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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 1059-1066
Nitric Oxide-Mediated Induction of Ferritin Synthesis in J774
Macrophages by Inflammatory Cytokines: Role of Selective Iron
Regulatory Protein-2 Downregulation
By
Stefania Recalcati,
Donatella Taramelli,
Dario Conte, and
Gaetano Cairo
From Cattedra di Gastroenterologia I, Istituto di Scienze Mediche,
IRCCS Ospedale Maggiore, Istituto di Microbiologia Medica,
Università degli Studi di Milano, and Centro di Studio sulla
Patologia Cellulare CNR, Milano, Italy.
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ABSTRACT |
Cytokine-treated macrophages represent a useful model to unravel the
molecular basis of reticuloendothelial (RE) iron retention in
inflammatory conditions. In the present study, we showed that stimulation of murine macrophage J774 cells with interferon
(IFN)- /lipopolysaccharide (LPS) resulted in a nitric
oxide-dependent modulation of the activity of iron regulatory proteins
(IRP)-1 and 2, cytoplasmic proteins which, binding to RNA motifs called
iron responsive elements (IRE), control ferritin translation.
Stimulation with cytokines caused a small increase of IRP-1 activity
and a strong reduction of IRP-2 activity accompanied by increased
ferritin synthesis and accumulation. Cytokines induced only a minor
increase of H chain ferritin mRNA, thus indicating that IRP-2-mediated
posttranscriptional regulation plays a major role in the control of
ferritin expression. This was confirmed by direct demonstration that
the translational repression function of IRP was impaired in stimulated
cells. In fact, translation in cell-free extracts of a reporter
transcript under the control of an IRE sequence was repressed less
efficiently by IRP-containing lysates from cytokine-treated cells than
by lysates from control cells. Our findings throw light on the role of
IRP-2 showing that: (1) this protein responds to a stimulus in opposite
fashion to IRP-1; (2) when abundantly expressed, as in J774 cells,
IRP-2 is sufficient to regulate intracellular iron metabolism in living cells; and (3) by allowing increased ferritin synthesis, IRP-2 may play
a role in the regulation of iron homeostasis in RE cells during
inflammation.
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INTRODUCTION |
IRON METABOLISM and physiology of cells
of the mononuclear phagocyte system are closely connected. Macrophages,
being both storage and recycling compartments of the metal, play a
central role in iron metabolism. Indeed, alterations of iron handling by reticuloendothelial (RE) cells may also be involved in the pathogenesis of disorders of iron metabolism, such as genetic hemochromatosis.1 In turn, modifications of iron
homeostasis are central to the function of RE cells in the inflammatory
response. In fact, diversion of iron from the circulation to RE stores
contributes to the production of reactive oxygen species essential for
macrophage cytotoxic activity and, at the same time, limits iron
availability to invading microorganisms.2
Intracellular iron homeostasis is controlled by iron regulatory
proteins (IRP)-1 and 2, cytosolic proteins that bind iron-responsive elements (IRE) in the untranslated regions of ferritin, the major iron
storage protein, and transferrin receptor (TfR) mRNA, which mediates
iron uptake.3-5 Under conditions of limited iron supply, IRP binding to IRE blocks ferritin mRNA translation and at the same
time stabilizes TfR mRNA. This results in increased cellular iron
availability. Conversely, when cellular iron levels are high, IRP lose
their binding activity, permitting ferritin mRNA translation and
degradation of TfR mRNA, thus lowering iron levels in the free pool. In
this condition, IRP-1 contains a 4Fe-4S cluster and functions as a
cytoplasmic aconitase.6 When iron is scarce, the cluster is
disassembled, aconitase activity is lost, and apo-IRP acquires
RNA-binding activity. IRP-2 lacks the amino acids present at the active
site of mitochondrial aconitase and consequently does not show
enzymatic activity. IRP-1 and IRP-2 bind IRE with similar affinities
and repress ferritin synthesis to the same extent, but their
sensitivity to in vitro reducing agents and their mode of regulation by
iron are different. In addition, they seem to bind distinct sets of RNA
sequences. Furthermore, the varying abundance of the two forms in
different tissues suggests that differential expression of the two IRP
can play an important role in cellular iron metabolism (reviewed in
Hentze and Kuhn,5 Henderson,7 and Harrison and
Arosio8).
In addition to iron levels, other signals such as oxidative stress and
nitric oxide (NO) can modulate the activity of both IRP and thus
influence cellular iron metabolism (reviewed in Hentze and
Kuhn,5 Harrison and Arosio,8 Pantopoulos et
al,9 Drapier and Bouton,10 Rouault and
Klausner,11 Hentze,12 Richardson and
Ponka,13 and Domachowske14). In particular, NO,
which targets metalloproteins, interacts with the 4Fe-4S cluster disrupting aconitase function and, accordingly, enhanced IRE-binding activity after induction of the NO pathway has been observed in several
cellular systems (see Weiss,2 Pantopoulos et
al,9 Drapier and Bouton,10 Richardson and
Ponka,13 and Domachowske14 for review). A
connection between NO, an important mediator of the inflammatory
response, and IRP, the key regulator of iron homeostasis, may have
important pathophysiologic ramifications in RE cells during the
inflammatory response, as also demonstrated by the finding of an
autoregulatory loop between iron metabolism and the NO pathway in
activated macrophages.15 However, the NO-mediated
activation of IRP that has been detected in interferon (IFN)-/lipopolysaccharide (LPS)-treated macrophages16-18
is difficult to reconcile with the modifications of RE iron metabolism
that occur in inflammatory states. In fact, increased IRP activity reduces ferritin synthesis17,18; it is expected that this,
in turn, impairs the iron storage capacity of RE cells, whereas
increased iron retention in the RE system is a well-known feature of
inflammation.2,19
In the present study, we found that treatment of the murine J774
macrophages with cytokines triggers a selective, NO-mediated, downregulation of IRP-2 activity that results in increased synthesis and accumulation of ferritin.
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MATERIALS AND METHODS |
Reagents.
Minimum Essential Medium (MEM), fetal calf serum, ( -32P)
uridine triphosphate (UTP), 32P deoxy cytidine
triphosphate (CTP), 35S methionine and
TRAN35S-LABEL were purchased from ICN Biomedicals (Opera
MI, Italy), desferrioxamine (DFO), NG-monomethyl-L-arginine
monoacetate (L-NMMA), S-nitroso-N-acetyl-D,L-penicillamine (SNAP),
N-acetyl-D,L-penicillamine (NAP), LPS from Escherichia coli
serotype 0111:B4 and ferric ammonium citrate were purchased from Sigma
Chemical Co (Milano, Italy), and antiserum to mouse macrophage
inducible nitric oxide synthase (iNOS) from Alexis Corp (Inalco S.p.A.,
Milano, Italy). Mouse recombinant IFN- and mouse recombinant tumor
necrosis factor (TNF-) were supplied by Genzyme srl. (Cinisello,
Italy), Hybond membranes and ECL Plus were supplied by Amersham Co
(Milano, Italy). The kits for in vitro transcription and wheat germ
extract were purchased from Promega Corp (Firenze, Italy).
Cell cultures and treatments.
The mouse macrophage cell line J774A.1 was grown in MEM supplemented
with 10% heat-inactivated fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin and 0.1 ng/mL streptomycin at 37°C in 5%
CO2. Medium and all reagents were endotoxin-free. Near
confluent cells (1.5 × 106) were stimulated for
different time periods (4 to 24 hours) initially with various
concentrations of LPS (0.1 to 10 µg/mL) and IFN- (10 to 200 U/mL)
and then routinely with 1 µg/mL LPS plus 100 U/mL IFN- in the
presence or absence of 250 µmol/L NMMA or 50 µmol/L DFO. Iron
overload was obtained by culturing cells in the presence of 50 µg/mL
ferric ammonium citrate for 24 hours. Cells were also exposed to 0.5 mmol/L SNAP or NAP for various time periods as described above. At the
end of the treatments cells were harvested, pelletted, and stored at
 80°C. TNF- and nitrite levels in the supernatant were
measured as previously described20 using the Wehi 164 Clone
13 cell line in an MTT tetrazolium cytotoxicity assay and
the Griess reaction, respectively.
Western blot analysis.
Aliquots of the cytosolic extracts used for the determination of IRP
activity containing equal amounts of proteins were electrophoresed in
10% acrylamide-sodium dodecyl sulfate (SDS) gels, electroblotted to
Hybond polyvinylidene difluoride (PVDF) membranes
(Amersham Co) and incubated with a 1:1,000 dilution of antiserum to
mouse iNOS (Alexis Corp). iNOS was detected by chemiluminescence using an immunodetection kit (ECL Plus, Amersham Co) according to the manufacturer's instructions.
Generation of RNA transcripts in vitro.
Probes for bandshift assays were transcribed from linearized pSPT-fer
containing the IRE of human ferritin H chain21 or CG28422 plasmids using T7 RNA polymerase in the presence of -32P UTP using a commercially available kit (Promega
Corp) as previously described.23 Unlabeled
I-12.chloramphenicol acetyl transferase (CAT) and I-19.CAT
mRNA,24 containing, respectively, the wild-type or mutated
ferritin IRE sequence upstream of the chloramphenicol-acetyltransferase (CAT) open reading frame, were transcribed from linearized plasmids in
the presence of m7GpppG using the same kit.
RNA-protein gel retardation assay.
Cells were lysed in the buffer described by Leibold and
Munro,25 the lysate was centrifuged at 16,000g for
5 minutes and the supernatant was used for RNA-protein bandshift
assays. Equal amounts of protein (2 µg as determined using the Bio
Rad protein assay kit) were incubated, in the absence or presence of
2% 2-mercaptoethanol, with a molar excess of IRE probe and treated
sequentially with RNase T1 and heparin as described
previously.23 To detect IRP-2, 10 µg proteins were
incubated with up to 2 × 105 cpm of
32P labeled mutated CG284 probe in the presence of 300 ng
tRNA and 1 mg/mL heparin.22 After separation on 6%
nondenaturing polyacrylamide gels, RNA-protein complexes were
visualized by autoradiography. For quantitation of IRP activity,
radioactivity of bands excised from dried gels was determined by liquid
scintillation counting.
Ferritin synthesis.
At the end of the treatments with cytokines or SNAP, 5 × 106 cells were incubated in methionine and cysteine-free
medium for 2 hours at 37°C in the presence of 50 µCi/mL of
35S methionine plus cysteine (TRAN35S-LABEL,
ICN). Cells were then homogenized in 20 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, and 1 mmol/L
phenylmethylsulfonylfluoride, and equal amounts of labeled proteins
were immunoprecipitated using antibodies against recombinant mouse H
and L ferritin subunits (see below). Immunoprecipitation products were
run on 15% polyacrylamide gels and radioactivity in ferritin was
revealed by fluorography and quantitated by densitometric scanning.
Analysis of ferritin mRNA levels.
Total cellular RNA was isolated as described elsewhere26
and equal amounts of RNA were electrophoresed under denaturing conditions. To confirm that each lane contained equal amounts of total
RNA, the ribosomal RNA content in each lane was estimated in ethidium
bromide-stained gels. RNA was transferred to Hybond-N filters (Amersham
Co), which were sequentially hybridized with the following
32P-labeled DNA probes: rat ferritin L subunit pRLFL3
cDNA,27 rat ferritin H subunit H 1110 cDNA,28
and mouse transferrin receptor cDNA clone pTfR-2.29 For
quantitative determinations, autoradiographic bands in the linear range
were scanned with a densitometer, and the values were calculated after
normalization to the amount of ribosomal RNA.
In vitro translation.
Capped in vitro transcribed RNA (10 ng) was added to wheat germ extract
(Promega Corp) in the presence or absence of different amounts of
cytoplasmic extracts (10 or 20 µg protein) and incubated for 1 hour
at 25°C in the presence of 15 µCi of 35S methionine.
Translation products were analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) and fluorography as described above.
Determination of ferritin content.
The intracellular concentration of H and L subunit rich ferritins was
determined in aliquots of the cytoplasmic extracts used for bandshift
assays using immunoassays based on rabbit polyclonal antibodies against
recombinant mouse H and L ferritins. The same two proteins were used as
standards. The two assays did not give cross-reactivity in the
concentration range used.30
Statistical analysis.
Values are expressed as means ± standard deviation (SD). The
significance of differences was evaluated with t-test using the Stata Statistical Software (Stata Corporation, College Station, TX).
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RESULTS |
NO modulates IRP-1 and IRP-2 activity differentially in
cytokine-treated J774 cells.
In a first series of experiments, we investigated the IRE-binding
activity of IRP in cytoplasmic extracts prepared from mouse macrophage
J774 cells; IRP-2 accounted for 50% or more of the total IRE binding
activity in these cells (Fig 1).
Stimulation with 1 µg/mL LPS plus 100 U/mL IFN- for 24 hours
slightly enhanced IRP-1 activity, but strongly reduced that of IRP-2.
Preliminary experiments (not shown) indicated no differences in
response with doses ranging between 50 and 200 U/mL IFN- and 1 to 10 µg/mL LPS. The extent of activation of IRP-1 and of inhibition of
IRP-2 was somewhat variable as shown by representative RNA-bandshift assays (Figs 1 and 2), but the pattern of response was consistent and
reproducible; Table 1 summarizes the
results of all experiments. As expected on the basis of previous
work,3-5 binding activity of both IRP was reduced in
iron-loaded cells used as a reference (Fig 1A); cytokine treatment
downregulated IRP-2 activity to the same extent as iron overload.
Treatment with cytokines in the presence of the iron chelator DFO
prevented the decrease of IRP-2 activity (Fig 1B) suggesting that
cytokines may act indirectly by increasing intracellular iron levels.
Time course experiments (Fig 2 and Table 1) showed a
prompt activation of IRP-1 and a progressive downregulation of IRP-2.
Differences in IRP-1 binding activity were eliminated by treatment of
cell extracts with 2% 2-mercaptoethanol, which completely activates
IRP-1 activity in vitro,3-5 thus indicating equal loading
of all samples (Figs 1 and 2, lower panels). After cytokine treatment,
increased production of TNF-, a typical marker of inflammatory
response, was consistently observed.
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| Fig 1.
Modulation of IRP-1 and IRP-2 activity in
cytokine-treated macrophage J774 cells. J774 cells were left untreated
or treated for 24 hours with cytokines (100 U/mL IFN- plus 1 µg/mL
LPS); where indicated, the iron chelator DFO (0.05 mmol/L) was also added. Iron loading was obtained by incubating cells with 50 µg/mL ferric ammonium citrate for 24 hours (lane 3). A total of 2 µg protein of cytoplasmic extracts was analyzed for IRE-binding activity by RNA gel retardation assay with excess 32P-labeled RNA
transcribed from the pSPT-fer probe containing the IRE of the ferritin
H mRNA in the absence (upper panels) or presence (lower panels) of 2%
2-mercaptoethanol. TNF- and nitrite production was assayed in the
culture medium as described in Materials and Methods. TNF- units
were calculated from a standard curve with rTNF- used as internal
standard for each test.
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| Fig 2.
Time course of IRP activity in cytokine-treated
macrophage J774 cells. J774 cells were left untreated (lane 1) or
treated for 4 (lane 2) or 24 hours (lanes 3,4) with cytokines (100 U/mL IFN- plus 1 µg/mL LPS); where indicated, the iNOS inhibitor NMMA (0.1 mmol/L) was also present (lane 4). A total of 2 µg protein of
cytoplasmic extracts was analyzed for IRE-binding activity by RNA gel
retardation assay with excess 32P-labeled probe containing
IRE sequences in the absence (upper panel) or presence (lower panel) of
2% 2-mercaptoethanol. TNF- and nitrite production was assayed in
the culture medium as described in the legend to Fig 1.
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Induction of NO production by IFN-/LPS is well
documented,2 indeed, an increased concentration of nitrite
in the culture medium was found in cytokine stimulated J774 cells (Figs
1 and 2). Enhanced levels of NO metabolites were the result of
increased levels of iNOS, as shown by Western blot analysis of cell
extracts (Fig 3). These results suggested the
involvement of the NO pathway in the modulation of IRP activity.
Indeed, the addition of NMMA, an iNOS inhibitor, prevented the
modifications of both IRP-1 and IRP-2 activity induced by stimulation
with cytokines (Fig 2, lane 4). To confirm the data indicating that NO
has a role in modulation of IRP activity, we treated J774 cells with
the NO donor SNAP. The addition of SNAP, but not of its inactive
nonnitrosylated counterpart NAP (data not shown), had a remarkable
effect on IRP activity, similar to that of treatment with IFN-/LPS
(Fig 4A and Table
1).
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| Fig 3.
Western blot analysis of iNOS content in cytokine-treated
macrophage J774 cells. Cytoplasmic extracts were prepared from murine J774 macrophages untreated and treated with IFN-/LPS for 24 hours as
described in Fig 1. Equal amounts (50 µg) of denatured proteins were
electrophoresed in an SDS 10% polyacrylamide gel, electroblotted to
filters, and incubated with anti-iNOS antibody (1:1,000 dilution) followed by secondary antibody as described in Materials and Methods. Bands were visualized by chemiluminescence. Migration of molecular mass
markers (myosin, phosphorylase B, and glutamic dehydrogenase, 250, 148, and 60 kD, respectively), loaded on the same gel, is shown on the left.
The results shown are representative of six separate experiments.
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| Fig 4.
Effect of NO on IRP-1 and IRP-2 activity of macrophage
J774 cells. (A) J774 cells were left untreated or treated with the NO
donor SNAP (0.5 mmol/L) for 24 hours as indicated. IRP activity in
cytoplasmic extracts was determined by bandshift assay using the
pSPT-fer probe as described in the legend to Fig 1. The results shown
are representative of seven separate experiments. (B) The mutant CG284
probe, which is specific for IRP-2, was incubated with lysates (10 µg
protein) of J774 cells untreated and treated with 0.5 mmol/L SNAP for
24 hours as indicated. The pSPT-fer probe was incubated with lysates (2 µg protein) of untreated cells. Formation of RNA/protein complexes
was estimated by bandshift assay as described in the legend to Fig 1.
The autoradiogram shown is representative of four separate
experiments.
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IRE-like sequences specifically recognized by IRP-2 have recently been
described.22 An RNA probe encompassing one such mutated IRE
sequence (CG284) incubated with J774 extracts formed an RNA/protein complex migrating close to the position of the faster moving complex detected by the pSPT-fer probe (Fig 4B). Binding activity was remarkably decreased in SNAP-treated cells (Fig 4B). The same results
were observed in cytokine-treated cells (data not shown). The
specificity of CG284 RNA for IRP-2 has previously been demonstrated by
competition experiments.22
Effect of cytokine stimulation on ferritin expression.
We then investigated the effect of the concomitant increase of IRP-1
and reduction of IRP-2 on ferritin accumulation. In J774 cells treated
with IFN-/LPS or with SNAP for 24 hours, H-rich-ferritin content was
significantly enhanced, whereas L-rich ferritin concentration was
somewhat increased, but the difference did not reach statistical significance (Table 1). To investigate the molecular mechanisms underlying the increase of ferritin content, we assessed ferritin synthesis: J774 cells were pulse labeled with radioactive amino acids
and ferritin was immunoprecipitated and analyzed on SDS gels. A strong
induction of H subunit synthesis (2.5-fold) was detected on stimulation
with IFN-/LPS (Fig 5) and SNAP (data not shown),
whereas L subunit synthesis was induced to a lower extent (1.6-fold).
As previously reported,31 the mouse H subunit had a higher
electrophoretic mobility than the L subunit. The finding that IRP-2
downregulation is accompanied by increased ferritin expression suggests
that posttranscriptional regulation by IRP-2 plays an important role in
the control of ferritin expression in this context.
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| Fig 5.
Ferritin synthesis by cytokine-treated macrophage J774
cells. J774 cells untreated or treated with IFN-/LPS for 24 hours were incubated in the presence of 35S methionine plus
cysteine (TRAN35S-LABEL) for 2 hours. Equal amounts of
labeled proteins were then immunoprecipitated with antibodies against
recombinant mouse H and L ferritin subunits (lanes 3 and 4); an aliquot
of the supernatants containing total proteins was also loaded on the
gel (lanes 1 and 2). Total proteins and immunoprecipitated ferritin
chains were separated on 15% SDS/polyacrylamide gels and visualized by fluorography. The results shown are representative of four separate experiments.
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However, because modulation of mRNA coding for proteins of iron
metabolism in cytokine-treated RE cells has been
reported,17,18,32 a role for transcriptional mechanisms in
increased ferritin synthesis cannot be ruled out. Thus, we investigated
the expression of ferritin mRNAs by Northern blot analysis. As shown in
Fig 6, after treatment with cytokines for 24 hours, L
subunit mRNA remained at the control level, whereas H ferritin subunit
mRNA was slightly increased (1.3-fold). Conversely, TfR mRNA was
decreased after stimulation of cells with IFN-/LPS.
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| Fig 6.
Effect of cytokine treatment on ferritin and transferrin
receptor mRNA levels in macrophage J774 cells. Total RNA isolated from
J774 cells untreated or treated with IFN-/LPS for 24 hours as
indicated was run in denaturing agarose gels, transferred to filters,
and hybridized with 32P-labeled DNA probes for H and L
ferritin subunits and TfR as described in Materials and Methods. The
autoradiograms shown are representative of four separate experiments.
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Effect of cytokine treatment on IRE-mediated translational
regulation.
The slight increase of H chain mRNA would not completely account for
the strong activation of ferritin synthesis, thus supporting the idea
that the induction of ferritin expression by inflammatory cytokines is
mainly controlled at the posttranscriptional level. To confirm the role
of translational control in the induction of ferritin synthesis by
IFN-/LPS, we studied the function of IRP as translational regulator
by assessing the effect of the addition of cytoplasmic extracts from
control or cytokine-treated J774 cells on the translation of a reporter
mRNA under the control of an IRE sequence. A chimeric transcript
composed of wild-type IRE sequence in front of a reporter CAT coding
sequence (I.12-CAT) was translated in a cell-free system derived from
wheat germ, which is devoid of endogenous IRP.33 As shown
in Fig 7 (left panel), the translation of I.12-CAT was
inhibited efficiently by addition of increasing amounts of
IRP-containing extracts from control cells, whereas repression by
extracts from stimulated cells was quantitatively less pronounced. The
same result was observed in SNAP-treated cells (data not shown). The
specificity of this translational control was assessed by analysis of a
control mRNA; in fact, translation of I-19.CAT mRNA, which has a
mutation in the IRE motif that prevents IRE/IRP
interaction,24 was not significantly affected by addition
of extracts from control (Fig 7, right panel) or stimulated (not shown)
J774 cells. This finding indicates that cytokine-mediated
downregulation of IRP-2 binding activity to an IRE sequence in
bandshift assays is also reflected in an impaired functional activity
in a translation repression assay.
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| Fig 7.
Effect of cytokine treatment on translational
repression activity of IRP in macrophage J774 cells. I-12.CAT (left
panel) and I-19.CAT (right panel) mRNA, containing, respectively, the
wild-type or mutated ferritin IRE sequence upstream of the CAT open
reading frame, were translated in wheat germ extract in the presence of 35S methionine. The effect on translational activity of the
addition of increasing amounts of lysate (10 to 20 µg protein) from
J774 cells untreated (lysate C) or treated with IFN-/LPS for 24 hours (lysate IFN-/LPS) was evaluated. Translation products were
separated on 10% SDS/polyacrylamide gels and visualized by
fluorography. The fluorogram shown is typical of four separate
experiments.
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DISCUSSION |
The characterization of two IRP with structural and functional
similarities, but significant differences in their mode of regulation
and pattern of tissue expression has raised the question of why cells
evolved two IRP. The finding that, in addition to consensus IRE, they
bind a different array of selected RNA targets indicates that a
possible reason for the presence of two IRP could be to enhance the
versatility of the IRE/IRP regulatory system, expanding its influence
to cellular processes not immediately related to intracellular iron
homeostasis. However, the complexity of the system may also be
increased if the two IRP respond preferentially to different stimuli.
In fact, we have demonstrated preferential regulation of IRP-2 activity
and consequent changes of the expression of the major proteins of
intracellular iron metabolism during liver cell
proliferation34 and oxidative stress.26 IRP
have been shown to be also a target of NO. Whereas IRP-1 activity has been found to be induced by NO (see Weiss,2
Pantopoulos,9 Drapier and Bouton,10 Richardson
and Ponka,13 and Domachowske14 for review), the
effect of NO on the activity of IRP-2 is less clearcut. Indeed, IRP-2
activity has been reported to be enhanced by NO in
macrophages,16-18 fibroblasts,35 and the liver
during the acute phase reaction,36 but not affected in
hepatoma cells.37
In the present study, we showed that stimulation by cytokines weakly
enhanced IRP-1 activity, but strongly reduced that of IRP-2 in the
mouse macrophage cell line J774. In contrast with the present data, a
coordinated increase of both IRP-1 and IRP-2 activity resulting in
decreased ferritin translation has been reported in
IFN-/LPS-treated J774 cells.17,18 We have no immediate explanation for this discrepancy, which apparently does not depend on
trivial technical or experimental reasons. In fact, we used time
periods and cytokine doses similar to those reported by Weiss et
al.17,18 However, an evident downregulation of
the fast-migrating band detected by the pSPT-fer probe, which
corresponds to the IRE/IRP-2 complex,5,7 was a consistent
finding in all of our experiments; in addition, the decrease of IRP-2
binding activity was confirmed by experiments using the IRP-2-specific
CG284 probe. Moreover, decreased IRP-2 activity was further indicated
by results obtained in a functional assay showing that the
translational repressor capacity of IRP was impaired in
cytokine-treated cells (see Fig 7). Evidence for the role of NO in the
modulation of IRP activity in cytokine-treated J774 cells was provided
by experiments in which the formation of NO by a physiologic source
(ie, cytokine-stimulated iNOS, see Fig 3) was specifically prevented
and by other experiments in which a compound that produces NO in
solution (SNAP) was added directly to cells. As to the mechanistic
aspects of the effects of NO on IRP activity, the reactivity of this
compound with iron-containing proteins suggests that IRP-1 activation
may depend on direct interaction of NO with the 4Fe-4S
cluster.9-14 On the other hand, IRP-2, lacking the cluster,
is not directly targeted by NO, which may instead downregulate the
activity of this protein indirectly by increasing intracellular iron
availability, as indicated by the demonstration that treatment with an
iron chelator prevented the fall of IRP-2 activity (see Fig 1B).
Interestingly, the opposite modulation of IRP-1 and IRP-2 activity in
stimulated J774 cells was accompanied by increased ferritin synthesis
and accumulation, indicating that IRP-2 downregulation can act as the
predominant effector of cellular iron homeostasis in this system. This
is further demonstrated by the cytokine-dependent decrease of TfR mRNA
levels found in the present study (see Fig 6) and
previously.18 It should be noted that IRP-2 is particularly abundant in J774 cells in which it represents at least 50% of total
IRP activity.
The prominent role of posttranscriptional mechanisms, ie, IRP-2
inactivation, in the control of ferritin induction is also supported by
the finding that cytokine stimulation, in agreement with previous
reports,17,18 induced only a minor increase of ferritin
mRNA, which would not fully account for the increase of ferritin
synthesis. The importance of posttranscriptional control in the
induction of ferritin synthesis was confirmed by experiments that
assessed translational regulation directly. In fact, IRP in lysates
from cytokine-treated cells showed a reduced capacity to repress
translation of IRE-containing transcripts in cell-free extracts. Thus,
the decreased IRP-2 binding activity detected in gel retardation assays
was reflected in impaired translational repression function. From a
quantitative viewpoint, the slight activation of IRP-1 might counteract
IRP-2 downregulation, thus explaining why induction of ferritin
expression is less prominent than IRP-2 inactivation.
Although the two IRP differ in their mechanisms of
regulation,5,7,8 they have been considered to be
coordinately regulated7; to our knowledge, the present
results are the first demonstration of an opposite response of IRP-1
and IRP-2 to a stimulus. In vitro experiments have indicated equal
binding affinity and equal functional capacity of the two
IRP,5,7,8 and we have shown specific modulation of IRP-2 in
the absence of significant changes of IRP-1 in
vivo.26,34,36 However, as IRP-1 usually accounts for the major part of IRE-binding activity, the role of IRP-2 has been generally somewhat underestimated. Here we show that when IRP-2 is
abundantly expressed, as in J774 cells, modulation of its activity is
sufficient to dictate intracellular iron metabolism. This also represents the first evidence that IRP-2 controls translation in living
cells. The recent finding of a cell line without detectable IRP-1
supports the idea that IRP-2 can function as the sole mediator of
intracellular iron metabolism.38
Diversion of iron traffic from the circulation into the storage sites
within the RE system is pivotal in inducing the hypoferremia that
accompanies inflammatory diseases. Previous reports,17,18 which showed NO-mediated IRP activation and consequent inhibition of
ferritin synthesis, are difficult to reconcile with the recognized changes of iron metabolism occurring under inflammatory
conditions.2,19 The present study, by demonstrating an
IRP-2-mediated increase of ferritin synthesis in cytokine-treated J774
cells, provides molecular evidence supporting previous findings in
experimental models of inflammation, which showed that iron retention
in RE cells was accompanied by increased ferritin
expression.19 Advances in the knowledge of the molecular
mechanisms underlying increased ferritin synthesis in cytokine-treated
RE cells could also give insights into the pathophysiology of the
anemia of chronic disease in which iron retention by macrophages limits
iron availability for hematopoiesis.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted September 23, 1997.
Supported by Ministero Università e Ricerca Scientifica e
Tecnologica (MURST), Consiglio Nazionale delle Ricerche (CNR), and
IRCCS Ospedale Maggiore, Milano, Italy and from the UNDP/World Bank/WHO
Special Programme for Research and Training in Tropical Diseases (TDR)
(to D.T.).
Address reprint requests to Dr Gaetano Cairo, Centro di
Studio sulla Patologia Cellulare CNR, Via Mangiagalli 31, 20133 Milano, Italy.
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.
| |
ACKNOWLEDGMENT |
We thank L. Kuhn for providing pSPT-fer and CG284 plasmids, K. Pantopoulos and M. Hentze for the generous gift of I-12.CAT and
I-19.CAT constructs, and P. Santambrogio for determination of ferritin
content.
| |
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Abstract
Introduction
Abstract