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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4321-4332
Impaired Ferritin mRNA Translation in Primary Erythroid
Progenitors: Shift to Iron-Dependent Regulation by the v-ErbA
Oncoprotein
By
Wolfgang Mikulits,
Matthias Schranzhofer,
Anton Bauer,
Helmut Dolznig,
Lioba Lobmayr,
Anthony A. Infante,
Hartmut Beug, and
Ernst
W. Müllner
From the Institute of Molecular Biology and Institute of Molecular
Pathology, Vienna Biocenter, University of Vienna, Vienna, Austria; and
the Department of Molecular Biology and Biochemistry, Wesleyan
University, Middletown, CT.
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ABSTRACT |
In immortalized cells of the erythroid lineage, the iron-regulatory
protein (IRP) has been suggested to coregulate biosynthesis of the iron
storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity,
IRP binds to an iron-responsive element (IRE) located in ferritin and
eALAS mRNA leaders, causing a block of translation. In contrast,
IRP-IRE interaction is reduced under high iron conditions, allowing
efficient translation. We show here that primary chicken erythroblasts
(ebls) proliferating or differentiating in culture use a drastically
different regulation of iron metabolism. Independently of iron
administration, ferritin H (ferH) chain mRNA translation was massively
decreased, whereas eALAS transcripts remained constitutively associated
with polyribosomes, indicating efficient translation. Variations in
iron supply had minor but significant effects on eALAS mRNA polysome
recruitment but failed to modulate IRP-affinity to the ferH-IRE in
vitro. However, leukemic ebls transformed by the
v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an
iron-dependent reduction of IRP mRNA-binding activity, resulting in
mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of
ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the
respective normal cellular homologues (c-ErbA/TR , c-ErbB/EGFR). It
turned out that v-ErbA, a mutated class II nuclear hormone receptor
that arrests erythroid differentiation, caused the change in ferH mRNA
translation. Accordingly, inhibition of v-ErbA function in these
leukemic ebls led to a switch from iron-responsive to iron-independent
ferH expression.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IRON IS AN ESSENTIAL nutrient required by
every proliferating cell for the function of multiple iron-containing
enzymes and hemoproteins.1,2 Because free iron is a
potential source for harmful oxygen radicals, rapid incorporation of
intracellular iron into proteins is crucial to keep the element in a
nontoxic form.3 Excess iron is detoxified by sequestration
into the iron storage protein ferritin, a multimer composed of 24 ferritin heavy (ferH) and light (ferL) subunits.4,5
A generally accepted standard model has emerged to describe how
different tissues and established erythroleukemic cell lines control
homeostasis of iron internalized via transferrin receptor (TfR)-mediated endocytosis. This model supposes that the expression of
TfR, ferritin, and the erythroid delta-aminolevulinate synthase (eALAS), a rate-limiting enzyme in heme biosynthesis exclusively present in erythroid cells, is coordinately regulated.6-8
Control is mainly exerted by the iron-regulatory protein 1 (IRP1),9-12 a trans-acting factor binding to
stem-loop structures in cognate mRNAs, termed iron-responsive elements
(IREs).13 A second isoform of similar function, IRP2, has
been described, which is ubiquitously expressed, with highest levels
found in neuronal tissues.14 At low intracellular iron
levels, IRPs interact with multiple IREs within the
3'-untranslated region (UTR) of TfR mRNA, thereby strongly
increasing mRNA half-life and TfR protein expression at the cell
surface.15,16 Concurrently, the binding of IRPs to IREs in
the 5'-UTR of ferritin and eALAS transcripts17-20
interferes with the assembly of functional 80S translation initiation
complexes and prevents protein synthesis.21,22 In contrast,
high iron abundance diminishes IRP-affinity to IREs, resulting in rapid TfR mRNA degradation and efficient translation of ferritin and eALAS
transcripts. However, this model fails to explain how primary erythroblasts (ebls) undergoing terminal differentiation cope with the
paradoxical situation that low IRP-activity, allowing efficient eALAS
biosynthesis should also favor ferritin production, thereby forming a
futile iron storage at times of massive iron demand for heme biosynthesis.
Until recently, studies on iron metabolism during erythropoiesis could
only be performed in erythroleukemic cell lines.19,20,23 All of these transformed cell types share common drawbacks with respect
to erythroid differentiation: they (1) require nonphysiological stimuli
to initiate differentiation, (2) mature incompletely or aberrantly
(insufficient hemoglobin accumulation, abnormal morphology, lack of
enucleation24), and (3) exhibit nontypical cell cycle control during differentiation.25 These erythroleukemic
cells show regulation of iron metabolism according to the standard
model.26 However, their abnormalities allow no reliable
conclusions on the regulation of iron metabolism upon maturation of
primary ebls.
Recently, culture conditions were established allowing the mass
cultivation of erythroid progenitors derived from chick bone marrow.
After a limited period of factor-dependent proliferation, termed
self-renewal, these cells can be induced to highly synchronous differentiation, resulting in cells virtually indistinguishable from
erythrocytes in peripheral blood.27,28 Hence, this system appeared ideally suited to study the regulation of iron utilization and
storage under in vivo-like conditions. Moreover, transformation of
these bone marrow-derived ebls with the avian erythroblastosis virus
(AEV)-generated immortalized cell lines (eg, HD3)29,30 as
well as mortal clones of AEV-infected ebls. These cell types allowed us
to address the question of whether there are differences in the
regulation of iron metabolism between normal versus transformed erythroid cells.
We show here that primary ebls exhibit a mode of iron-dependent gene
regulation clearly distinct from the standard model. Under low as well
as high exogenous iron supply, ferH mRNA remained essentially absent
from polyribosomes and was translated at low levels, whereas the
coexpressed eALAS transcripts were mainly bound to polysomes under both
conditions. These differences in the mode of ferH versus eALAS mRNA
translation could not be observed in HD3 leukemic cells. Extensive
analysis showed that v-ErbA, 1 of the 2 oncoproteins expressed by AEV,
induced and maintained regulation of iron metabolism according to the
standard model in primary ebls. The idea that v-ErbA was sufficient for
this molecular phenotype was supported by functional inactivation of this oncoprotein in AEV-transformed ebls: loss of v-ErbA activity resulted in a reversal to iron-independent ferritin production.
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MATERIALS AND METHODS |
Cell culture and retroviral infection.
The chicken hepatoma cell line LMH-2A was grown in Waymouth medium
supplemented with 10% fetal calf serum (FCS), 2 mmol/L L-glutamine,
and antibiotics at 37°C and 5% CO2. Tissue culture plates were coated with 0.1% gelatine 4 hours before use. Human HeLa
cervical carcinoma cells were grown in Dulbecco's modified Eagle's
medium (DMEM) plus 10% FCS and antibiotics.
Primary chicken erythroid progenitors were isolated and cultivated as
described earlier,25,27,28,31 starting from 2 × 108 normal bone marrow cells of 3- to 7-day-old Spafas
chicks. Briefly, 5 × 107 purified cells were plated
into 10 mL modified colony-forming unit-erythrocyte (CFU-E)
medium32 supplemented with 100 ng/mL recombinant avian stem
cell factor (SCF)31 and 40 ng/mL recombinant human
insulin-like growth factor (IGF-1; Sigma, St Louis, MO). These so-called SCF-progenitors were expanded at 37°C and 5%
CO2 to numbers between 2 × 108
and 5 × 108. Before harvesting or induction of
differentiation, spontaneously maturing erythroid cells were removed by
Percoll purification.
To induce terminal differentiation of 3- to 4-day-old SCF-progenitors,
self-renewing cells were washed twice with serum-free DMEM and cultured
in differentiation medium for 4 days, maintaining a density between 2 and 3 × 106 cells/mL. The differentiation medium
represents a modified CFU-E medium without chicken serum (Epotest
medium) supplemented with 1.4 nmol/L insulin and 3% anemic chicken
serum as a source of avian erythropoietin (Epo).31
To infect primary ebls, retrovirus-producing chicken embryo fibroblasts
(CEFs) were generated by cotransfection of the retroviral construct
carrying the gene of interest with helper virus DNA and G418
selection.33 Subsequently, freshly prepared chick bone marrow cells were cocultivated with mitomycin-C-treated CEFs producing the particular recombinant retroviruses, essentially as
described.34
Ebls expressing v-ErbA alone or in combination with the murine Epo
receptor (EpoR)35 were expanded under self-renewing
conditions in CFU-E medium supplemented with 100 ng/mL avian SCF, 40 ng/mL human IGF-1, 1 µmol/L estradiol (E2), and 3 µmol/L of the
glucocorticoid-antagonist ZK 112993.36 In case of c-ErbA
expression on its own or plus exogenous murine EpoR, the same factors
were added; however, the CFU-E medium was stripped of endogenous
thyroid hormone and retinoids.37 ts34A2-ebls, expressing a
temperature-sensitive mutant of the v-ErbB oncoprotein at
37°C,38,39 were kept for proliferation in CFU-E medium
containing 100 ng/mL avian SCF, 40 ng/mL human IGF-1, 1 µmol/L E2,
and 1 µmol/L dexamethasone (Dex), whereas CER-2 ebls expressing
c-ErbB40 were additionally supplemented with 20 ng/mL transforming growth factor  (TGF; Promega, Madison, WI).
All AEV-transformed ebls, ie, the primary clone ts34A6,41
the established cell line HD3E11,42 and the cell line
HD3E11 ectopically expressing the murine EpoR, termed
HD3E22,43 were cultured under proliferation conditions in
either standard growth medium consisting of DMEM supplemented with 8%
FCS, 2% chicken serum, 10 mmol/L HEPES, pH 7.2, and antibiotics or in
Epotest medium plus 40 ng/mL human IGF-1. Differentiation of HD3E22
ebls was induced by incubation in differentiation medium supplemented with 2 U/mL recombinant human Epo, 1.4 nmol/L insulin, 1 µmol/L of
the estrogen antagonist ICI 164384, 1 µmol/L ZK 112993, the ErbB
inhibitor PD 153035,44 and, where appropriate, with the c-Kit antagonist EXBW 50 (a kind gift of Boehringer Ingelheim, Ingelheim, Germany; H. Beug, unpublished
data). For all types of ebls, cell size and number were
monitored daily in an electronic cell analyzer (CASY;
Schärfe-System, Reutlingen, Germany).
To achieve iron deprivation, cells were incubated for 24 hours with 50 µmol/L of the specific iron chelator desferrioxamine (Des). High iron
supply was accomplished by supplementing culture media with either 1 mg/mL iron-saturated chicken ovotransferrin (Tf, conalbumin; Sigma) or
50 µg/mL ferric ammonium citrate (FeCit).
Assays for differentiation parameters.
Cell populations induced to differentiate were daily analyzed for their
stage of maturation by photometrical determination of hemoglobin
concentrations as well as by cytocentrifugation onto slides and
subsequent staining with neutral benzidine blue plus histological dyes
as described previously.25
Cloning of chicken ferH IRE cDNA.
By using the forward primer 5'-GTC GAA TTC CAG AGC GCG TCG GCG
AGG CTG-3' and the reverse primer 5'-GGT TGG TCA CAT GGT
CAC CCA GCT GC-3', both derived from the genomic chicken ferritin H subunit sequence,45 a 597-bp DNA fragment
(EcoRI-BstEII) was amplified from total chicken liver
RNA by reverse transcription-polymerase chain reaction (RT-PCR). The
sequence of the isolated PCR product was 100% identical to the cDNA
sequence predicted from the chicken ferH gene, spanning a region from
the transcription start site in exon I up to a single BstEII
restriction site in exon IV. This EcoRI-BstEII ferH
cDNA fragment was ligated into a pGEM-T vector (Promega) and termed
pGEM-cferH597. Religation after removal of an Apa I fragment of
pGEM-cferH597 (comprising the C-terminal part of exon I and exon II-IV)
resulted in the plasmid pGEM-cferH-IRE, which carried 78 bp of the
5'-UTR (5'-CAGAGCGCGT CGGCGAGGCT GAGCGGAGCG GGTTCCTGCG TCAACAGTGC TTGGACGGAA CCGGCCGCGC
TCGGGCCC-3'), including the entire IRE stem-loop structure; the
characteristic terminal hexaloop-sequence and the unpaired bulged
C-residue in the stem are underlined.
The 44 bp of the 5'-UTR of chicken eALAS cDNA
(5'-ATTCCAGTGC GTTCGTCCTC AGCGCGGGGC AACGGCACAG GACG-3')
containing the entire IRE hairpin motif according to the published
sequence46 was produced by oligonucleotide synthesis,
inserted via BamHI linkers into pGEM3Zf( ) (Promega),
resulting in the plasmid pGEM-eALAS-IRE, and verified by sequencing.
Sucrose gradient analysis.
The distribution of ferH chain and eALAS mRNA between ribosome-free and
polyribosome-associated compartments was determined by
separating cytoplasmic extracts from 2 × 107 LMH-2A
hepatoma cells or 4 to 5 × 107 ebls in linear 15% to
40% sucrose gradients as described.47,48 Each gradient was
harvested into 18 fractions and the extracted RNA analyzed by
electrophoresis on denaturing 1% formal dehyde agarose gels and
subsequent Northern blotting. After UV-cross-linking of RNA to the
nylon membranes, the distribution of 18S and 28S rRNA was visualized by
staining of filters with methylene blue. The membranes were
sequentially hybridized with a random primed 32P-labeled
0.45-kb fragment (Nco I-BstEII) of the chicken ferH chain cDNA and a 1.4-kb fragment (Pst I-Pst I) of the
chicken eALAS cDNA.49 Signals on the autoradiographs were
scanned and quantified with a laser densitometer (Molecular Dynamics,
Sunnyvale, CA).
Western blotting.
The harvested cells were washed in ice-cold phosphate-buffered saline
and lysed in an isotonic buffer containing 10 mmol/L HEPES, pH 7.6, 40 mmol/L KCl, 5% glycerol, 1 mmol/L dithiotreitol, and 0.2% of the
nonionic detergent NP40.48 Protein concentrations were
determined by a Coomassie Brilliant Blue assay (Bio-Rad, Hercules,
CA). Ten to 20 µg of cell extract from each sample was separated in sodium dodecyl sulfate (SDS) polyacrylamide gels and
transferred to nitrocellulose (Protran; Schleicher & Schuell, Keene,
NH). Reversible staining of the membranes with acidic
Ponceau S solution (Merck, Darmstadt, Germany) was used to visualize
proper protein transfer. After blocking with 5% low fat dry milk, the filters were probed with the respective antisera: antihorse spleen ferritin, raised in rabbit by using the affinity isolated antigen (Sigma); antirat IRP1 (a kind gift of R.S. Eisenstein)50;
or antirat IRP2 (a kind gift of E.A.
Leibold).12 Immunoreactive signals were
detected by enhanced chemoluminescence (Amersham, Uppsala, Sweden) and
quantified by laser densitometry.
Analysis of IRP protein abundance and mRNA binding activity.
The affinity of IRPs to the cis-acting palindromic IRE sequence
in chicken ferH mRNA was analyzed by gel retardation assays essentially
as described previously.48,51 The preparation of cytoplasmic extracts and quantitation of protein concentrations were
performed as outlined above for Western blotting.
32P-radiolabeled in vitro transcripts were produced by T7
RNA polymerase after linearization of the plasmid pGEM-cferH-IRE with
Apa I. A total of 1.3 × 106 dpm (~1.0 ng)
of labeled IRE-containing in vitro transcript was incubated with 2 µg
of protein for 30 minutes at room temperature and subsequently treated
with RNAse T1 and heparin. RNA-protein complexes were visualized after
separation in 6% nondenaturing polyacrylamide gels that were fixed by
drying onto a DEAE ion exchange paper and subjected to autoradiography.
Signals corresponding to IRP-IRE complexes were quantified by
phosphoimaging (Molecular Dynamics).
To analyze IRP1 protein abundance, extracts were incubated for 30 minutes with 2% 2-mercaptoethanol before the assay to reduce IRP1 for
the determination of total RNA binding activity.51
For antibody-mediated supershift experiments, extracts were
preincubated with 1 or 3 µL of the specific rat IRP1 or IRP2 antisera or an unspecific antibody (IgG) for 2 hours at 4°C before the binding reaction with 32P-labeled chicken ferH-IREs.
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RESULTS |
Iron-dependent translational control of ferritin and eALAS mRNAs in
erythroleukemic and hepatoma cell lines.
Both ferH and eALAS mRNAs harbor single palindromic IREs in their
leader region and thus are considered as targets for iron-dependent mRNA binding of IRP.6 However, previous studies focused
either exclusively on the IRP-interaction with the mRNAs of ferritin subunits in different cell lines and tissues or on IRP-complex formation with eALAS mRNA in erythroleukemic cells.17-20
Therefore, we examined the potential of IRP to actually coregulate
translation initiation of ferH and eALAS transcripts. For this
analysis, we initially used chicken erythroblasts (ebls) transformed by
the cooperating AEV oncoproteins v-ErbB and v-ErbA and
expressing the murine Epo receptor (EpoR) to allow terminal
differentiation of these cells (termed HD3E22)43 with
recombinant factors. Sucrose gradient analysis was performed with cells
either iron-depleted by exposure to the specific chelator
desferrioxamine (Des) or treated with physiologically high levels of
iron-saturated transferrin (Tf; 1 mg/mL; L. Lobmayr, unpublished
data), the physiological iron source.2 By
this technique, untranslated mRNPs (<80S region) are separated from
polyribosome-associated mRNAs, which are engaged in protein synthesis
and sediment further down into the gradient.47
As expected, ferH mRNA was restricted to the ribosome-free pool
(fractions 1-9) in iron-depleted HD3E22 ebls, whereas a significant mobilization into the polysome-bound compartment was observed under
high Tf concentrations (Fig 1A and B). This
shift was comparable to the one in exponentially growing chicken
hepatoma cells (LMH-2A). It should be noted that differences in iron
concentrations did not affect the overall ferH mRNA abundance in these
cells. Similar results were obtained in primary chicken embryo
fibroblasts (data not shown) and in previous studies on ferL mRNA
translation in human HeLa cervical carcinoma cells.48 This
indicated that the relative proportion of ferH mRNA bound to polysomes
at high iron concentrations (10% to 20%) was comparable in these
different cell types. To our surprise, already in iron-deprived HD3E22
ebls, a significant portion of eALAS transcripts was found in polysome fractions. However, the eALAS transcripts shifted onto even larger polysomes upon administration of high Tf levels, suggesting
iron-dependent translational control to a certain extent (Fig 1A and
B).
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| Fig 1.
Iron-dependent translational control of ferH and eALAS
mRNA in LMH-2A hepatoma cells and AEV-transformed ebls (HD3E22). Cells
were incubated with the iron chelator desferrioxamine (DES; 50 µmol/L) or iron-loaded transferrin (TF; 1 mg/mL iron-saturated
chicken ovotransferrin) for 24 hours before harvesting. (A) Cytoplasmic
extracts were separated in linear 15% to 40% sucrose
gradients47 and the RNA was isolated from 18 fractions
analyzed by Northern blotting. Fraction 1 corresponds to the top and
fraction 18 to the bottom of the gradient. The filters were hybridized
with 32P-labeled probes specific for chicken ferH and, in
case of HD3E22 ebls, subsequently with a chicken eALAS
cDNA.49 The lowest panel displays the profile obtained by
staining of the RNA with methylene blue (mb). The emergence of a
constant molar ratio between 28S and 18S RNA (upper and lower band,
respectively) around fraction 9 indicates the assembly of 80S
initiation complexes and marks the boundary between the ribosome-free
and polyribosome-bound compartment. Signals between gradients from the
same cell type are directly comparable, because (1) the same aliquot of
RNA from each fraction was blotted, (2) both filters were hybridized
with the very same probe, and (3) exposed for the same time. Thus, the
sum of signals over all the fractions corresponds to total cytoplasmic
mRNA levels. (B) Quantitative analysis of hybridization signals from
Northern blotting by laser densitometry. (Open symbols) Cells depleted
of iron by Des; (solid symbols) cells under high Tf. To facilitate
comparison between different experiments, the RNA content of each
fraction was expressed as a percentage of the total amount of RNA
contained in the gradient. (C) Ferritin protein expression in HD3E22,
LMH-2A, and HeLa cells was determined by Western blotting as described
in Materials and Methods. Numbers below each pair of lanes represent
the factors of iron-dependent upregulation of ferritin expression (fold
induction).
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Concomitant with the iron-induced translocation of ferH mRNA from the
ribosome-free to the polysome-associated compartment, we observed a
regulation at the level of ferritin protein. This effect was strongest
in HD3E22 ebls, where a 25-fold increase of ferritin abundance was
found under high iron (Fig 1C; see also, Fig 3). A significant but more
moderate change in ferritin levels (4×) occurred in LMH-2A (Fig
1C), because these cells retained some more ferH mRNA in the
ribosome-containing fractions (9-11) under iron depletion as compared
with HD3E22 (Fig 1A and B). Two other AEV-transformed ebls, both
lacking the murine EpoR, ie, the established cell line
HD3E1142 and the clonal strain ts34A6,41 yielded comparable results as obtained in HD3E22 cells
(Table 1). These results are in line with
the expected coregulation of ferH and eALAS mRNA, although the fraction
of polysome-associated eALAS mRNA in the iron-deprived HD3E22 ebls was
surprisingly high.
Translation of ferH mRNA is impaired at high iron concentrations in
primary erythroblasts.
Erythroleukemic cell lines differentiate aberrantly and are defective
in mature erythrocyte functions. Therefore, we became interested to
investigate the translational control of ferH and eALAS transcripts in
primary ebls, in which coregulation of these mRNAs would be
functionally neutral or disadvantageous and thus should be futile. The
erythroid progenitors used for this analysis were either maintained in
the proliferative stage by stem cell factor (SCF; SCF-ebls) or induced
to synchronous differentiation by Epo and insulin.30 During
96 hours of maturation, the cells increased their hemoglobin content to
the level attained in peripheral blood erythrocytes, drastically
reduced their cell volume, and showed a typical erythrocyte morphology
after cytocentrifugation and staining to visualize hemoglobin (data not
shown).25 As in the preceding experiments, cytoplasmic cell
extracts were fractionated in sucrose gradients to monitor the
ribosomal recruitment of iron-responsive target mRNAs.
In sharp contrast to the standard model, ferH mRNA remained almost
exclusively ribosome-free in these cells under low as well as high Tf
supply, during self-renewal and after 48 or 96 hours of terminal
differentiation (Fig 2). Moreover, this
translational inhibition was independent of the iron source: even under
high concentrations of ferric ammonium citrate, ferH transcripts were not translated more efficiently (48 hours differentiating SCF-ebls; Fig
2, lowest panel). This phenotype suggested that the translation of ferH
transcripts should be diminished, independently of iron abundance and
progress into erythroid maturation.
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| Fig 2.
Iron-independent reduction of ferH mRNA translation in
proliferating and differentiating primary ebls. The SCF-dependent
erythroid progenitors were grown from chicken bone marrow as
described.25 Maturation was induced by removal of
self-renewal factors (SCF and IGF-1) and addition of avian Epo
(high-titer anemic serum) and insulin to the medium (see Materials and
Methods). Self-renewing SCF-ebls and cells induced to differentiate for
48 or 96 hours were treated with low (0.03 mg/mL) or high levels of
iron-saturated Tf (1 mg/mL) or high ferric ammonium citrate
concentrations (FeCit; 50 µg/mL, lowest panel at 48 hours) for 24 hours. Sucrose gradient analysis and densitometrical quantitation were
performed as described in the legend to Fig 1. ( ) ferH mRNA; ( )
eALAS mRNA.
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E-ALAS mRNA was strongly bound to polyribosomes at low iron levels, and
a moderate mobilization into larger polyribosomes was observed in
response to high iron levels. This was true for proliferating cells as
well as for those induced to differentiate for 48 and 96 hours. Thus,
the ability for iron-dependent translational control of eALAS mRNA was
maintained in primary SCF-ebls, comparable to the observations in
transformed HD3 ebls (see Fig 2).
Taken together, these results indicated the existence of an
iron-insensitive mechanism specific for the inhibition of ferH protein
synthesis, which did not affect translation of eALAS transcripts. The
restriction of ferH mRNA to the ribosome-free compartment correlated
well with the lack of iron-dependent regulation of ferritin protein
levels in normal SCF-ebls, although protein expression was not entirely
eliminated (Fig 3B). Thus, ferH as well as
eALAS mRNAs were differently used in committed erythroid cells,
although their 5'-UTRs contain functional IREs, which in theory
should have resulted in iron-responsive coregulation. In the following, the mechanism governing the differential translational control of ferH
versus eALAS mRNA will be referred to as the erythroid mode of
regulation of iron metabolism.
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| Fig 3.
Retroviral expression of v-ErbA or v-ErbB in primary ebls
and the consequences for ferritin biosynthesis. Bone marrow-derived,
primary ebls expressing either the v-ErbA or the v-ErbB oncoprotein
(see Materials and Methods) were kept for 24 hours under self-renewing
conditions in the presence of 50 µmol/L Des to achieve iron depletion
or were incubated with 1 mg/mL Tf to induce full iron saturation. (A)
Distribution of ferH mRNA as determined by sucrose gradient analysis
(for details, see legend to Fig 1). (Insets) The ribosome-bound mRNA
compartment (fractions 9-18) is shown with an extended ordinate to
highlight the difference between v-ErbA and v-ErbB expressing cells.
(Open symbols) Des; (solid symbols) Tf. (B) Ferritin protein levels as
detected by Western blotting in self-renewing primary ebls (SCF),
v-ErbA- or v-ErbB-expressing ebls (v-ErbA, v-ErbB), and
AEV-transformed red blood cells (HD3E22) under iron scarcity (Des) or
high iron abundance (Tf). In case of SCF-progenitors, low Tf (0.03 mg/mL) instead of Des was used to induce IRP-activity. Numbers below
each pair of lanes represent the factors of iron-dependent upregulation
of ferritin expression (fold induction).
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Translational inhibition of ferH mRNA in primary SCF-progenitors is
abolished by the v-ErbA oncoprotein.
As shown above, AEV-transformed ebls conferred iron-dependent
translational regulation according to the standard model (ie, coregulation of eALAS and ferH mRNA translation), whereas primary ebls
exhibited the iron-insensitive erythroid mode. This raised the question
of which of the 2 AEV oncogenes was responsible for this effect. In
addition, we tested whether the induction of standard mode regulation
was restricted to cells expressing the oncogenic ErbB and/or ErbA
variant(s) or could also be induced by the respective, nonmutated
cellular proto-oncogenes. Suitable retroviral vectors expressing these
proteins were used to infect primary bone marrow ebls, either alone or
in various combinations.31
The analysis showed that v-ErbA, a mutated version of the cellular
thyroid hormone receptor (TR), on its own was capable to restore
iron-dependent regulation of ferH protein synthesis in self-renewing
ebls. Sucrose gradient analysis showed that v-ErbA-expressing erythroid cells exhibited a shift of ferH transcripts towards the
polysomal compartment under high Tf concentrations. This mobilization was associated with an 8-fold upregulation in the level of ferritin protein (Fig 3A and B). Thus, v-ErbA-expressing primary ebls
correspond to AEV-transformed ebls in their mode of translational
regulation of ferH mRNA (see Figs 1 and 3B). Similar results were
obtained using ebls coexpressing v-ErbA and the murine EpoR (Table 1).
In contrast to v-ErbA, the v-ErbB oncoprotein, a mutant of the cellular
epidermal growth factor receptor (EGFR), failed to alter the erythroid
mode of regulation of iron metabolism. Under conditions of iron
depletion and excess, ferH mRNA was mainly restricted to the
ribosome-free compartment (Fig 3A), and in consequence, ferritin
protein levels were not modulated (Fig 3B). A comparable result was
obtained in ebls overexpressing c-ErbB, the normal cellular EGFR (Table
1). Similar to HD3E22 and SCF-ebls, the translational efficiency of
eALAS mRNA moderately responded to depletion and excess of iron in both
v-ErbA- and v-ErbB-expressing cells, as detected by sucrose gradient
analysis (data not shown). This confirmed the data from SCF-ebls that
translational control of eALAS mRNA is iron-dependent, whereas the
utilization of ferH mRNA is differentially affected by the v-ErbA oncoprotein.
These results raised the question of whether the switch from the
erythroid to the standard mode of ferritin biosynthesis by v-ErbA could
also be obtained with c-ErbA/TR. Upon overexpression, this nuclear
class II hormone receptor promotes proliferation of ebls and
differentiation arrest in a fashion indistinguishable to v-ErbA when
completely withdrawn from ligand.37 To our surprise, c-ErbA/TR had no effect on the inhibition of ferH mRNA translation in primary ebls, neither alone nor in combination with the murine EpoR
(Table 1). This was true for both the inability of ferH mRNA to bind to
polysomes in these cells and to produce increased levels of ferritin
protein in response to high iron levels.
In conclusion, the v-ErbA oncoprotein was required to confer standard
type regulation of iron metabolism in primary self renewing ebls.
Neither its nonmutated form c-ErbA/TR nor the EGFR in its normal
(c-ErbB) or mutated oncogenic form (v-ErbB) were able to abrogate the
translational inhibition of ferH mRNA, typical for the erythroid mode.
V-ErbA expression restores iron-dependent modulation of IRP-binding
activity to the ferH-IRE.
To obtain first insights into the mechanism responsible for the
erythroid mode of ferritin regulation, we studied the role of IRP in
the interference with ferH mRNA translation. To this purpose, IRP
abundance and IRE-affinity were determined in vitro from cytosolic
extracts by gel retardation assays.47,48 To avoid potential
artifacts arising from the binding of chicken IRP to mammalian
IRE-templates, a chicken ferH cDNA containing the entire 5'-UTR
and including the regulatory IRE stem-loop structure was cloned from
total chicken liver RNA by RT-PCR as delineated from the published
genomic sequence.45
Surprisingly, IRP-mRNA binding activity could not be modulated in
response to iron in primary SCF-progenitors
(Fig 4 and L. Lobmayr, unpublished
observation). In contrast, leukemic HD3E22 ebls displayed
an 8-fold increase in IRP-IRE complex formation under iron depletion,
as predicted by the standard model. Moreover, the total IRP abundance
as monitored by the activation of nonbinding IRP molecules with
2-mercaptoethanol52 was low in the primary progenitors
compared with their leukemic counterparts (Fig 4A). Similar results
were obtained in 2 other strains of AEV-transformed ebls (HD3E11,
ts34A6; see Table 1 and data not shown). In good agreement with the
data obtained by sucrose gradient and Western blot analysis, we found
that retrovirally expressed v-ErbA restored iron-dependent modulation
of IRP-IRE binding affinity, as indicated by a greater than 7-fold
increase in the formation of IRP-IRE complexes under conditions of iron
deprivation. The abundance of IRP and the extent of its regulation
closely corresponded to those observed in ebls expressing both AEV
oncogenes (HD3E22; Fig 4A). Similar results were obtained with extracts
of cells expressing v-ErbA in combination with murine EpoR (Table 1 and data not shown). On the contrary, exogenous v-ErbB or c-ErbB were unable to induce iron-dependent modulation of IRP-IRE complex formation
in primary ebls. Cells overproducing c-ErbA/TR, either alone or in
combination with murine EpoR, were as iron-insensitive as the primary
SCF-ebls (Table 1). From these data we concluded that the functional
impact of the v-ErbA oncogene but not its cellular counterpart, c-ErbA,
was sufficient to confer iron-dependent IRP modulation in
SCF-progenitors.
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| Fig 4.
Iron-dependent modulation of IRP-binding activity to the
ferH-IRE in primary versus transformed ebls. (A) IRP-IRE-binding
activity was determined in vitro by a gel retardation assay as outlined
in Materials and Methods. Aliquots of the cell extracts from
self-renewing ebls used for immunoblotting (see Fig 3) were incubated
with an excess of radiolabeled ferH-IRE transcripts and the resulting
RNA-protein complexes were separated in nondenaturing polyacrylamide
gels. Where indicated, maximal IRP1-IRE binding activity was estimated
by treatment of the extracts with 2-mercaptoethanol (2-ME) in vitro.
The amount of radioactivity in the IRP-IRE complexes was quantified by
phosphoimaging. Numbers below the pair of corresponding lanes represent
the fold induction of IRP activity. (B) Levels of IRP1 (left panel) and
IRP2 (right panel) in transformed versus primary ebls cultivated under
different iron supply, as detected by immunoblotting. (C) IRP1 versus
IRP2 binding activity to the ferH-IRE in extracts of transformed and
primary ebls treated for 24 hours with Des. Gel retardation assays in
combination with antibody-mediated supershifts were performed as
described in detail under Materials and Methods. As a control (no
extract), lysis buffer was incubated with 3 µL antibody and the
radioactive ferH-IRE probe. -IRP1 and -IRP2, IRP1 and IRP2
specific antisera, respectively; IgG, nonspecific antibody.
|
|
To assess whether IRP1 and/or IRP2 was responsible for the observed
mobility shift of the chicken ferH-IRE probe and the modulation of
ferritin expression in the various erythroid cell types tested, Western
blot analyses were performed. The amount of IRP1 expression was greatly
increased in the AEV-transformed HD3E22 cells as compared with normal
SCF erythroid cells (Fig 4B). This was not the case for IRP2 protein,
whose abundance is even slightly decreased in the transformed cells. To
our knowledge, this result also provided the first evidence for the
existence of IRP2 in chicken. No iron-dependent variation in amounts of
IRP1 protein was observed in the cell types tested, in accordance with
previous reports,8 which showed that IRP1 activity is
mainly governed by modifications of the FeS cluster near the RNA
binding domain. In contrast, IRP2 expression was massively reduced by
iron administration, again in agreement with data from the literature
demonstrating a reduction of IRP2 protein stability in the presence of
iron.53,54
The apparent coexpression of IRP1 and IRP2 in erythroid cells raised
the question of which of these RNA binding proteins was responsible for
the electrophoretic mobility shift described in Fig 4A. Moreover, the
emergence of a double band might have suggested that each band
corresponded to one of the IRPs. Hence, supershift assays were
performed in which cellular extracts from HD3E22 as well as SCF-ebls
were incubated with antisera specific for IRP1 or IRP2, respectively,
before the IRP-IRE binding reaction. In the HD3E22 cells, the majority
of both bands of the doublet could be supershifted with an antibody
against IRP1; however, some supershifted complexes were produced by the
IRP2-specific antibody (Fig 4C). In contrast, SCF cells appeared to
contain less IRP1 activity and a slightly higher amount of IRP2 than
the transformed cells. These data correlate well with the observations
from the Western blot experiments (Fig 4B).
Taken together, these analyses strongly suggested that both IRP1 and
IRP2 are responsible for both bands in the electrophoretic mobility
shift with the chicken ferH-IRE. This was verified with nonerythroid
chicken cells as well as mouse fibroblasts (data not shown), indicating
that the double band is not cell type, but probe-specific. Thus, the
lower intensity of the IRP-IRE complex formation in SCF cells seems to
be a true reflection of reduced expression of IRP1 and IRP2 combined.
Reversion of standard to erythroid mode of ferritin expression by
functional inactivation of v-ErbA in leukemic AEV-erythroblasts.
Earlier work has shown that the v-ErbA oncoprotein requires the
cooperation of the receptor tyrosine kinase (RTK) c-Kit (or other
tyrosine kinases such as v-ErbB) to display its biological activity,
ie, continuous proliferation coupled with a tight differentiation arrest in primary erythroid progenitors.30,36 In the
absence of cooperating RTK, v-ErbA is biologically inactive. We
therefore asked the question of whether the standard-type regulation of ferH translation in leukemic HD3E22 ebls could be abolished by functional inactivation of v-ErbA via abrogation of RTK activity.
HD3E22 ebls express both v-ErbB and c-Kit. The constitutively
active v-ErbB protein can be inhibited by the low molecular weight inhibitor PD 153035 specific for the EGFR.44 On the
other hand, c-Kit is inactivated by withdrawal of its ligand, SCF,
together with inhibition of its kinase activity by the low
molecular weight c-Kit inhibitor EXBW 50. When HD3E22 cells are
sub- jected to this combined treatment, they differentiate
terminally in the presence of human Epo plus insulin (H. Beug,
unpublished data).
After differentiation induction by the above-described treatment,
HD3E22 cells drastically reduced their cell size, showed a greater than
10-fold increase in hemoglobin content, and displayed a typical
erythrocyte morphology within 96 hours. In contrast, cells grown in the
absence of Epo/insulin and inhibitors continued to proliferate and
showed differentiation arrest (data not shown). Both cell types were
analyzed for their expression of ferritin protein by Western blot after
exposure to Des or high Tf levels. Interestingly, cells induced to
differentiate for 48 to 96 hours by the above-described treatment
failed to upregulate ferritin expression in response to iron,
resembling the phenotype described for primary SCF-ebls
(Fig 5; see also, Fig 3B). Ferritin was
still expressed after 48 hours of differentiation, but the levels were similar under depletion and excess of iron. Ninety-six hours after differentiation induction, ferritin protein was downregulated under
both conditions. In contrast, proliferating HD3E22 ebls that contained
an active v-ErbA (ie, in the absence of inhibitors) exhibited the
typical greater than 10-fold elevation in ferritin protein under high
Tf conditions (Fig 5; see also, Fig 3B). HD3E22 ebls induced to
differentiate for 48 hours by SCF withdrawal and v-ErbB inhibition by
PD153035 only (ie, without adding the c-Kit inhibitor EXBW 50) showed
iron-dependent modulation of ferritin expression to an extent
comparable to proliferating cells (data not shown). This suggests that
a residual c-Kit activity (or a closely related RTK) may have been
sufficient to activate v-ErbA function with respect to alter
regulation of ferH translation. These results support the
hypothesis that the activity of the v-ErbA oncoprotein is sufficient to
abolish the iron-independent inhibition of ferH translation in primary
SCF-dependent erythroid progenitors.
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| Fig 5.
Functional inactivation of the v-ErbA oncoprotein
switches AEV-transformed ebls from iron-dependent to iron-independent
ferritin expression. AEV-transformed ebls (HD3E22) were cultivated in
Epotest medium either under proliferation conditions (IGF-1) or induced
to terminal differentiation by supplementation with 2 U/mL human Epo,
1.4 nmol/L insulin, 1 µmol/L each of the steroid antagonists ICI
164384 and ZK 112993, the ErbB inhibitor PD 153035, and the c-Kit
antagonist EXBW 50 for up to 96 hours (see Materials and Methods).
Twenty-four hours before harvesting for Western blot analysis, ebls
were switched to identical media containing either Des (50 µmol/L) or
Tf (1 mg/mL). Numbers below each pair of lanes represent the fold
induction of iron-dependent ferritin expression.
|
|
| |
DISCUSSION |
The current investigation provides the first evidence for differential
translational regulation of ferH versus eALAS mRNA during normal
erythropoiesis. Using primary ebls, we show that ferritin mRNA
translation is not subject to regulation by iron: binding of ferritin
mRNA to polysomes is specifically impaired under low as well as high
iron concentrations. In contrast, eALAS transcripts are already bound
to polysomes under iron scarcity, but shift to larger polysomes in
response to high Tf, corroborating previous evidence for translational
control of eALAS. We further demonstrate that the hypothetical
regulatory loop responsible for the inhibition of ferH protein
synthesis in primary ebls can be efficiently shut off by the v-ErbA
oncoprotein, restoring iron-responsive regulation of ferH mRNA
translation, as observed in leukemic AEV-transformed ebls. The
specificity of v-ErbA as a molecular switch between the standard and
the erythroid mode of ferritin regulation could be confirmed by
abolishing v-ErbA activity in erythroleukemic ebls, causing a change
from iron-dependent to iron-independent ferritin expression typical for
the erythroid mode of regulation of iron metabolism.
The moderate level of eALAS mRNA translational control might correspond
to a variation in the hairpin motif of the chicken eALAS-IRE. As
previously shown by sequence analysis of the chicken eALAS genomic DNA
as well as mRNA,46 the consensus element
5'-CAGUGN-3' representing the hypercritical loop region of
the IRE is changed to 5'-CAGCGN-3'. A similarly aberrant
sequence 5'-CAGGGN-3' was independently
reported.55 These naturally occurring variants as well as
other point mutations generated in vitro have been described to impair
the interaction with IRP.56,57 Preliminary results suggest
that the alteration observed in the chicken eALAS IRE is indeed
responsible for a 30- to 40-fold reduced affinity of IRP towards this
particular element (data not shown).
In the chicken system, efficient retroviral vectors facilitate
ectopical (over)expression of cellular proto-oncogenes and their
corresponding viral counterparts.29,30 Our results from retroviral overexpression experiments clearly demonstrated that v-ErbA
oncoprotein alone is sufficient to reverse the inhibition of ferH mRNA
translation, resulting in standard-type regulation of iron storage as
observed in AEV-transformed chicken ebls (Figs 3 and 4A). Similar to
v-ErbA, overexpression of the cellular homologue c-ErbA/TR has been
shown to induce proliferation and to arrest differentiation of ebls,
when allowed to cooperate with c-Kit and deprived of all ligands (T3
and retinoids activating the heterodimerization partner RXR). Hence,
v-ErbA was proposed to represent a c-ErbA/TR permanently frozen in
the nonliganded conformation that is able to repress transcription
around some or all of the c-ErbA/TR-responsive target
genes.37 However, as opposed to v-ErbA, exogenous
c-ErbA/TR did not influence the reduction of ferH mRNA translation
efficiency (Table 1). This indicates that the mutations present in
v-ErbA must endow it with additional features important for
IRP-dependent translational regulation.
Leukemias are characterized by hyperproliferation of immature or
partially mature hematopoietic progenitor cells. In vitro, normal bone
marrow-derived SCF-progenitors undergo short-term self renewal for 8 to
10 generations until they differentiate or become apoptotic. However,
overexpression of exogenous ErbA causes long-term self renewal (>25
generations), mimicking an erythroleukemic phenotype.30 Our
data provide a potential correlation between the expression of ferritin
under high Tf concentrations and the life span of erythroid
progenitors. Whereas short-term proliferating SCF-ebls display low
ferritin abundance at high iron levels, long-term self renewal is
accompanied by an upregulation of ferritin expression in ebls
overexpressing v-ErbA, reaching levels typical for the leukemic HD3
cells. These findings support the hypothesis that the ability to
detoxify free iron via ferritin, to avoid the emergence of harmful
radicals and reactive oxygen species, is essential for continuously
growing cells. The idea is consistent with previous findings that
rapidly proliferating tumor cells express high levels of
ferritin.58,59 Thus, SCF-progenitors might not require
high-level ferritin expression for iron detoxification, because these
cells (1) use all available iron for hemoglobin production and (2) are
eliminated after a relatively short time due to their limited number of
cell divisions in which no long-term accumulation of damage caused by
iron can occur.
At present, the molecular mechanisms governing the differential use of
ferH and eALAS mRNAs in self-renewing as well as differentiating primary SCF-ebls remain essentially unknown. The phenotype cannot be
explained by a differentiation-associated increase in the abundance of
these mRNAs (exceeding the number of available IRP molecules), because
differential translational control was already observed in
self-renewing cells. In addition, analysis of the corresponding mRNA
levels showed no massive alterations upon terminal erythropoiesis (data
not shown).
Moreover, the inhibition of ferH mRNA translation cannot be due to
alterations in IRP abundance. v-ErbA expressing transformed ebls with
their relatively high IRP abundance (Fig 4A) allow translation of ferH
mRNA under high iron, whereas normal SCF progenitors with their
relatively low IRP expression reduce use of this mRNA independently of
the iron status. What may cause the nonresponsiveness to iron? At least
2 possibilities are conceivable. First, in primary erythroid cells, IRP
may be subjected to a posttranslational modification resulting in
constitutive binding to ferH-IREs. Recent studies support the idea that
phosphorylation of IRP can affect function.60 The
translational control of eALAS transcripts, also presumably mediated by
IRP, may well remain iron-sensitive in the same cells (SCF-progenitors)
due to the alterations in the IRE consensus sequence mentioned above.
Second, a putative factor X might specifically bind to ferH mRNA,
freezing IRPs to their cognate ferH IREs by direct protein-protein
interaction. Alternatively, complex formation between this putative
factor X and ferH mRNA could inhibit ferH translation in an
IRP-independent manner. Such an inhibitory factor should be either
directly or indirectly inactivated by v-ErbA, in line with the ability
of v-ErbA to transcriptionally repress distinct target genes in
proliferating ebls.36,39,61
The possibility to compare 2 closely matched erythropoietic cell
systems (AEV-transformed and primary chicken ebls) for their mode of
iron use and storage enabled us to address the generally important
question of how high ferritin levels can be compatible with high eALAS
abundance in normal and aberrant ebls. In contrast to previous
investigations in erythroleukemic cell lines,6,7 primary
ebls displayed a differential translational control of ferH versus
eALAS mRNA. This erythroid mode of iron metabolism offers a clear-cut
rationale how a new balance is reached between (high) iron utilization
and (low) storage capacity upon normal erythropoiesis: eALAS expression
is on to ensure heme biosynthesis in the erythropoietic process of
hemoglobinization, which requires tremendous amounts of iron, whereas
de novo ferritin expression is off and iron storage capacity is low but
still sufficient to avoid potential iron toxicity.
In conclusion, our work has shown that the standard model of iron
metabolism represents just one of several possibilities particularly
suitable for undifferentiated, rapidly proliferating cells. They
require moderate Tf-iron levels for growth and survival, but need an
effective mechanism to protect them from toxic radicals and reactive
oxygen species generated by free iron. From a functional aspect, it is
not surprising that a different mode of iron regulation is operative in
erythroid cells that are transiently proliferating but exhibit a very
high iron demand before and during differentiation. Here, regulation of
iron-responsive genes according to the standard model would cause
serious problems, which are circumvented by the erythroid mode of regulation.
| |
ACKNOWLEDGMENT |
The authors acknowledge the kind gift of IRP1 antiserum from Richard S. Eisenstein (University of Wisconsin, Madison, WI) and IRP2 antiserum
from Elizabeth A. Leibold (University of Utah, Salt Lake City, UT).
LMH-2A cells were kindly provided by Marcela Hermann and N. Erwin
Ivessa. The authors thank Lukas C. Kühn for stimulating
discussions and Eva Maria Deiner for expert technical assistance.
| |
FOOTNOTES |
Submitted March 15, 1999; accepted August 18, 1999.
Supported by grants from the "Herzfeld Family Foundation" and the
"Fonds zur Förderung der Wissenschaftlichen Forschung," Austria.
The sequence data have been submitted to the EMBL/GenBank databases
under accession no. Y14698.
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 Ernst W. Müllner, PhD, Institute of
Molecular Biology, Vienna Biocenter, University of Vienna, Dr.
Bohr-Gasse, A-1030 Vienna, Austria; e-mail: em{at}mol.univie.ac.at.
| |
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ABSTRACT
INTRODUCTION