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Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2128-2134
The Cellular Labile Iron Pool and Intracellular Ferritin in K562 Cells
By
Abraham M. Konijn,
Hava Glickstein,
Boris Vaisman,
Esther G. Meyron-Holtz,
Itzchak N. Slotki, and
Z. Ioav Cabantchik
From the Department of Human Nutrition and Metabolism, Faculty of
Medicine, and Department of Biological Chemistry, Institute of Life
Sciences, Hebrew University of Jerusalem and Nephrology Unit, Shaare
Zedek Medical Center, Jerusalem, Israel.
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ABSTRACT |
The labile iron pool (LIP) harbors the metabolically active and
regulatory forms of cellular iron. We assessed the role of intracellular ferritin in the maintenance of intracellular LIP levels.
Treating K562 cells with the permeant chelator isonicotinoyl salicylaldehyde hydrazone reduced the LIP from 0.8 to 0.2 µmol/L, as
monitored by the metalo-sensing probe calcein. When cells were reincubated in serum-free and chelator-free medium, the LIP partially recovered in a complex pattern. The first component of the LIP to
reappear was relatively small and occurred within 1 hour, whereas the
second was larger and relatively slow to occur, paralleling the decline
in intracellular ferritin level (t1/2= 8 hours). Protease inhibitors such as leupeptin suppressed both the changes in ferritin levels and cellular LIP recovery after chelation. The changes in the
LIP were also inversely reflected in the activity of iron regulatory
protein (IRP). The 2 ferritin subunits, H and L, behaved qualitatively
similarly in response to long-term treatments with the iron chelator
deferoxamine, although L-ferritin declined more rapidly, resulting in a
4-fold higher H/L-ferritin ratio. The decline in L-ferritin, but not
H-ferritin, was partially attenuated by the lysosomotrophic agent,
chloroquine; on the other hand, antiproteases inhibited the degradation
of both subunits to the same extent. These findings indicate that,
after acute LIP depletion with fast-acting chelators, iron can be
mobilized into the LIP from intracellular sources. The underlying
mechanisms can be kinetically analyzed into components associated with
fast release from accessible cellular sources and slow release from
cytosolic ferritin via proteolysis. Because these iron forms are known
to be redox-active, our studies are important for understanding the
biological effects of cellular iron chelation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MAMMALIAN CELLS maintain steady levels of
metabolically active iron through the regulation of iron uptake and
storage.1-9 The metabolically active forms of intracellular
iron are components of a cytosolic labile iron pool (LIP), classically
referred to as the chelatable iron pool.10 This pool
harbors forms of iron loosely associated with macromolecular complexes
and, possibly, also harbors low molecular weight
forms.10-12 The levels of these LIP forms are thought to be
both sensed and homeostatically controlled by iron responsive proteins
(IRPs) that, by cell iron sensing, coordinately regulate the expression
of the transferrin receptor (TfR) and of ferritin.1-9 The
accepted view is that increased cellular iron levels appear initially
in the LIP, but that excess iron is eventually safely sequestered into
ferritin molecules. The ferritins are macromolecules that display a
robust iron-binding capacity (up to 4,500 iron atoms per polymer
molecule) with properties dictated by the protein subunit composition.
Ferritin is essentially hetero-polymeric, being composed of different
combinations of heavy (H = 21 kD) and light (L = 19 kD) subunits that
are characteristic of tissue or cell type.13 However, in
addition to its iron-scavenging capacity, ferritin seemingly serves
also as a potential source of the metal for the synthesis of heme (and,
possibly, iron-containing enzymes)12,14 and for catalysis
of reactive oxidant species when the metal interacts with pro-oxidant
species.15-17 The presumed mechanism of iron transfer from
ferritin to heme, originally observed in early human erythroid
precursor cells that mature in a liquid culture, was tentatively
localized to an acidic cell compartment and was attributed to ferritin
shell degradation by acid proteases.14 However, it has not
yet been determined whether the process is associated with the normal
turnover of the ferritin protein or with specific signals that respond
to cellular requirements, such as a posttranslational upregulation of
LIP levels via enhanced ferritin degradation. To study the potential
contribution of ferritin as a donor of iron to other molecules, we have
looked at the LIP as possibly the first compartment of iron released
from ferritin. For that purpose, we investigated the temporal and
quantitative relationships between intracellular ferritin levels and
the in situ LIP levels and their impact on IRP activity as well as iron homeostasis in K562 cells.
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MATERIALS AND METHODS |
Cell cultures.
Human K-562 cells were cultured in  -minimal essential medium
(-MEM) containing 7% fetal calf serum (FCS),
L-glutamine, and antibiotics as described previously.18
Cellular LIP was measured with the fluorescent metalosensor calcein-AM
as previously described.19 In brief, for calcein loading, 1 × 107/mL cells were incubated with 250 nmol/L
calcein-AM for 5 minutes at 37°C in -MEM (without bicarbonate),
washed, resuspended at room temperature at a density of 1 × 106/mL in -MEM containing 1% bovine serum
albumin, and used within 1 hour. Before use, the cells were washed and
finally resuspended (1 × 106/mL) in Na-HEPES (20 mmol/L) buffered saline (HBS; 145 mmol/L NaCl, pH 7.2, 37°C).
Fluorescence (488 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
stabilization of the baseline, anticalcein antibodies were added to
quench extracellular probe fluorescence and the amount of intracellular
metal bound to calcein (CA-Fe) was assessed by (1) addition in excess
(100 µmol/L) of the fast permeating chelator isonicotinoyl
salicylaldehyde hydrazone (referred to in figures as ISAH when used for
cellular iron depletion and SIH when used for measuring the LIP; kindly
donated by Dr Prem Ponka, Montreal, Quebec, Canada); (2) superimposing
the calibration curve of standard free acid-calcein added to a cell
suspension; and (3) calculation of LIP (given as [CA  Fe + [Fe]),12,18 using the experimental kd value
of CA-Fe obtained in cells and the number of cells measured in the
experimental setup.
Electromobility RNA gel retardation assays.
Cells were washed twice in ice-cold phosphate-buffered saline (PBS) and
then lysed for 30 minutes on ice with a buffer containing protease and
RNAse inhibitors (lysis buffer: 25 mmol/L Tris-HCl, pH 8, containing
1% Triton X-100 [Pierce, Rehovot, Israel], 40 mmol/L KCL, 10 µg/mL
leupeptin, 1 µmol/L pepstatin, 10 µg/mL chymostatin, 0.023 trypsin
inhibitor units [TIU]/mL aprotinin [all from Boehringer Mannheim,
Mannheim, Germany], 10 µg/mL benzamidine, 3.7 µg/mL N-tosyl-L-phenylalanine chloromethyl ketone [TPCK], 3.7 µg/mL N-tosyl-L-lysine chloromethyl ketone [TLCK], 0.25 mmol/L
phenylmethylsulphonyl fluoride [PMSF] [all from Sigma-Israel,
Rehovot, Israel], and 10 µL/mL ACE-RNAse inhibitor). Cell nuclei and
debris were precipitated by centrifugation at 10,000g for 15 minutes, and the protein content of the supernatant was analyzed by the
BCA protein assay (Pierce, Rockford, IL). Freshly prepared lysates were
incubated with the iron-responsive element (IRE) probe (kindly provided
by Dr Tracey Rouault, National Institutes of Health, Bethesda, MD) that
was transcribed and labeled with 32P as described by Haile
et al.20 For each lane of the polyacrylamide gel, 10 µg
protein of a freshly prepared lysate was incubated for 10 minutes at
room temperature with 15 µL 32P-"IRE Probe"
cocktail (containing 30,000 cpm IRE-Probe, 2 µg t-RNA, 3.3%
Glycerol, and 1 µL ACE-RNAse inhibitor) in the presence or absence of
2%  -mercaptoethanol. The reaction mixture was applied onto an 8%
native polyacrylamide gel and electrophoresis was allowed to proceed
for 2 hours at 180 V. To standardize the 32P-IRE-IRP
migration, recombinant IRP was used (obtained from Dr Tracey Rouault),
and gels were fixed and subsequently dried. The intensity of the label
of the 32P-IRE/IRP complex was determined by phosphorimaging.
Ferritin measurements.
Cells were harvested, washed, and lysed on ice in solubilization buffer
containing 1% Triton X-100, 10 µg/mL aprotinin,10 µg/mL leupeptin,
1 µmol/L pepstatine, 10 µg/mL benzamidine, 3.7 µg/mL TPCK, 3.7 µg/mL TLCK, 0.25 mmol/L PMSF, and 0.02% sodium azide in 10 mmol/L
Tris-HCl, pH 7.4. After heating for 10 minutes at 70°C followed by
immediate cooling on ice, the lysates were centrifuged at
10,000g for 10 minutes and the supernatants collected and
stored at 80°C until use. Ferritin standards either were isolated from human term placenta or from the spleen of a thalassemic patient obtained at surgery, as previously described.21,22 Measurements of cellular ferritin were performed on cell
lysates using a fluorogenic enzyme-linked immunosorbent assay
(ELISA).23 The antiferritin antibodies were raised either
against human spleen (L-subunits) or placental (H+L subunits) ferritin.
H-ferritin levels were considered to be the difference between the
results obtained with ELISAs conducted with anti-total placental
ferritin and those performed with antispleen ferritin antibodies.
H-ferritin levels thus obtained were identical to those found when
measured by a recently developed specific ELISA for H-ferritin using
H-ferritin-specific monoclonal antibodies.24
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RESULTS |
The LIP of K562 cells was assessed by the calcein-based method in
conjunction with the fast permeating chelator isonicotinoyl salicylaldehyde hydrazone (SIH),19 a tridentate,
fast-permeating, and high-affinity chelator of iron and other metals.
We have shown previously that SIH exhaustively extracts
55Fe from the chelatable fraction of
55Fe-labeled cells and from calcein in calcein-loaded
cells.12 Calcein, which contains an EDTA-like binding
moiety, binds to and is quenched by Fe(II) and (III) as well as Cu(II),
Ni(II), and Co(II). However, we believe that calcein, used in
conjunction with SIH, is a reliable and specific indicator of cellular
LIP for 2 reasons: (1) in comparison with Fe, the total available pools
of the above-noted metals are negligible in viable living cells; and
(2) deferoxamine (DFO), which has a high affinity for Fe but a low
affinity for the other metals mentioned above, abrogates the
calcein-detectable LIP.12 The calcein-based method for LIP determination is as follows: fluorescent calcein, formed
intracellularly upon cellular loading with the nonfluorescent precursor
calcein-AM, binds to a fraction of the cellular iron associated with
the LIP, thereby quenching its fluorescence. That fraction of iron that is exposed by the addition of the iron chelator SIH is indicated after
the arrow in Fig 1 (left half). The
increase in the fluorescence signal elicited by SIH reflecting cellular
iron was used for calculating the values of LIP (shown as micromoles of
iron per liter on the right), as described in Materials and Methods.
Anticalcein fluorescence-quenching antibodies were added to the medium
to bind extracellular calcein and thereby to ascertain that the
fluorescence changes elicited by SIH are associated with intracellular
iron.
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| Fig 1.
Short-term recovery of LIP levels in iron-depleted K562
cells. K562 cells were incubated in HBS (2 × 106/mL)
alone or in the presence of 100 µmol/L SIH for 10 minutes at 37°C
to examine acute iron depletion. Cells were washed free of chelator and
incubated for an additional recovery period either in Dulbecco's
modified Eagle's medium (DMEM) alone or DMEM
supplemented with 20 µg/mL leupeptin, 50 µg/mL transferrin, or 10%
FCS. At the end of the incubation period, the cells were washed with
HBS and processed for LIP measurements by loading with calcein-AM (CA),
washed, and resuspended in HBS-medium. Fluorescence (left panels) was
continuously monitored as described in Materials and Methods. When
fluorescence readings were steady, anti-CA antibodies (ab) were added
(to quench extracellular fluorescence), followed by 100 µmol/L SIH,
to attain maximum recoverable fluorescence. Shown are tracings obtained
after recovery periods of 0 to 6 hours (ISAH-0-6 h). The maximum value,
which was equivalent to the concentration of CA-bound iron [CA-Fe],
was used for calculating LIP values (in micromoles per liter). LIP
values are shown in the right panels for the different treatments: (A)
recovery in DMEM alone (recov), (B) recovery in DMEM supplemented with
leupeptin (recov.+leup), or (C) recovery in DMEM supplemented with
transferrin (Recov. + Tf) or FCS (Recov.+ FCS).
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Cell iron dynamics in K562 cells after fast iron depletion and
subsequent recovery.
K562 cells grown in standard culture conditions at exponential phase
show steady-state LIP values of 0.7 to 0.9 µmol/L as reported
earlier.18 After 10 minutes of treatment with SIH, the LIP
is quickly reduced to 0.2 to 0.25 µmol/L (labeled "ISAH-0h" and
"recov. 0 h" in Fig 1A; a representative experiment is shown in
Fig 1 and pooled data are shown in Fig 2).
However, after removal of the chelator and resuspending the cells in an
essentially iron-free medium, LIP gradually recovered over the
following 3 to 6 hours (labeled "ISAH-3h"/"recov. 3h" and
"ISAH-6h"/"recov. 6h," respectively, in Fig 1A). After an
8-hour recovery period, LIP apparently reached a new steady-state value
of 0.54 to 0.6 µmol/L, which is somewhat lower than that of the
control (not shown). The recovery of LIP after the 10 minutes of iron
depletion by the chelator was markedly inhibited by leupeptin (Fig 1B)
and other antiproteases such as chymostatin and pepstatin (not shown).
These results indicate that the process of LIP recovery was apparently
dependent on intracellular proteolysis. When the recovery from
the initial iron depletion was performed in the presence of an
extracellular iron source, either in the form of diferric transferrin
(Tf) or FCS, the LIP was restored more quickly. Thus, when Tf was
added, LIP increased to levels considerably higher than those found in
iron-free medium (Fig 1C). However, with 10% FCS in the cell medium,
the LIP level attained was similar to that observed in stock cultures.
It should be noted that we could not rule out the possibility that
trace amounts of the chelator remained associated with the cell after washing. However, if this were the case, the values of LIP depicted in
Fig 1, as well as the general trend in the changes of LIP with time,
would be somewhat underestimated. Hence, the interpretation of these
findings would be essentially unchanged.
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| Fig 2.
Long-term recovery of LIP levels in K562 cells after
acute iron depletion. Calcein-loaded cells were resuspended in HBS and
the fluorescence was monitored for 10 to 20 minutes. At 0 time, 100 µmol/L SIH was added for 10 minutes (labeled as ISAH); cells were
then washed free of chelator and resuspended in recovery medium lacking
SIH. The cells were incubated either alone (recov: con.) or
containing the indicated additives, as in Fig 1. In some experiments,
cells were incubated with SIH for 30 minutes before washing (ISAH pre:
30' recov: Con; ISAH pre 30' recov: Leup.). At the indicated time
points, intracellular LIP concentration was measured as described.
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The time dependence of LIP recovery after initial depletion with SIH is
shown in more detail in Fig 2, which combines data from Fig 1 with
additional experiments. When cells were not pretreated with SIH and
maintained in an essentially iron-free medium in both the pretreatment
and recovery periods (indicated as none/none), the LIP levels decreased
somewhat (~30% ± 7%) and then remained stable over
the following 18 hours. After a brief (10 minutes) pretreatment with
SIH (indicated as "ISAH pre.10min," Fig 2), LIP was rapidly
reduced from 0.85 ± 0.07 µmol/L to 0.20 ± 0.08 µmol/L (n = 5). Upon removal of SIH, LIP recovered with time in a biphasic mode.
The initial phase was completed within 2 to 10 minutes, with LIP
reaching a value of 0.33 ± 0.07 µmol/L for virtually all of the
cells after a brief pretreatment with the chelator. The second phase of
recovery showed a peak LIP value and time response that were dependent
on the presence of additives in the medium. In serum-free medium, the
second phase of LIP recovery was slow as compared with the first phase
(t1/2 of ~8 hours), reaching about 70% of the original
steady-state value within 18 hours. In the presence of leupeptin
(replenished twice during the 18-hour recovery period), the second
phase of recovery was markedly delayed, but the initial, relatively
small recovery was unaffected. The rate of recovery and the magnitude
of LIP in the second phase were markedly enhanced if either human
diferric-Tf or FCS was present. Tf elicited a typical overshoot in the
LIP level in the first hour and a subsequent relaxation to steady-state
levels, with the latter being significantly higher than those obtained with FCS. We tentatively attributed the initial phase of LIP recovery to the replenishment of LIP by iron released from intracellular macromolecular stores that were apparently unaffected by the relatively short chelator treatment. Indeed, if the chelator treatment was prolonged from 10 to 30 minutes (indicated as ISAH-30', Fig 2), LIP levels were reduced to less than 0.12 ± 0.08 µmol/L and
remained at that level if the medium included leupeptin, which
suppressed only the second phase of LIP recovery. Thus, the second
phase of LIP recovery might be associated primarily with proteolytic events leading to release of iron into the LIP. An additional contribution of iron to be considered is that of a contaminant taken up
from the nominally iron-free medium by a leupeptin-sensitive mechanism.
However, this possibility seem unlikely, because supplementing the
culture medium of a murine erythroid cell line (MEL) with 5 µmol/L
iron(III)-citrate [a concentration much greater than estimated
contaminating levels of iron(III)] did not have a significant effect
on their LIP as measured by the calcein method (data not shown).
The changes in LIP levels after chelator-induced iron depletion and
their susceptibility to antiproteases were compared with changes in IRP
levels (Fig 3, upper panel). Indeed, after
either a 10-minute chelator treatment (ISAH-DMSO) or an 8-hour
treatment with leupeptin (Con-leup.), the cells showed increased IRP
activities, concomitant with the decreases in LIP. Compared with the
control (Con-DMSO), IRP activity increased by 50% and 20% for
chelator- and leupeptin-treated cells, respectively. However, the
active/total IRP ratio did not differ significantly from the control
after incubation with leupeptin alone but increased by 20% after the treatment with the chelator. From Fig 3 it is apparent that part of the
increased IRP activity can be attributed to an increase in total
cellular IRP content. Moreover, when cells were treated with both
chelator for 10 minutes and leupeptin for 8 hours (ISAH-leup.), LIP was
maintained at a relatively low level and IRP activity increased to its
highest level. Thus, an 80% increase in IRP activity was observed
compared with the control and the active/total IRP ratio increased by
44%; concomitantly, there was a 25% increase in total cellular IRP.
We interpret the results to indicate that rapid iron depletion by a
fast-acting chelator results in the depletion of iron in the LIP. The
accompanying change in IRP levels is the result of a LIP-sensitive
process. In this process, leupeptin inhibits the degradation of
intracellular ferritin and the release of iron from ferritin, as we
have shown previously,14 and thereby prevents the
replenishment of the cellular LIP. On the other hand, cells treated
with chelator and then incubated in culture medium containing FCS
rapidly regained their normal LIP levels via membrane-associated transport mechanisms and IRP activity responded accordingly. However, the total cellular IRP level remained elevated.
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| Fig 3.
Correlation between total ferritin (ferritin), IRP, and
LIP levels in K562 cells after acute iron depletion. K562 cells
incubated for 30 minutes without additives (Con) or with 100 µmol/L
SIH (ISAH) were allowed to "recover" for 8 hours at 37°C in
bicarbonate-free DMEM containing the indicated additives: 0.1%
dimethyl sulfoxide (DMSO) final (Con-DMSO, ISAH-DMSO), 20 µg/mL leupeptin (Con-Leup, ISAH-leup), or 10% FCS (ISAH-FCS).
`Untreated' cells (left bar) were grown in full medium without
additives for 8 hours. After the 8-hour period, aliquots of cells were
taken for determination of LIP levels by the calcein method as in Fig 1
(lower panel,  ), ferritin by ELISA (lower panel,  ), and
IRP by electromobility gel retardation assay (upper panel). LIP levels
are given in micromoles per liter, ferritin values are expressed
relative to control (which contained ~130 ng/mg protein), and IRP is
given in terms of densitometry tracings (OD) of gel shift bands
relative to control. All samples in the gel were obtained from
equivalent numbers of cells. Rec-IRP refers to recombinant IRP, which
is used here as standard for identification purposes. The second row of
the upper panel depicts gel shifts performed after treatment with
-mercaptoethanol (ME) representing total (fully activated)
cellular IRP.
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Cellular ferritin levels after LIP depletion.
The ferritin levels of K562 cells in exponentially growing cultures
were 706 ± 157 ng/mg protein for H-subunits and 100 ± 33 ng/mg protein for L-subunits (Table 1).
After a 24-hour incubation with fresh medium, the respective levels of
H-subunits and L-subunits increased to 1,010 ± 174 and 123.5 ± 36 ng/mg protein, respectively. In the presence of 100 µmol/L DFO,
ferritin levels decreased by approximately 60% for H-subunits and 90%
for L-subunits. In contrast, the antiproteases, leupeptin and
chymostatin, induced increases in both H- and L-subunit levels of about
70% and 90%, respectively. The leupeptin-induced increase in total
ferritin is already apparent after 8 hours and closely correlates with
inhibition of LIP recovery by this agent
(Fig 4).
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Table 1.
Effect of DFO, Protease, and Lysosomal Inhibitors on
Intracellular H-Ferritin and L-Ferritin Levels and H/L-Ferritin
Ratios in K562 Cells
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| Fig 4.
Time dependence of LIP recovery and total ferritin levels
in K562 cells after iron chelation. K562 cells were treated for 10 minutes with 100 µmol/L SIH and then incubated in serum-free growth
medium alone () or containing leupeptin 20 µg/mL (added at 0 time
and at 8 hours;  ). At the indicated times, cells were washed and
analyzed for LIP (by the calcein method) and total ferritin levels (by
ELISA) as described in Fig 3. Ferritin levels (upper panel) are given
relative to untreated cells (extreme left bar) and LIP levels (lower
panel) expressed in micromoles per liter.
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The lysosomotropic agent chloroquine had only a minor effect on the
ferritin subunits when added alone (not statistically significant).
However, in combination with DFO, chloroquine partially reversed the
inhibitory effect of the chelator on L-ferritin, but had no apparent
significant effect on H-ferritin levels (Table 1). On the other hand,
DFO and the antiproteases, which individually produced opposing effects
on the 2 ferritin subunits, produced no net effect on subunit levels
when added together. Such countereffects might indicate a possible
common site of action on ferritin levels. However, the lack of a
distinct effect of chloroquine alone would seemingly exclude an acidic
compartment as the putative common site.
As we have shown, both ferritin subunits generally reacted towards the
various agents in qualitatively and quantitatively similar fashions.
However, the susceptibility of L-subunits to DFO alone or in
combination with chloroquine was considerably greater (5- and 3-fold,
respectively) than that of H-subunits. (Alternately, the H-subunits
might be more resistant to degradation than the L-subunits.)
This point is strikingly illustrated in Table 1, depicting H/L ratios
that are markedly increased after DFO treatment either alone or in
combination with chloroquine. It is also clear from Table 1 that
chloroquine partially reversed the suppressive effect of DFO on the H/L
ratio. This was due to a selective effect of chloroquine on L-ferritin,
as described above (Table 1). This might suggest not only different
metabolic fates, but also possibly different mechanisms of degradation
of the 2 ferritin subunits after chelation.
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DISCUSSION |
Despite the various roles attributed to the LIP in cell iron
homeostasis and in possible toxic effects, very few studies have attempted to relate these properties quantitatively with the LIP per
se. Recently, it has become possible to determine the LIP in living
cells with a novel fluorescence-based method.19 This method
was used here for assessing some of the cellular factors involved in
the maintenance of LIP within physiological limits in K562 cells. Those
factors include both the translational control of ferritin and TfR
expression that are IRP-dependent and ferritin degradation that is
IRP-independent.
Measurements of the LIP with a method based on calcein equilibration
with intracellular iron19 showed that the cellular LIP
apparently consists of at least 2 kinetic components. The first, which
is the major contributor to the LIP under physiological conditions (0.6 to 0.8 µmol/L), is evidently in rapid equilibrium with calcein and
easily accessible to chelators. This is seen in Figs 1 and 2, which
show that most of the intracellular calcein-quenchable fluorescence can
be rapidly restored by a short treatment with the highly cell-membrane
permeate chelator isonicotinoyl salicylaldehyde hydrazone (SIH). The
second component is relatively minor (0.1 to 0.2 µmol/L); it appears
after removal of SIH and increases and levels off in less than 1 hour.
This minor LIP fraction is apparently in a relatively slow equilibrium
with calcein and is relatively less accessible to SIH than the major
one (Figs 1A and 2). This kinetically slow component can be eliminated
either by pretreatments of cells with SIH or by increasing the chelator concentration (Glickstein and Cabantchik, unpublished
observations). We can infer from the present studies that
the slow component (which we refer to as "cryptic") includes
forms of iron that are loosely associated with cell constituents but
not easily accessible to relatively large probes such as calcein or the
chelator SIH. An analogous form of iron, which is inaccessible to
chelators such as transferrin, DFO, or deferriprone (L1), has recently
been detected in sera of iron-overloaded patients (some with even
<70% transferrin iron saturation).25 If either human
diferric-Tf or FCS was present in the cell medium, the rate of recovery
and the magnitude of LIP in the second phase were markedly enhanced, with Tf evoking a typical overshoot. In serum-free medium, the second
phase of LIP recovery was relatively slow, thus showing the high
efficiency of iron delivery to the cell by Tf. Another cellular iron
component, which is not in equilibrium with calcein but can contribute
substantially to the LIP, is shown after iron starvation. This
component replenishes approximately 50% to 70% of the original LIP,
and its contribution to the LIP can be markedly reduced by protease
inhibitors such as leupeptin. Thus, the major replenishment of iron in
the LIP after chelation treatment might be associated with degradation
of putative iron-containing proteins such as ferritin. The half-life of
this component was approximately 8 hours, which is considerably shorter
than that reported previously for ferritin in K562 cells.26
However, the lack of an external iron source in a serum-free medium
might explain the shortened half life of the ferritin. Further evidence
that ferritin is a possible source for the LIP iron is inhibition of
both the recovery of the LIP and the decrease in cellular ferritin
levels by the protease inhibitors leupeptin and chymostatin.
It was assumed that in K562 cells the predominant mechanism for release
of iron from ferritin is through the constitutive degradation of the
protein in lysosomes.26 Furthermore, the presumed mechanism
for iron release from ferritin and its transfer to hemoglobin
originally observed in early human erythroid precursor cells that
mature in a liquid culture was tentatively localized to an acidic cell
compartment. The mechanism proposed was ferritin shell degradation by
acid proteases, because ferritin degradation, as well as the transfer
of its iron to heme, was inhibited by chloroquine and by the acid
protease inhibitors chymostastin and leupeptin.14 We showed
here that leupeptin inhibited long-term as well as short-term LIP
recovery after LIP depletion by SIH and that it also inhibited the
decrease in cellular ferritin levels (Figs 3 and 4). This means that,
when there are no extracellular iron resources, ferritin is the
intracellular source for iron and the iron is released by its
proteolytic degradation.
Like all intracellular proteins, the level of ferritin is determined by
the degree of its degradation and biosynthesis. Although ferritin
degradation and the release of its iron is a proteolytic process14,17,26,27 the mechanism by which this process is regulated is still an enigma.
Cytosolic ferritin has been assumed to be degraded in
lysosomes,14,17,26,27 and we have shown that in human
erythroid cells developing in a liquid culture chloroquine inhibited
the release of iron from ferritin and its transfer to
heme.14 We have found here that this is not the case in
K562 cells after chelator treatment, insofar as chloroquine had a
minimal effect on preventing ferritin degradation (Table 1).
The question of whether IRPs control the cell LIP levels and, vice
versa, whether LIP levels dictate IRP activities has been dealt with
extensively.3,4,6-8 However, both concepts have been only
inferred due to experimental difficulties in estimating the
LIP.2 In the current work, IRP activity was inversely
correlated with the LIP in various experimental conditions, although
generally over a long time period (8 hours). This includes treatment of cells with SIH (cell iron depletion) and prevention of iron
replenishment into the LIP by controlling intracellular protein
degradation (Figs 3 and 4). However, in all of these studies, despite
the demonstrable changes in LIP levels that are induced by chelation, there were no detectable changes in IRP activity up to 2 hours after
treatment (data not shown). In fact, no substantial changes in IRP were
observed earlier than 8 hours posttreatment. Therefore, we suggest
that, of the 2 parameters associated with the recovery of cell iron,
the LIP provides a quicker indicator than the IRP of the iron available
to the cell for its metabolic needs at any given point in time.
Part of the increased IRP activity after LIP manipulation can be
attributed to an increase in total cellular IRP content (Fig 3).
Moreover, when cells were treated with a combination of chelator and
leupeptin, LIP was at its lowest level and IRP activity increased by
80% compared with the controls incubated with DMSO alone. The active/total IRP ratio increased by 44%. Thus, 25% of the increased IRP activity could be ascribed to the augmentation in total cellular IRP content. From the data given above, we suggest that part of the IRP
changes can be attributed to IRP 2, the synthesis and oxidation-dependent degradation of which is determined by the cellular
iron availability and is not posttranslationally
activated.28,29
The additional parameter tested, ferritin, also showed a pattern of
behavior complementary to that of LIP and IRP after iron chelation
treatment (Fig 4). Again, leupeptin, by inhibiting ferritin degradation, apparently reduced the recovery of LIP after cell iron
depletion when no extracellular source of iron was available.
In general, inhibition of intracellular proteolysis by protease
inhibitors resulted in parallel increases in both H and L subunits of
ferritin (Table 1), whereas chelator treatment of cells led to
decreases in the cell levels of both subunits. However, although the
suppressive effect of DFO on L-ferritin subunit levels in K562 cells
was relatively greater than that on H-subunits, the resulting H/L ratio
in DFO-treated cells was significantly higher than in untreated cells
(Table 1). These results indicate that the DFO-induced iron depletion
differentially affects L-ferritin levels either by inhibiting
L-ferritin synthesis (via IRP) or by stimulating L-ferritin
degradation. A similar differential behavior was also observed when DFO
was used in combination with chloroquine. Whereas chloroquine alone
hardly affected either H- or L-subunit levels, the combination with DFO
differentially reduced the L-subunit, leading to an increased H/L
ratio. However, the H/L ratio after this combined treatment was still
significantly lower than after DFO alone, due to selective reversal by
chloroquine of the DFO-induced L-ferritin suppression. Because DFO has
weak base properties, it is plausible that chloroquine counteracted DFO
effects on L-subunit levels via a lysosomotropic effect, suggesting different modes of processing of each subunit.
In conclusion, this work shows that, in the absence of an extracellular
iron source, K562 cells replenish their LIP by various mechanisms that
include a fast release of iron from immediate intracellular resources
followed by a slow release from ferritin. The release of iron from
ferritin into the LIP is associated with a proteolytic event, inasmuch
as protease inhibitors prevent it from occuring. LIP levels apparently
dictate the IRP activity, although a considerable gap in time is found
before those levels are sensed and the corrective mechanisms are
eventually set in motion.
| |
FOOTNOTES |
Submitted October 29, 1998; accepted May 18, 1999.
Supported in part by the Israel Research Fund, the EEC Biomed 2, National Institutes of Health Grant No. AI-20342, and the Mirsky
Foundation for Cancer Research.
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 Abraham 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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