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RED CELLS, IRON, AND ERYTHROPOIESIS
Iron control of erythroid development by a novel aconitase-associated regulatory
pathway
Grant C. Bullock,1 Lorrie L. Delehanty,1 Anne-Laure Talbot,1 Sara L. Gonias,1 Wing-Hang Tong,2 Tracey A. Rouault,2
Brian Dewar,3 Jeffrey M. Macdonald,3 Jason J. Chruma,4 and Adam N. Goldfarb1
1Department of Pathology, University of Virginia School of Medicine, Charlottesville; 2Molecular Medicine Program, National Institute of Child Health and Human
Development, Bethesda, MD; 3Department of Biomedical Engineering, University of North Carolina, Chapel Hill; and 4Department of Chemistry, University of
Virginia, Charlottesville
Human red cell differentiation requires
the action of erythropoietin on committed
progenitor cells. In iron deficiency, committed erythroid progenitors lose responsiveness to erythropoietin, resulting in
hypoplastic anemia. To address the basis
for iron regulation of erythropoiesis, we
established primary hematopoietic cultures with transferrin saturation levels
that restricted erythropoiesis but permitted granulopoiesis and megakaryopoiesis. Experiments in this system identi-
fied as a critical regulatory element the
aconitases, multifunctional iron-sulfur
cluster proteins that metabolize citrate to
isocitrate. Iron restriction suppressed mitochondrial and cytosolic aconitase activity in erythroid but not granulocytic or
megakaryocytic progenitors. An active
site aconitase inhibitor, fluorocitrate,
blocked erythroid differentiation in a manner similar to iron deprivation. Exogenous isocitrate abrogated the erythroid
iron restriction response in vitro and re-
versed anemia progression in irondeprived mice. The mechanism for aconitase regulation of erythropoiesis most
probably involves both production of metabolic intermediates and modulation of
erythropoietin signaling. One relevant signaling pathway appeared to involve protein kinase C␣/␤, or possibly protein kinase C␦, whose activities were regulated
by iron, isocitrate, and erythropoietin.
(Blood. 2010;116(1):97-108)
Introduction
Red cell production results from erythropoietin (Epo)–driven
survival, proliferation, and maturation of committed bone
marrow progenitor cells. This process critically depends on
cellular uptake of adequate bioavailable iron, provided in the
form of diferric transferrin. Compromise in iron uptake or
intracellular trafficking results in iron-restricted erythropoiesis,
characterized by diminished marrow responsiveness to Epo.1
Epo acts during an early phase of erythroid development,
between late (erythroid burst-forming unit) and early pronormoblast stages, before the initiation of hemoglobin synthesis.1-3
Therefore, iron restriction serves as a checkpoint to restrain
Epo-driven progenitor expansion in the face of limited iron
stores. It has been documented in zebrafish, in which defects in
intracellular iron utilization block early erythroid differentiation,4 and in mammals, in which dietary iron deficiency impairs
the transition from erythroid colony-forming unit to pronormoblast.5 In the clinical setting, iron-restricted erythropoiesis
underlies many of the anemias that are refractory to Epo
treatment (eg, anemias of chronic renal disease and
inflammation).1
The lineage- and stage-selective nature of the erythroid iron
restriction response has suggested involvement of a specialized
signaling pathway distinct from the iron depletion response that
occurs in a wide variety of cell types treated with chelators.
Supporting this notion, chelator-induced iron depletion in MCF-7
cells causes cell-cycle arrest in G1 phase followed by apoptosis,6
neither of which is seen in iron-restricted erythropoiesis in vivo.2,6,7
Thus, to identify mechanisms applicable to the erythroid iron
restriction response, an in vitro model was created in which
primary human hematopoietic progenitors underwent culture in
medium with transferrin saturation levels that allowed normal
granulopoiesis and megakaryopoiesis but precluded normal erythropoiesis. In this system, early erythroid progenitors showed
impaired growth and maturation in response to Epo but no increase
in apoptosis.
Intracellular iron response pathways use several kinase cascades, hypoxic response factors, Smad proteins, Notch signaling,
and iron-response proteins 1 and 2 (IRP1 and IRP2).8-16 In the
best-characterized iron regulatory system, IRP1 functions as cytosolic aconitase (ACO1) under high-iron conditions and converts to
an RNA-binding factor under low-iron conditions. This latter
function, involving engagement of cis-acting iron response elements in specific mRNAs (eg, ferritin and transferrin receptor 1),
has been widely accepted to mediate many features of the
mammalian iron deprivation response.15
The current studies examined signaling alterations associated
with the erythroid iron deprivation response, based on the hypothesis that a discrete pathway may connect iron availability with Epo
responsiveness. From screening known iron response pathways, we
identified aconitase enzymatic activity as correlating most closely
with the lineage-specific iron restriction response. A functional role
for these enzymes was supported by multiple experimental approaches, including enzymatic inhibition and provision of exogenous metabolic product. In the latter experiments, isocitrate
strikingly reversed the erythroid iron restriction response in vitro as
Submitted October 28, 2009; accepted April 13, 2010. Prepublished online as
Blood First Edition paper, April 20, 2010; DOI 10.1182/blood-2009-10-251496.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
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BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
BULLOCK et al
well as in vivo, raising the possibility for a novel therapeutic
approach to Epo-resistant anemias.
Possible mechanisms for erythroid regulation by aconitase and
isocitrate include production of adenosine triphosphate (ATP),
reduced form of nicotinamide adenine dinucleotide (NADH),
nicotinamide adenine dinucleotide phosphate (NADPH), and succinyl coenzyme A (CoA), the last of which serves as a precursor for
heme biosynthesis. However, multiple independent experimental
approaches supported a novel signaling function. In particular, the
iron-aconitase-isocitrate pathway played a critical role in Epomediated down-regulation of protein kinase C␣/␤ (PKC␣/␤)
activation and also affected PKC␦ activation status. Accordingly,
hyperactivation of PKC induced by iron deprivation contributed to
multiple aspects of the erythroid iron restriction response. A model
for iron regulation of erythropoiesis is presented that integrates
these findings with the recent report of physical and functional
interaction between PKC and aconitase.17
Methods
Cell cultures and treatments
Purified human CD34⫹ progenitors derived from granulocyte-colony stimulating
factor–mobilized peripheral blood cells of healthy donors were provided by the
Hematopoietic Cell Processing Core of the Fred Hutchinson Cancer Research
Center as previously described.18 The cells were grown at 37°C with 5% CO2 in
serum-free medium consisting of Iscove modified Dulbecco medium with
␤-mercaptoethanol, lot-tested bovine serum albumin, and the indicated cytokines
(PeproTech). To establish cultures with defined transferrin saturations, the
medium was supplemented with defined ratios of the insulin/transferrin/selenium
A and B preparations from StemCell Technologies. After thawing, the CD34⫹
progenitors first underwent approximately 48 hours of prestimulation in Iscove
modified Dulbecco medium with ␤-mercaptoethanol, BIT 9500 supplement
(StemCell Technologies), and cytokines consisting of 100 ng/mL stem cell factor
(SCF), 100 ng/mL FMS-like tyrosine kinase 3 ligand, 100 ng/mL thrombopoietin, and 50 ng/mL interleukin-3. After prestimulation, the cells were washed and
resuspended in unilineage differentiation medium at 1.5 ⫻ 105 cells/mL. Erythroid medium contained 4.5 U/mL Epo and 25 ng/mL SCF; granulocytic
medium contained 10 ng/mL granulocyte colony-stimulating factor, 10 ng/mL
interleukin-3, and 25 ng/mL SCF; and the megakaryocytic medium contained
40 ng/mL thrombopoietin, 100 ng/mL SDF1␣, and 25 ng/mL SCF with cells
grown on fibronectin-coated plates. Cell numbers and viabilities were determined
manually with the use of a hemacytometer and trypan blue staining. Barium
fluorocitrate (Sigma-Aldrich) was converted to sodium fluorocitrate with the use
of the approach of Paulsen et al.19 L⫹- and D⫺-isocitrate trisodium salts were
obtained by saponification of the corresponding enantiopure isocitric acid
lactones (3 equivalents NaOH, 80°C, 4 hours). The L⫹- and D⫺-isocitric acid
lactones were synthesized from dimethyl L- and D-malate, respectively, by the
method of Schmitz et al.20 The cell-permeable ␣-ketoglutarate derivative,
trifluoromethyl benzyl-␣-ketoglutarate (TaKG), was synthesized as previously
described.21 Racemic sodium isocitrate, sodium citrate, sodium ␣-ketoglutarate,
and chloramphenicol were all purchased from Sigma-Aldrich. Small-molecule
inhibitors, including bisindolylmaleimide I hydrochloride and Gö6983 were
purchased from EMD/Calbiochem. Knockdown of ACO2 expression in K562
and CD34⫹ cells used the LMP retroviral vector (Open Biosystems) containing
short hairpin RNA (shRNA) template subcloned from the pSM2c construct
V2HS_231437 (Open Biosystems). Transduction followed by analysis of green
fluorescent protein–positive (GFP⫹) cells was performed as previously described.22
Flow cytometry
Flow cytometric analysis of erythroid differentiation by costaining for
glycophorin A (GPA) and CD41 was performed as previously described.18,22 Staining of cells with annexin V–phycoerythrin and 7-aminoactinomycin D used the Annexin 5-phycoerythrin Apoptosis Detection Kit I
(BD PharMingen). For cell-cycle analysis, cells underwent fixation with
ethanol followed by staining with propidium iodide in the presence of
RNAase. Analyses were performed on a FACSCalibur instrument (BD
Biosciences) with the use of FlowJo software Version 8.6.3 (TreeStar Inc).
Aconitase, IRP, and metabolic assays
Assays for aconitase activites of ACO1/ACO2, iron-responsive element
(IRE)–binding activities of IRP1/IRP2, and IRP2 protein levels were
performed as previously described.23 For detailed information on aconitase
assays, as well as on measurements of cellular ATP, adenosine monophosphate (AMP), isocitrate, citrate, NADH, oxidized form of NADPH
(NADP), and NADPH, see supplemental Experimental Procedures (available on the Blood Web site; see the Supplemental Materials link at the top of
the online article).
In vivo models
The animal experiments were approved by the University of Virginia Animal
Care and Use Committee. The mice consisted of C57BL/6 purchased from The
Jackson Laboratory. In initial experiments to assay for toxicity, iron replete,
6-week-old female mice received sodium DL-isocitrate by daily intraperitoneal
injection at 200 mg/kg/d for 5 days. To induce iron deficiency anemia,
3-week-old male weanlings were placed on the Iron Deficient Diet from Harlan
Teklad (product no. TD 30 396), which contains 2 to 5 ppm of iron. For
measurements of complete blood counts (CBCs), retro-orbital blood samples
were analyzed on the Hemavet 850 FS automated CBC analyzer (Drew
Scientific). Starting at 1 month of the diet, CBCs were regularly monitored.
Treatments, initiated at onset of anemia (approximately day 40 of diet), consisted
of daily intraperitoneal isocitrate or ␣-ketoglutarate (200 mg/kg) or equivalent
volume of saline for 6 to 7 consecutive days.
Statistical analysis
Pairwise comparisons of values in treatment groups used a 2-tailed Student
t test. P values less than or equal to .05 were considered significant.
Immunoblot
Whole-cell lysates underwent sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE) followed by immunoblotting as previously
described.18 Antibodies included mouse anti–␤-actin, and mouse antitubulin (Sigma-Aldrich); rabbit anti-ACO2 (generously provided by Dr Luke
Szweda, Oklahoma Medical Research Foundation); rabbit antiferritin
heavy chain and anti–5-aminolevulinate synthase e (ALAS-e; Santa Cruz
Biotechnology); and rabbit anti–globin (Accurate Chemical & Scientific).
Rabbit polyclonal antibodies to phosphorylated and total PKC isozymes
were purchased from Cell Signaling Technology, with the exceptions of
antibodies to phospho-PKC⑀ and phospho-PKC␩ (Millipore).
Results
An in vitro model of iron-restricted erythropoiesis
An important aspect of iron-restricted erythropoiesis in vivo
consists of lineage selectivity, manifesting as an isolated anemia
with normal numbers of white cells and normal to elevated platelet
counts.24 To recapitulate this phenomenon in vitro, we identified a
critical threshold of transferrin saturation that allowed human
CD34⫹ hematopoietic progenitors to undergo granulopoiesis and
megakaryopoiesis but not erythropoiesis, with all cultures initiated
at a density of 1.5 ⫻ 105 cells/mL. In particular, various transferrin
saturations from 100% to 25% had no effect on any lineage,
whereas a transferrin saturation of 15% caused an erythroid
lineage-specific growth arrest and gradual decline in viability
(Figure 1A). By contrast, 1% transferrin saturation decreased
growth and viability in all 3 lineages (Figure 1A). Thus, transferrin
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BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
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Figure 1. A primary human model system for iron restriction of erythropoiesis. (A) Effect of transferrin saturation (Sat) on growth and viability of hematopoietic lineages.
CD34⫹ cells underwent unilineage culture in serum-free media. Results show mean ⫾ SEM for 3 to 5 independent experiments. (B) Inhibition of early erythroid differentiation
by iron restriction. Erythroid cultures with 100% or 15% transferrin saturation underwent flow cytometry (fluorescence-activated cell sorting [FACS]) with gating on viable cells.
CD34/CD36 expression was analyzed on day 4. Indicated are percentages of cells in various quadrants. (C) Minimal apoptosis associated with erythroid iron restriction. Cells
from panel B were analyzed by FACS for 7-amino-actinomycin D (7-AAD) and annexin V staining with gating on all cells. Whole cell lysates from day 5 cultures underwent
immunoblotting (IB) for caspase 3 (bottom; arrow indicates cleavage product). (D) Minimal effect of erythroid iron restriction on cell-cycle parameters. Day 5 cultures stained
with propidium iodide underwent FACS. Indicated are percentages of cells in various fractions, including subdiploid (Sub).
saturation levels at 15% elicited an erythroid iron-restriction
response and at 1% produced a non–lineage-specific response.
In a rodent model, iron-restricted erythropoiesis has been
characterized by a developmental block at the transition from
erythroid colony-forming unit to early erythroblast.5 Erythroid
maturation of human progenitors can be monitored by flow
cytometric quantitation of GPA, a lineage-specific surface marker
whose expression increases as a direct function of cellular differentiation; in addition, the earliest detectable events of human
erythroid lineage commitment are characterized by simultaneous
down-regulation of CD34 and up-regulation of CD36.25 In vitro
iron restriction in our system disrupted early erythroid differentiation, inhibiting the up-regulation of GPA and CD36, as well as the
down-regulation of CD34 (Figure 1B). Previous studies comparing
erythroid versus megakaryocytic output of primary human CD34⫹
cell cultures have used combined staining for GPA and CD41.26 By
this approach, the erythroid blockade observed with 15% transferrin saturation was associated with increased proportions of
CD41⫹ GPA⫺ cells, most probably representing megakaryocytic
precursors, a conclusion further supported by their structure on
Wright-stained cytospins (see Figure 3C). The iron-restricted erythroid
cultures also showed a slight increase in cell death. Importantly, this cell
death appeared to be nonapoptotic, with no increase in annexin V
staining among 7-amino-actinomycin D–negative cells and with no
increase in caspase 3 cleavage (Figure 1C). Cell-cycle analysis showed
no evidence for G1 or G2/M arrest as the basis for growth inhibition in
Figure 1A, nor was a significant subdiploid (Sub) population seen
(Figure 1D). Additional multiparametric flow cytometric studies confirmed the absence of annexin V staining and cell-cycle arrest within the
GPA⫹ erythroid fraction (supplemental Figure 1). Furthermore, treatment of cells with a caspase inhibitor failed to alleviate the block in
erythropoiesis caused by iron restriction (supplemental Figure 1).
Characterization of relevant pathways: a novel role for
aconitase
To identify mediators of the erythroid iron-restriction response
shown in Figure 1, candidate iron-regulated pathways were analyzed by 3 approaches: determination of signaling perturbations,
pharmacologic pathway activation or blockade, or retroviral/
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BULLOCK et al
genetic blockade. These approaches provided no evidence for
involvement of hypoxia-inducible factor, Notch, c-Jun N-terminal
kinase, p38, extracellular signal-regulated kinase, Janus kinase 2,
caspase, heat shock protein 70, ferritin heavy chain, or bone morphogenetic protein–Smad (for details, see supplemental Tables 1-2).
Although aconitase inactivation occurs in vitro during cellular
iron depletion,23 iron-deficient animals have shown little evidence
for declines in specific activity in nonhematopoietic tissues.
Specifically, Meyron-Holtz et al27 showed identical cytosolic
(ACO1) and mitochondrial (ACO2) aconitase activites in liver
extracts from iron-replete and iron-deficient mice, despite a clear
increase in IRP2 activity in the iron-deficient samples. Along
similar lines, comparison by Chen et al28 of iron-replete and
-deficient rat livers showed no change in ACO1 activity and a 25%
decrease in ACO2 activity that was attributable to a decrease in
ACO2 protein levels. We examined the effect of iron restriction on
aconitase activities in primary hematopoietic progenitors in a
gel-based assay that resolves the mitochondrial and cytosolic
isoforms. In these experiments, iron restriction reproducibly caused
60% decreases in both mitochondrial and cytosolic aconitase
activities in erythroid samples (P ⬍ .05) but no significant changes
in megakaryocytic or granulocytic enzyme activities (Figure 2AB). The protein levels of mitochondrial aconitase remained unaffected by erythroid iron restriction (Figure 2A), despite the
pronounced decrease in enzymatic activity. Cytosolic aconitase
protein was below the limit of immunoblot detection in these cells.
Consistent with the decrease in aconitase activities, iron-restricted
BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
erythroblasts showed a 3-fold decrease in intracellular isocitrate
levels (P ⫽ .02) (supplemental Figure 2A). In 2 of 2 experiments,
increased intracellular citrate occurred with iron restriction, but this
finding was not statistically significant because of the variable
magnitude of the change (supplemental Figure 2B). Concomitant
activation of ATP-citrate-lyase (ACL), which promotes metabolic
shunting of citrate to acetyl-CoA, may have prevented major citrate
buildup during iron deprivation (supplemental Figure 2C). We also
examined the activities of isocitrate dehydrogenase (IDH) and
succinate dehydrogenase, enzymes which act downstream of
aconitase. With the use of gel-based enzymography, 2 NADPdependent and no NAD-dependent IDH activities were identified in
erythroid cells, neither of which diminished with iron restriction; in
fact, the upper species/activity appeared slightly increased with
iron restriction (supplemental Figure 2E). succinate dehydrogenase, which contains an iron-sulfur cluster, showed no change in
activity due to iron restriction (supplemental Figure 2F).
To determine whether loss of aconitase activity could contribute
to impairment in erythropoiesis, erythroid cultures with 100%
transferrin saturation underwent treatment with the active site
inhibitor fluorocitrate. In vivo and in vitro pharmacologic studies,
as well as x-ray crystallography, have all established aconitase
enzymes as the main target of fluorocitrate action.29 The mechanism of inhibition occurs through conversion of fluorocitrate to
4-hydroxy-trans-aconitate, which then stably occupies the active
site; however, at high levels fluorocitrate may also block citrate
transport.30 Fluorocitrate treatment reproducibly blocked erythroid
Figure 2. Implication of aconitase activity in iron regulation of erythropoiesis. (A) Erythroid-specific aconitase inactivation during iron restriction. Enzymography (Acon
Activity) was performed on extracts from day 4 cultures in erythroid (Ery), megakaryocytic (Mk), or granulocytic (Gran) media with 100% or 15% transferrin saturation; controls
consisted of K562 cells expressing shRNAs targeting ACO2 or ACO1. Expression of mitochondrial aconitase protein in samples (bottom panel, IB: ACO2). (B) Summary of
3 independent experiments as in panel A. The y-axis is the ratio of scanned signals obtained with 15% transferrin saturation to those obtained with 100% transferrin saturation,
expressed as mean percentage ⫾ SEM. **P ⬍ .001 for Ery ACO1 changes and 0.03 for Ery ACO2 changes. Mk and Gran signals showed no significant changes. (C) Direct
aconitase inhibition impairs erythroid differentiation in a manner similar to iron restriction. Erythroid cultures with 100% transferrin saturation were supplemented with
fluorocitrate (FC) and underwent FACS assessment of differentiation (top panels) and viability (forward [FSC] and side scatter [SSC] in bottom panels) on day 5. Where
indicated, 10mM citrate was also included in culture. (D) Summary of 3 independent experiments as in panel C, using 200␮M fluorocitrate.
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BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
maturation in a dosage-dependent manner and was reversed by
inclusion of excess citrate (Figure 2C-D). Although the highest
dose of fluorocitrate (200␮M) also diminished cellular viability by
approximately 23%, this effect was less than the differentiation
block of approximately 50%. Concerns about nonspecific toxicity
due to residual barium in our fluorocitrate preparation also
prompted us to test the effects of fluoroacetate, provided as a highly
pure, ready-to-use sodium salt. Fluoroacetate, which undergoes
conversion in cells to fluorocitrate, potently blocked erythroid
differentiation and caused no decrease in cell viability (supplemental Figure 3A). Therefore, the effects of aconitase inhibition on
erythroid differentiation are not attributable to nonspecific toxicity.
To determine whether ACO1 contributed to erythroid maturation, we compared the responses of wild-type and knockout
mice31 to phenylhydrazine-induced hemolytic anemia, with no
differences observed. Therefore, the role of ACO2 was assessed
in erythroid progenitors in high-iron cultures with the use of
shRNA targeting. ACO2 knockdown that used the retroviral
construct from Figure 2A caused a reproducible albeit modest
block in GPA expression (supplemental Figure 3B-C). Attempts
at combined shRNA knockdown of both enzymes, followed by
standard culture in normal erythroid medium with adequate iron
and no exogenous isocitrate, yielded too few transfectants for
meaningful analysis.
Isocitrate reversal of the erythroid iron-restriction response
In an attempt to bypass aconitase defects associated with iron
deprivation, erythroid cultures were supplemented with exogenous
DL-isocitrate at dosages similar to those routinely used for
short-chain fatty acid induction of fetal hemoglobin in erythroid
progenitors.32 Strikingly, DL-isocitrate reversed all of the phenotypic features of iron-restricted erythropoiesis, including abnormalities in the GPA/CD41 ratio, cellular structure, hemoglobinization,
and globin expression (Figure 3A-E). The 5-mM dose reproducibly
restored erythroid GPA expression to 84% of the levels seen with
100% transferrin saturation (Figure 3B). Maximal rescue of
erythropoiesis, with essentially complete restoration of all parameters, was observed with 20mM isocitrate. Importantly, DLisocitrate did not reverse the decline in ferritin levels associated
with iron restriction (Figure 3E), suggesting that it does not affect
cellular iron stores. However, ferritin levels may also be regulated
at the transcriptional level. Therefore, direct assays of IRP activity
were conducted as well. Analysis of IRP activity showed that 15%
transferrin saturation activated both IRP1 and IRP2 (Figure 3F-G).
Interestingly, DL-isocitrate reversed the activation of IRP1 by
erythroid iron restriction but had minimal effects on activity or
expression of IRP2, the more reliable sensor of intracellular iron.31
Trace iron contamination of the isocitrate was also ruled out by
mass spectrometry and atomic emission spectroscopy. Furthermore, in erythroid cultures with limiting amounts of Epo,
isocitrate failed to reverse the effects of iron restriction,
confirming that its action occurs independently of iron delivery
to cells (see Figure 5A-B). To rule out potential artifacts caused
by isocitrate chelation of extracellular calcium, cells were
treated with the extracellular calcium chelator EGTA (ethylene
glycol tetraacetic acid), which showed no effects at doses
ranging from 5␮M to 1.5mM (not shown). As an additional
specificity control, cells underwent treatment with citrate.
Unlike isocitrate, citrate at 20mM exacerbated the effects of iron
restriction (supplemental Figure 4).
IRON REGULATION OF ERYTHROPOIESIS BY ACONITASE
101
Regulation of erythropoiesis by isocitrate: metabolic and
nonmetabolic pathways
A possible mechanism for isocitrate regulation of erythropoiesis
consists of its metabolic processing through the Krebs cycle to
yield ATP, NAD(P)H, and succinyl CoA. Succinyl CoA provides a
critical building block for heme synthesis through conjugation with
glycine by ALAS-2, an erythroid enzyme deficient in patients with
X-linked sideroblastic anemia.33 Evidence against such a mechanism was provided by analysis of cellular ATP, NADH, and
NADPH levels, none of which showed significant change as a
function of iron restriction or isocitrate rescue (Figure 4A). AMP
kinase activation, which reflects cellular energy balance, was not
enhanced by iron restriction; in addition, intracellular AMP levels
were not increased (supplemental Figure 5A-B). Furthermore,
exogenous ␣-ketoglutarate, provided as the bioactive cellpermeable ester TaKG,21 failed to reverse the erythroid ironrestriction response (supplemental Figure 5C). TaKG, like exogenous hemin, induced erythroid differentiation in K562 cells,
consistent with its uptake and metabolism to heme (supplemental
Figure 5D). As additional evidence suggesting a nonmetabolic
mechanism of action, isocitrate, but not hemin, relieved an
erythroid-differentiation blockade caused by chloramphenicol, an
inhibitor of mitochondrial biogenesis (supplemental Figure 5E-F).
Finally, enantiopure preparations of D- and L-isocitrate were
compared for rescue of iron-restricted erythropoiesis. Only
D-isocitrate can be metabolized by isocitrate dehydrogenase.34
Surprisingly, L-isocitrate restored cellular viability with an efficiency equivalent to that of D-isocitrate (Figure 4B). L-isocitrate
also rescued erythroid differentiation (Figure 4C), albeit not as
efficiently as D-isocitrate. In aggregate, these results suggest that
isocitrate regulation of erythropoiesis may occur, at least in part,
through a nonmetabolic mechanism.
Coupling of iron and isocitrate to Epo modulation of PKC
To determine the involvement of Epo signaling in isocitrate
regulation of erythropoiesis, rescue experiments as in Figures 3 and
4 were conducted with a variety of Epo doses. Notably, a
concentration of 0.05 U/mL Epo still permitted partial erythroid
differentiation under iron-replete conditions but completely precluded isocitrate rescue of differentiation under iron restriction
(Figure 5A-B). In the course of screening Epo-associated signal
transduction pathways, an association was identified between
responsiveness to isocitrate and the status of PKC␣/␤ activation
(T638 phosphorylation). In particular, in cultures with high levels
of Epo (4.5 U/mL), iron restriction induced PKC␣/␤ hyperactivation, which was reversed by inclusion of isocitrate (immunoblot in
Figure 5C; scanning densitometry in supplemental Figure 6). By
contrast, cells cultured with low levels of Epo (0.05 U/mL) showed
PKC␣/␤ hyperactivation regardless of iron or isocitrate levels.
These findings were highly reproducible in 3 independent experiments and were specific for PKC␣/␤. Most other PKC isozymes
such as ␪ (Figure 5C), ⑀ (supplemental Figure 8A), ␩ (not shown),
and ␭ (not shown) showed no changes in activation as a function of
iron, isocitrate, or Epo levels. Two notable exceptions consisted of
PKC␦, which was consistently hyperactivated by iron restriction
but unaffected by Epo deprivation, and PKC␮, whose activation
diminished with Epo deprivation but not with iron restriction
(supplemental Figure 8A). These assays cannot distinguish between activation of the highly homologous PKC␣ and PKC␤
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BULLOCK et al
BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
Figure 3. Isocitrate abrogation of the erythroid ironrestriction checkpoint. (A) Isocitrate (IC) reverses defects
in differentiation and viability. Five-day erythroid cultures
with 15% transferrin saturation included trisodium isocitrate
(IC) at 5mM. FACS analysis of GPA and CD41 expression
with gating on viable fraction. (B) Summary of 3 independent experiments as in panel A. (C) Isocitrate reverses
structural changes associated with iron restriction. Light
microscopy of Wright-stained cytospins from cultures in
panel A (400⫻ magnification). Images were acquired with
the use of an Olympus BX51 microscope equipped with an
Olympus DP70 digital camera. The objective lens consisted
of Uplan Fl 40⫻/0.75 NA. Image acquisition and processing
used Adobe Photoshop, CS3/10.0 and CS2/9.0, respectively. (D) Isocitrate enhances hemoglobinization. Erythroid
cultures with 100% or 15% transferrin saturation were
supplemented with 20mM isocitrate. Photograph of day 5
cell pellets. (E) Isocitrate augments globin chain expression. Whole cell lysates from panel D underwent immunoblotting. (F) IRE-binding activity of IRP1 and IRP2: influences of transferrin saturation and isocitrate. RNA gel-shift
assays were performed on extracts from day 4 CD34⫹
cultures in erythroid medium with 100% or 15% transferrin
saturation and 20mM isocitrate. (G) IRP2 stabilization by
iron deprivation: minimal effect of isocitrate. Cellular extracts from panel F underwent immunoblotting. FSC indicates forward scatter; and SSC, side scatter.
isoforms, but only the expression of the former protein could be
ascertained by immunoblot.
Time course analysis determined whether the kinetics of
PKC␣/␤ hyperactivation were consistent with a causal role in the
erythroid iron-restriction response. Notably, PKC␣/␤ hyperactivation occurred 2 days after initiating iron restriction and persisted
through day 4 (immunoblot in Figure 5D; scanning densitometry in
supplemental Figure 7). The phenotypic response was first manifested between days 3 and 4 (Figure 1A-C). Therefore, PKC␣/␤
hyperactivation preceded the growth, viability, and differentiation
effects of iron restriction. To further examine the role of PKC␣/␤
hyperactivation, iron-restricted cells underwent treatment with
0.5␮M bisindolylmaleimide I (BIM), a regimen that inhibits PKC
but is not isozyme selective.35 Remarkably, BIM treatment completely reversed the decline in cellular viability associated with iron
restriction (Figure 5E-F). With regard to differentiation, BIM
diminished the proportion of megakaryocytic cells but did not alleviate
the block in erythroid differentiation. To address concerns that BIM
might also inhibit PKC isozymes that support erythroid differentiation,
for example, PKC⑀,36,37 similar experiments were conducted with a
more selective inhibitor, Gö6983, which targets classical isozymes as
does its congener Gö6976 but also spares PKC␮.38 Gö6983 (0.5␮M)
significantly restored erythroid differentiation (P ⫽ .01) and diminished
megakaryocytic differentiation (P ⫽ .003) in the iron-restricted cultures
(Figure 5G). These results show an Epo-signaling requirement for
isocitrate rescue of erythroid differentiation under conditions of iron
restriction and specifically implicate signals involved in the downmodulation of PKC␣/␤ and ␦ activation. Failure of this downmodulation may contribute, at least in part, to the phenotypic features of
the erythroid iron-deprivation response.
To more specifically examine the contribution of PKC␣ to the
erythroid iron-deprivation response, lentiviral shRNA constructs
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IRON REGULATION OF ERYTHROPOIESIS BY ACONITASE
103
Figure 4. Participation of a nonmetabolic isocitrate signaling pathway. (A) Cellular ATP, NADH, and NADPH levels in day 5 erythroid cultures. Shown are means ⫾ SEMs
for 3 independent experiments. (B) Viability effects of L- versus D-isocitrate in erythroid iron restriction. Erythroid cultures received 1mM, 5mM, and 10mM isocitrate (IC)
enantiomers, with FACS analysis on day 5. Graph summarizes 3 independent experiments with the use of 10mM isocitrate. **P ⫽ .012 for D-IC ⫹ 15% versus 15% and 0.017
for L-IC ⫹ 15% versus 15%. No significant difference was seen for D-IC ⫹ 15% versus L-IC ⫹ 15%. (C) Effects of L- versus D-isocitrate on erythroid differentiation. FACS
analysis of GPA and CD41 expression in cultures from panel B, with gating on viable fraction. **P ⫽ .04 for D-IC ⫹ 15% versus 15%; *P ⫽ .05 for L-IC ⫹ 15% versus 15%. In
addition, P ⫽ .05 for D-IC ⫹ 15% versus L-IC ⫹ 15%.
were used to knock down expression in primary erythroid progenitors. As shown in supplemental Figure 8B, 1 of 3 constructs used,
sh 692, effectively knocked down erythroid PKC␣ expression. The
knockdown of PKC␣ did not have a major effect on GPA
expression during iron deprivation (supplemental Figure 8C).
These results suggest functional redundancy in which PKC␤ and
PKC␦ may also contribute to the iron-deprivation response.
Influence of isocitrate on erythropoiesis in vivo
In normal adult C57BL/6 mice, intraperitoneal injection of a
trisodium DL-isocitrate solution at 200 g/kg/d for 5 consecutive
days produced no evidence of toxicity on the basis of signs/
symptoms and necropsy studies. Therefore, this regimen was tested
in a murine model of dietary iron deficiency anemia. In this model,
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104
BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
BULLOCK et al
Figure 5. Crosstalk of iron and isocitrate with Epo signaling: PKC␣/␤ as common target. (A-B) Epo levels influence isocitrate rescue. FACS analysis of 5 day cultures
with indicated doses of Epo plus 25 ng/mL SCF; 100% or 15% transferrin, and 20mM isocitrate (IC) were included as indicated. Graph summarizes results of 3 independent
experiments. (C) Iron restriction and Epo deprivation both induce PKC␣/␤ hyperactivation. Epo levels influence the capacity of isocitrate to reverse the hyperactivation. Whole
cell lysates from 4 day erythroid cultures underwent immunoblotting. (D) Kinetics of PKC␣/␤ hyperactivation associated with erythroid iron restriction. (E-F) Complete reversal
of viability defects by pan-PKC inhibitor. Erythroid cultures with 100% or 15% transferrin were treated with 0.5␮M BIM, with FACS analysis on day 5. Graph represents
3 independent experiments. (G) Partial reversal of differentiation defects with selective PKC inhibitor. Cultures as in panel E were treated with either 0.5␮M BIM or with 0.5␮M
Gö6983. Graphs summarize 3 independent experiments. DN indicates GPA⫺ CD41⫺ double-negative cells. **P ⫽ .02 for DN percentage in BIM ⫹ 15% versus 15%. **P ⫽ .01
for GPA percentage in Gö6983 ⫹ 15% versus 15%.
male C57BL/6 weanlings placed on a low-iron diet reproducibly
developed a progressive anemia that began approximately between
days 30 and 45 of the diet. In 2 independent experiments, isocitrate
treatment completely reversed the progression of anemia, as
assessed by red blood cell number and hematocrit (Figure 6).
Examination of full hematologic indexes (supplemental Table 3)
indicated that the stimulatory effects of isocitrate were essentially
confined to these parameters. The lack of increase in mean
corpuscular volume and the decline in mean corpuscular hemoglobin concentration may have resulted from isocitrate driving red cell
production in the face of ever-decreasing iron stores. To address the
possibility that isocitrate might be acting as a nonspecific energy
source, an additional experiment compared equivalent doses of
isocitrate and ␣-ketoglutarate in mice with iron deficiency anemia.
In contrast to results obtained with isocitrate, progression of
anemia was unaffected by ␣-ketoglutarate administration (Figure
6). Administration of exogenous citrate to mice has not been
performed because of the potential for its conversion to isocitrate
by nonerythroid marrow cells.
Discussion
Iron regulation of erythroid differentiation provides a checkpoint to
prevent inappropriate iron utilization when body stores are limited.
In the absence of such a checkpoint, erythropoiesis could potentially divert iron from vital functions in other tissues.15 To function
in a protective manner, this checkpoint must act at stages before
Epo-mediated expansion of the progenitor pool and subsequent
hemoglobinization. In addition, it must act in a rheostatic manner to
calibrate erythropoietic output to iron availability.
Comparison of iron deficiency anemia with other human disorders of hemoglobin synthesis highlights some distinctive features
of the erythroid iron-restriction pathway. Primary disorders of
globin and heme synthesis, for example, thalassemias and ALAS-2
deficiency, display erythroid hyperplasia in the bone marrow.33,39
By contrast, marrows from patients with iron deficiency anemia
lack erythroid hyperplasia,40 despite markedly increased (⬃ 4-fold)
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BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
IRON REGULATION OF ERYTHROPOIESIS BY ACONITASE
105
Figure 6. Influence of isocitrate on erythropoiesis in vivo.
Isocitrate (IC) inhibits anemia progression during iron deprivation. Peripheral red blood cell (RBC) number and hematocrit
(HCT) in mice on iron-deficient diets. Treatment with isocitrate
(dashed lines) versus either normal saline (NS) or ␣-ketoglutarate
(␣KG; solid lines) was initiated on days indicated by arrows.
Each point represents mean ⫾ SEM for 6 mice/group. **Experiment 1 day 54, isocitrate versus saline, P values are .007 and
.001 for RBC number and HCT. ##Experiment 2 day 45,
isocitrate versus saline, P values are .015 and .018 for
RBC number and HCT. †Comparison of isocitrate with ␣ketoglutarate, day 56, P values are .044 and .046 for RBC
number and HCT.
serum Epo levels.41 Furthermore, defective conversion of protoporphyrin IX to heme by ferrochelatase occurs in both iron deficiency
anemia and erythropoietic protoporphyria, but only a minority of
patients with erythropoietic protoporphyria develop anemia.42
These clinical distinctions imply that iron restriction of erythropoiesis in humans does not result simply from defects in globin or
heme production.
The clinical relevance of this regulatory pathway derives from its
role in anemias associated with inflammation, renal disease, malignancy, and myelodysplasia, in which defects in iron uptake or processing render erythroid progenitors resistant to Epo stimulation.1,43 In these
anemias, treatment of patients with intravenous iron may partially
restore Epo responsiveness, but this measure cannot be applied over the
long term because of risks of iron overloading. Of additional clinical
importance, this regulatory pathway has been exploited in the therapy of
polycythemia vera, in which purposeful induction of iron deficiency by
phlebotomy remains the treatment of choice in restraining overactive
erythropoiesis.44
In the current studies, the human progenitor model captured
several key features of in vivo iron-restricted erythropoiesis, that is,
lineage specificity, inhibition of early differentiation, and growth
suppression, while lacking features of the more generic cellular
iron-depletion response, that is, the G1 arrest and apoptosis seen
with iron chelation. The growth inhibition occurred in the absence
of phase-specific arrest and may result from generalized slowing of
the entire cell division cycle, as has recently been described in
erythroid progenitors treated with desferrioxamine.45 Interestingly,
the threshold of transferrin saturation required to achieve this
effect, 15%, correlates with the anemic threshold seen in patients
with iron deficiency.46 Normal transferrin concentrations in human
serum (2-3 mg/mL) greatly exceed those in our cell culture system
(0.05 mg/mL), but differences in bioavailability, matrix compositions, target cell densities, and competing tissue demands complicate in vivo versus in vitro comparisons of absolute levels.
However, using percentage saturation as a point of comparison may
conceivably correct for many of these differences.
Several distinct approaches have implicated aconitase activity as a
control element within this pathway. First, the aconitase enzymes
underwent inactivation in an erythroid-selective manner in response to
15% transferrin saturation. The protein levels of ACO2 were not
significantly affected by iron restriction, indicating a posttranslational
mechanism rather than IRE-mediated translational control.16,47 Why
ACO2 protein levels did not decrease with iron restriction is not clear,
but one possibility is that its IRE may require a greater degree of cellular
iron depletion than was induced by our iron restriction. Second,
fluorocitrate inhibition of aconitase activity elicited a response similar to
that of iron restriction, that is, inhibition of erythroid growth and
differentiation. Attempts to determine which of the 2 isoenzymes
regulates erythropoiesis did not yield definitive results. ACO1 knockout
had no effect on steady-state or stress erythropoiesis in mice, and ACO2
knockdown caused only partial inhibition of GPA up-regulation in
human CD34⫹ cells. One possibility is that the 2 isoenzymes may
function semiredundantly,ACO2 perhaps contributing more thanACO1.
However, another probable possibility, discussed in Figure 7, is that the
simple loss of enzymatic function may not fully explain the role of
aconitase in the erythroid response to iron restriction. The mechanism
for aconitase inactivation during iron restriction may result from
destabilization of the iron-sulfur clusters within the active sites, a notion
supported by the activation of IRP1 RNA binding activity associated
with erythroid iron restriction (Figure 3F).
A potential confounding factor is that iron deprivation alters the
cellular composition of the cultures, and this alteration could lead
in a secondary manner to diminished aconitase activity. Several
lines of evidence argue against this possibility. First, the aconitase
assays were conducted on day 4 of culture, a point at which the
megakaryocytic composition differs only by 20% in iron-replete
versus iron-restricted cultures (Figure 1B). Second, erythroid
aconitase activity does not increase as a function of cellular
maturation (see supplemental Figure 2D), ruling out a role for the
maturation block caused by iron restriction. Third, ample precedent
exists for direct inactivation of cellular aconitase by iron deprivation.
Additional evidence implicating aconitase in the erythroid
response to iron restriction is the striking ability of isocitrate to
abrogate this response in vitro and in vivo. The mechanism
underlying this phenomenon most probably involves both nonmetabolic and metabolic pathways. The participation of a nonmetabolic
pathway is suggested by the ability of the metabolically inert
L-isomer to completely rescue viability and partially rescue
differentiation. The ability of D-isocitrate to contribute to heme
biosynthesis may account for its greater potency in restoring
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106
BULLOCK et al
erythroid differentiation. However, in addition to direct metabolic
conversion, isocitrate could also contribute to heme synthesis in an
indirect manner by restoring the expression of ALAS-2 (eALAS) in
iron-deprived erythroid cells (see Figure 5C). That isocitrate
metabolism alone is insufficient is suggested by the lack of rescue
by ␣-ketoglutarate in vitro and in vivo. The prevention of IRP1
activation by isocitrate in Figure 3F is consistent with an ability to
enter cells, bind aconitase, and stabilize active site iron-sulfur
clusters, in keeping with described substrate protective effects.48
Figure 7 provides a model in which aconitase/isocitrate integrates signals from Epo, iron levels, and potentially other stimuli.
With their active-site iron-sulfur clusters, aconitases may function
as sensors for iron levels. Their production of isocitrate contributes
to erythropoiesis in part through the metabolic generation of heme
(Metabolic Flux Pathway). During iron restriction (Figure 7B),
destabilization of the iron-sulfur cluster (see Figure 3F) is predicted
to induce conformational changes and enhanced assembly of
signaling complexes that include PKC. Along these lines, several
recent studies have provided evidence for aconitase-kinase interactions, including ACO2 binding to conventional PKC.17,49,50 Provision of exogenous isocitrate (Figure 7C) has 3 predicted consequences that are nonmutually exclusive. The first, bypassing of the
metabolic blockade, may permit heme biosynthesis but does not
suffice to reverse the iron-restriction response, as discussed earlier.
BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
The second, stabilization of the iron-sulfur cluster (see Figure 3F),
is predicted to restore aconitase to its “high iron” conformation and
thereby dissociate the PKC-associated signaling complexes whose
hyperactivation impairs Epo responsiveness. The third, binding to
IDH, is probably relevant because L-isocitrate, although incapable
of undergoing metabolic conversion, still retains the capacity for
IDH binding.34 IDH has also been identified in complexes with
signal-transducing proteins49 and represents an excellent candidate
for participating, along with aconitase and PKC, in an ironrestriction signalosome, a possibility currently under investigation.
Acknowledgments
We thank Luke Szweda (Oklahoma Medical Research Foundation)
for providing antibodies, Eyal Gottlieb and Dan Tennant (Beatson
Institute for Cancer Research) for providing the compound TaKG,
and Klaus Ley and the University of Virginia Cardiovascular
Research Center for the use of the Hemavet analyzer.
This work was supported by a Ruth L. Kirschstein National
Research Service Award, by the National Institutes of Health (grant
F32 HL0860046), and by a University of Virginia K12 Clinical
Translational Career Development Award (G.C.B.); by the University of Virginia Cancer Center through the James and Rebecca
Figure 7. Model for erythropoietic regulation by an ironaconitase-isocitrate pathway. In the absence of iron restriction
(A; ⬎ 15% Transferrin Saturation) aconitase enzymes possess
intact iron-sulfur clusters and function mainly in the conversion of
citrate to isocitrate by the Metabolic Flux Pathway. In the
presence of iron restriction (B; 15% Transferrin Saturation),
destabilization of the aconitase iron-sulfur clusters induces assembly of a repressive signalosome which may act in part through
PKC hyperactivation. In addition, diminished metabolic flux may
compromise heme production and lead to shunting of citrate by
activated ATP-citrate-lyase (P-ACL) to oxaloacetate (OAA) and
acetyl-CoA. Isocitrate rescue (C; 15% Transferrin Saturation with
Exogenous Isocitrate) may prevent assembly of a repressive
signalosome by stabilization of aconitase iron-sulfur clusters and
by binding to isocitrate dehydrogenase (IDH) enzymes. In addition, exogenous D- but not L-isocitrate may support heme
biosynthesis. However, exogenous isocitrate does not prevent
shunting of citrate by activated ATP-citrate-lyase.
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BLOOD, 8 JULY 2010 䡠 VOLUME 116, NUMBER 1
IRON REGULATION OF ERYTHROPOIESIS BY ACONITASE
Craig Cancer Research Scholars Award and the NCI Cancer Center
Support (grant P30 CA44579), by the Biomedical Innovation Fund
of the Ivy Foundation (J.J.C.); by the National Institute of Diabetes
and Digestive and Kidney Diseases (grant R01 DK079924; A.N.G.);
and by the Roche Foundation for Anemia Research.
Authorship
Contribution: G.C.B. designed the research, performed experiments, and wrote the manuscript; L.L.D., A.-L.T. and S.L.G.
107
designed the research, performed experiments, and analyzed data;
W.-H.T. and T.A.R. designed, performed, and interpreted studies of
IRP1 and IRP2; B.D. and J.M.M. designed, performed, and
interpreted NMR metabolomic studies; J.J.C. synthesized and
analyzed vital new reagents (enantiopure isocitrates); and A.N.G.
designed the research, analyzed data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Adam N. Goldfarb, University of Virginia
School of Medicine, PO Box 800904, Charlottesville, VA 22908;
e-mail: [email protected].
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2010 116: 97-108
doi:10.1182/blood-2009-10-251496 originally published
online April 20, 2010
Iron control of erythroid development by a novel aconitase-associated
regulatory pathway
Grant C. Bullock, Lorrie L. Delehanty, Anne-Laure Talbot, Sara L. Gonias, Wing-Hang Tong, Tracey
A. Rouault, Brian Dewar, Jeffrey M. Macdonald, Jason J. Chruma and Adam N. Goldfarb
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