Iron is essential for all life, yet can be dangerous under certain conditions. Iron storage by the 24-subunit protein ferritin renders excess amounts of the metal non-reactive and, consequentially, ferritin is crucial for life. Although the mechanism detailing the storage of iron in ferritin has been well characterized, little is known about the fate of ferritin-stored iron and whether it can be released and reutilized for metabolic use within a single cell. Virtually nothing is known about the use of ferritin-derived iron in non-erythroid cells. We therefore attempted to answer the question of whether iron from ferritin can be used for haem synthesis in the murine macrophage cell line RAW 264.7 cells. Cells treated with ALA (5-aminolaevulinic acid; a precursor of haem synthesis) show increased haem production as determined by enhanced incorporation of transferrin-bound 59Fe into haem. However, the present study shows that, upon the addition of ALA, 59Fe from ferritin cannot be incorporated into haem. Additionally, little 59Fe is liberated from ferritin when haem synthesis is increased upon addition of ALA. In conclusion, ferritin in cultivated macrophages is not a significant source of iron for the cell's own metabolic functions.

INTRODUCTION

Iron is indispensable for life and functions as a metal cofactor of numerous proteins [1,2]. Iron-containing proteins utilize iron mostly in the form of haem prosthetic groups or iron–sulfur clusters, but other almost innumerable forms of iron prosthetic groups exist. Haemoproteins carry out the vital tasks of oxygen binding, oxygen metabolism and electron transfer. In addition, non-haem iron-containing proteins catalyse important reactions in energy metabolism and DNA synthesis.

The chemical properties that render iron so versatile for life, however, also create dangerous problems for living organisms. Namely, excess ‘free’ cellular iron in its ferrous form can catalyse the generation of ROS (reactive oxygen species) via the Fenton reaction [3]. It is therefore crucial that iron is safely sequestered and stored in the cell [4], a task exclusively performed by the ubiquitous iron-storing protein ferritin.

Ferritin is a 24-subunit protein (430–460 kDa) consisting of heavy (21 kDa) and light (19 kDa) ferritin chains [1,5] and is capable of storing up to 4500 atoms of iron in the form of ferric-oxyhydroxide phosphate [5]. Although the characterization of iron incorporation into ferritin has been well documented [5], the fate of iron once it has been stored in ferritin is poorly understood. It is well established that extensive phlebotomies can mobilize hepatic stores of ferritin-iron which is then, via Tf (transferrin), efficiently delivered into erythroblasts whose numbers are greatly elevated under these conditions [6,7]. However, it has never been shown whether ferritin-iron can serve as a source of bioavailable iron within a single cell.

Some previous studies have suggested that in developing red blood cells, ferritin-iron can be used for haem synthesis [811], although there is strong evidence to the contrary [6,1218]. Developing red blood cells, which can take up iron exclusively via the TfR1 (Tf receptor 1)/Tf pathway [2,19], synthesize approx. 85% of organismal haem for haemoglobin production. It has previously been reported that in erythroid precursors, a cytoplasmic chelator, which cannot enter endosomes or mitochondria, is unable to capture Tf-derived iron during its path to mitochondria (sites of haem synthesis) and that a highly efficient delivery of iron for haem synthesis requires an interaction of Tf-containing endosomes with mitochondria [20,21]. Importantly, these cells contain relatively low levels of ferritin [13] and extremely low amounts, if any, of iron outside haemoglobin [2]. Additionally, MEL (murine erythroleukaemic) cells constitutively expressing ferritin exhibit significantly decreased iron incorporation into haemoglobin, suggesting that high levels of ferritin ‘steal’ iron destined for haemoglobin [22]. Finally, the conditional deletion of ferritin heavy-chain in adult mice did not cause any decrease in haematocit or haemoglobin levels [22a]. The above observations are not compatible with the conclusion that ferritin is an intermediate in iron delivery to haemoglobin. We therefore investigated whether ferritin-iron donation and its utilization for haem synthesis can occur in cultured macrophages which express relatively high amounts of ferritin.

Macrophages of the RES (reticuloendothelial system) phagocytose senescent or damaged red blood cells, recycle haemoglobin-iron and release iron back into the circulation making it available for erythropoiesis and other iron-dependent processes. RES macrophages can temporarily store iron in ferritin and, under certain pathophysiological conditions, will contribute to ACD (anaemia of chronic disease) by inhibiting iron release into plasma. In ACD, inflammatory cytokines stimulate the release of hepcidin [23,24] which subsequently interacts with the iron exporter ferroportin [2527], resulting in its degradation and a resultant retention of iron within macrophages. It has also been shown that nitrogen monoxide species generated during inflammation can stimulate macrophage iron retention through hepcidin-independent mechanisms [28,29]. On the other hand, when plasma iron turnover increases, RES macrophages can reduce their retention of iron in order to satiate an increased organismal demand for functional iron [30]. Hence, macrophage cells can store considerable amounts of iron in a regulated manner. The aim of the present study was to determine whether these cells can draw upon this iron reserve to meet their own cellular requirements.

We investigated the utilization of iron in the murine macrophage cell line RAW 264.7, by stimulating haem synthesis using the haem-precursor ALA (5-aminolaevulinic acid). The generation of ALA from succinyl-CoA and glycine is catalysed by the enzyme ALA-S (ALA synthase). This is the first and rate-limiting step of the eight-step haem synthesis pathway in non-erythroid cells, which express the ubiquitous ALA-S isoform (ALA-S1) [31]. Therefore addition of ALA to culture medium led to increased 59Fe incorporation into haem while concomitantly increasing cellular 59Fe uptake, indicating a close relationship between haem synthesis and iron uptake in these non-erythroid cells. In contrast, 59Fe2–Tf pre-labelling experiments (in which macrophages were first labelled with 59Fe2–Tf, washed and then re-incubated in order to examine the utilization of ferritin-iron) revealed that ALA was unable to mobilize 59Fe from 59Fe-labelled ferritin. As expected [20], the inhibition of iron release from Fe2–Tf complexes by bafilomycin A1 inhibited 59Fe incorporation into haem in both untreated and ALA-treated macrophages. We provide in the present paper compelling evidence that challenges the generally accepted notion [32] that ferritin-iron is available for intracellular iron use (at least for haem synthesis) in cells that contain relatively high levels of ferritin, such as macrophages. Our results indicate that ferritin-iron is not available for haem synthesis. Perhaps the only way ferritin-iron can be released in macrophages is by ferritin degradation followed by iron export out of the cell by ferroportin [33]. In addition, treatment of RAW 264.7 macrophages with bafilomycin A1, an inhibitor of iron release from Fe2–Tf in endosomes, abrogates haem synthesis, similarly to developing erythrocytes [20] indicating that Fe2–Tf is the major, if not the sole, source of iron for haem synthesis in cultured macrophages.

MATERIALS AND METHODS

Chemicals

DMEM (Dulbecco's modified Eagle's medium), FBS (fetal bovine serum), penicillin, streptomycin and glutamine were from Invitrogen. ALA and SnPPIX [Sn-PPIX (protoporphoryrin IX)] were obtained from Frontiers Scientific. Bafilomycin A1 was obtained from BioShop. [35S]Methionine was obtained from PerkinElmer. [32P]UTP was obtained from Amersham Biosciences. 59Fe2–Tf was made from 59Fe-Cl3 obtained from PerkinElmer as described previously [34,35]. All other chemicals used were obtained from Sigma. SIH (salicylaldehyde isonicotinoyl hydrazone) and Fe–SIH were synthesized as described previously [16,36].

Cells

RAW 264.7 murine macrophages were obtained from the American Type Culture Collection. Cells were grown in 60 cm2 plastic culture dishes (Falcon) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C in DMEM containing 10% FBS, extra L-glutamine (300 μg/ml), sodium pyruvate (110 μg/ml), penicillin (100 units/ml) and streptomycin (100 μg/ml).

Macrophage 59Fe2–Tf labelling strategies

Experiments using 59Fe2–Tf were conducted in two different ways depending on the desired characteristics to be studied. First, in order to ascertain the rate of haem synthesis and/or total 59Fe2–Tf uptake, cells were incubated under various conditions simultaneously with 0.05 μM (saturating concentration) 59Fe2–Tf following which samples were washed twice with ice-cold PBS and collected by centrifugation (9300 g for 1 min at 4 °C); these experiments are referred to as ‘co-labelling experiments’. The measurement of 59Fe levels associated with haem or present in other cell compartments are described below.

Secondly, in order to investigate intracellular 59Fe movements (i.e. from ferritin to haem) cells were incubated in culture medium containing 0.05 μM 59Fe2–Tf for 48 h. Cells were then washed twice with ice-cold PBS and re-incubated in culture medium without 59Fe2–Tf. Following the re-incubation under various conditions, the samples were collected and analysed as described below. These experiments are referred to as ‘pre-labelling experiments’ and resemble, in principle, ‘pulse–chase experiments’.

Importantly, the tissue culture medium used in all experiments contains non-radioactive bovine serum–Tf.

Measurement of 59Fe in haem, non-haem fractions and ferritin

Measurements of 59Fe in haem and non-haem fractions were carried out as described previously by an acid precipitation method [12,20]. Cells were collected by centrifugation (9300 g for 1 min), lysed in water and boiled in 1 ml of 0.2 M HCL; the samples were then transferred to an ice-bath and 59Fe–haem-containing proteins were precipitated with ice-cold 7% TCA (trichloroacetic acid) solution and collected by centrifugation (1300 g for 5 min at 4 °C). Precipitated proteins (containing 59Fe in haem) were washed twice with 7% TCA post-centrifugation; supernatants contained non-haem 59Fe. Following collection of supernatants into separate tubes, measurements of 59Fe radioactivities in haem and non-haem fractions were carried out in a Packard Cobra gamma counter (PerkinElmer). In addition, radioactivities of immunoprecipitated 59Fe–ferritin were similarly measured (see below for the description of the immunoprecipitation method).

Western blot analysis

Cells were lysed at 4 °C in 80 μl of Munro (+) buffer [10 mM Hepes (pH 7.5), 3 mM MgCl2, 40 mM NaCl, 5% glycerol, 1 mM dithiothreitol and 0.2% Nonidet P40]. Samples were then diluted with 2 vol. of Munro (−) buffer [10 mM Hepes (pH 7.5), 3 mM MgCl2, 40 mM NaCl, 5% glycerol and 1 mM dithiothreitol]. Cell debris was cleared by centrifugation (9300 g for 1 min at 4 °C) and the protein concentration was measured using Bradford reagent (Bio-Rad). Cell lysates (15 μg) were resolved by SDS/PAGE and proteins transferred on to nitrocellulose filters. The blots were blocked with 10% (w/v) non-fat dried skimmed milk in PBS containing 0.1% Tween 20 (BioShop) and probed with anti-TfR (1:4000 dilution; Zymed Laboratories), anti-ferritin (1:500 dilution; Dako), anti-HO-1 (haem oxygenase-1, 1:10000 dilution; Stressgen) or anti-(β-actin) (1:500 dilution; Sigma) antibodies at 4 °C overnight. After three washes with PBS containing 0.1% Tween 20, the blots were further incubated for 2 h at room temperature (25 °C) with goat anti-(rabbit IgG) (1:5000 dilution; Sigma) and, for TfR, anti-(mouse IgG) (1:5000 dilution; Sigma). Detection of the peroxidase-coupled secondary antibodies was performed using the ECL (enhanced chemiluminescence) method as described by the manufacturer (Amersham Biosciences). The blots were quantified by densitometry.

Metabolic labelling and immunoprecipitation

Cells were labelled for 1 h with 100 μCi/ml [35S]methionine in methionine-free DMEM, washed three times with ice-cold PBS, after which they were lysed with RIPA buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate and 0.1% SDS] for 30 min at 4 °C. Pulse–chase experiments were carried out similarly, albeit with [35S]methionine pre-labelling prior to various treatments. Anti-ferritin IgG, obtained from Roche, was added to lysates which contained equivalent amounts of protein, according to the method of Bradford, and incubated overnight at 4 °C, then 60 μl of Protein A–Sepharose was added for 3 h at 4 °C to precipitate the immune complexes. The beads were washed three times with ice-cold RIPA buffer and then boiled with SDS loading dye. Immunoprecipitated protein was resolved using SDS/PAGE (12.5% gel). The gel was dried and analysed by autoradiography.

Gel-retardation assay

The gel-retardation assay used to measure the interaction between IRPs (iron regulatory proteins) and the IRE (iron response element) was carried out as described previously [37]. Briefly, 6×106 cells were washed with ice-cold PBS and lysed at 4 °C in 80 μl of Munro (+) buffer. Samples were then diluted with 2 vol. of Munro (−) buffer to a protein concentration of 1 μg/μl, and 10 μg aliquots were analysed for IRP binding by incubating them with an excess amount of 32P-labelled pSRT-fer RNA transcript (kindly provided by Dr Lukas Kühn, Swiss Institute for Experimental Cancer Research, CH-1015 Lausanne, Switzerland), which contains one IRE. This RNA was transcribed in vitro from linearized plasmid template using T7 RNA polymerase in the presence of [32P]UTP. To form RNA–protein complexes, cytoplasmic extracts were incubated for 10 min at room temperature with an excess amount of labelled RNA. Heparin (5 mg/ml) was added for a further 10 min to prevent non-specific binding. RNA–protein complexes were analysed in 6% non-denaturing polyacrylamide gels. In parallel, duplicate samples were treated with 2% 2-mercaptoethanol before the addition of the RNA probe.

Haem assay

The intracellular haem content was assayed as described previously [38]. Briefly, following counting, the macrophages were resuspended in 500 μl of concentrated formic acid. The haem concentration was determined spectrophotometrically at 395 nm in a Varian Cary 13e spectrophotometer. The resulting absorbances were compared against a standard curve of haemin.

PPIX assay

The intracellular PPIX content was measured according to a previously published method [39]. Briefly, macrophages were resuspended in 100 μl of a suspension of 5% celite and 0.9% NaCl. A 2 ml aliquot of a 4:1 ethyl acetate/acetic acid were then added. The samples were mixed by vortex-mixing for 10 s and then centrifugation for 20 s at 500 g. The supernatants were then transferred to another set of tubes to which 2 ml of 1.5 M HCl was added. Samples were then vortex-mixed for 10 s and then centrifuged for 20 s at 500 g. Then, 2 ml of the lower phase was transferred into a quartz cuvette. The fluorescence was then measured at an excitation wavelength of 405 nm and an emission wavelength of 610 nm using a PerkinElmer Luminescence Spectrometer, LS 55.

Photography

Pictures of cell pellets were taken using a digital camera in ambient light or a dark room with UV light. The UV light was produced by a EGUV-58 Mineralight Multiband UV lamp (115 V, 60 Hz, 47 A).

Preparation of reticulocytes

Adult female CD1 mice (Charles River) were injected intraperitoneally with neutralized phenylhydrazine at a dose of 50 mg/kg of body weight per day for three continuous days. On the third or fourth day following the last injection, mice were anaesthetized using 1 ml of 2.5% avertin, and blood was collected via cardiac puncture. After three washes with ice-cold PBS, cells (approx. 45% reticulocytes, as determined by new Methylene Blue staining) were resuspended in modified Eagle's medium [containing 25 mM Hepes, 10 mM NaHCO3 and 1% BSA (pH 7.4)], and incubated at 37 °C with shaking. All usage of animals was performed in accordance with McGill University's animal use protocols and ethical guidelines.

Data analysis

All results presented are representative of three or more repeated experiments. All experiments using 59Fe2–Tf were done using triplicates for each sample. Values are means±S.D. ImageJ software was used for all densitometric analysis. Statistical significance was always calculated against control samples using the Student's t test.

RESULTS

ALA increases 59Fe incorporation into haem in macrophages

Before determining the possible availability of ferritin-iron for haem synthesis in macrophages, we first needed to examine whether ALA levels can affect iron incorporation from Tf into haem. We therefore treated the cells with 59Fe2–Tf together with ALA for 6 or 24 h and determined the amount of 59Fe present in the cells as the haem or non-haem fractions (Figure 1). Importantly, 59Fe incorporation into haem increased significantly upon addition of ALA, demonstrating that ALA treatment enhances haem synthesis (Figure 1B). Moreover, cellular 59Fe uptake was also increased in parallel with 59Fe incorporation into haem (Figure 1A). These results indicate that haem synthesis in cultured macrophages exerts a significant influence on iron uptake in contrast with the scenario seen in reticulocytes in which added ALA does not increase iron uptake from Tf [40].

ALA treatment increases 59Fe incorporation into haem and its cellular uptake

Figure 1
ALA treatment increases 59Fe incorporation into haem and its cellular uptake

RAW 264.7 cells were simultaneously labelled with 0.05 μM 59Fe2–Tf and treated with or without 1 mM ALA for 6 and 24 h. Cells were then lysed and incubated at 100 °C in 0.2 M HCl. After boiling, proteins containing 59Fe–haem were precipitated using TCA. (A) Total cellular 59Fe levels; calculated by combining measurements of pellets and supernatants. (B) 59Fe levels in haem. (C) Ambient light photograph of cell pellets of samples treated without or with 1 mM ALA. (D) Histogram of haem levels measured spectrophotometrically [using parallel samples as those shown in (C)]. (E) UV light photograph of the same samples photographed in (C). (F) Histogram of PPIX levels measured spectrofluorimetrically [using parallel samples as (C)]. PPIX levels in untreated RAW 264.7 cells were extremely low or non-measurable. (G) As a positive control of PPIX assays, mouse reticulocytes were treated with 1 mM ALA, 1 mM SA or 100 μM SIH for 2 h following which PPIX levels were measured spectrofluorimetrically. Ctrl, control.

Figure 1
ALA treatment increases 59Fe incorporation into haem and its cellular uptake

RAW 264.7 cells were simultaneously labelled with 0.05 μM 59Fe2–Tf and treated with or without 1 mM ALA for 6 and 24 h. Cells were then lysed and incubated at 100 °C in 0.2 M HCl. After boiling, proteins containing 59Fe–haem were precipitated using TCA. (A) Total cellular 59Fe levels; calculated by combining measurements of pellets and supernatants. (B) 59Fe levels in haem. (C) Ambient light photograph of cell pellets of samples treated without or with 1 mM ALA. (D) Histogram of haem levels measured spectrophotometrically [using parallel samples as those shown in (C)]. (E) UV light photograph of the same samples photographed in (C). (F) Histogram of PPIX levels measured spectrofluorimetrically [using parallel samples as (C)]. PPIX levels in untreated RAW 264.7 cells were extremely low or non-measurable. (G) As a positive control of PPIX assays, mouse reticulocytes were treated with 1 mM ALA, 1 mM SA or 100 μM SIH for 2 h following which PPIX levels were measured spectrofluorimetrically. Ctrl, control.

After 24 h of labelling with 59Fe, the percentage of the total cellular 59Fe in ferritin and haem was 50% (results not shown; see also Figure 5C) and 10% respectively, and remained unchanged upon addition of ALA (Figures 1A and 1B). In other words, the increase in 59Fe incorporation into haem observed after ALA treatment is due to an increase in iron uptake and not a redistribution of intracellular iron stores, as would be expected if ferritin-iron was used for haem synthesis.

Interestingly, ALA-treated RAW 264.7 cell pellets were distinctly brown indicating an observable increase in their haem content (Figure 1C). More importantly, haem levels measured spectrophotometrically were increased following addition of ALA (Figure 1D). Whereas 24 h treatment of RAW 264.7 cells with ALA increased their haem levels only 1.7-fold, the increase in PPIX levels was much more substantial, from virtually immeasurable levels to more than 20 nmol/mg of protein (Figures 1E and 1F). This experiment is congruent with the conclusion that iron is not available for haem synthesis in RAW 264.7 cells when their PPIX levels are experimentally increased.

ALA treatment alters cellular iron homoeostasis

To examine the effects of ALA treatment on cellular iron metabolism, we examined the levels of several proteins and/or their activities following treatment of macrophages with ALA over the course of 24 h (Figures 2 and 3). TfR levels were increased (Figures 2A and 2B), matching the increased rates in cellular 59Fe uptake (Figure 1A). HO-1, which is inducible by haem and catabolizes haem into biliverdin and carbon monoxide, also releases iron from the tetrapyrrole macrocycle [41]. HO-1 is dramatically and very rapidly increased in ALA-treated cells (Figure 2C), further supporting the conclusion that an increase in haem production occurs upon addition of ALA to macrophages. Interestingly, ferritin protein levels were decreased in ALA-treated cells (Figure 2D).

ALA treatment leads to increased TfR and HO-1 expression and decreased ferritin levels

Figure 2
ALA treatment leads to increased TfR and HO-1 expression and decreased ferritin levels

(A) Western blot of TfR, HO-1 and ferritin following 1 mM ALA treatments over time. (BD) Densitometric analysis of (B) TfR, (C) HO-1 and (D) ferritin levels respectively; all histograms were normalized to β-actin. Gels shown here are from a representative experiment; each blot is from a single gel and lanes were rearranged for conceptual clarity. Values are based on control levels at each time interval. Ctrl, control.

Figure 2
ALA treatment leads to increased TfR and HO-1 expression and decreased ferritin levels

(A) Western blot of TfR, HO-1 and ferritin following 1 mM ALA treatments over time. (BD) Densitometric analysis of (B) TfR, (C) HO-1 and (D) ferritin levels respectively; all histograms were normalized to β-actin. Gels shown here are from a representative experiment; each blot is from a single gel and lanes were rearranged for conceptual clarity. Values are based on control levels at each time interval. Ctrl, control.

The RNA-binding proteins IRP1 and IRP2 are key sensors of intracellular iron levels and regulate TfR and ferritin levels in response to changes in cellular iron status [1,2]. Under iron-depleted conditions, both IRPs bind to IREs, nucleotide stemloop sequences located in the 5′-UTR (5′-untranslated regions) of ferritin mRNA and the 3′-UTR of TfR mRNA. Such binding leads to translational repression of ferritin mRNA and stabilization of the TfR message, thereby increasing iron uptake from Tf. Conversely, under iron-replete conditions, overall IRP-binding activity is decreased, leading to TfR mRNA destabilization and efficient ferritin mRNA translation. This reciprocal regulation allows increased iron storage together with reduced iron uptake [37,42,43].

Treatment of macrophages with ALA led to modulation of IRP-binding activities as assessed by electromobility band-shift assays (Figure 3). IRP1 binding increased (Figure 3B), possibly explaining increased TfR levels (Figure 2B) and blunted ferritin expression (Figure 2D). Decreased IRP2 activity, on the other hand, probably reflects an increase in haem [44] or intracellular iron levels following increased iron uptake in ALA-treated cells (Figure 1A). Interestingly, ALA has been reported to mediate IRP1 activation [45] via pro-oxidative mechanisms and may lead to such changes here as well.

ALA treatment conversely affects IRP1 and IRP2 binding activities

Figure 3
ALA treatment conversely affects IRP1 and IRP2 binding activities

(A) IRP levels following 1 mM ALA treatment over time. Electromobility band-shift assay using a radiolabelled IRE probe. 2-Mercaptoethanol (β-ME)-treated cell lysates were used as a loading control. (B and C) Densitometric analysis of IRP1 and IRP2 binding activity respectively; normalized to β-ME-treated samples. Values are based on control levels at each time interval. Ctrl, control.

Figure 3
ALA treatment conversely affects IRP1 and IRP2 binding activities

(A) IRP levels following 1 mM ALA treatment over time. Electromobility band-shift assay using a radiolabelled IRE probe. 2-Mercaptoethanol (β-ME)-treated cell lysates were used as a loading control. (B and C) Densitometric analysis of IRP1 and IRP2 binding activity respectively; normalized to β-ME-treated samples. Values are based on control levels at each time interval. Ctrl, control.

Ferritin synthesis, but not stability, is decreased by ALA treatment

We further examined the effects of ALA on ferritin synthesis and stability by pulsing the cells with [35S]methionine after (ferritin synthesis) or before (ferritin stability) ALA treatment and quantitatively immunoprecipitating ferritin (Figure 4). Ferritin synthesis was dramatically decreased in ALA-treated cells (Figure 4B), suggesting that elevated IRP activities may be blocking ferritin synthesis. On the other hand, ferritin stability did not change upon ALA treatments (Figure 4C), in contrast with what has been reported previously [45]. These results suggest that a decrease in ferritin synthesis, and not stability, upon ALA treatment leads to the reduced protein levels seen in Figure 2(D).

Ferritin synthesis, but not stability, is decreased following ALA treatment

Figure 4
Ferritin synthesis, but not stability, is decreased following ALA treatment

(A) (i) Ferritin synthesis. Cells were treated for the times indicated with 1 mM ALA, washed and then labelled with [35S]methionine for 1 h. Cells were then washed and lysed, following which ferritin was immunoprecipitated using anti-ferritin antibodies and Protein A–Sepharose beads. The samples were analysed using SDS/PAGE and autoradiography. (ii) Ferritin stability. Cells were first pulsed with [35S]methionine for 1 h, washed and then treated for the times indicated with ALA. Cells were lysed and ferritin immunoprecipitated as in (i). (B and C) Densitometry analysis of autoradiographs of ferritin synthesis and stability respectively. The radiograph shown here is from a representative experiment. Each radiograph is from a single gel and lanes were rearranged for conceptual clarity. Ctrl, control.

Figure 4
Ferritin synthesis, but not stability, is decreased following ALA treatment

(A) (i) Ferritin synthesis. Cells were treated for the times indicated with 1 mM ALA, washed and then labelled with [35S]methionine for 1 h. Cells were then washed and lysed, following which ferritin was immunoprecipitated using anti-ferritin antibodies and Protein A–Sepharose beads. The samples were analysed using SDS/PAGE and autoradiography. (ii) Ferritin stability. Cells were first pulsed with [35S]methionine for 1 h, washed and then treated for the times indicated with ALA. Cells were lysed and ferritin immunoprecipitated as in (i). (B and C) Densitometry analysis of autoradiographs of ferritin synthesis and stability respectively. The radiograph shown here is from a representative experiment. Each radiograph is from a single gel and lanes were rearranged for conceptual clarity. Ctrl, control.

Ferritin-iron is unavailable for haem synthesis in macrophages

In order to examine whether ferritin-iron stores are decreased upon stimulation of haem synthesis, we pre-labelled cells with 0.05 μM 59Fe2–Tf for 48 h following which cells were washed and subsequently treated with or without ALA for 24 h. Ferritin was then immunoprecipitated and the 59Fe content of ferritin was measured. Interestingly, our results indicate that, upon the addition of ALA, neither ferritin-associated 59Fe (Figure 5B) nor the percentage of total 59Fe associated with ferritin changes (Figure 5C), despite decreases in ferritin protein levels (Figure 2D). This strongly argues against the hypothesis that ferritin-iron can be mobilized and used for haem synthesis.

Ferritin-iron stores remain unchanged following ALA treatment

Figure 5
Ferritin-iron stores remain unchanged following ALA treatment

Cells were first labelled with 0.05 μM 59Fe2–Tf for 48 h. Cells were then washed and re-incubated for 24 h with and without 1 mM ALA. Cells were lysed and ferritin was immunoprecipitated as described in Figure 4(A). (A) Total cellular 59Fe; calculated by combining measurements of immunoprecipitated protein and supernatants. (B) 59Fe in immunoprecipitated ferritin. (C) The percentage of the total cellular 59Fe in ferritin. Ctrl, control; kcpm, 1000 c.p.m.

Figure 5
Ferritin-iron stores remain unchanged following ALA treatment

Cells were first labelled with 0.05 μM 59Fe2–Tf for 48 h. Cells were then washed and re-incubated for 24 h with and without 1 mM ALA. Cells were lysed and ferritin was immunoprecipitated as described in Figure 4(A). (A) Total cellular 59Fe; calculated by combining measurements of immunoprecipitated protein and supernatants. (B) 59Fe in immunoprecipitated ferritin. (C) The percentage of the total cellular 59Fe in ferritin. Ctrl, control; kcpm, 1000 c.p.m.

Treatment with ALA increased 59Fe incorporation into haem when macrophages were co-labelled with 59Fe2–Tf (Figure 1B). However, this experiment does not address whether iron can be mobilized from ferritin and used for haem synthesis upon addition of ALA. We therefore pre-labelled macrophages with 59Fe2–Tf for 48 h (in a similar manner as in the above experiment shown in Figure 5), washed and then treated the cells with or without ALA for 24 h (Figure 6). Importantly, both 59Fe incorporation into haem (Figure 6B) and 59Fe retention (Figure 6A) in ALA-treated cells did not differ from untreated cells, despite the strong stimulation of haem production (Figure 1A). These results unequivocally demonstrate that neither ferritin- nor non-ferritin-iron is available for haem synthesis in this model. This conclusion is further supported by our finding that there is neither a decrease in 59Fe–ferritin (Figure 5) nor an increase in 59Fe-labelled haem following treatment with ALA (Figure 6B).

Intracellular iron stores cannot donate iron for haem synthesis in macrophages

Figure 6
Intracellular iron stores cannot donate iron for haem synthesis in macrophages

Cells were first labelled with 0.05 μM 59Fe2–Tf for 48 h. Cells were then washed and re-incubated for 24 h with and without 1 mM ALA and 50 μM SnPPIX. Cells were then lysed in acid and haem was precipitated using TCA as described in Figure 1. (A) Total cellular 59Fe levels; calculated by combining measurements of 59Fe radioactivities in haem and non-haem iron proteins. (B) 59Fe levels in haem. (C) The percentage of the total cellular 59Fe in haem. Ctrl, control.

Figure 6
Intracellular iron stores cannot donate iron for haem synthesis in macrophages

Cells were first labelled with 0.05 μM 59Fe2–Tf for 48 h. Cells were then washed and re-incubated for 24 h with and without 1 mM ALA and 50 μM SnPPIX. Cells were then lysed in acid and haem was precipitated using TCA as described in Figure 1. (A) Total cellular 59Fe levels; calculated by combining measurements of 59Fe radioactivities in haem and non-haem iron proteins. (B) 59Fe levels in haem. (C) The percentage of the total cellular 59Fe in haem. Ctrl, control.

HO-1 activity does not ‘mask’ mobilization of intracellular iron for haem synthesis

59Fe–haem levels remain unchanged in pre-labelled cells following ALA treatment (Figure 6). However, HO-1 levels were significantly increased upon addition of ALA (Figure 2C). It is therefore conceivable that in our experiments, in which the cells were pre-labelled with 59Fe before ALA addition, increased HO-1 activity could liberate 59Fe from haem, which could then be used for de novo haem synthesis. Therefore we treated 59Fe- pre-labelled cells with the HO-1 inhibitor SnPPIX (Figure 6). SnPPIX decreased both the degradation of 59Fe-labelled haem (Figure 6B) and the export of 59Fe from cells (Figure 6A), indicating that SnPPIX treatment successfully inhibited HO-1 activity. Importantly, neither 59Fe–haem levels (Figure 6B) nor 59Fe retention (Figure 6A) differed between SnPPIX-treated and ALA+SnPPIX-treated cells, indicating that HO-1 activity is not ‘masking’ intracellular iron mobilization for haem synthesis. As expected, treatment with SnPPIX increased the percentage of intracellular 59Fe in haem (Figure 6C). These results indicate that iron liberated from haem by HO-1 is not a source for haem synthesis, further confirming our conclusion that no source of intracellular iron is available for haem synthesis in these cells.

Haem synthesis in cultured macrophages is dependent on iron uptake from Tf

We have established that neither intracellular nor ferritin-iron stores represent a significant source of iron for haem synthesis in macrophages (Figure 6). Likewise, ferritin-iron in erythroid cells cannot be used for haem synthesis (reviewed in [13]); instead, these cells have an absolute requirement for Tf-bound iron [13,19]. Indeed, the incubation of reticulocytes with bafilomycin A1, an inhibitor of the v-ATPase (vacuolar-type H+-ATPase) proton pump responsible for endosomal acidification (and thus the release of iron from Tf), led to an almost complete abrogation of haem synthesis [20].

We therefore treated macrophages with bafilomycin A1 along with ALA in an attempt to elucidate whether macrophages are also dependent on Fe2–Tf for haem synthesis (Figure 7). Bafilomycin A1 treatment alone led to a reduction in both 59Fe incorporation into haem (Figure 7B) and 59Fe uptake (Figure 7A), documenting that efficient haem synthesis in cultured macrophages depends on the availability of Tf-borne iron. This conclusion is further supported by our finding that ALA-stimulated incorporation of 59Fe into haem can be significantly inhibited by bafilomycin A1 (compare ALA with ALA+Baf in Figure 7B).

59Fe incorporation into haem is abrogated when iron release from transferrin is blocked by bafilomycin A1

Figure 7
59Fe incorporation into haem is abrogated when iron release from transferrin is blocked by bafilomycin A1

Cells were simultaneously labelled with 0.05 μM 59Fe2–Tf and treated with or without the treatments indicated [1 mM ALA; 10 nM bafilomycin A1 (Baf) or 1 mM SA] for 24 h. Cells were then lysed in acid and haem was precipitated using TCA as described in Figure 1. (A) Total cellular 59Fe levels; calculated by combining measurements of 59Fe radioactivities in haem and non-haem iron proteins. (B) 59Fe levels in haem.

Figure 7
59Fe incorporation into haem is abrogated when iron release from transferrin is blocked by bafilomycin A1

Cells were simultaneously labelled with 0.05 μM 59Fe2–Tf and treated with or without the treatments indicated [1 mM ALA; 10 nM bafilomycin A1 (Baf) or 1 mM SA] for 24 h. Cells were then lysed in acid and haem was precipitated using TCA as described in Figure 1. (A) Total cellular 59Fe levels; calculated by combining measurements of 59Fe radioactivities in haem and non-haem iron proteins. (B) 59Fe levels in haem.

As a positive control for inhibition of haem synthesis we treated cells with SA (succinylacetone), a competitive inhibitor of ALA dehydratase, the second enzyme in the haem synthesis pathway. Indeed, SA treatment of cells decreased both 59Fe incorporation into haem (Figure 7B) and 59Fe uptake (Figure 7A) to a similar degree as bafilomycin A1. In contrast, SA increases iron uptake in reticulocytes [16], further highlighting the fundamental differences that exist between erythroid [12,16,18] and macrophage iron metabolism.

DISCUSSION

Although iron is absolutely essential for life, it is potentially harmful because of its capacity to catalyse the production of ROS [3]. Therefore it is crucial for organisms to tightly control iron levels. The ferritin nanocage can store up to 4500 atoms of iron where it is safely sequestered. The fate of iron once stored in ferritin has generally been assumed [32,46] to be a ready intracellular source of iron for the synthesis of proteins that require iron or haem for their function. Whereas ferritin can release iron, albeit very slowly [47], to the best of our knowledge no conclusive evidence to date has been reported that ferritin can effectively donate iron intracellularly for the synthesis of iron-dependent proteins.

Approx. 85% of organismal haem is made in immature erythroid cells for haemoglobin synthesis [31]. Some studies, building on the observation that ferritin levels decrease with the onset of haemoglobin synthesis in developing erythrocytes, suggested that ferritin-iron in haemoglobinizing cells is used for haem synthesis [811]. Previously, we collected compelling evidence against a role of both extra- and intra-cellular ferritin-iron donation for erythrocyte haem synthesis and asserted that Fe2–Tf is the exclusive source of iron for erythroid cells [13]. Pertaining to ferritin as a possible intermediate in donating iron for haemoglobin synthesis, several studies have failed to show ferritin-derived iron incorporation into haem in erythroid cells [6,12,1418]. Of tantamount importance to this discussion is the specific relationship of transferrin to erythroid cells as the only physiologically relevant donor of iron [19] and the fact that the rate of iron uptake from Tf by developing erythroid cells almost exactly matches the rate of haemoglobin production [7,13,31].

The aforementioned discussion has focused on ferritin-poor but haem-rich erythroid cells. However, utilization of ferritin-iron for haem synthesis, assuming it occurs, should be investigated using ferritin-rich cells such as macrophages that play an indispensable role in the recycling, and often storage, of haemoglobin-derived iron [7,23]. Indeed, in a normal healthy individual, macrophages of the RES can store up to 10% of total organismal iron, whereas in individuals suffering from ACD, an even greater retention of iron is observed in these cells [23,24]. We therefore carried out a series of experiments in RAW 264.7 macrophages in order to examine the relationship between intracellular stores of iron, ferritin and haem synthesis in these cells.

We first ascertained that ALA, a haem precursor, is capable of increasing 59Fe incorporation into haem (Figure 1B) and simultaneously also increasing 59Fe uptake, indicating that treatment with ALA increases haem synthesis (as also noted by the brown colour of ALA-treated cell pellets in Figure 1C). Additionally this experiment revealed that in these cells, a tight positive relationship exits between haem synthesis and iron uptake. This is in contrast with what happens in developing erythroid cells where cellular iron uptake is the limiting step of haem sythesis [31,35]. As expected, we also observed an increase in TfR and HO-1 (Figure 2), which is of utmost importance for haemoglobin recycling in macrophages, along with changes in the IRP/IRE binding status (Figure 3). It cannot be ignored, however, that ALA and the resultant elevation of free PPIX may introduce oxidative stress which contributes to the activation of IRP1 and induction of HO-1 protein expression. We have previously shown that non-haem HO-1 induction in RAW 264.7 cells still affords protection against oxidative stress and, in line with the results of the present study, does not induce ferritin production [48]. Furthermore, the dramatic increase in PPIX synthesis may provide a sink for iron in the mitochondria, decreasing its availability for iron–sulfur protein biogenesis, thereby increasing IRP1-binding activity [49].

Interestingly, ferritin protein levels (Figure 2C) and synthesis (Figure 4B) decreased upon addition of ALA. As ferritin stability is not altered in these conditions (Figure 4C), the decrease of ferritin protein levels is due to a decrease in ferritin synthesis. Moreover, and most importantly, ferritin-iron stores are not decreased upon stimulation of haem synthesis by ALA treatment (Figure 5) as would be expected if ferritin-iron was delivered to mitochondria for insertion into PPIX to make haem. This is reminiscent of the irresponsiveness of some patients to erythropoietin therapy, a condition referred to as ‘relative’ or ‘functional iron deficiency’ [50]. In both situations, the stimulus for increased haemoglobinization could be, at least in part, counteracted by a low efficiency in the mobilization of stored iron from macrophages. Indeed, Finch and colleagues [30] described in their in vivo studies that after an initial phase of rapid iron release to the circulation (t1/2 of 34 min) from haemoglobin processing in RES macrophages, there was a slow period of iron discharge from macrophages that had a t1/2 of 7 days; the second period probably represents a slow phase of iron mobilization from ferritin that may involve its degradation [33].

At first glance one may argue that the decrease in ferritin synthesis is evidence that ferritin-iron can be delivered for haem synthesis. Therefore we pre-labelled macrophages with 59Fe2–Tf and then treated cells with or without ALA (Figures 5 and 6). We observed that ALA treatment did not increase 59Fe–haem levels (Figure 6B) nor 59Fe retention (Figure 6A) by macrophages. However, there is a possibility that the increased levels of HO-1 following ALA treatment (Figure 2C) could be masking the utilization of intracellular stores of iron for haem synthesis by causing a rapid degradation of de novo synthesized haem. We therefore inhibited HO-1 with the addition of SnPPIX and found that, whereas this treatment partially prevented the degradation of pre-labelled 59Fe–haem, it did not affect 59Fe–haem levels following ALA treatment (Figure 6B). These results, along with a failure of ALA treatments to mobilize 59Fe–ferritin for haem synthesis (Figure 5), strongly suggest that intracellular stores of iron cannot be used for haem synthesis in macrophages, despite the abundance of ferritin-iron in these cells.

We have previously reported a stringent requirement of Fe2–Tf for haem synthesis in reticulocytes by showing that an inhibitor of endosomal iron release from Tf, bafilomycin A1, blocked iron incorporation into haem [20]. We therefore attempted to examine whether haem synthesis in macrophages was also dependent on the availability of Fe2–Tf by incubating macrophages with bafilomycin A1 and 59Fe2–Tf. We observed a remarkable inhibition of 59Fe incorporation into haem upon bafilomycin A1 treatment, despite an abundance of iron already present within the cells inside ferritin, suggesting that macrophages cultivated in vitro are also dependent on Fe2–Tf for their haem synthesis (Figure 7B).

Taken together, the results described above provide considerable evidence that ferritin-iron stores are not a significant source of iron for haem synthesis in macrophages, similarly to what has previously been observed in developing erythroid cells. The stringent requirement of Tf-bound iron for haem synthesis also occurs in cells that have the capacity to store considerable amounts of iron by virtue of their role in recycling haemoglobin-iron. However, further research is needed to investigate the source of iron for haem synthesis in myoglobin-producing skeletal and cardiac myocytes, and in hepatocytes that contain high levels of cytochromes.

Abbreviations

     
  • ACD

    anaemia of chronic disease

  •  
  • ALA

    5-aminolaevulinic acid

  •  
  • ALA-S

    ALA synthase

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • FBS

    fetal bovine serum

  •  
  • HO-1

    haem oxygenase-1

  •  
  • IRE

    iron response element

  •  
  • IRP

    iron regulatory protein

  •  
  • PPIX

    protoporphoryrin IX

  •  
  • RES

    reticuloendothelial system

  •  
  • ROS

    reactive oxygen species

  •  
  • SA

    succinylacetone

  •  
  • SIH

    salicylaldehyde isonicotinoyl hydrazone

  •  
  • TCA

    trichloroacetic acid

  •  
  • Tf

    transferrin

  •  
  • TfR

    Tf receptor

  •  
  • UTR

    untranslated region

AUTHOR CONTRIBUTION

Marc Mikhael carried out all experiments and wrote the manuscript. Marc Mikhael, Alex Sheftel and Prem Ponka conceived experiments and edited the manuscript before acceptance.

We thank Dr Tariq Roshan for his help in preparing mouse reticulocytes.

FUNDING

This research was supported by grants from the Canadian Institutes for Health Research (CIHR) [grant numbers 7331 and 141000] and the Canadian Blood Services awarded to P. P.

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