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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3205-3211
By
From the Departments of Human Nutrition and Metabolism, and
Hematology, Faculty of Medicine; Department of Biological Chemistry,
Institute of Life Sciences, The Hebrew University of Jerusalem;
Department of Medicine, Shaare Zedek Medical Center, Jerusalem, Israel;
and Cell Biology and Metabolism Branch, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda,
MD.
Human erythroid precursors grown in culture possess membrane
receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron
by the cell. Normal human erythroid precursors were grown in a 2-phase
liquid culture that supports the proliferation, differentiation, and
maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with 59Fe- and/or
125I-labeled acid isoferritin and chased. The
125I-labeled ferritin protein was rapidly degraded and only
50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, 59Fe decreased in ferritin and
increased in hemoglobin. Extracellular holoferritin uptake elevated the
cellular labile iron pool (LIP) and reduced iron regulatory protein
(IRP) activity; this was inhibited by leupeptin or chloroquine.
Extracellular apoferritin taken up by the cell functioned as an iron
scavenger: it decreased the level of cellular LIP and increased IRP
activity. We suggest that the iron from extracellular is metabolized in
a similar fashion by developing erythroid cells as is intracellular
ferritin. Following its uptake, extracellular ferritin iron is released
by proteolytic degradation of the protein shell in an acid compartment.
The released iron induces an increase in the cellular LIP and
participates in heme synthesis and in intracellular iron regulatory pathways.
DEVELOPING ERYTHROID CELLS (DEC) take up
substantial amounts of iron, mainly for heme synthesis. Iron in excess
of immediate cellular requirements is stored in ferritin. Ferritin functions as an iron-storage protein and is essential for cellular iron
homeostasis. The amount of iron in cells exceeds its solubility, and it
is necessary to concentrate excess iron and maintain its solubility.
Excess cellular iron is stored in a soluble and nontoxic form in
ferritin. Isoferritins are heteropolymers, consisting of 24 subunits of
two types: H (heavy) and L (light), with a molecular mass of
approximately 21 kD and 19 kD, respectively. The relative ratio of the
two subunits is characteristic of tissue and cell type.1
Isoferritins rich in H-type subunits have a lower isoelectric point
(pI) than isoferritins composed predominantly of L-type subunits (basic
iso-Fts). The ferritin protein shell surrounds a central iron core that
contains up to 4,500 iron atoms.1-4 Recent evidence
suggests that ferritin is not simply an "iron sink," but plays a
dynamic role in cellular iron metabolism. In addition to its
iron-scavenging capacity, ferritin serves also as a potential source of
iron for the synthesis of heme5 and iron-containing
enzymes6 and for replenishing the labile iron pool
(LIP)7 believed to be identical to the chelatable iron pool.8 The presumed mechanism of ferritin iron
mobilization is protein degradation by acid proteases in an acidic
compartment of the cell.5
The critical need for iron in all living cells on the one hand, and its
toxicity on the other is underlined by a subtle coordinated mechanism
of regulation of iron sensing, acquisition, storage, and utilization,
which is particularly important when iron is in great demand, such as
in hemoglobin-synthesizing cells. Transferrin receptor (TfR)
expression, which is the major route of iron uptake, is
postranscriptionally regulated through the stability of its mRNA. The
biosynthesis of ferritin subunits and that of erythroid Iron delivery to mammalian cells in general and to erythroid cells in
particular is largely attributed to diferric transferrin. In a previous
report, we have shown that developing human erythroid cells possess on
their surface, in addition to TfR, receptors that bind specifically and
internalize acid isoferritin.14 This was performed by a
specific, saturable process, distinct from the uptake of iron
associated with albumin. It was highly regulated by the iron status of
the cell and by its degree of maturation. Internalized ferritin-iron is
utilized for heme synthesis and, thus, this process could represent a
physiologic pathway for iron assimilation.15 Ferritin
receptors that internalize ferritin are also found on cells of the
human T-cell line MOLT4.16
In another study, we have shown that intracellularly
synthesized ferritin released its iron by a proteolytic process in a lysosome-like compartment.5 We hypothesized that
extracellular ferritin, once internalized by the cell, is
indistinguishable from intracellular ferritin in its iron release
pathway and its effect on cellular iron metabolism. To test this
hypothesis, we monitored the course of the internalized ferritin-iron
and ferritin-protein in human DEC grown in liquid culture and studied
the effects of leupeptin, a reversible inhibitor of trypsin-like and
cysteine proteases, and chloroquine, a weak, acidophylic base known to inhibit lysosomal and siderosomal function by rising their
pH.17 We found that both extracellular holoferritin and
apoferritin were rapidly internalized by the cells and were degraded by
proteolysis in an acidic cellular compartment. This was followed by
iron transfer from the internalized ferritin into hemoglobin. Treatment
with the above inhibitors prevented ferritin degradation, as well as transfer of its iron. The effect of internalized ferritin on
intracellular iron metabolism was studied by measuring the levels of
LIP and the activity of IRP. The results indicated that internalized
holoferritin increased cellular LIP and decreased IRP, whereas
apoferritin had an opposite effect, which suggests that internalized
holoferritin is an iron donor, while apoferritin behaves like an iron chelator.
The results of the present study show that extracellular ferritin and
apoferritin taken up by developing human erythroid cells grown in
liquid culture modify intracellular iron metabolism. Iron from
extracellular holoferritin is released by a proteolytic mechanism and
represses IRP activity, while apoferritin chelates cellular iron,
decreases cellular LIP, and activates IRP. Extracellular ferritin-iron
once taken up is processed by the cell in the same way as intracellular
ferritin-iron.
Erythroid cell cultures.
The 2-phase liquid culture was used as previously
described.18,19 Briefly, mononuclear cells were isolated
from peripheral blood samples of normal donors by Ficoll-Hypaque
density gradient centrifugation and seeded in alpha-minimal essential
medium ( Ferritin.
Ferritin was isolated from human term placenta and fractionated into
isoferritins as previously described.20 The isoferritin used in the present experiments was the "acid I" fraction, which has the highest H to L subunit ratio of the placental isoferritins and
as such the most acidic pI, designated in this report as
"ferritin."
Preparation of apoferritin.
Apoferritin was prepared as described previously,15,21 by
the reduction and the subsequent chelation of the iron core of the
ferritin. In brief, human acid I isoferritin was dialyzed overnight
against a 500-fold excess (vol/vol) of 0.2 mol/L acetate buffer, pH
5.5, containing 1% thioglycolic acid and 10 mmol/L 2,2'-bipyridyl, at
4°C. It was subsequently dialyzed 4 times against 0.2 mol/L
Na-acetate buffer, pH 5.5, and twice against 10 mmol/L 3-(N-morpholino)
propanesulfonic acid (MOPS) buffer, pH 7.0.
Preparation of 59Fe-labeled ferritin.
Apoferritin was labeled with 59Fe in 0.2 mol/L MOPS buffer,
pH 7.0, with no additional Fe-binding ligands, using a mixture of 5%
59Fe(III), as 59FeCl3 (Dupont-NEN,
Boston, MA) and 95% 56Fe(II) as ferrous sulfate.
Equilibration between Fe(II) and Fe(III) was achieved in 0.1N HCl. The
iron loading was performed essentially as described by Levi et
al.22 MOPS buffer was chosen over 2-[N-Morpholino] ethanesulfonic acid (MES) buffer, since the auto-oxidation rate of iron
in MOPS was slower than in any of several other "Good" buffers
tested, thereby decreasing the tendency for the formation of
non-ferritin-bound ferric-hydroxypolymers. Labeling was performed in
the presence of 100 U/mL catalase to reduce harmful free radical reactions. Aliquots of the 59Fe solution, each providing a
final concentration of 0.1 mmol/L iron, were added to a solution that
contained 0.25 mg/mL apoferritin, to give a total of 800 to 1,000 iron
atoms per ferritin molecule. Ferritin labeling was followed
spectroscopically at 310 nm. Aliquots of Fe(II) were added only after
the previous Fe(II) was completely oxidized. Small amounts of iron
polymers and aggregated ferritin were removed by filtration
before the use of the ferritin preparations.15 Incorporation of the ferritin-associated iron into the ferritin iron
core under the conditions employed has been firmly
established.23
Preparation of 125I-labeled ferritin.
Ferritin was iodinated by solid-phase enzymatic radioiodination with
Enzymobead (Bio-Rad Laboratories, Richmond, CA) according to the
procedure supplied by the manufacturer as described previously.14
125I-ferritin was separated from free iodine on a Bio-Spin 30 column (Bio-Rad).
Ferritin uptake and degradation by erythroid precursor cells.
Cells were washed 3 times with phosphate-buffered saline (PBS) and then
incubated in serum-free phase II medium supplemented with 3% BSA,
named "incubation medium." Cells were incubated for 30 minutes
with 4 nmol/L ferritin labeled with either 59Fe or
125I, or with 4 nmol/L apoferritin labeled with
125I (pulse labeling). The cells were then washed once with
incubation medium and incubated again with incubation medium that
contained 400 nmol/L unlabeled ferritin or apoferritin. Incubation was
continued for indicated times and cell lysates were prepared for
sodium-lauryl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Where indicated, cells were preincubated for 60 minutes, before pulse
labeling, in serum-free medium with 15 µmol/L chloroquine or 20 µg/mL leupeptin. Inhibitors were added again after each change of
medium and fresh leupeptin, which has a short half-life at 37°C,
was added every 12 hours. Cells were harvested at indicated times,
washed 4 times with PBS, and lysed on ice in solubilization buffer
containing 1% Triton X-100 (Pierce, Rockford, IL), 10 µg/mL
aprotinin, 10 µg/mL leupeptin (both from Boehringer Mannheim,
Mannheim, Germany), 10 µg/mL benzamidine, 3.7µg/mL
N-tosyl-L-phenyl-alanine-chloromethyl-ketone (TPCK), 1 µmol/L N-tosyl-L-lysine-chloromethyl-ketone
(TLCK), 0.25 mmol/L phenylmethylsulfonylfluoride (PMSF), and 0.02%
sodium azide (all from Sigma-Israel, Rehovoth, Israel) in 10 mmol/L
Tris-HCl, pH 7.4. The lysates were centrifuged at 10,000g for
15 minutes. The supernates were collected and stored at Electromobility RNA gel retardation assay.24
Cells were washed twice with ice-cold PBS and then lysed by incubation
on ice for 30 minutes with solubilization buffer that contained 25 mmol/L Tris-HCl, pH 8, 40 mmol/L KCl, and the previously specified
protease inhibitors. Cell nuclei were precipitated at 10,000g
for 15 minutes. Protein content of the supernatant was measured
with the "BCA Protein Assay Reagent" (Pierce).
Measurement of the cellular LIP.
Cellular LIP was measured with the fluorescent metal sensor
calcein-aceto-methory (AM) as previously described.25 Cells were incubated without (control) or with either 40 nmol/L H-rich holoferritin or 40 nmol/L apoferritin for 64 hours. Cells were then
incubated for 5 minutes at 37°C in MEM-BSA containing 250 nmol/L
calcein-AM. After calcein loading, the cells were washed 3 times and
resuspended in 20 mmol/L Na-HEPES, 145 mmol/L NaCl, pH 7.2, 37°C.
Fluorescence (488 nm excitation, 517 nm emission) was measured in a
continuous mode using a PTI fluorescence station (PTI, New Brunswick,
NJ) with the cells constantly stirred and kept at 37°C. After
attaining a stable baseline, anticalcein antibodies were added (for
quenching extracellular probe fluorescence) and the amount of
intracellular metal, bound to calcein (CA-Fe), was assessed by addition
of 100 µmol/L of the fast permeating chelator isonicotinoyl-salicyl-aldehyde-hydrazone (SIH, kindly donated by Dr
Prem Ponka, Montreal, Canada).
Iron release from internalized ferritin.
We have previously shown that H-subunit-rich extracellular ferritin is
taken up by human DEC in culture by a receptor-mediated process and its
iron is incorporated into heme.15 Likewise, iron from
intracellular ferritin is released in a lysosome-like compartment
by a proteolytic process.5 Proteolysis in acid compartments could correspondingly be involved in the release of iron from ferritin internalized by DEC. To test this hypothesis, cells were treated with leupeptin, a reversible inhibitor of
trypsin-like and cysteine proteases, or with chloroquine, a weak,
acidophilic base known to inhibit lysosomal and siderosomal function by
raising their pH.17
The effect of extracellular ferritin on the LIP.
The cellular compartment representing the metabolically active form of
iron is thought to be a weakly bound low molecular fraction that
possibly connects between cytosolic ferritin and hemoglobin. This
compartment is referred to as the chelatable iron or LIP.6
If iron is released from internalized extracellular ferritin and is
transferred from there to hemoglobin, it would probably be channeled
through the LIP. Therefore, we measured the LIP in DEC after incubation
with either 40 nmol/L ferritin or 40 nmol/L apoferritin for 64 hours
beginning on day 6 of phase II. Ferritin uptake by DEC led to a 200%
increase in intracellular LIP compared with control cells, whereas
apoferritin caused a decrease in LIP to 50% of controls (Fig
5).
Effect of extracellular ferritin on IRP activity.
If IRP activity is regulated by the magnitude of intracellular
LIP, the addition of extracellular ferritin or apoferritin, modifying
cellular LIP levels (as shown in Fig 5), should affect IRP
activity. Cells were incubated on day 6 of phase II for 64 hours with
either 40 nmol/L ferritin, 40 nmol/L apoferritin, 100 µmol/L ferric
ammonium citrate (FAC) or 50 µmol/L deferoxamine (DFO). The relative
activities of IRP are shown in Fig 6.
Ferritin caused a reduction in IRP activity similar to the decline in
activity observed by incubating the cells for the same time period with FAC. Incubation with apoferritin led to increased IRP activity similar
to that observed after incubation with DFO. Because identical amounts
of protein were applied from cellular lysates, it seems that total IRP
levels were affected as well, as shown by IRP activity in the presence
of
Effect of protease inhibitors on the regulation of IRP activity by
ferritin.
Ferritin downregulates IRP activity (Fig 6), presumably by release of
ferritin iron causing an increase in the cellular LIP. As apoferritin
enhanced IRP activity (Fig 6), inhibiting iron release from ferritin
might be expected to abolish the decrease in IRP activity. Cells,
beginning at day 6 of phase II, were incubated with 40 nmol/L ferritin
for 64 hours and, where indicated, leupeptin or chloroquine was added.
Both leupeptin and chloroquine inhibited ferritin protein degradation
and suppressed iron release from ferritin (Fig 2). Figure
7 shows that, indeed, both leupeptin and
chloroquine prevented the downregulation of IRP activity by ferritin.
Relationship of LIP concentration to IRP activity.
If IRP activity is affected by LIP concentration, LIP concentration and
the IRP activity should be inversely correlated. Indeed, ferritin by
releasing its iron, and apoferritin, presumably by binding iron,
altered the magnitude of LIP and the activity of IRP in an inversely
correlated manner (Fig 8).
Cellular ferritin has been long thought to serve as an iron storage
protein that protects cells against the toxic effects of free iron. We
have previously shown that in DEC, ferritin may also function as a
metabolically active reservoir and donate its iron for heme
synthesis.5 Cellular ferritin in DEC is derived from 2 sources: (1) de novo, intracellularly synthesized ferritin, which is
regulated by the iron status of the cell through the activity of
IRP10-13; and (2) extracellular ferritin, the uptake of
which is mediated through specific surface receptors.14,15 By studying the turnover of the de novo synthesized ferritin in DEC, we
have shown that in order to release its iron to LIP, representing the
metabolically active form of cellular iron, and subsequently to heme,
this ferritin should be proteolytically degraded in an acid compartment
(eg, lysozomes) of the cell.5,7
Submitted April 16, 1999; accepted July 2, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to A.M. Konijn, PhD, Department of Human
Nutrition and Metabolism, The Hebrew University, Faculty of
Medicine, PO Box 12272, Jerusalem 91120, Israel; e-mail:
konijn{at}md2.huji.ac.il.
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This article has been cited by other articles:
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TOP
ABSTRACT
INTRODUCTION
-aminolevulonic acid synthase are also regulated by cellular iron
status. mRNAs for TfR carry at the 3', and for ferritin at the
5' end, specific stem-loop structures, or iron-responsive elements (IRE), which reversibly bind iron-regulatory proteins (IRP-1
and IRP-2).
-MEM) supplemented with 10% fetal calf serum (FCS) (both
from Biological Industries, Beit-Haemek, Israel), 1 µg/mL
cyclosporine A (Sandoz, Basel, Switzerland), and 10% conditioned
medium from the 5637 bladder carcinoma cell line. The cultures were
incubated at 37°C, under an atmosphere of 5% CO2 in
air, with extra humidity. After 7-day incubation in this phase I
culture, the nonadherent cells were harvested, washed, and recultured
in fresh medium composed of 
5 mol/L 
-mercaptoethanol,
15 mmol/L glutamine, 10
80°C
until use. Lysates were analyzed for the whole ferritin molecule by
SDS-PAGE on 6% gels without heating the samples under nonreducing
conditions. For analysis of subunits, samples were heated for 3 minutes
at 96°C under reducing conditions, and analyzed on 10% to 15%
SDS-PAGE gradient gels. Gels were dried and exposed to phosphor-imaging (Fujix Bas 1000 Fuji, Japan) and/or autoradiography. Quantitation of
the radioactivity in the gels was done by scanning with the phosphor imager.
3Prime Inc, West Chester, PA]). Incubation
was performed for 5 minutes at room temperature without (active IRP) or
with 2% 
