Iron regulatory protein-independent regulation of ferritin synthesis by nitrogen monoxide Marc Mikhael 1,2 , Sangwon F. Kim 2 , Matthias Schranzhofer 3 , Shan S. Lin 1,4 , Alex D. Sheftel 1,2 , Ernst W. Mullner 3 and Prem Ponka 1,2 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 incubated 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. 3828 FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS [3,6,7]. Hence, virtually all organisms can synthesize the icosikaitetrameric protein, ferritin, which can safely house thousands of iron at oms 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-nitrosylation 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 3 264.7 cells (a 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 M. Mikhael et al. IRP-independent effects of NO + on Ft synthesis FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS 3829 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 [Na 2 Fe(CN) 5 NO], 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.) 100μ M SNP 0 15 30 60 120 180 (min) IRP 1 IRP 2 +β-ME A B 100μM 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 l M) for the indicated time intervals and were then washed and pulse labeled (1 h) with [ 35 S]-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]. IRP-independent effects of NO + on Ft synthesis M. Mikhael et al. 3830 FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 ,18h) 4 or SNP (100 lm, 3 h). Figure 6 Fig. 4. SNP releases minimal amounts of iron during incubation with DFO. SNP (100 l M) or ferric citrate (FC) (100 lM) were incuba- ted (37 °C) with or without DFO (100 l M) 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 l M) 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 analyzed by SDS ⁄ PAGE followed by autoradiography. M. Mikhael et al. IRP-independent effects of NO + on Ft synthesis FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS 3831 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, K 3 Fe(CN) 6 , KCN, NaCN, NaNO 3 ] for 3 h, following which they were washed and then pulse-labeled (1 h) with [ 35 S]-methionine and harvested. Fer- ritin was immunoprecipitated by using anti-ferritin 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 l M) for 3 h and blotted onto nylon membranes. The filters were hybridized sequentially with [ 32 P]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). IRP-independent effects of NO + on Ft synthesis M. Mikhael et al. 3832 FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 SNP- 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 stimulated iron incorporation into ferritin [37], which would likely decrease iron lev- els in the labile iron pool 6 . In this study, we have 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 M. Mikhael et al. IRP-independent effects of NO + on Ft synthesis FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS 3833 [ 35 S]-methionine was from Perkin–Elmer (Boston, MA); [ 32 P]-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 cm 2 plastic culture dishes (Falcon, Franklin Lakes, NJ) in a humidified atmosphere of 95% air and 5% CO 2 at 37 °C 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 l g ÆmL )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 · 10 6 cells were washed with ice-cold NaCl ⁄ P i and lyzed at 4 °Cin80lL of lysis(+) buffer (10 mm Hepes, pH 7.5, 3 mm MgCl 2 ,40mm 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 lgÆ l L -1 , and 10 lg aliquots were analyzed for IRP binding by incubating them with an excess amount of 32 P-labeled pSRT-fer RNA tran- 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 [ 32 P]-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 methionine-free RPMI media, washed three times with cold NaCl ⁄ P i , 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 4 °C. Anti-ferritin IgG obtained from Roche (Indianapolis, IN) was added to the lysates and incubated overnight at 4 °C, then 60 lL of protein A–Sepharose was added for 3 h at 4 °C 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 4 °C in extraction buffer (10 mm Tris ⁄ HCl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl 2 , 0.5% Nonidet P-40 and 500 UÆmL )1 RNAsin), and nuclei were removed by centrifugation (12 000 g ,10s,4°C). The super- natant was supplemented with 20 mm dithiothreitol, 150 lgÆmL )1 cycloheximide, 1.5 mgÆmL )1 heparin and 1 mm phenylmethylsulfonyl fluoride and centrifuged (12 000 g, 5 min, 4 °C) 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 MgCl 2 ,10mm dithiothreitol, 100 lgÆmL )1 cycloheximide, and 0.5 mgÆmL )1 heparin) and centrifuged in a SW41Ti rotor (Beckman, Palo Alto, CA) (178 305 g, 120 min, 4 °C) 7 without brake. Fractions (550 lL) were collected and digested with 150 l g Æ mL )1 pro- teinase K in 1% SDS and 10 mm EDTA (30 min, 37 °C). 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 [ 32 P]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% CO 2 at 37 °C 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. IRP-independent effects of NO + on Ft synthesis M. Mikhael et al. 3834 FEBS Journal 273 (2006) 3828–3836 ª 2006 The Authors Journal compilation ª 2006 FEBS Acknowledgements This work was supported by a grant (to PP), a fellow- ship (to SFK), and a scholarship (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. References 1 Eisenstein RS (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu Rev Nutr 20, 627–662. 2 Klausner RD, Rouault TA & Harford JB (1993) Regu- lating the fate of mRNA: the control of cellular iron metabolism. Cell 72, 19–28. 3 Munro HN & Linder MC (1978) Ferritin: structure, biosynthesis, and role in iron metabolism. Physiol Rev 58, 317–396. 4 McCord JM (1998) Iron, free radicals, and oxidative injury. 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Iron regulatory protein-independent regulation of ferritin synthesis by nitrogen monoxide Marc Mikhael 1,2 , Sangwon F. Kim 2 , Matthias Schranzhofer 3 ,. effect on ferritin synthesis. More- over, acute regulation of ferritin synthesis by NO + is accomplished by a rapid mobilization of polysome-free ferritin