Biochem. J. (2013) 451, 61–67 (Printed in Great Britain)
61
doi:10.1042/BJ20121649
Nitrogen monoxide inhibits haem synthesis in mouse reticulocytes
Marc R. MIKHAEL*†, Shan SOE-LIN*‡, Sameer APTE* and Prem PONKA*†‡1
*Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Côte Ste-Catherine Road, Montréal, QC, Canada, H3T 1E2, †Department of Physiology, 3655 Promenade Sir
William Osler, Montréal, QC, Canada, H3G 1Y6, and ‡Department of Experimental Medicine, McGill University, Lady Meredith House, 1110 Pine Avenue West, Montréal, QC, Canada,
H3A 1A3
AI (anaemia of inflammation) often manifests in patients with
chronic immune activation due to cancer, chronic infections,
autoimmune disorders, rheumatoid arthritis and other diseases.
The pathogenesis of AI is complex and involves cytokinemediated inhibition of erythropoiesis, insufficient erythropoietin
production and diminished sensitivity of erythroid progenitors to
this hormone, and retention of iron in haemoglobin-processing
macrophages. NO (nitric oxide) is a gaseous molecule produced
by activated macrophages that has been identified as having
numerous effects on iron metabolism. In the present study,
we explore the possibility that NO affects iron metabolism in
reticulocytes and our results suggest that NO may also contribute
to AI. We treated reticulocytes with the NO donor SNP (sodium
nitroprusside). The results indicate that NO inhibits haem
synthesis dramatically and rapidly at the level of erythroidspecific 5-aminolaevulinic acid synthase 2, which catalyses the
first step of haem synthesis in erythroid cells. We also show that
NO leads to the inhibition of iron uptake via the Tf (transferrin)–
Tf receptor pathway. In addition, NO also causes an increase in
eIF2α (eukaryotic initiation factor 2α) phosphorylation levels and
decreases globin translation. The profound impairment of haem
synthesis, iron uptake and globin translation in reticulocytes by
NO raises the possibility that this gas may also contribute to AI.
INTRODUCTION
proliferation [8,9]. In addition, cytokine-mediated inhibition
of erythroblast proliferation and differentiation and reduced
sensitivity of erythroblasts to erythropoietin have also been
identified as important contributing factors leading to inefficient
erythropoiesis in AI patients [5].
Activated macrophages are also known to produce enormous
amounts of the diatomic gas NO (nitric oxide) via the induction
of iNOS (inducible NO synthase) [10–13]. Interestingly, NO has
been implicated in the inhibition of ALA-S (5-aminolaevulunic
acid synthase) [14–16], the first enzyme of haem synthesis and
ferrochelatase [17–19] and the last enzyme of haem biosynthesis.
In addition, NO is also known to increase the synthesis of
ferritin, the major iron-storing protein [5,20–25], thus possibly
contributing to RES iron retention in AI.
Haem is a key regulator of protein translation in erythroid
cells, exercising its regulatory effects via the activity of eIF2α
(eukaryotic initiation factor 2α) kinase, also known as HRI (haemregulated inhibitor) [26,27]. When active, HRI phosphorylates
eIF2α, thereby inactivating it which, in turn, leads to translational
inhibition [27]. In the presence of haem, however, haem binds
and inactivates HRI [27] thus maintaining eIF2α activity (i.e.
dephosphorylated eIF2α) and ensuring that globin translation
proceeds when haem is present. Interestingly, NO has been shown
to increase eIF2α phosphorylation and inhibit translation [28].
Subsequent research revealed that NO activates HRI [29,30]
thereby inhibiting translation by phosphorylating eIF2α.
We therefore explored the possibility that NO may also
affect reticulocyte iron metabolism and globin translation. In
the present paper, we report that SNP (sodium nitroprusside)
treatment decreases the synthesis of haem and 59 Fe uptake in
Iron is the most abundant transition metal in vertebrate organisms
and is used as a cofactor of numerous proteins involved in
many processes including oxygen transport, energy metabolism
and DNA replication. Although iron is present in all cells in
vertebrates, the erythrocyte’s oxygen transporter haemoglobin is
the most abundant iron-containing protein and contains almost
80 % of the body’s iron [1,2]. Excess iron is stored primarily in
hepatocytes and macrophages of the spleen and liver that comprise
the RES (reticuloendothelial system).
Iron is lost from the organism by non-specific mechanisms
(e.g. cell desquamations) and is compensated for by intestinal
iron absorption [1]. In humans, under normal conditions, the
body’s iron content is maintained by the daily absorbance
of 1 mg of iron by duodenal enterocytes. RES macrophages
phagocytose senescent and damaged erythrocytes. The greatest
flow (∼25 mg/day) of iron therefore occurs from macrophages
of the RES to the circulation where iron is tightly bound to the
principal plasma iron carrier Tf (transferrin), and is then destined
primarily to developing erythrocytes [3,4].
Prolonged activation of reticuloendothelial macrophages can
result in perturbed iron metabolism leading to AI (anaemia of
inflammation), also known as anaemia of chronic disease, a
condition that often arises in patients suffering from chronic
infections, autoimmune diseases, rheumatoid arthritis or cancer
[5]. AI is not the consequence of decreased body iron stores,
but rather the inappropriate retention of iron by the RES
[6,7]; a mechanism that is thought to limit the bioavailability
of iron to pathogens and tumour cells, thus hampering their
Key words: anaemia of infection, haem synthesis, iron
metabolism, nitric oxide (NO), reticulocyte.
Abbreviations used: AI, anaemia of inflammation; ALA, 5-aminolaevulunic acid; ALA-S, ALA synthase; DMEM, Dulbecco’s modified Eagle’s medium;
eIF2α, eukaryotic initiation factor 2-α; eIF2α-P, phosphorylated eIF2α; HRI, haem-regulated inhibitor; iNOS, inducible NO synthase; IRP, iron-regulatory
protein; RES, reticuloendothelial system; SA, succinyl acetone; SIH, salicylaldehyde isonicotinoyl hydrazone; SNAP, S -nitroso-penicillinamine; SNP, sodium
nitroprusside; TCA, trichloroacetic acid; Tf, transferrin; TTBS, Tris-buffered saline containing 0.1 % Tween 20.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
62
M. R. Mikhael and others
reticulocytes incubated with radiolabelled diferric Tf (59 Fe–Tf),
in a time- and concentration-dependent manner. In addition,
the inhibition of haem synthesis by NO can be reversed
by adding ALA (5-aminolaevulinic acid), suggesting that NO
inhibits the activity of ALA-S2 (the erythroid-specific ALAS). NO also decreases 59 Fe incorporation into haem when 59 Fe–
SIH (salicylaldehyde isonicotinoyl hydrazone), an iron-chelator
complex, is administered instead of 59 Fe–Tf. Interestingly, we
found that the effects of NO also depends on the redox form of
NO produced. In addition, SNP treatment of reticulocytes led to
increased levels of eIF2α phosphorylation and decreased globin
translation.
We therefore conclude that NO inhibits haem synthesis at the
levels of ALA-S2, but also exerts additional effects on eIF2α
activity and globin synthesis. We discuss the implication of
these results for AI and the possibility that NO contributes
to the pathophysiology of this disease by adversely affecting
haemoglobinization in developing erythrocytes.
MATERIALS AND METHODS
Western blotting
Cells were lysed in RIPA buffer [50 mM Tris/HCl, 150 mM
NaCl, 1 % Nonidet P40, 0.5 % sodium deoxycholate and 0.1 %
SDS (pH 7.4)] for 30 min at 4 ◦ C. Cell debris was cleared by
centrifugation (9300 g)] and protein concentration was measured
with the Bradford reagent (Bio-Rad Laboratories). Cell lysates
containing 45 μg of protein were resolved by SDS/PAGE (12 %
gel) and proteins were transferred on to nitrocellulose filters.
The blots were saturated with 10 % non-fat dried skimmed
milk powder in TTBS (Tris-buffered saline containing 0.1 %
Tween 20) and probed with 1:1000 dilution anti-eIF2α-P
(phosphorylated eIF2α) antibody (New England Biolabs) and
1:500 dilution anti-β-actin antibody (Sigma) at 4 ◦ C overnight.
After three washes with TTBS, the blots were incubated for
2 h at room temperature (25 ◦ C) with 1:5000 diluted goat anti(rabbit immunoglobulin) (Sigma). Detection of the peroxidasecoupled secondary antibodies was performed using the ECL (GE
Healthcare) method according to the manufacturer’s instructions.
The blots were quantified by densitometry. Representative results
were chosen out of three or more experiments for presentation.
Chemicals
35
DMEM (Dulbecco’s modified Eagle’s medium), penicillin and
streptomycin were obtained from Invitrogen. ALA and haemin
were obtained from Frontiers Scientific, SNAP (S-nitrosopenicillinamine) was from Alexis Biochemicals and [35 S]methionine was from PerkinElmer. Radiolabelled diferric Tf was
made from 59 Fe-Cl3 obtained from PerkinElmer and prepared
as described previously [31,32]. 59 Fe–SIH was generated as
described previously for PIH (pyridoxal isonicotinoyl hydrazone)
[33]. All other chemicals were obtained from Sigma.
Cells were labelled for 2 h with 100 μCi·ml − 1 [35 S]methionine in
DMEM, washed three times with ice-cold PBS, after which they
were lysed with RIPA buffer for 30 min at 4 ◦ C. Samples were then
boiled with SDS loading dye and resolved by using SDS/PAGE
(12.5 % gel). The gel was dried and analysed by autoradiography.
Preparation of reticulocytes
Adult female CD1 mice (Charles River, Saint-Constant,
QC, Canada) were injected intraperitoneally with neutralized
phenylhydrazine at a dose of 50 mg/kg per day for 3 days
continuously. On day 3 or 4, following the last injection, mice
were anaesthetized using 1 ml of 2.5 % avertin, and blood was
collected via cardiac puncture. After three washes with ice-cold
PBS, cells (∼45 % reticulocytes, as determined by new Methylene
Blue staining) were resuspended in DMEM [containing 25 mM
Hepes, 10 mM NaHCO3 and 1 % BSA (pH 7.4)] and incubated at
37 ◦ C with moderate shaking (New Brunswick Scientific) for 2 h.
59
Fe-labelling of reticulocytes
In order to measure and determine the rate of haem synthesis and
total 59 Fe uptake, cells were incubated under various conditions
simultaneously with 1 μM 59 Fe–Tf or 5 μM 59 Fe–SIH, after
which samples were washed twice with ice-cold PBS and
their radioactivity counted in a Packard Cobra gamma counter
(PerkinElmer).
Measurement of [59 Fe]haem
Measurements of [59 Fe]haem levels were carried out by an acid
precipitation method as described previously [34,35]. Briefly,
following incubation with 59 Fe–Tf, reticulocytes were collected,
washed, lysed in distilled water and boiled following the addition
of HCl. [59 Fe]haem containing proteins (mainly haemoglobin)
were precipitated with ice-cold 7 % TCA (trichloroacetic acid)
solution. Samples were washed twice with 7 % TCA and counted
for 59 Fe radioactivity.
c The Authors Journal compilation c 2013 Biochemical Society
S-Metabolic labelling
Data analysis
All results presented are representative of three or more repeated
experiments. All experiments using 59 Fe2 –Tf were carried out
using triplicates for each sample. Error bars represent the S.D.
ImageJ software (NIH) was used for all densitometric analysis.
Statistical significance was calculated always against control
samples using Student’s t test.
RESULTS
NO decreases 59 Fe uptake and incorporation into haem
In order to examine the effects of NO on reticulocyte
iron metabolism, murine reticulocytes were incubated with
the NO donor SNP for different time intervals and at
different concentrations in the presence of 59 Fe–Tf (Figure 1).
Remarkably, 59 Fe incorporation into haem was rapidly and
dramatically inhibited in SNP-treated samples. In addition, low
concentrations of SNP potently reduced 59 Fe incorporation into
haem (Figure 1B). Interestingly, 59 Fe uptake was also reduced
significantly in SNP-treated samples (Figure 1). In order to
exclude the possibility that SNP-decomposition products led
to these observed effects, we also treated reticulocytes with
another NO donor, SNAP. Indeed, treatments of SNAP also
led to decreased 59 Fe incorporation into haem and 59 Fe uptake
in reticulocytes (Figure 1). Both SNP and SNAP produce the
NO redox species NO䊉 and NO + ; however, SNP is known to
produce primarily NO + [36]. These results confirm that NO is
indeed capable of decreasing iron uptake and haem synthesis in
reticulocytes.
NO inhibits haem synthesis at the levels of ALA-S2
NO has previously been reported to inhibit haem synthesis in
various cell types by inactivating ALA-S or ferrochelatase, the
first and last enzyme of the haem synthesis pathway respectively
Nitric oxide inhibits haem synthesis in reticulocytes
63
Figure 1 SNP and SNAP treatment of reticulocytes decreases 59 Fe uptake
and incorporation into haem in a time- and concentration-dependent manner
Reticulocytes were incubated with DMEM incubation medium containing 1 μM 59 Fe–Tf. Cells
were then lysed and, after boiling samples, [59 Fe]haem-containing haemoglobin was precipitated
using TCA. Total cellular 59 Fe levels and 59 Fe levels in haem were measured using a gamma
counter. (A) Reticulocytes were treated with 100 μM SNP or SNAP and collected at different
time intervals. (B) Reticulocytes were treated with increasing concentrations of SNP or SNAP
and collected after 2 h of treatment. Ctrl, control.
[14–19]. Hence, to investigate whether NO affects ALA-S2, we
treated reticuolocytes with SNP and ALA, the product of the first
reaction of haem synthesis catalysed by ALA-S2 in haemoglobinsynthesizing cells, in 59 Fe–Tf-containing medium (Figure 2A),
in order to determine whether NO disrupted haem synthesis
at the level of ALA-S2. When cells were treated with SNP in
the presence of ALA, 59 Fe incorporation into haem was greatly
increased, indicating that NO inhibits haem synthesis at the level
of ALA-S2. However, the inhibition of total 59 Fe uptake by SNP in
reticulocytes was unaffected when ALA was added to the medium.
SA (succinyl acetone) reverses NO-mediated inhibition of 59 Fe
uptake by reticulocytes
Reticulocyte iron uptake from Tf is unique in that it is inhibited
when intracellular levels of ‘uncommitted’ haem are high and
vice versa, and can be experimentally observed in these cells
by the addition of exogenous haem or increasing intracellular
haem levels [37–40]. SA is a known inhibitor of haem synthesis
that exerts its effects by inhibiting ALA dehydratase [33,41], the
second enzyme of the haem synthesis pathway. This indicates that
SA treatment increases iron uptake from Tf in reticulocytes by
decreasing intracellular levels of haem [33].
We therefore hypothesized that the decrease in 59 Fe uptake
observed in NO-treated reticulocytes could be due to an increase
Figure 2 NO-mediated inhibition of haem synthesis and iron uptake is
reversed by ALA and SA respectively
Reticulocytes were incubated in DMEM containing 1 μM 59 Fe–Tf. Cells were then lysed and, after
boiling samples, [59 Fe]haem-containing haemoglobin was precipitated using TCA. Total cellular
59
Fe levels and 59 Fe levels in haem were measured using a gamma counter. (A) Reticulocytes
were treated with 100 μM SNP alone or in the presence of 5 mM ALA for 2 h. (B) Reticulocytes were treated with 100 μM SNP alone or in the presence of 1 mM SA for 2 h. Ctrl, control.
in ‘free’ intracellular ‘uncommitted’ haem levels that occurred
despite reduced haem synthesis. To this end, we co-treated
reticulocytes with SNP and SA in the presence of 59 Fe–Tf and
measured 59 Fe uptake and incorporation into haem (Figure 2B).
Importantly, SA alone dramatically decreased 59 Fe incorporation
into haem and, as expected, slightly elevated 59 Fe uptake. In
addition, co-treatments of SNP and SA further reduced 59 Fe
incorporation into haem. Remarkably, the inhibition of cellular
59
Fe uptake by NO was significantly reversed in NO donor
and SA co-treated reticulocytes, suggesting that NO-mediated
decreases in 59 Fe uptake may be due to a paradoxical increase in
‘uncommitted’ haem levels.
NO inhibits 59 Fe incorporation into haem, but not total uptake in
59
Fe–SIH-labelled cells
In order to further investigate the mechanism of NO mediated
inhibition of 59 Fe uptake in reticulocytes, we labelled cells
with 59 Fe–SIH instead of 59 Fe–Tf in the presence of SNP
(Figure 3). 59 Fe–SIH, which crosses membranes easily and
c The Authors Journal compilation c 2013 Biochemical Society
64
M. R. Mikhael and others
Figure 3 SNP decreases 59 Fe incorporation into haem, but not iron uptake
when 59 Fe–SIH is the source of iron
Reticulocytes were labelled with DMEM containing 5 μM 59 Fe–Tf or 5 μM 59 Fe–SIH in the
presence or absence of 100 μM SNP and collected after 2 h. Total cellular 59 Fe levels and 59 Fe
levels in haem were measured using a gamma counter. Ctrl, control.
efficiently, provides iron for metabolic uses independently of the
Tf–Tf receptor pathway [32]. SNP was found to decrease 59 Fe
incorporation into haem, once again confirming that NO inhibits
the haem synthesis pathway, but total 59 Fe uptake from 59 Fe–SIH.
These results suggest that inhibition of 59 Fe uptake in SNP-treated
reticuloctyes (Figure 1B) is most likely to be due to an inhibition
of iron uptake from Tf.
SNP decreases eIF2α activity and globin synthesis
The translation of globin in erythroid cells is dependent on
haem. Its deficiency inhibits protein synthesis due to activation
of HRI, which is a cAMP-independent kinase that specifically
phosphorylates and inactivates the α-subunit of eIF2. Haem
binding to HRI inhibits the phosphorylation of eIF2α by
Figure 4
HRI, resulting in efficient translation of globin [27]. The
level of haem synthesis regulates globin mRNA translation
in reticulocytes due to haem-mediated regulation of HRI, and
consequently eIF2α activity. We therefore examined eIF2α-P
levels in reticulocytes treated with SNP (Figure 4A). As expected,
haem and SA decreased and increased eIF2α-P levels respectively.
Interestingly, SNP treatments resulted in significant increases in
eIF2α-P levels (Figure 4A).
As eIF2α phosphorylation correlates directly with globin
translation in reticulocytes, we examined the effects of NO on
globin synthesis as well (Figure 4B). As expected, experiments
with reticulocytes labelled with [35 S]methionine revealed that
haemin greatly increases globin synthesis, whereas SA has the
opposite effect. Interestingly, SNP treatments led to a marked
concentration-dependent decrease in globin synthesis (Figure 4B)
that corresponds to increased eIF2α-P levels (Figure 4A). These
results suggest that inhibition of haem synthesis in SNP-treated
reticulocytes (Figure 1) leads to an inhibition of eIF2α activity
(Figure 4A) and, consequently, to a repression of globin synthesis
as well (Figure 4B).
SNP-mediated increase in eIF2α-P levels and decreased globin
synthesis are reversed by ALA co-treatment
In order to further characterize the inhibition of eIF2α activity
(Figure 4A) and globin synthesis (Fig 4B), which occurs in
reticulocytes treated with SNP, we co-treated reticulocytes with
SNP and ALA and examined eIF2α-P levels and globin synthesis
(Figure 5). If the effects of SNP on eIF2α phosphorylation
and globin synthesis are due to inhibited haem synthesis, then
ALA treatments should reverse these effects, indicating that
the decrease in eIF2α activity is haem-dependent. Indeed, the
addition of ALA to SNP-treated samples dramatically reversed
both the increase in eIF2α-P levels and globin synthesis and
markedly decreased eIF2α-P levels and increased translation of
globin (Figure 5). This complete reversal also correlates with 59 Fe
incorporation into haem in reticulocytes co-treated with SNP and
ALA (Figure 2A). These results suggest that the effects of SNP
SNP increases eIF2α-P levels and decreases globin synthesis
(A) eIF2α-P Western blot of reticulocytes incubated in DMEM containing 1 μM non-radioactive Fe–Tf in the presence of various concentrations of SNP, 250 μM haemin or 1 mM SA. Cells were
collected after 2 h of treatment. The histogram represents a densitometric analysis of eIF2α-P levels normalized to β-actin levels (not shown). Mean values for non-control samples are 130, 141,
29 and 154 %. (B) Reticulocytes were incubated in [35 S]methionine containing DMEM with 1 μM non-radioactive Fe–Tf in the presence of various concentrations of SNP, 250 μM haemin or 1 mM
SA. Cells were collected and lysed after 2 h of treatment and analysed using SDS/PAGE and autoradiography. The histogram represents a densitometric analysis of globin synthesis. The radiograph
is of the same gel; however, the last two lanes are rearranged for conceptual reality. The mean values for non-control samples are 68, 56, 55, 200 and 31 %. Ctrl, control.
c The Authors Journal compilation c 2013 Biochemical Society
Nitric oxide inhibits haem synthesis in reticulocytes
Figure 5 ALA reverses the effects of SNP on eIF2α-P levels and globin
synthesis
eIF2α-P Western blot and 35 S–globin SDS/PAGE autoradiography of reticulocytes incubated in
DMEM containing 1 μM non-radioactive Fe–Tf in the presence of 100 μM SNP with or without
5 mM ALA, 5 mM ALA alone or 250 μM haemin. For the globin synthesis experiment, cells were
incubated in the same medium and under the same conditions with [35 S]methionine. Cells
were collected and lysed after 2 h of treatment. The histogram represents a densitometric
analysis of eIF2α-P levels (normalized to β-actin; not shown) and globin synthesis. The mean
values for non-control samples for eIF2α-P are 123, 65, 45 and 24 %, and for 35 S–globin are
74, 146, 187 and 282 %. Ctrl, control.
on eIF2α and globin synthesis are mediated by the reduction of
haem, which is likely to lead to increased HRI activity and thus
translational repression.
DISCUSSION
AI often affects patients with chronic immune activation that
occurs as a result of chronic infections, cancer, autoimmune
disorders and other conditions. AI is caused by at least
three factors: inefficient production of erythropoietin, cytokineand acute-phase protein-mediated inhibition of erythroblast
proliferation and differentiation, and iron retention within the
RES (reviewed by Weiss [5]).
Although macrophages are capable of producing massive of
amounts of NO under immunological stimuli via iNOS [10–13],
little has been done to address the possible effects of such high
levels of macrophage-derived NO on haemoglobinization [42].
Erythrocytes are produced in the bone marrow in close proximity
to macrophages called ‘nurse cells’. Developing erythrocytes
at various stages of differentiation surround these nurse cells
to form erythroblastic islands. The close proximity of these
macrophages to developing erythrocytes highlights the possibility
that macrophage-derived NO can affect the growth and maturation
of erythrocyte precursors [43,44]. Indeed, several studies have
shown that NO has adverse effects on haem synthesis [14–19],
but, to the best of our knowledge, there are no reports on the
effects of nitric oxide on iron metabolism or haem synthesis in
erythroid cells.
65
We therefore examined the effects of the NO donor SNP
on murine reticulocyte haem synthesis, iron uptake and globin
translation. We found that the NO donors SNP and SNAP
rapidly and potently inhibited haem synthesis in reticulocytes
(Figure 1B). Importantly, the concentrations of NO donors used
in the present study to repress haem synthesis are comparable
with those that generate pathophysiological concentrations of NO
in macrophages, i.e. in the micromolar range [12,13].
In order to elucidate the nature of NO-mediated repression
of haem synthesis observed in the present study, we treated
reticulocytes with SNP in the presence of ALA (Figure 2A), the
product of the first reaction of haem synthesis that is catalysed by
ALA-S2 in erythroid cells. The addition of ALA to SNP-treated
cells prevented the inhibition of haem synthesis (Figure 2A) and
restored the percentage of total cellular 59 Fe incorporation into
haem (results not shown) strongly suggesting that NO inhibits
ALA-S2 activity. Indeed, we exclude the transcriptional downregulation of ALA-S2 by NO because reticulocytes do not possess
nuclei. Furthermore, it is unlikely that NO inhibits translation of
ALA-S2 by modulating the activities of the RNA-binding IRPs
(iron-regulatory proteins), because IRP RNA-binding activities
were not significantly altered by SNP or SNAP treatments under
the experimental conditions used in the present study (results not
shown). We hypothesize that NO may inhibit ALA-S2 activity via
S-nitrosylation, a post-translational, widespread and reversible
modification of proteins. S-nitrosylation of specific cysteine
residues by the NO + species, produced by iNOS, SNP and SNAP,
leads to alterations of enzymatic activities [45].
Interestingly, although SNP treatment decreased 59 Fe uptake
from 59 Fe–Tf (Figure 1), the addition of ALA to such cells
did not prevent a decrease in the uptake of 59 Fe (Figure 2A).
However, co-treatments of SNP with SA, an inhibitor of the
second enzyme of haem synthesis, ALA dehydratase, restored
59
Fe uptake levels, although haem synthesis was further inhibited
(Figure 2B). This result is probably due to the fact that
‘uncommitted’ haem in reticulocytes inhibit 59 Fe uptake as was
demonstrated previously by adding exogenous haem or increasing
intracellular haem levels by inhibiting globin translation [37–
40]. In our case, the restoration of 59 Fe uptake during SNP
treatments with SA may be due to the near total elimination
of haem synthesis that is achieved when these combinations
of haem synthesis inhibitors (i.e. SNP and SA) are added to
cells. Therefore we hypothesize that NO inhibits iron uptake by
acting directly on the Fe–Tf-uptake pathway independently of its
effect on haem synthesis. Thus, in our experiments in which we
added SA and SNP together to reticulocytes, haem synthesis is
greatly inhibited so as to sufficiently lower haem levels, thus
eliminating any residual haem-mediated ‘suppression’ of iron
uptake. Interestingly, the marked reduction of 59 Fe incorporation
into haem that occurs in the presence of SNP is distinct from SNP’s
effect on 59 Fe uptake as shown by our experiments using 59 Fe–SIH
(Figure 3).
In reticulocytes, haem is essential for the efficient translation
of globin [46–48], thus co-ordinating protein synthesis with haem
incorporation to form haemoglobin. This is achieved through the
activity of HRI [26]; when haem levels are low, HRI is active
and phosphorylates eIF2α, leading to an inhibition of globin
translation, which is virtually the sole protein that is synthesized in
reticulocytes [48,49]. In contrast, when haem levels are abundant,
haem binding to HRI leads to its inactivation [50], resulting
in the efficient translation of globin. We therefore investigated
the effects of SNP on both HRI phosphoregulation and globin
synthesis. The SNP treatment of reticulocytes led to an increase
in eIF2α phosphorylation, indicating that NO inhibited translation
in these cells. Indeed, when reticulocytes were treated with SNP in
c The Authors Journal compilation c 2013 Biochemical Society
66
M. R. Mikhael and others
[35 S]methionine-containing medium, globin synthesis decreased
(Figure 4B) in accordance with elevated eIF2α-P levels.
Our further investigation revealed that the effects of SNP on
eIF2α phosphorylation and globin synthesis can be prevented by
ALA (Figure 5). These results strongly suggest that SNP-derived
NO exerts its effects on eIF2α and globin synthesis indirectly by
inhibiting haem synthesis, most probably at the level of ALAS2 (Figures 1 and 2A). Thus, in SNP-treated reticulocytes, the
decreased levels of haem (Figure 1) most probably leads to an
activation of HRI and therefore an inhibition of eIF2α activity
(Figure 4A) and globin synthesis (Figure 4B).
In conclusion, the production of NO by activated macrophages
in AI [51] may also be a contributing factor responsible for
anaemia.
AUTHOR CONTRIBUTION
Marc Mikhael carried out all experiments and wrote the paper. Shan Soe-Lin and Sameer
Apte prepared mice for the aforementioned experiments. Marc Mikhael and Prem Ponka
conceived the experiments and edited the paper before submission.
FUNDING
This research was supported by grants from the Canadian Institutes for Health Research
(CIHR) and the Canadian Blood Services (CBS) [grant numbers 2309 and 8314 respectively
(to P.P.)].
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Received 26 October 2012/18 January 2013; accepted 30 January 2013
Published as BJ Immediate Publication 30 January 2013, doi:10.1042/BJ20121649
c The Authors Journal compilation c 2013 Biochemical Society