HepG2 cells which secrete Cp in an amount sufficient to promote iron
uptake (23), K562 cells do not secrete Cp (or transferrin, Ref. 31)
thereby permitting more precise control of conditions. Cp stimulated
55Fe-NTA uptake by iron-deficient K562 cells by about
2-3-fold (Fig. 1A).
Half-maximal stimulation was at about 10 µg/ml Cp and maximal uptake
was at about 30 µg/ml, a level much lower than the unevoked human
plasma concentration of 300 µg/ml. The stimulation of iron uptake by
Cp was observed in iron-deficient cells but not in iron-sufficient cells (Fig. 1B), a finding consistent with studies of HepG2
cells (23). Cp stimulated iron uptake by decreasing the apparent
Km of 55Fe-NTA uptake from about 2 µM to about 0.5 µM iron, without changing the maximum uptake rate (Fig. 1B). This result indicated
that the number of active transporters was not altered by Cp, but
rather that the affinity of the transporter for iron was increased or that the amount of substrate was increased by Cp.
Several control experiments were done to show that Cp stimulated iron
entry into the cell, and thus exclude the possibility that Cp increased
extracellular binding of iron to the cell surface (e.g. by
ferroxidase-dependent conversion of soluble
Fe2+ to insoluble Fe3+). First, we measured
iron uptake using a procedure specifically designed to remove complexes
of surface-bound iron (32). According to this method, cells are rinsed
with a solution containing 5 µM sodium dithionite to
reduce Fe3+ to Fe2+ and with 5 µM
bathophenanthroline disulfonate to specifically chelate
Fe2+. This procedure gave results essentially identical to
those obtained with the normal rinse solution containing 10 µM EDTA; Cp increased iron uptake by 2-3-fold in
iron-deficient K562 cells when measured using either the "reducing"
wash or the normal wash (data not shown). To verify iron
internalization, intracellular and membrane-bound iron was measured.
K562 cells were incubated with 59Fe-NTA, the cells were
lysed, and membrane and cytosol fractions isolated by centrifugation.
In this experiment Cp stimulated about a 2-fold increase in
radioactivity measured in intact (and iron-deficient) cells, and nearly
all of this increase was accounted for by 59Fe in the
cytosolic fraction (Fig. 1C); 10% or less of the total cell
radioactivity was bound to membranes under any condition. To verify the
intracellular localization of 59Fe, incorporation into
protein was determined by SDS-PAGE and autoradiography. Radiolabeled
protein was not detected in the membrane fraction of any samples (Fig.
1, D and E). However, the cytosolic fraction
exhibited a single major 59Fe-labeled band that co-migrated
with ferritin (Fig. 1F). Identification of the
59Fe-labeled band as ferritin was confirmed by
immunodepletion with anti-ferritin IgG, but not by a nonspecific
antibody (Fig. 1F). Cp increased incorporation of
59Fe into ferritin in lysates made from iron-deficient
cells by about 90%; equal protein loading of the gel lanes was shown
by Coomassie Blue stain (Fig. 1G). These results demonstrate
that the increase in iron associated with cells in the presence of Cp
is due to cellular uptake and entry into a metabolically active intracellular iron pool.
The finding that Cp did not stimulate iron uptake by iron-sufficient
cells (Fig. 1B) suggested that a factor required for uptake,
e.g. the transporter itself, was induced or activated by
iron deficiency. To test whether this factor must be synthesized de novo, K562 cells were treated with actinomycin D or
cycloheximide prior to iron depletion. Cp-stimulated iron uptake was
completely blocked by both inhibitors suggesting transcriptional
up-regulation of an iron transporter (or a regulatory protein) by iron
deficiency (Fig. 1H). To determine whether Cp stimulates
iron efflux as well as influx, K562 cells were preloaded with
55Fe-NTA, and Cp-stimulated iron release was measured.
Under these conditions, Cp did not enhance iron release (Fig.
1I). The addition of apo-transferrin or NTA to the medium as
Fe3+ acceptors did not alter this result. This result was
consistent with our previous findings in HepG2 cells (23), but not with results of Young et al. (33) who reported a small
Cp-stimulated increase in iron efflux from HepG2 cells. The difference
may be due to experimental conditions, e.g. our methods
measure the effects of Cp on initial rates whereas the 18-h efflux
intervals of Young et al. (33) may reflect equilibrium
processes shifted by Cp.
Transport Mechanism of Cp-stimulated Iron Uptake--
The
conversion of Fe2+ to Fe3+ by Cp ferroxidase
activity is thought to facilitate the binding of iron to apotransferrin
(34). It is thus possible that Cp stimulates cellular iron uptake by stimulating the formation of Fe2-transferrin which delivers
Fe3+ by transferrin receptor-mediated endocytosis. Although
K562 cells are not known to secrete transferrin (31), this pathway was tested by addition of a rabbit polyclonal anti-human transferrin antibody to the incubation medium. The antibody only slightly inhibited
Cp-stimulated iron uptake but completely blocked 55Fe
uptake mediated by transferrin (Fig.
2A). Essentially identical results were obtained using a monoclonal anti-human transferrin receptor antibody (not shown). These results show that Cp-stimulated iron uptake was transferrin receptor-independent, but did not exclude
the possibility that other receptor-mediated endocytic processes were
involved, e.g. endocytosis of the putative Cp receptors (35,
36) or other iron-protein receptors. The lysosomatropic inhibitor,
amantadine, was used to inhibit receptor-mediated endocytosis (37).
Amantadine had essentially no effect on Cp-stimulated 55Fe-NTA uptake, but was a very effective inhibitor of
transferrin-mediated iron uptake (Fig. 2B). Comparable
results were obtained using ammonium chloride (38) to inhibit
receptor-mediated endocytosis (data not shown).
Cation Specificity and Role of Ferroxidase Activity in
Cp-stimulated Iron Uptake--
The cation specificity of Cp-stimulated
cell transport was investigated. Multiple divalent cations, including
Ca2+, Mg2+, Cu2+, or
Zn2+ (and Mn2+ and Co2+, not
shown), added at a 70-fold molar excess did not substantially compete
for Cp-stimulated 55Fe uptake (Fig.
3). In the same experiment
Fe2+ (FeCl3 plus ascorbic acid) in the presence
of Cp completely suppressed uptake. This result suggests that Cp
stimulates a transporter with absolute specificity for Fe2+
or a trivalent cation transporter; the latter is consistent with a
requirement for ferroxidase activity of Cp.
The role of Cp ferroxidase activity was examined using
ferroxidase-defective Cp. We took advantage of the ability of
thiomolybdate to bind copper and irreversibly inhibit Cp ferroxidase
activity (29). Treatment of Cp with ammonium tetrathiomolybdate
completely blocked its iron uptake activity (Fig.
4A). Thiomolybdate did not
inhibit iron uptake by inactivation of the cellular transport pathway
since the addition of untreated Cp to cells incubated with
thiomolybdate-treated Cp fully restored iron uptake. The inactivation
by thiomolybdate treatment was confirmed by an in-gel assay for
ferroxidase activity (Fig. 4B). The integrity of apoCp was
tested by SDS-PAGE analysis and Coomassie Blue staining which revealed
a 132-kDa undegraded protein (Fig. 4C). To verify the role
of Cp ferroxidase activity in iron transport, ferroxidase-defective apoCp was prepared by removal of copper by complexing with cyanide under reducing conditions (27). ApoCp did not stimulate
55Fe influx into iron-deficient K562 cells (Fig.
4D). The absence of apoCp ferroxidase activity was confirmed
by an in-gel assay (Fig. 4E), and the structural integrity
of the protein was shown by SDS-PAGE and Coomassie Blue staining (Fig.
4F). To further demonstrate the integrity of apoCp, it was
reconstituted to holoCp by incubation with copper in the presence of
GSH as a reducing agent. The reconstituted Cp regained about 80% of
its ferroxidase activity and about 70% of its iron uptake enhancing
activity (Fig. 4, D and E). The complete
inactivity of two ferroxidase-defective forms of Cp is persuasive
evidence that ferroxidase activity is necessary for Cp-stimulated iron
uptake, and suggests that the Fe3+ product is transported
into the cell by a trivalent cation transporter. The results also
suggest that Cp increases iron uptake by increasing available substrate
(by formation of Fe3+), not by activation of the
transporter itself.
To test whether the transporter is Fe3+-specific or if it
transports multiple trivalent cations, we examined the ability of Al3+, Cr3+, and Ga3+ to compete for
iron uptake. All three trivalent cations inhibited Cp-stimulated iron
cellular uptake in a concentration-dependent manner,
reaching greater than 90% inhibition at about 10 µM
(Fig. 5A). Since trivalent
cations may inhibit Cp ferroxidase activity (39), we investigated
whether the effects of Al3+, Cr3+, and
Ga3+ on iron uptake were due to inhibition of ferroxidase
activity rather than by competition for a transporter. Cr3+
and Al3+, at a concentration that blocks iron uptake (10 µM), did not inhibit Cp ferroxidase activity, although
some inhibition was found at higher ion concentrations (Fig. 5,
B and C). Ga3+, even at 100 µM, did not inhibit ferroxidase activity (Fig.
5D). Together these data show that trivalent cations do not
inhibit Cp-stimulated iron uptake by inhibition of Cp ferroxidase
activity. More likely, these ions may block iron uptake by competing
with iron for a binding site on a trivalent cation-specific
transporter.
The inhibition of iron transport by multiple trivalent cations
indicates a nonspecific trivalent cation transporter. However, the
competition results do not prove that these competing cations are
actually transported; they may compete with Fe3+ for the
binding site on the transporter without subsequent entry into the cell.
To test this possibility, 51CrCl3 uptake by
K562 cells was measured. 51Cr3+ uptake was high
and exhibited two properties consistent with uptake by the transporter
used for Cp-stimulated iron uptake (Fig. 6). First, 51Cr uptake was
much higher in iron-deficient K562 cells than in iron-sufficient cells.
Second, 51Cr uptake was inhibited by Fe3+ (and
by Cr3+ and Al3+), and by Fe2+ plus
Cp, but not by Fe2+ alone (Fig. 6). Cp did not increase
51Cr uptake, thus providing additional evidence that Cp
does not directly activate the transporter. Together these results show that the transporter mediating Cp-stimulated Fe3+ uptake is
a high affinity trivalent cation transporter.
Our results show that Cp stimulates cellular iron uptake by
ferroxidase-dependent conversion of Fe2+ to
Fe3+ and transport by a trivalent cation-specific
transporter. This uptake mechanism recalls the newly elucidated iron
uptake pathway in S. cerevisiae and S. pombe
shown in Fig. 7A (24).
According to this model the yeast ferrireductase Fre1p/Fre2p initiates
the uptake pathway by one-electron reduction of Fe3+ to
Fe2+. The reduced metal is reoxidized to Fe3+
by Fet3p (or Fio1p), a Cp homologue with ferroxidase activity. Finally,
Fe3+ is transported into the cell by Ftr1p (or Fip1p), a
six-spanning membrane transporter that physically interacts with Fet3p
(Fio1p). These yeast proteins are all transcriptionally up-regulated
during iron deficiency by activation of the Aft1p transcription factor (40). By analogy we propose a model in which Cp is positioned in the
center of an analogous iron transport pathway for mammalian cells (Fig.
7B). In this model extracellular iron is first reduced by a
surface ferrireductase, re-oxidized by Cp, and finally transported into
the cell by a trivalent cation-specific transporter. The evidence for
the mammalian pathway elements, and the similarities and differences
with their yeast counterparts, are addressed below.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-counting. To determine incorporation of
59Fe into individual proteins, aliquots (20 µg of
protein) of membrane and cytosolic fractions were subjected to SDS-PAGE
(6.5%) under nonreducing conditions and autoradiography (Hyperfilm,
Amersham, Buckinghamshire, United Kingdom). To evaluate total protein
in these fractions, the gel was stained with Coomassie Blue. In a control experiment, ferritin in the cytosolic fraction was
immunodepleted by incubation for 18 h with rabbit anti-human
ferritin IgG (1:1000) or with a nonspecific antibody (rabbit anti-human
Gq
IgG; Santa Cruz Biotechnology, Santa Cruz, CA), then
with protein A-Sepharose, and centrifuged at 16,000 × g.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
The effect of Cp on iron uptake in K562
cells. A, 55Fe-NTA (1.4 µM)
was incubated with iron-deficient K562 cells in the presence of
purified human Cp for 20 min at 25 °C, and 55Fe uptake
was measured by a filtration assay. B, iron-deficient
(Fe-def.; circles) or iron-sufficient
(Fe-suff.; triangles) K562 cells were incubated with
55Fe-NTA in the presence (closed symbols) or
absence (open symbols) of Cp (30 µg/ml) for 20 min at
25 °C and 55Fe uptake was measured. C,
iron-sufficient K562 cells were incubated with 59Fe-NTA
(1.4 µM) in the absence (open hatched bars) or
presence (closely hatched bars) of Cp (30 µg/ml) for 20 min at 25 °C. Iron-deficient cells were similarly incubated in the
absence (dark hatched bars) or presence (black
bars) of Cp. The cells were lysed, and the membrane fraction was
separated from the cytosol-containing fraction by centrifugation.
Amounts of 59Fe were determined by -ray scintillation
counting. D, aliquots of membranes (20 µg of protein)
described in C were subjected to SDS-PAGE under non-reducing
conditions and autoradiography. E, Coomassie Blue stain of
the gel in D. F, aliquots of lysates (20 µg of
protein) described in C were subjected to SDS-PAGE under
non-reducing conditions and autoradiography. As a control, ferritin was
removed from the cytosolic fraction by immunoprecipitation
(IP) with anti-ferritin IgG (anti-ferr.; 1:1000)
or nonspecific IgG (1:1000). G, Same as F but the
gel was stained with Coomassie Blue. The position of ferritin was
determined using unlabeled ferritin as a standard (Ferr.
std.; 25 µg) and is indicated by an arrow.
H, K562 cells were treated with medium only (open
hatched bars), 10 µg/ml actinomycin D (black bars),
or 10 µg/ml cycloheximide (closely hatched bars) for
1 h and then made iron-deficient by treatment with
bathophenanthroline. The cells were incubated for 20 min in the
presence or absence of 30 µg/ml Cp and 55Fe uptake was
measured. I, K562 cells were preloaded with
55Fe-NTA (11.4 µM) for 30 min at 25 °C.
Cells were then washed and reincubated for 20 min with apotransferrin
(apoTf, 30 µg/ml), NTA (0.2 µM), or Cp (30 µg/ml) as indicated. Release of 55Fe into the medium was
measured.
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Fig. 2.
Characterization of the Cp-stimulated iron
transport mechanism. A, iron-deficient K562 cells were
pretreated for 30 min with medium only (open hatched bars),
with a polyclonal antibody to human transferrin (1:1000, solid
bars), or with an unrelated polyclonal antibody (1:1000,
closely hatched bars). Cells were then incubated for 20 min
with 55Fe-NTA (1.4 µM) in the presence of
medium only, 30 µg/ml Cp, or 30 µg/ml transferrin (and the same
antibody included during the preincubation), and iron uptake was
measured. B, iron-deficient K562 cells were pretreated for
20 min in the absence (hatched bars) or presence
(solid bars) of 5 mM amantadine. The cells were
then incubated with 55Fe-NTA (1.4 µM) in the
presence of Cp (30 µg/ml) or transferrin (30 µg/ml), and iron
uptake was measured after 20 min.
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Fig. 3.
Effect of divalent cations on Cp-induced iron
uptake. Iron-deficient K562 cells were incubated for 20 min with
55Fe-NTA (1.4 µM) in the presence
(solid bars) or absence (hatched bars) of 30 µg/ml of Cp and with 100 µM chloride salts of
Fe2+ (FeCl3 in the presence of 1 mM
ascorbate), Ca2+, Mg2+, Cu2+, or
Zn2+.
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Fig. 4.
Requirement for ferroxidase activity in
Cp-induced iron uptake. A, Cp was reacted with ammonium
tetrathiomolybdate (CpTM) as described under
"Experimental Procedures." Iron-deficient K562 cells were incubated
with 55Fe-NTA (1.4 µM) in the presence of Cp
(30 µg/ml), CpTM (30 µg/ml), or CpTM (30 µg/ml) plus Cp (30 µg/ml; 1 × Cp) or plus Cp (150 µg/ml;
5 × Cp), or with thiomolybdate alone (2.35 µM).
Iron uptake was measured after 20 min. B, ferroxidase
activity of 5 µg of Cp and CpTM were measured by an
in-gel assay. C, Cp and CpTM (5 µg) were
subjected to SDS-PAGE and Coomassie Blue staining. D, apoCp
was prepared by complexing copper with cyanide as described under
"Experimental Procedures." Cp was reconstituted with copper
(Cpreconst.) by incubation with
Cu1+-GSH. Iron-deficient K562 were incubated with
55Fe-NTA (1.4 µM) in the presence of medium
only, Cp, apoCp, or Cpreconst. for 20 min, and then
55Fe uptake was measured. E, ferroxidase
activity of 5 µg of Cp, apoCp, and Cpreconst. was
measured by an in-gel assay. F, integrity of Cp, apoCp, and
Cpreconst. was examined by SDS-PAGE (4-12% gel) and
Coomassie Blue staining.
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Fig. 5.
Effect of trivalent cations on Cp-stimulated
iron uptake. A, iron-deficient K562 cells were
incubated with 1.4 µM 55Fe-NTA, 1 mM ascorbate, and 0-100 µM FeCl3
( ), AlCl3 (
), CrCl3 (
), or
GaCl3 (
) in the presence of 30 µg/ml Cp. In a negative
control experiment cells were similarly incubated in the absence of Cp
(×). 55Fe uptake was measured after 20 min. B,
ferroxidase activity of Cp (5 µg) incubated with 0-100
µM CrCl3 for 30 min at 37 °C was measured
by in-gel assay. C, same as in B but Cp incubated
with 0-100 µM AlCl3. D, same as
in B but Cp incubated with 100 µM
GaCl3.
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Fig. 6.
Chromium uptake by K562
cells. 51Cr-NTA (0.14 µM) was incubated
with iron-sufficient (open hatched bars) or iron-deficient
(closely hatched bars) K562 cells in the presence or absence
of Cp (30 µg/ml). In other wells, Fe-deficient cells were incubated
with 100 µM chloride salts of Cr3+,
Al3+, Fe3+ (added in the absence of 1 mM ascorbate), or Fe2+ (FeCl3 in
the presence of 1 mM ascorbate) in the presence or absence
of Cp (30 µg/ml). 51Cr uptake was measured after 20 min
by the same method used for 55Fe-NTA uptake except that
51Cr was determined by -ray scintillation
counting.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 7.
Models of transferrin-independent iron uptake
in yeast and mammalian cells. A, iron uptake pathway in S. cerevisiae and S. pombe (adapted from Kaplan and
O'Halloran (24)). B, proposed Cp-stimulated iron uptake
pathway in mammalian cells utilizing a trivalent cation
(Me3+)-specific transporter. Reagents used in cell culture
studies are indicated in parentheses.
The requirement for a surface ferrireductase activity for transferrin-independent iron uptake has been shown for human HeLa (16), HepG2 (9), and K562 cells (15). Our studies have not addressed the role of ferrireductase activity in Cp-stimulated iron uptake; however, the requirement for the reductant ascorbate (not shown) is consistent with an important role of a ferrireductase under physiological conditions (i.e. in the absence of exogenous reductant). The identity of the mammalian ferrireductase is not known, but the phagocyte NADPH oxidase complex may be involved since Fre1p has partial sequence homology to its gp91-phox subunit (19).
Our results show that Cp stimulates 55Fe uptake by iron-deficient K562 cells by up to 2-3-fold. An absolute requirement for ferroxidase activity for Cp-stimulated iron uptake was shown using two ferroxidase-deficient preparations: thiomolybdate-treated Cp and copper-depleted apoCp. This part of the pathway is analogous to the yeast pathway since Fet3p (and Fio1p), a protein with some sequence homology to Cp, has been implicated in high affinity iron uptake (20). Fet3p, like Cp, is a multicopper oxidase with O2-dependent ferroxidase activity (21). A requirement for Fet3p ferroxidase activity in iron uptake has been suggested by the correlation between ferroxidase activity (and Fet3p expression) and iron uptake (20, 22). A recent study has shown directly, by site-directed mutagenesis, that the ferroxidase activity of Fet3p is required for high affinity iron uptake by yeast (41). A similarity between the yeast and mammalian systems is found in the transcriptional up-regulation by iron deficiency of Fet3p in S. cerevisiae (20) and Cp in HepG2 cells (23). K562 cells do not produce Cp, indicating tissue-specific expression (not shown). A species-specific difference is seen in the cellular localization of the ferroxidase. In yeast, Fet3p is membrane-bound and physically interacting with a membrane transporter (21). In contrast, mammalian Cp has a signal sequence and is efficiently secreted. This difference may reflect the paracrine nature of iron homeostasis in mammalian systems that is absent in yeast. The spatial relationship between Cp and the trivalent cation transporter is not known. One possibility is that Cp transiently associates with the transporter to increase the efficiency of Fe3+ delivery. The identification of putative Cp-binding proteins on the surface of multiple cell types (36), which are up-regulated by iron deficiency (42), is consistent with this mechanism. Alternatively, Fe3+ provided by Cp may be delivered to the transporter by diffusion.
The ion specificity of the mammalian non-transferrin iron uptake pathway(s) is uncertain, due in part to differences between the cells and uptake assays used in various laboratories. Our experiments show that the Cp-stimulated iron transporter in K562 cells is trivalent cation-specific based on the lack of inhibition by divalent cations, the inhibition by trivalent cations, the transport of 51Cr, and the requirement for ferroxidase activity in iron transport. Others have suggested that Fe2+ is the principle transport form based on the requirement for reducing agents and inhibition by Fe2+-specific chelators (9). However, in light of recent studies showing the participation of both reduced and oxidized iron in the transport pathway, these approaches do not give specific information on the actual ion transported. Further evidence for Fe2+ transport has been provided by studies showing that non-transferrin iron uptake is blocked by divalent cations (43). However, studies in K562 cells show an inability of divalent cations to compete, a finding in agreement with our own (10). Two previous reports suggest that Ga3+ and other trivalent cations regulate iron uptake (44, 45), but there is no direct evidence for iron uptake via a trivalent cation-specific pathway. To our knowledge the ion specificity of the yeast iron transporters Ftr1p and Fio1p have not been reported.
The identity of the transporter facilitating Cp-stimulated iron uptake is not known. Identification of the specific genes encoding the two yeast transporters has not been helpful since homologous mammalian cDNA's have not been identified. Several mammalian iron transporters have recently been discovered and are pathway candidates. One such iron transporter is DCT1, a member of the Nramp gene family (14). However, divalent cations are efficiently transported by DCT1 suggesting it is not linked to Cp-stimulated transport. A possible role for SFT, an iron transporter cloned from phorbol ester-treated K562 cells, is supported by the fact that the SFT-mediated iron transport is not inhibited by most divalent cations. However, its localization in recycling endosomes, and the stimulated uptake of both transferrin-bound iron and non-transferrin iron uptake, indicate a probable role in intracellular iron transport (13).
The presence of iron reduction and oxidation steps in a single pathway is surprising. In fact, early models of the yeast uptake pathway described an extracellular ferrireductase and an intracellular ferroxidase, thus avoiding a "futile" cycle (20, 46). This model has since been modified by biochemical studies, and the ferroxidase domain of Fet3p, like Cp, is extracellular (21). The function of the ferrireductase is most likely to release soluble Fe2+ from Fe3+ complexes (Fe3+-NTA in our experiments, possibly non-transferrin bound iron in vivo). The function of the ferroxidase is clearly to convert the solubilized Fe2+ to the Fe3+ form that is recognized by the downstream trivalent cation transporter. The advantage of a two-step pathway of iron transport rather than a simpler one-step pathway of direct Fe2+ transport via a divalent cation-specific transporter is uncertain. One possibility is that the two-step pathway gives higher cation specificity. For example, a trivalent cation-specific transporter could greatly improve specificity because Fe3+ will compete successfully with the relatively rare trivalent cations found in tissues. In the case of a divalent cation transporter, Fe2+ would have a much more difficult task competing with abundant ions such as Mg2+, Zn2+, etc. According to this proposed mechanism, the ferrireductase activity improves iron solubility and the ferroxidase activity improves transport specificity.
The precise role of Cp in iron homeostasis in vivo is poorly
understood. The data presented here and elsewhere suggest that alterations in plasma Cp concentration or activity may alter iron homeostasis, and could contribute to human disease. The finding of
hemochromatosis in aceruloplasminemia patients is generally considered
as evidence that the normal function of Cp is to mediate iron efflux
from cells and tissues (47, 48). However, these studies may also be
consistent with a role of Cp in cellular iron uptake. For example, iron
taken into cells by the Cp-mediated pathway might reduce subsequent
iron uptake more efficiently than iron taken in by other pathways. In
this case, Cp may act in effect as a negative regulator of iron uptake,
and iron-loading would occur in its absence. Alternatively, the defect
in Cp-mediated iron influx may lead to extracellular accumulation of an
iron form to a concentration that permits nonspecific, low affinity uptake by cells, thus explaining the observed tissue overload. A more
detailed understanding of the function of Cp in cellular iron metabolic
pathways will put us in a better position to answer questions on the
role of Cp in iron homeostasis in physiological and pathological states.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL29582 and HL52692 (to P. L. F.) and a Fellowship of the American Heart Association of Northeast Ohio (to C. K. M.).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.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-8053; Fax: 216-444-9404; E-mail: foxp{at}cesmtp.ccf.org.
The abbreviations used are: HH, hereditary hemochromatosis; Cp, ceruloplasmin; GSH, reduced glutathione; Nramp, natural resistance-associated macrophage protein; NTA, nitrilotriacetic acid; PAGE, polyacrylamdie gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid.
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