Báo cáo khoa học: Iron regulatory protein-independent regulation of ferritin synthesis by nitrogen monoxide pot

The regulation of ferritin synthesis is largely accom-plished via an elegant regulatory system that tightly controls intracellular iron levels.. Results NO+-mediated induction of ferriti

synthesis by nitrogen monoxide

Marc Mikhael1,2, Sangwon F Kim2, Matthias Schranzhofer3, Shan S Lin1,4, Alex D Sheftel1,2, Ernst W Mullner3and Prem Ponka1,2

1 Department of Physiology, McGill University, Montreal, Canada

2 Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada

3 Department of Medical Biochemistry, Division of Molecular Biology, Max F Perutz Laboratories, Medical University of Vienna, Austria

4 Division of Experimental Medicine, McGill University, Montreal, Canada

Iron (Fe) is essential for life, functioning as a metal

cofactor for many proteins containing either heme

or nonheme iron [1–3] Hemoproteins have crucial

biological functions, such as oxygen binding, oxygen

metabolism, and electron transfer Many nonheme

iron-containing proteins catalyze key reactions involved

in energy metabolism and DNA synthesis However,

the chemical properties of iron which are exploited for a remarkable range of biological functions have created problems for living organisms In excess, cel-lular ‘free’ iron catalyzes the Haber–Weiss reaction that can lead to the production of cytotoxic oxygen radicals [4,5] The safe storage and sequestration of iron is therefore an absolute necessity within the cell

Keywords

ferritin; iron; iron regulatory proteins;

nitrogen monoxide; NO

Correspondence

1

P Ponka, Lady Davis Institute, McGill

University, 3755 Cote Ste-Catherine Road,

Montreal, Quebec, H3T 1E2, Canada

Fax: +1 514 340 7502

Tel: +1 514 340 8260

E-mail: prem.ponka@mcgill.ca

(Received 2 June 2006, revised 20 June

2006, accepted 22 June 2006)

doi:10.1111/j.1742-4658.2006.05390.x

The discovery of iron-responsive elements (IREs), along with the identifica-tion of iron regulatory proteins (IRP1, IRP2), has provided a molecular basis for our current understanding of the remarkable post-transcriptional regulation of intracellular iron homeostasis In iron-depleted conditions, IRPs bind to IREs present in the 5¢-UTR of ferritin mRNA and the 3¢-UTR of transferrin receptor (TfR) mRNA Such binding blocks the translation of ferritin, the iron storage protein, and stabilizes TfR mRNA, whereas the opposite scenario develops when iron in the intracellular tran-sit pool is plentiful Nitrogen monoxide (commonly designated nitric oxide; NO), a gaseous molecule involved in numerous functions, is known to affect cellular iron metabolism via the IRP⁄ IRE system We previously demonstrated that the oxidized form of NO, NO+, causes IRP2 degrada-tion that is associated with an increase in ferritin synthesis [Kim, S & Ponka, P (2002) Proc Natl Acad Sci USA 99, 12214–12219] Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase in ferritin synthesis that initially occurs without changes in the RNA-binding activities of IRPs Moreover, we demonstrate that the translational efficiency of ferritin mRNA is significantly higher in cells trea-ted with SNP compared with those incubatrea-ted with ferric ammonium cit-rate, an iron donor Importantly, we also provide definitive evidence that the iron moiety of SNP is not responsible for such changes These results indicate that SNP-mediated increase in ferritin synthesis is, in part, due to

an IRP-independent and NO+-dependent post-transcriptional, regulatory mechanism

Abbreviations

DFO, desferoxamine; FAC, ferric ammonium citrate; Ft, ferritin; hDFO, high molecular mass version of DFO; IFN, interferon; IRE, iron-responsive element; IRP, iron regulatory protein; LPS, lipopolysaccharide; NO, nitric oxide; PIH, pyridoxal isonicotinoyl hydrazone;

SIH, salicylaldehyde isonicotinoyl hydrazone; SNP, sodium nitroprusside; TfR, transferrin receptor.

[3,6,7] Hence, virtually all organisms can synthesize

the icosikaitetrameric protein, ferritin, which can safely

house thousands of iron atoms in a shell-like structure

Ferritin is a 430–460 kDa protein made up of 24

subunits of heavy (H; 21 kDa) and light (L; 19 kDa)

ferritin chains [3,8] While both H- and L-ferritin are

involved in incorporating iron, H-ferritin is several

times more efficient than L-ferritin This difference

appears to be due to a ferroxidase center associated

with the H-ferritin subunit that promotes the

oxida-tion of ferrous iron [9] By contrast, the L-subunit

has a higher capacity than the H-subunit to induce

iron-core nucleation [10,11], suggesting that both

ferritin chains cooperate in the overall uptake and

storage of iron

The regulation of ferritin synthesis is largely

accom-plished via an elegant regulatory system that tightly

controls intracellular iron levels The structurally

sim-ilar iron regulatory proteins 1 and 2 (IRP1 and 2)

function as iron sensors [4–7] In iron-depleted

condi-tions, IRPs are active and consequently bind specific

nucleotide sequences, iron-responsive elements (IRE),

located in the 5¢-UTR of ferritin mRNA and the

3¢-UTR of transferrin receptor (TfR) mRNA Such

binding leads to translational repression of ferritin

mRNA and stabilization of the TfR message

Con-versely, under iron-replete conditions, IRP binding

decreases, leading to TfR mRNA destabilization while

ferritin mRNA is efficiently translated IRP1 assumes

cytosolic aconitase activity in such iron-replete

condi-tions, whereas IRP2 is targeted for degradation via the

ubiquitin–proteasome pathway [1,2,12,13]

It is well established that IRP-binding activities are

also modulated by noniron stimuli such as hydrogen

peroxide, hypoxia, phosphorylation, and nitric oxide

(NO) [14–21] NO, in particular, has emerged as an

extraordinary signaling molecule [22,23] whose targets

differ depending on its redox state [24] The reduced

form of NO, the NO radical (NO•), transduces signals

primarily via direct interactions with the iron of heme

moieties in guanylyl cyclase [25–27]; NO• also binds to

iron in the iron–sulfur clusters of IRP1 [19,28] and

mitochondrial aconitase [29,30] Numerous

laborator-ies have shown that NO• increases the RNA-binding

activities of IRP1 in many cell types [14,15,18,

20,28,31] In contrast, oxidized NO, the nitrosonium

ion (NO+), reacts with thiol groups of cysteine

resi-dues, typically resulting in a reversible signaling

mech-anism known as S-nitrosylation [24,32] A multitude of

proteins have been identified as targets of

S-nitrosyla-tion [23,33,34] including IRP2, whose S-nitrosylaS-nitrosyla-tion

leads to its ubiquitination and subsequent proteosomal

degradation [35]

We have previously shown that macrophages acti-vated by lipopolysaccharide (LPS) and interferon-c (IFNc), a condition known to induce NO synthesis [36], exhibit NO-dependent IRP2 degradation accom-panied by an increase in ferritin synthesis [20,21,37] Moreover, sodium nitroprusside (SNP), a NO+donor, was also found to cause IRP2 degradation followed by

a dramatic induction of ferritin synthesis [20,35,37] Recently, Bourdon et al [38] proposed that SNP, a compound containing complexed iron, contributes to IRP2 degradation by supplying iron to cells In this study, we show that the iron component of SNP is not involved in the stimulation of ferritin synthesis More-over, we have discovered that, in RAW

macrophage cell line), SNP stimulates ferritin synthe-sis, at least in part, by a mechanism that does not require IRP2 degradation We also report a similar phenomenon in INFc⁄ LPS-treated macrophages

Results

NO+-mediated induction of ferritin synthesis precedes changes in IRP/IRE binding

We have previously shown that treatment of RAW 264.7 cells with the NO+ donor, SNP, causes the degradation of IRP2 associated with an increase of ferritin synthesis [20,37] Here, we examined the kinet-ics of ferritin synthesis and changes in RNA-binding activities of IRPs in response to SNP exposure for var-ious time intervals First, RAW 264.7 cells were trea-ted with 100 lm SNP for 15–180 min, after which the cells were thoroughly washed and incubated with [35 S]-methionine for 1 h Figure 1A shows that exposure of cells to SNP for a time interval as short as 30 min led

to a significant increase in ferritin synthesis levels Interestingly, IRP2 degradation was noticeable only

at 2 h following incubation of RAW 264.7 cells with SNP, whereas the RNA-binding activity of IRP1 remained largely unaffected (Fig 1B) Surprisingly, the rapid induction of ferritin synthesis by SNP

in RAW 264.7 cells occurred much earlier than any decrease of IRP activities could be detected (Fig 1A,B; 0–60 min) This strongly suggests that SNP-mediated induction of ferritin synthesis is, at least in part, inde-pendent of IRP⁄ IRE regulation

IFNc⁄ LPS-mediated ferritin synthesis occurs without changes in IRP activity

We have also previously shown that a combination of IFNc and LPS is able to increase ferritin synthesis

in macrophages in an IRP2-dependent manner via the

production of NO by inducible nitric oxide synthase

[21,37] Because SNP is able to mediate ferritin

synthe-sis before IRP activities are changed, we hypothesized

that endogenously produced NO is also able to

repro-duce such a phenomenon Indeed, when we treated

RAW 264.7 macrophages with both IFNc and LPS

for as little as 1 h, we observed a more than twofold

induction of ferritin synthesis (Fig 2A) As expected,

the increase in ferritin synthesis was accompanied

by NO production (nitrite concentrations, Fig 2A)

Importantly, IRP levels did not change during the first

two hours of IFNc⁄ LPS treatment (Fig 2B),

suggest-ing that, like SNP-derived NO, endogenously produced

NO is able to mediate changes in ferritin synthesis

prior to the modulation of IRP activities

SNP enhances ferritin synthesis even in the

absence of IRP activity

The above experiments indicate that SNP may increase

ferritin synthesis via an IRE⁄ IRP-independent

mechan-ism To find further support for this conclusion we

pretreated RAW 264.7 cells with an iron donor, ferric

ammonium citrate (FAC; 50 lgÆmL)1) for 18 h, washed and then incubated them with or without SNP for an additional 3 h As expected, pretreatment of RAW 264.7 cells with FAC for 18 h led to abolish-ment of IRP-binding activities (Fig 3A, lane 2) with

a concomitant increase in ferritin synthesis (Fig 3B, lane 2) The addition of SNP to FAC-pretreated cells augmented ferritin synthesis by more than twofold (Fig 3B, lanes 2 versus 3) despite similar levels of IRP-binding activity in both conditions (Fig 3A, lanes

2 versus 3) This indicates that SNP is able to augment ferritin synthesis beyond the levels capable solely by the classical IRE⁄ IRP system

The bioavailability of SNP iron is negligible SNP, which contains iron [Na2Fe(CN)5NO], is a well-established NO+ donor [24,39] that reacts with thiol groups leading to S-nitrosylation of target proteins [32,40] We have previously shown that SNP causes S-nitrosylation of Cys178 in IRP2, which, in turn, trig-gers the ubiquitination and degradation of the protein [35] It has, however, been suggested that the ability of SNP to both stimulate IRP2 degradation and induce ferritin synthesis is accomplished through its iron moi-ety [38] Hence, we examined whether SNP releases

A

B

Fig 2 Effects of IFNc ⁄ LPS on ferritin (Ft) synthesis (A) and IRP-binding activities (B) in RAW 264.7 cells (A) Cells were incubated

in the presence of IFNc (100 UÆmL)1) and LPS (5 lgÆmL)1) for the indicated time intervals and were then washed and pulse labeled (1 h) with [ 35 S]-methionine and harvested Ferritin was immunopre-cipitated by using anti-ferritin IgG and analyzed by SDS ⁄ PAGE fol-lowed by autoradiography DA, densitometric analysis, in arbitrary units (B) Cells were treated with IFNc ⁄ LPS as in (A) and the pro-tein extracts assayed for IRE-binding activities using gel-retardation assays [20] Nitrate was assayed by using the Greiss reagent as described by Green et al [52].

35 S H+L

1.0 1.4 3.2 7.1 15.7 24.8 (D.A.)

100 μ M SNP

0 15 30 60 120 180 (min)

IRP 1

IRP 2

+ β-ME

A

B

100 μ M SNP

0 15 30 60 120 180 (min)

IRP 1

IRP 2

Fig 1 Effects of SNP on ferritin synthesis (A) and IRP-binding

activities (B) in RAW 264.7 cells (A) Cells were incubated in the

presence of SNP (100 lM) for the indicated time intervals and were

then washed and pulse labeled (1 h) with [35S]-methionine and

har-vested Ferritin was immunoprecipitated by using anti-ferritin IgG

and analyzed by SDS ⁄ PAGE followed by autoradiography DA,

den-sitometric analysis, in arbitrary units (B) Cells were treated with

SNP as in (A) and the protein extracts assayed for IRE-binding

activ-ities using gel-retardation assays [20], performed in the absence or

presence of 2% b-mercaptoethanol (b-ME), a condition that reveals

total RNA binding activity of IRP1 [51].

iron which could be responsible for the SNP-mediated

induction of ferritin synthesis To do so, equimolar

amounts of either ferric citrate or SNP were incubated

with desferoxamine (DFO) in tissue culture medium at

different time intervals during which the absorption

spectra were recorded (Fig 4A) Iron-laden chelators

exhibit a characteristic absorption pattern (Fig 4A)

The amount of iron transferred from SNP to the

chelator gradually increased but was extremely slow

(Fig 4B) Importantly, there was no detectable loss

of iron from SNP in 3 h as no Fe–DFO complexes

were observed at this time (Fig 4B) Identical results

were obtained using other chelators such as

pyrid-oxal isonicotinoyl hydrazone (PIH), salicylaldehyde

isonicotinoyl hydrazone (SIH) and a high molecular

mass version of DFO (hDFO; data not shown) These

results were corroborated by the experimental

out-come that IRP1 levels are not decreased after the

treatment of RAW 264.7 cells with SNP for 3 h

(Fig 1A)

Further support for our conclusion that SNP is not

a source of chelatable iron comes from our earlier

observation that DFO, which is commonly used to

intercept intracellular iron, was unable to attenuate

SNP-induced degradation of IRP2 [20] Here we

exploited hDFO, which is unable to penetrate cell membranes, to show that hDFO was unable to prevent SNP-mediated increases in ferritin synthesis (Fig 5A), indicating that SNP does not donate iron to the cell culture medium Moreover, neither the SNP-like com-pound, potassium ferricyanide, nor cyanide and nitrate compounds were able to increase ferritin synthesis (Fig 5B) further indicating that it is NO+ that is responsible for SNP-mediated induction of ferritin synthesis

NO+enhances the efficiency of ferritin mRNA translation

To elucidate the mechanism by which NO+ derived from SNP induces ferritin synthesis independent of the IRP⁄ IRE system, we examined the levels of ferritin mRNA associated with polysomes in untreated RAW 264.7 cells or those treated with either FAC (50 lgÆmL)1, 18 h)

Fig 4 SNP releases minimal amounts of iron during incubation with DFO SNP (100 lM) or ferric citrate (FC) (100 lM) were incuba-ted (37 C) with or without DFO (100 lM) for various time intervals following which the Fe–DFO complexes were detected using spec-trophotometric analysis at wavelengths 350–550 nm (A) Represen-tative spectrophotometric profiles of FC, DFO and DFO + FC at time 0 h (B) Relative levels of Fe–DFO formation for media with

FC, DFO + FC and DFO + SNP Absorbance measurements were taken at 410 nm; the peak that corresponds to Fe–DFO complexes

as observed in (A).

9 Fig 3 Effect of SNP on IRP-binding activities (A) and ferritin

syn-thesis (B) in control or FAC-pretreated cells RAW 264.7 cells were

incubated with either control medium or FAC (50 lgÆmL)1) for 18 h,

washed with cold NaCl ⁄ P i and incubated with either control

med-ium or with SNP (100 lM) for 3 h (A) Gel-retardation analysis of

protein (10 lg) extracted from RAW 264.7 cells after different

treat-ments (B) RAW 264.7 cells, treated as in (A), were pulse labeled

(2 h) with [ 35 S]-methionine, and [ 35 S]-ferritin was

immunoprecipitat-ed (by using anti-ferritin IgG) and analyzimmunoprecipitat-ed by SDS ⁄ PAGE followed

by autoradiography.

shows that in cells incubated with FAC, 10% of the

ferritin message can be found in a polysome-bound

form However, SNP treatment yielded a significantly

elevated fraction of ferritin mRNA associated with

polysomes (50%), indicating that NO+ increases the

efficiency of ferritin translation significantly above the

levels that can be achieved with iron

Discussion

It is well known that the inflammatory signals cause macrophages to produce NO [36,41] We have pre-viously shown that IFNc⁄ LPS-mediated activation of murine macrophages caused NO-dependent IRP2 de-gradation [21], and that such changes led to an increase

in ferritin synthesis [20,37] Moreover, preventing the degradation of IRP2 by proteasomal inhibitors also blocked the ferritin synthesis increase [37], indicating that inflammatory signals in murine macrophages can activate ferritin synthesis via the degradation of IRP2 Our laboratory reported that SNP, a NO+donor, was also able to trigger IRP2 degradation followed by an increase in ferritin synthesis [37] Such NO-dependent IRP2 degradation was caused by the S-nitrosylation of Cys178 which led to ubiquitination of the protein fol-lowed by its degradation in the proteosome [35] These results suggest that NO-mediated IRP2 degradation is largely responsible for the increase in ferritin synthesis

in both SNP and IFNc⁄ LPS-treated macrophages

In this report, we show that SNP enhances ferritin synthesis not only by the mechanism involving IRP2 degradation, but also by an IRP⁄ IRE-independent pro-cess We show that treatment of RAW 264.7 cells with SNP increases ferritin synthesis much faster than IRP activity decreases (Fig 1) In addition, we also show that IFNc⁄ LPS treatment for as little as 1 h is able

to produce a similar phenomenon, whereby ferritin

5

levels increase more than twofold without any signifi-cant change in IRP⁄ IRE-binding activities (Fig 2)

Fig 5 hDFO does not block the induction of ferritin synthesis in

SNP-treated RAW 264.7 cells (A); effects of various control

com-pounds on ferritin synthesis are also shown (B) Cells were

incuba-ted with SNP or various other reagents [hDFO, K3Fe(CN)6, KCN,

NaCN, NaNO 3 ] for 3 h, following which they were washed and

then pulse-labeled (1 h) with [35S]-methionine and harvested

Fer-ritin was immunoprecipitated by using anti-ferFer-ritin IgG and analyzed

by SDS ⁄ PAGE followed by autoradiography.

Fig 6 Polysome profiles of mRNAs isolated

by sucrose gradient fractionation RNA was extracted from RAW 264.7 cells treated with either FAC (50 lgÆmL)1) for 18 h or SNP (100 lM) for 3 h and blotted onto nylon membranes The filters were hybridized sequentially with [32P]dCTP[aP]-labeled probes specific for H-ferritin 18S and 28S rRNA profiles from a representative polysome gradient are shown as control for RNA integrity (loading).

Importantly, IFNc⁄ LPS treatment was also

accom-panied by an increase in NO production (Fig 2A)

Moreover, SNP is able to enhance ferritin synthesis

above the levels seen following the pretreatment of cells

with the iron donor, FAC This occurs despite the fact

that similar levels of IRP-binding activity are detectable

in samples treated with FAC alone and those exposed

to both FAC and SNP together (Fig 3, lane 2 versus 3)

These results suggest the existence of a yet unidentified

regulatory mechanism of ferritin translation that can

operate independently of the IRE⁄ IRP system

It has been proposed that the active effector

compo-nent of SNP is iron [38], even though SNP has been

extensively used as a NO donor by many laboratories

[24,26,27,42–44] Bourdon et al [38] claimed that SNP

is capable of donating iron to cells even though there is

no chemical evidence for iron release from SNP [39,45]

Indeed, we have shown that iron transfer from SNP to

DFO and other chelators is negligible under our

experi-mental conditions in which SNP causes an increase of

ferritin synthesis Moreover, we showed that

ferricya-nide, an iron complex similar to SNP, did not affect

IRP2 levels [20] or ferritin synthesis (Fig 5B)

Bourdon et al also reported that the iron chelator

DFO was able to prevent both SNP-mediated IRP2

degradation and the induction of ferritin synthesis; the

authors concluding that it is SNP-derived iron, rather

than NO, which is responsible for such changes [38]

However, we previously reported [20] that neither

DFO nor EDTA (a cell-impermeable iron chelator)

added together with SNP were able to attenuate

mediated IRP2 degradation, indicating that

SNP-derived iron was not responsible for IRP2 degradation

This conclusion is also supported by our finding that

IRP1 levels remain unchanged during 10 h of

treat-ment of RAW 264.7 cells with SNP [20] The

discrep-ancy between our results and those of Bourdon et al

[38] may be because we examined an acute response to

SNP (3–10 h), whereas Bourdon et al incubated cells

with SNP or SNP and DFO for 18 h It is known that

SNP has a short half-life (0.5–1 h) [20,46] and the

effect of DFO is rather slow due to its poor membrane

permeability [47] Therefore, it can be expected that in

the study by Bourdon et al DFO did not actually

block the effect of SNP per se but rather decreased

intracellular iron levels when the effect of SNP expired,

and an increase in IRP2 levels, that suppressed ferritin

synthesis, resumed

In order for mRNA to be translated into protein,

the message has to become associated with ribosomes,

forming polysomes IRP binding to the IRE on the

5¢-UTR of ferritin mRNA prevents translation of the

protein In this report we demonstrate that treatment

of RAW 264.7 cells with iron (50 lgÆmL)1 FAC, 18 h) and the resulting decrease in IRP activity will cause

 10% of the total ferritin message to become poly-some associated (Fig 6) Importantly, SNP treatment

of the cells for only 3 h redistributed as much as 50%

of the ferritin mRNA from the polysome-free form to the polysome-bound form These data, along with the fact that we were unable to detect any transcriptional changes in ferritin expression by SNP treatment (data not shown), are congruent with our observations that translational upregulation of ferritin synthesis is rap-idly and dramatically achieved to levels greater than those attainable by iron loading when RAW 264.7 cells are exposed to the nitrosonium ion donor To the best

of our knowledge, this is the first report showing that

NO can regulate ferritin synthesis in a manner that is,

at least in part, independent of the IRP⁄ IRE system

In conclusion, we have previously shown that chem-ically produced NO+, which causes S-nitrosylation of the thiol groups of proteins, decreased the RNA-bind-ing activity of IRP2 followed by IRP2 degradation and an increase in ferritin synthesis [6,20,35,37] We have also provided strong evidence that the iron com-ponent of SNP is not responsible for IRP2 degrada-tion We showed that: (a) the effect of SNP on IRP2 degradation was not prevented by EDTA or DFO [20]; (b) SNP did not decrease the RNA-binding activ-ity of IRP1, which would be expected if iron was liber-ated [20]; and (c) SNP stimulliber-ated iron incorporation into ferritin [37], which would likely decrease iron lev-els in the labile iron pool

defin-itively demonstrated that the effect of SNP is not due

to its integrated iron moiety and that NO+from SNP

is responsible for its effect on ferritin synthesis More-over, acute regulation of ferritin synthesis by NO+ is accomplished by a rapid mobilization of polysome-free ferritin mRNA that occurs much more efficiently than

in iron-treated cells It is likely that S-nitrosylation

of a protein(s) involved in the activation of ferritin translation is the mechanism underlying our findings, therefore further research is needed to delineate the players involved in NO+-mediated, IRP2-independent stimulation of ferritin mRNA translation

Experimental procedures

Chemicals

Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Wisent Inc (Saint-Jean-Baptiste de Rouville, Canada); fetal bovine serum, penicillin, streptomycin, and glutamine were from Invitrogen Corp (Carlsbad, CA) SNP, FAC, and LPS were from Sigma (St Lous, MO); and

[35S]-methionine was from Perkin–Elmer (Boston, MA);

[32P]-UTP was from Amersham Biosciences (Little Chalfont,

UK) The iron chelators PIH and SIH were synthesized as

described previously [39]; DFO was obtained from Pharma

Science (Montreal, Canada); hDFO was obtained from

Bio-medical Frontiers Inc (Minneapolis, MN) IFNc was

obtained from Roche (Indianapolis, IN) All other

chemi-cals were obtained from Sigma, unless specified otherwise

Cells

RAW 264.7 murine macrophages were obtained from

American Type Culture Collection Cells were grown in

60 cm2plastic culture dishes (Falcon, Franklin Lakes, NJ)

in a humidified atmosphere of 95% air and 5% CO2at 37C

in DMEM containing 10% fetal bovine serum, extra

l-gluta-mine (300 lgÆmL)1), sodium pyruvate (110 lgÆmL)1),

peni-cillin (100 unitsÆmL)1), and streptomycin (100 lgÆmL)1)

Gel-retardation assay

The gel-retardation assay used to measure the interaction

between IRPs and IREs was carried out as described

previ-ously [20] Briefly, 6· 106

cells were washed with ice-cold NaCl⁄ Pi and lyzed at 4C in 80 lL of lysis(+) buffer

(10 mm Hepes, pH 7.5, 3 mm MgCl2, 40 mm NaCl, 5%

gly-cerol, 1 mm dithiothreitol, and 0.2% Nonidet P-40) After

lysis, the samples were centrifuged for 5 min at 10 000 g to

remove the nuclei Samples of cytoplasmic extract were

dilu-ted with two volume of lysis(–) buffer (without 0.2%

Noni-det P-40) to a protein concentration of 1 lgÆlL-1, and 10 lg

aliquots were analyzed for IRP binding by incubating them

with an excess amount of32P-labeled pSRT-fer RNA

script, which contains one IRE [49] This RNA was

tran-scribed in vitro from linearized plasmid template using T7

RNA polymerase in the presence of [32P]-UTP To form

RNA–protein complexes, cytoplasmic extracts were

incuba-ted for 10 min at room temperature with excess amount of

labeled RNA Heparin (5 mgÆmL)1) was added for another

10 min to prevent nonspecific binding RNA–protein

com-plexes were analyzed in 6% nondenaturing polyacrylamide

gels In parallel, duplicate samples were treated with 2%

b-mercaptoethanol before the addition of the RNA probe

Metabolic labeling and immunoprecipitation

Cells were labeled for 1 h with (100 lCiÆmL)1) [35

S]-methi-onine in methiS]-methi-onine-free RPMI media, washed three times

with cold NaCl⁄ Pi, after which they were lyzed with RIPA

buffer (50 mm Tris⁄ HCl, 150 mm NaCl, 1% Nonidet P-40,

0.5% sodium deoxycholate, 0.1% SDS) for 30 min at 4C

Anti-ferritin IgG obtained from Roche (Indianapolis, IN)

was added to the lysates and incubated overnight at 4C,

then 60 lL of protein A–Sepharose was added for 3 h at

4C to precipitate the immune complexes The beads were

washed three times with cold RIPA buffer and then boiled with SDS loading dye Immunoprecipitated protein was resolved by using 12.5% SDS⁄ PAGE The gel was dried and analyzed by autoradiography

Analysis of ferritin mRNA association with polysomes

Sucrose-gradient fractionation was performed essentially as described [50] Extracts from resting and activated cells were prepared by lysis at 4C in extraction buffer (10 mm Tris⁄ HCl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl2, 0.5% Nonidet P-40 and 500 UÆmL)1 RNAsin), and nuclei were removed by centrifugation (12 000 g, 10 s, 4C) The super-natant was supplemented with 20 mm dithiothreitol,

150 lgÆmL)1cycloheximide, 1.5 mgÆmL)1heparin and 1 mm phenylmethylsulfonyl fluoride and centrifuged (12 000 g,

5 min, 4C) to eliminate mitochondria The supernatant was layered onto a 10 mL linear sucrose gradient (15–40% sucrose w⁄ v supplemented with 10 mm Tris ⁄ HCl, pH 7.5,

140 mm NaCl, 1.5 mm MgCl2, 10 mm dithiothreitol,

100 lgÆmL)1cycloheximide, and 0.5 mgÆmL)1heparin) and centrifuged in a SW41Ti rotor (Beckman, Palo Alto, CA) (178 305 g, 120 min, 4C)

(550 lL) were collected and digested with 150 lgÆmL)1 pro-teinase K in 1% SDS and 10 mm EDTA (30 min, 37C) RNAs were then recovered by phenol⁄ chloroform ⁄ isoamyl alcohol extraction, followed by ethanol precipitation RNAs were analyzed by electrophoresis on denaturing 1.2% for-maldehyde agarose gels and subsequent northern blotting After RNA transfer to nylon membranes (GeneScreen, NEN, Boston, MA) and UV cross-linking, the distribution

of 18S and 28S rRNAs was visualized by methylene blue staining of the filters [35] The membranes were sequentially hybridized with various [32P]dCTP[aP]-labeled random-primed ferritin cDNA probes or antisense [32 P]UTP[aP]-labeled RNA probes After washing and autoradiography, signals were quantified by PhosphorImaging (Molecular Dynamics, Sunnyvale, CA)

Iron transfer from SNP to iron chelators

Equimolar amounts of either SNP or ferric citrate (100 lm) were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37C in DMEM containing 10% fetal bovine serum, extra l-glutamine (300 lgÆmL)1), sodium pyruvate (110 lgÆmL)1), penicillin (100 UÆmL)1), and streptomycin (100 lgÆmL)1), with or without iron chelators for various time intervals Experiments were done using DFO, hDFO, PIH and SIH as iron chelators

Statistics

Experiments were repeated at least three times and the representative data are presented

This work was supported by a grant (to PP), a

fellow-ship (to SFK), and a scholarfellow-ship (to ADS) from the

Canadian Institutes of Health Research (CIHR) and the

‘Fonds zur Fo¨rderung der Wissenschaftlichen Forschung’

(FWF), Austria, grant SFB F-28 (to EWM) and the

Hertzfelder Family Foundation (to EWM) We thank

Biomedical Frontiers for their generous gift of hDFO

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