|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3205-3211
Regulation of Intracellular Iron Metabolism in Human Erythroid
Precursors by Internalized Extracellular Ferritin
By
E.G. Meyron-Holtz,
B. Vaisman,
Z.I. Cabantchik,
E. Fibach,
T.A. Rouault,
C. Hershko, and
A.M. Konijn
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.
 |
ABSTRACT |
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.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
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  -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).9,10 Low cellular iron levels activate IRP-1 by
removing an attached iron-sulfur cluster10 and the
half-life of IRP-2 is prolonged under these conditions,11
causing them to bind to the ferritin-IRE and to inhibit translation.
When cellular iron levels rise, IRP-1 is inactivated and IRP-2 is
degraded following oxidation and ubiquitination.12,13 The
absence of IRP binding to IRE allows continued apoferritin translation.
It is suggested that the regulation of intracellular iron metabolism by
the above mechanisms is through cytosolic LIP. The level of the LIP is
thus both sensed and homeostatically controlled by IRPs.7
Iron newly acquired by the cell emerges initially in the LIP and excess
iron is subsequently sequestered within the ferritin molecule.
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.
| |
MATERIALS AND METHODS |
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 ( -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 -MEM, 30% FCS, 1% deionized bovine
serum albumin (BSA), 10 5 mol/L  -mercaptoethanol,
15 mmol/L glutamine, 106 mol/L dexamethasone, and 1 U/mL human recombinant erythropoietin (Ortho Pharmaceutical, Raritan,
NJ). This part of the culture is referred to as phase II. After 5 or 6 days of incubation, erythroblasts were purified by centrifugation on
45% Percol (density, 1.0585 g/mL; Pharmacia, Uppsala, Sweden). The
upper layer, which contained mainly proerythroblasts and basophilic
normoblasts, was collected, washed, and resuspended in the original
medium for future incubation. Cell samples were analyzed between the
sixth and twelfth days of phase II. Viability of the cells was
determined by trypan blue exclusion and was higher than 95%. The
majority of erythroid cells were basophilic normoblasts on day 6 of
phase II, polychromatophilic normoblasts on days 8 to 10, and
orthochromatic normoblasts on day 12.
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  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.
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).
Freshly prepared lysates containing 10 µg protein were incubated for
10 minutes at room temperature with 15 µL of an
32P-labeled IRE Probe cocktail (containing 30,000 cpm/lane
IRE Probe 24, 2 µg/lane tRNA, 3.3% glycerol, 1 µL/lane ACE-RNAse
inhibitor [5Prime 3Prime Inc, West Chester, PA]). Incubation
was performed for 5 minutes at room temperature without (active IRP) or
with 2% -mercaptoethanol (total IRP). The reaction mixture was
applied to an 8% native polyacrylamide gel and electrophoresis was
allowed to proceed for 2 hours at 180 V. As a marker for the
32P-IRE-IRP migration, recombinant IRP was used. Gels were
fixed by immersing them in a mixture containing 40% methanol and 10% acetic acid in water and subsequently dried. Gels were exposed to
phosphor imaging and/or autoradiography.
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).
| |
RESULTS |
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
59Fe-labeled ferritin ("acid I" placental
isoferritin,20 see Materials and Methods) was used to
follow the fate of ferritin iron internalized by DEC. Cells on day 8 of
phase II were pulse-labeled for 30 minutes with 4 nmol/L
59Fe-ferritin and chased with unlabeled ferritin as
described in Materials and Methods. Incubation was continued for times
as indicated. The distribution of radioiron between ferritin and
hemoglobin in the lysates was followed by SDS-PAGE under nonreducing
conditions, followed by phosphor imaging and scanning in the phosphor
imager. Figure 1 shows that already after
the 30-minute pulse, some radioiron appeared in hemoglobin.
Internalized 59Fe-ferritin released its iron and a decrease
in 59Fe in ferritin was observed concomitant with an
increase of 59Fe in hemoglobin. Thus, in control cells at
time 0 (after a 30-minute pulse and chase), 28% of the radioiron was
in hemoglobin, and, at 48 hours, 60% was in hemoglobin. The effect of
leupeptin or chloroquine was measured by preincubating the cells for 60 minutes with either 20 µg/mL leupeptin or 15 µmol/L chloroquine
followed by labeling with a 30-minute pulse with 4 nmol/L
59Fe-ferritin and chasing with unlabeled ferritin as
described earlier. The inhibitors were present during the entire
experiment; leupeptin was renewed every 12 hours.
View larger version (26K):
[in this window]
[in a new window]
| Fig 1.
Distribution of radioiron between internalized ferritin
and hemoglobin (Hb) following inhibition of protein degradation. DEC on
day 8 of phase II were pulse labeled for 30 minutes with 4 nmol/L
59Fe-ferritin, then washed and chased with 400 nmol/L
unlabeled ferritin. Inhibition of protein degradation was initiated 60 minutes before the 59Fe-ferritin pulse by 20 µg/mL
leupeptin or 15 µmol/L chloroquine, and inhibitors were present
throughout the experiment. Leupeptin, which has a short half-life at
37°C, was added every 12 hours. Equal amounts of protein from cell
lysates were analyzed on SDS-PAGE under nonreducing conditions followed
by phosphor imaging and scanning with the phosphor imager.
59Fe-Hb was expressed as percentage of the label in Hb and
ferritin. [59Fe-Hb/(59Fe-Hb + 59Fe-ferritin)] × 100. Unlabeled Hb was used as a marker
(not shown).
|
|
Compared with controls, considerably less radioiron was observed in
hemoglobin following incubation with either leupeptin or chloroquine.
Thus, only 16% to 19% and 25% to 26% of the radioiron was in
hemoglobin 30 minutes and 48 hours after the chase, respectively. Thus,
inhibition of proteolysis by leupeptin as well as inhibition of
lysosome-like activity by chloroquine caused radioiron to remain in the
internalized ferritin and inhibited its transfer to hemoglobin.
The suppression of radioiron release from internalized ferritin by
leupeptin and by chloroquine implies that ferritin protein degradation
is necessary for its iron release. To clarify this point, cells on day
8 of phase II were treated with leupeptin or chloroquine and
pulse-labeled with 4 nmol/L ferritin whose protein moiety was labeled
with 125I, followed by a chase with unlabeled ferritin, as
described earlier. Incubation was continued for indicated periods. The
cellular content of the 125I-ferritin was followed by
SDS-PAGE under reducing conditions. Following its uptake,
125I-ferritin underwent proteolysis in untreated, control
cells, while in cells treated with leupeptin or chloroquine,
125I-ferritin protein degradation was inhibited (Fig
2). The half-life of the radioiodinated
ferritin-protein was calculated to be 3.5 hours. The radioiron content
of ferritin in the presence or absence of chloroquine correlated with
the decay of the 125I-label of the ferritin protein (Fig
3). Thus, release of ferritin iron is
dependent on proteolysis.
View larger version (52K):
[in this window]
[in a new window]
| Fig 2.
Effect of leupeptin or chloroquine on the degradation of
internalized 125I-ferritin protein. Human DEC were
incubated on day 8 of phase II for 60 minutes with either 20 µg/mL
leupeptin or 15 µmol/L chloroquine. Cells were pulse labeled for 30 minutes with 4 nmol/L 125I-ferritin then washed and chased
with 400 nmol/L unlabeled ferritin. Incubation was continued for 0, 3, 8, and 24 hours (A, lanes 1, 2, 3, and 4, respectively) or for 0, 3, 24, and 48 hours (B, lanes 5, 6, 7, and 8, respectively). Inhibitors
were refreshed after each wash and fresh leupeptin was added every 12 hours. Equal amounts of protein from cell lysates were analyzed on
SDS-PAGE under reducing conditions.
|
|
View larger version (23K):
[in this window]
[in a new window]
| Fig 3.
Effect of chloroquine on radiolabeled (125 I)
ferritin-protein degradation (A) and radioiron loss (B) from
extracellular ferritin taken up by DEC. Incubations were performed for
0, 5, 20, and 44 hours. Other experimental conditions were as described
in Figs 1 and 2. The intensity of the ferritin-label was measured by
scanning with the phosphor imager. Means ± SD of 3 independent
experiments.
|
|
To examine the necessity for iron to be present in ferritin for protein
degradation, DEC were incubated with 125I-apoferritin, in
the presence or absence of 20 µg/mL leupeptin or 15 µmol/L
chloroquine. Following a chase with apoferritin as above, analysis by
SDS-PAGE under reducing conditions showed that apoferritin was degraded
in DEC, with a half-life of approximately 2.5 hours. Leupeptin, as well
as chloroquine, inhibited apoferritin degradation, leupeptin being more
effective than chloroquine at the concentrations used (Fig
4). Hence, iron in ferritin is not essential for degradation of the protein.
View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
Radiolabeled apoferritin protein degradation following
inhibition by leupeptin or chloroquine. Cells were labeled with 4 nmol/L 125I-apoferritin and chased with 400 nmol/L
unlabeled apoferritin. Incubations were performed for 0, 5, 20, and 44 hours. Other experimental conditions were as described in Figs 1 and 2.
The intensity of the apoferritin-label was measured by scanning with
the phosphor imager of SDS-PAGE performed under reducing conditions.
One of 2 experiments with comparable results is shown.
|
|
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).
View larger version (21K):
[in this window]
[in a new window]
| Fig 5.
Effect of extracellular ferritin on the LIP.
Cellular LIP was measured with the fluorescent tracer calcein. DEC
were incubated with 40 nmol/L H-rich holoferritin or apoferritin for 64 hours, beginning at day 6 of phase II or with medium only (control).
Cells were loaded with 250 nmol/L calcein AM for 5 minutes at 37°C.
Fluorescence was measured before and after chelation with 100 µmol/L
SIH. Relative cellular LIP values are demonstrated.
|
|
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 -mercaptoethanol.
View larger version (38K):
[in this window]
[in a new window]
| Fig 6.
Regulation of IRP activity by extracellular ferritin.
Cells were incubated with medium only (control, lane 2), 100 µmol/L ferric ammonium citrate (FAC, lane 3), 40 nmol/L H-rich
holoferritin or apoferritin (lanes 4 and 5, respectively), or with 50 µmol/L DFO (lane 6) for 64 hours, beginning at day 6 of phase II.
Fresh cytoplasm extracts (10 µg protein) were analyzed for IRP
activity by electromobility-RNA gel retardation. In lane 1, a 100-fold
excess of unlabeled IRE was added as a specific competitor to the
labeled IRE. Cell lysates were analyzed without -mercaptoethanol
(2-ME) (top panel) or after incubation with 2% 2-ME for 5 minutes at
room temperature (bottom panel).
|
|
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.
View larger version (39K):
[in this window]
[in a new window]
| Fig 7.
Effect of leupeptin and chloroquine on the influence of
ferritin on IRP activity. DEC were incubated for 64 hours, beginning on
day 6 of phase II, with the following: medium only (lane 1), 40 nmol/L
ferritin (lane 2), 20 µg/mL leupeptin (lane 3), 40 nmol/L ferritin
together with 20 µg/mL leupeptin (lane 4), 15 µmol/L chloroquine
(lane 5), or 40 nmol/L ferritin together with 15 µmol/L chloroquine
(lane 6). Fresh cytoplasmic extracts (10 µg protein) were analyzed
for IRP activity by electromobility-RNA gel retardation. A control
containing 120 ng of recombinant IRP-1 was included (lane 7). Cell
lysates were analyzed without 2-ME (top panel) or after incubation with
2% 2-ME for 5 minutes at room temperature (lower panel).
|
|
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).
View larger version (15K):
[in this window]
[in a new window]
| Fig 8.
Effect of ferritin and apoferritin on LIP levels and IRP
activity in erythroid cells . Cellular LIP was measured with a
fluorescent tracer and cellular IRP activity by electromobility-RNA gel
retardation.
|
|
| |
DISCUSSION |
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
In the present study, we have monitored the course of extracellular
ferritin internalized by DEC. We hypothesized that if extracellular-derived ferritin internalized by the cells behaves like
intracellularly synthesized ferritin, it should have a similar effect
on cellular iron metabolism. To test this hypothesis, we incubated DEC,
at their basophilic normoblast stage, with acid ferritin containing
59Fe or ferritin whose protein was labeled with
125I. Our results showed that short (30-minute) incubation
was enough for internalization of discernable amounts of ferritin into
the cells. 59Fe was observed in hemoglobin immediately
following the pulse (Fig 1, Time 0); with time, 59Fe
decreased in ferritin and increased in hemoglobin. This was associated
with a decrease in 125I in ferritin. Leupeptin, a
reversible inhibitor of trypsin-like and cysteine proteases, and
chloroquine, a weak acidophylic base known to inhibit lysosomal and
siderosomal functions by increasing their pH 17, inhibited the rate of
release of ferritin iron and the decay of ferritin protein. These
results indicate that extracellular ferritin can be taken up by the
cells, and once internalized its iron is transferred into heme by a
process that involves proteolytic degradation in an acid compartment of
the cell. These results showed a pattern previously established for
endogenous, intracellular ferritin.5
When these experiments were repeated with apoferritin, the results
showed that apoferritin too is internalized and proteolytically degraded, with a half-life of 2.5 hours. Thus, in contrast to transferrin uptake, which is specific to iron-loaded transferrin, both
iron-loaded holoferritin and iron-free apoferritin can be taken up and
proteolytically degraded by DEC.
We further studied the effect of internalized ferritin and apoferritin
on cellular iron metabolism by measuring LIP levels and IRP activity.
We found that ferritin increased, whereas apoferritin depressed
cellular LIP. The latter results are in agreement with previous studies
in which mouse erythroleukemic cells that were transfected with and
overexpressed the H-ferritin subunit showed a significant lowering of
cellular LIP26,27 in association with decreased hemoglobin
production.27 IRP activity was measured by an RNA gel
retardation assay. Ferritin was found to decrease, and apoferritin to
increase IRP activity (Figs 6 through 8). These results demonstrated
that ferritin behaves as an iron donor (eg, ferric ammonium citrate).
Leupeptin, a reversible inhibitor of trypsin-like and cysteine
proteases, and chloroquine, a weak, acidophylic base known to inhibit
lysosomal and siderosomal functions by increasing their
pH,17 with or without added ferritin, caused an increase in
IRP activity apparently by inhibiting ferritin proteolysis and, as
such, decreasing LIP level.5,7 Apoferritin behaved in a
manner similar to an iron chelator (eg, deferoxamine).
The mechanism by which extracellular apoferritin may decrease cellular
LIP is an enigma, since it is difficult to conceive how could
apoferritin migrate from the endosome (if, indeed it is taken up into
an endosome) to the cytoplasm. However, apoferritin potentially could
prevent endosomal iron from being released into the cytoplasm.
Our results suggest an inverse relation between cellular LIP and IRP
activity. Moreover, leupeptin or chloroquine, by inhibiting proteolysis, causes an increase in IRP activity, presumably via a
decrease in the LIP levels.5,7 The concept of LIP levels regulating IRP activity and vice versa has been dealt with
extensively,7,28,29 but it has not been easy to
substantiate because of difficulties in measuring LIP.30
Recently, we have implicated such an association for K562 cells using
the calcein fluorescent method for LIP measurements.7 The
present studies, employing a novel fluorescence-based method for the
determination of LIP in living cells,25 strongly favor this concept.
Extracellular acid isoferritin may have a physiologic role in iron
acquisition by erythroid cells as its uptake is regulated by the iron
status of the cell.15 However, it is unlikely that serum
ferritin serves such a role, since serum ferritin is iron poor
(behaving rather like apoferritin31) and is of a basic nature, whereas DEC have a higher affinity for acid
ferritin.14,15 In the local microenvironment of the bone
marrow, erythroid precursors are associated with a central
reticuloendothelial cell in erythropoietic islands. Electron
microscopic studies have long suggested that ferritin may be
transferred from macrophages to erythroid cells.32 Recently, it was found that some mouse embryos lacking transferrin receptors might still produce near-normal amounts of erythrocytes early
in gestation, which suggests ferritin uptake directly from the
erythropoietic milieu or from macrophages.33 Preliminary studies in our laboratory using the 2-phase erythroid culture system
(unpublished data, May 1997) have shown the transfer of radioiron from
macrophage ferritin to erythroid cells and its incorporation into hemoglobin.
Our work shows that extracellular ferritin or apoferritin internalized
by DEC modify intercellular iron metabolism by donating iron to or
depriving iron from the LIP, resulting in modified IRP activity. Iron
is released from ferritin by a proteolytic event, most probably in an
acidic compartment, as indicated by the prevention of iron release and
suppression of IRP activity by added ferritin, following the use of
protease inhibitors and chloroquine. Internalized extracellular
ferritin is apparently metabolized in a manner identical with
endogenous, intracellular ferritin.5,7
| |
FOOTNOTES |
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.
| |
REFERENCES |
1.
Arosio P, Albertini A:
Isoferritins: Structure and immunology, in
Weatherall DJ,
Fiorelli G,
Gorini S
(eds):
Advances in Red Cell Biology. New York, NY, Raven, 1982, p 27
2.
Arosio P, Adelman TG, Drysdale JW:
On ferritin heterogeneity: Further evidence for heteropolymers.
J Biol Chem
253:4451, 1978[Abstract/Free Full Text]
3.
Munro HN, Linder M:
Ferritin: Structure, biosynthesis and role in iron metabolism.
Physiol Rev
58:317, 1978[Free Full Text]
4.
Theil E:
Ferritin: Structure, gene regulation, and cellular function in animals, plants, and microorganisms.
Ann Rev Biochem
56:289, 1987[Medline]
[Order article via Infotrieve]
5.
Vaisman B, Fibach E, Konijn AM:
Utilization of intracellular ferritin iron for hemoglobin synthesis in developing human erythroid precursors.
Blood
90:831, 1997[Abstract/Free Full Text]
6.
Breuer W, Epsztejn S, Cabantchik ZI:
Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron (II).
J Biol Chem
270:24209, 1995[Abstract/Free Full Text]
7.
Konijn AM, Glickstein H, Vaisman B, Meyron-Holtz EG, Slotki IN, Cabantchik ZI:
The cellular labile iron pool (LIP) and intracellular ferritin in K562 cells.
Blood
94:2128, 1999[Abstract/Free Full Text]
8.
Jacobs A:
Low molecular weight intracellular iron transport compounds.
Blood
50:433, 1977[Abstract/Free Full Text]
9.
Leibold EA, Guo B:
Iron-dependent regulation of ferritin and transferrin receptor expression by the iron responsive element binding proteins.
Ann Rev Nutr
12:345, 1992[Medline]
[Order article via Infotrieve]
10.
Klausner RD, Rouault TA, Harford JB:
Regulating the fate of mRNA: The control of cellular iron metabolism.
Cell
72:19, 1993[Medline]
[Order article via Infotrieve]
11.
Samaniego F, Chin J, Iwai K, Rouault TA, Klausner RD:
Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post-translational regulation.
J Biol Chem
269:30904, 1994[Abstract/Free Full Text]
12.
Iwai K, Klausner RD, Rouault TA:
Requirement for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2.
EMBO J
14:5350, 1995[Medline]
[Order article via Infotrieve]
13.
Iwai K, Drake SK, Wehr NB, Weissman AM, LaVaute T, Minato N, Klausner RD, Levine RL, Rouault TA:
Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: Implications for degradation of oxidized proteins.
Proc Natl Acad Sci USA
95:4924, 1998[Abstract/Free Full Text]
14.
Meyron-Holtz EG, Fibach E, Gelvan D, Konijn AM:
Binding and uptake of exogenous iso-ferritins by cultured human erythroid precursors.
Br J Haematol
86:635, 1994[Medline]
[Order article via Infotrieve]
15.
Gelvan D, Fibach E, Meyron-Holtz EG, Konijn AM:
Ferritin uptake by human erythroid precursors is a regulated iron uptake pathway.
Blood
88:3200, 1996[Abstract/Free Full Text]
16.
Moss D, Powell LW, Arosio P, Halliday JW:
Characterization of the ferritin receptors of human T lymphoid (MOLT-4) cells.
J Lab Clin Med
119:273, 1992[Medline]
[Order article via Infotrieve]
17.
Ohkuma S, Poole B:
Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.
Proc Natl Acad Sci USA
75:3327, 1978[Abstract/Free Full Text]
18.
Fibach E, Manor D, Oppenheim A, Rachmilewitz EA:
Proliferation and maturation of human erythroid progenitors in liquid culture.
Blood
73:100, 1989[Abstract/Free Full Text]
19.
Fibach E, Treves A, Rachmilewitz EA:
Growth of normal erythroid progenitors in liquid culture: A comparison with colony growth in semisolid culture.
Int J Cell Cloning
9:57, 1991[Abstract]
20.
Konijn AM, Tal R, Levi R, Matzner Y:
Isolation and fractionation of ferritin from human term placenta-A source for human isoferritins.
Analyt Biochem
144:423, 1985
21.
Konijn AM, Gelvan D, Meyron-Holtz E, Fibach E:
Can ferritin provide iron for hemoglobin synthesis? Response.
Blood
89:2613, 1997[Free Full Text]
22.
Levi S, Luzzago A, Cesareni G, Cozzi A, Fraceschinelli F, Albertini A, Arosio P:
Mechanism of ferritin iron uptake: Activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants.
J Biol Chem
263:18086, 1988[Abstract/Free Full Text]
23.
Treffry A, Gelvan D, Konijn AM, Harrison PM:
Ferritin does not accumulate iron oxidized by caeruloplasmin.
Biochem J
305:21, 1995
24.
Haile DJ, Hentze MW, Rouault TA, Harford JB, Klausner RD:
Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements.
Mol Cell Biol
9:5055, 1989[Abstract/Free Full Text]
25.
Epsztejn S, Kakhlon O, Breuer W, Glickstein H, Cabantchik ZI:
A fluorescence assay for the labile iron pool (LIP) of mammalian cells.
Anal Biochem
248:31, 1997[Medline]
[Order article via Infotrieve]
26.
Picard V, Renaudie F, Porcher C, Hentze MW, Grandchamp B, Beaumont C:
Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution.
Blood
87:2057, 1996[Abstract/Free Full Text]
27.
Picard V, Epsztejn S, Sanambrogio P, Cabantchik ZI, Beaumont C:
Role of ferritin in the control of the labile iron pool in murine erythroleukemia cells.
J Biol Chem
273:15382, 1998[Abstract/Free Full Text]
28.
Mascotti DP, Goessling LS, Rup D, Tach RE:
Effects of the ferritin open reading frame on translational induction by iron.
Prog Nucleic Acid Res Mol Biol
55:121, 1996[Medline]
[Order article via Infotrieve]
29.
Mascotti DP, Rup D, Tach RE:
Regulation of iron metabolism: Translational effects mediated by iron, heme, and cytokines.
Ann Rev Nutr
15:239, 1995[Medline]
[Order article via Infotrieve]
30.
Theil EC:
Iron regulatory elements (IREs): A family of mRNA non-coding sequences.
Biochem J
304:1, 1994
31.
Worwood M, Aherne W, Dawkins S, Jacobs A:
The characteristics of ferritin from human tissues, serum and blood cells.
Clin Sci Mol Med
48:441, 1975[Medline]
[Order article via Infotrieve]
32.
Bessis MC, Breton-Gorius J:
Iron metabolism in the bone marrow as seen by electron microscopy: A critical review.
Blood
19:635, 1962[Abstract/Free Full Text]
33.
Levy JE, Jin O, Fujiwara Y, Kuo F, Andrews NC:
Transferrin receptor is necessary for development of erythrocytes and the nervous system.
Nature Genet
21:396, 1999[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. Iwasaki, E. L. MacKenzie, K. Hailemariam, K. Sakamoto, and Y. Tsuji
Hemin-Mediated Regulation of an Antioxidant-Responsive Element of the Human Ferritin H Gene and Role of Ref-1 during Erythroid Differentiation of K562 Cells.
Mol. Cell. Biol.,
April 1, 2006;
26(7):
2845 - 2856.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. T. Chen, L. Li, D.-H. Chung, C. D.C. Allen, S. V. Torti, F. M. Torti, J. G. Cyster, C.-Y. Chen, F. M. Brodsky, E. C. Niemi, et al.
TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis
J. Exp. Med.,
October 3, 2005;
202(7):
955 - 965.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. A. Wingert, A. Brownlie, J. L. Galloway, K. Dooley, P. Fraenkel, J. L. Axe, A. J. Davidson, B. Barut, L. Noriega, X. Sheng, et al.
The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1
Development,
December 15, 2004;
131(24):
6225 - 6235.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Menier, M. Rabreau, J.-C. Challier, M. Le Discorde, E. D. Carosella, and N. Rouas-Freiss
Erythroblasts secrete the nonclassical HLA-G molecule from primitive to definitive hematopoiesis
Blood,
November 15, 2004;
104(10):
3153 - 3160.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Ned, W. Swat, and N. C. Andrews
Transferrin receptor 1 is differentially required in lymphocyte development
Blood,
November 15, 2003;
102(10):
3711 - 3718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Donovan, A. Brownlie, M. O. Dorschner, Y. Zhou, S. J. Pratt, B. H. Paw, R. B. Phillips, C. Thisse, B. Thisse, and L. I. Zon
The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1)
Blood,
December 15, 2002;
100(13):
4655 - 4659.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION