Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for βmajor-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/β MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.

INTRODUCTION

The mechanisms governing haemoglobin synthesis and the maturation of erythroid cells have been studied in great detail and serve as a paradigm for the regulation of gene expression and cellular differentiation [1]. Friend-virus-infected MEL (mouse erythroleukaemia) cells provide a valuable model to study the molecular events governing erythroid differentiation. MEL cells can be induced to undergo erythroid differentiation by a variety of agents including DMSO, HMBA (hexamethylene bisacetamide) and haemin [25]. MEL cell differentiation parallels the development from proerythroblasts to orthochromatic normoblasts in vivo and is manifested by the induction of proteins involved in globin expression, haem synthesis and iron uptake, as well as other erythroid markers including the Band 3 anion exchanger [610]. Terminal differentiation of MEL cells is shown by reduced cell growth and the production of large amounts of haemoglobin [5].

A series of studies suggested that MAPKs (mitogen-activated protein kinases) play an important role in erythroid differentiation. Eukaryotic cells possess multiple MAPK pathways, including the ERKs (extracellular-signal-regulated kinases), the p38 MAPKs and the JNKs (c-Jun N-terminal kinases). A range of stimuli, including cytokines, growth factors and environmental stress, have been shown to activate these pathways [11]. The involvement of MAPKs in the regulation of erythroid differentiation has been studied in mammalian cell lines using a variety of inducing agents, including EPO (erythropoietin), hydroxyurea, butyrate and haemin [1220]. For instance, down-regulation of p38 MAPK expression by antisense oligonucleotides leads to a block in differentiation in EPO-induced erythroleukaemic SKT6 cells [20]. In contrast, inhibition of ERK1/2 promoted EPO-induced erythroid differentiation in the leukaemia cell line UT-7/GM [16]. Butyrate-induced erythroid differentiation of K562 cells also involved the inhibition of ERK and the activation of the p38 MAPK pathways [15]. These and other studies have illustrated that MAPKs play an important role in erythrocyte maturation, but many aspects of MAPK function in the regulation of erythroid differentiation remain to be elucidated.

In the present study, we have investigated the role of ERK and p38 MAPK in the differentiation of MEL cells exposed to the inducer HMBA. Our results suggest that the ERK and p38 MAPK pathways play opposing roles in the regulation of erythroid differentiation through the modulation of genes involved in globin production, haem synthesis and iron uptake.

EXPERIMENTAL

Cells and culture conditions

MEL cells (clone 745 GM86) [21] were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum) with the addition of 100 units/ml penicillin and 100 μg/ml streptomycin. For experiments, 2×105 cells/ml were cultured for the indicated times with or without 4 mM HMBA. At 1 h before adding this inducing agent, 5 μM of the MEK1/2 (MAPK/ERK kinase 1/2) inhibitor U0126 (Promega) or 3 μM of the p38α/β MAPK inhibitor SB202190 (Calbiochem) was added to the cells.

Fluorimetric haem assay

The total cellular concentration of haem was determined fluorimetrically as described previously [7] with minor modifications. The method consists of the removal of haem iron under acid-reducing conditions, resulting in the conversion of haem into its fluorescent derivative porphyrin. MEL cells were pelleted at 2000 g for 5 min and washed with 1× PBS. Then, 2×105 cells were centrifuged at 3800 g for 10 min. The pellets were resuspended in 500 μl of fresh and filtered 2 M oxalic acid. The tubes were vortex-mixed vigorously and heated at 100 °C for 30 min. The samples were cooled, and fluorescence was measured in a PerkinElmer LS55 luminescence spectrometer equipped with an R-955 red-sensitive emission photomultiplier tube. The excitation wavelength was 404 nm and the emission wavelength was 655 nm [22]. A standard curve was generated using a 10 mM haemin stock solution prepared in 1% ethanolamine (pH 7.8) [23].

Haemoglobin assay

The haemoglobin content of MEL cells was determined using a spectrophotometric assay, as described previously [24]. The net absorbance at 413 nm (wavelength of maximum absorption for haemoglobin) was determined and normalized to the total protein concentration.

Northern blot analysis

Total RNA was extracted using the TRIzol® reagent (Invitrogen), according to the manufacturer's instructions. A portion (10 μg) of RNA was subjected to formalin/agarose gel electrophoresis (1% agarose), transferred on to a HYBOND-XL nylon membrane (Amersham Biosciences), and hybridized with 32P-labelled probes. The probes used for hybridization were as follows: a 185bp EcoRI/HindIII fragment of the pSP64Mb134 vector [25] for the Hbb-b1major-globin) gene, a 1 kb BamHI/XbaI fragment of the Mfrn1 {mitoferrin 1 [Slc25a37 (solute carrier family 25 member 37)]} cDNA in the pCS2+ vector [26], a PstI 600 bp fragment of the pBR332erythALAS vector (a gift from Dr Peter Curtis, Department of Microbiology, Thomas Jefferson University, Philadelphia, PA, U.S.A.) for the Alas2 (erythroid-specific 5-aminolaevulinic acid synthase 2) gene and a NotI/EcoRI 400 bp fragment of the pYX-AscMglrx5 vector (a gift from Professor Leonard Zon, Department of Biological and Biomedical Sciences, Harvard University, Boston, MA, U.S.A.) for the Glrx5 (glutaredoxin 5) gene. As a control, we performed hybridization with an end-labelled oligonucleotide (5′-CTTCCTCTAGATAGTCAAGTTCGACCGTCT-3′) specific for 18S rRNA.

Immunoblot analysis

Cell pellets were resuspended directly in Laemmli sample buffer or first extracted with whole-cell lysis buffer [10 mM Tris/HCl (pH 8.0), 420 mM NaCl, 250 mM sucrose, 2 mM MgCl2, 1 mM CaCl2 and 1% (w/v) Triton X-100] and then centrifuged at 9500 g for 3 min. Total proteins were separated by SDS/PAGE and transferred on to a PDVF membrane (Immobilon; Millipore). The membranes were incubated with antibodies specific for haemoglobin (MP Biomedicals), Tfr [Tf (transferrin) receptor] (Invitrogen), HSP70 (heat-shock protein 70) (Affinity BioReagents), phospho-ERK and total ERK (Cell Signaling). Secondary HRP (horseradish peroxidase)-conjugated antibodies (Pierce) were then applied. The proteins were detected using the Immobilon Western chemiluminescent reagents (Millipore) following the manufacturer's instructions. Restore Western Blot stripping buffer (Thermo Scientific) was used.

Iron uptake and incorporation into haem

59Fe2-labelled Tf was prepared from 59FeCl3 (PerkinElmer) and apo-Tf, as described previously [27]. Cells were incubated with 59Fe for the last 4 h of 48 h treatment. Thereafter, cells were collected and lysed in water. Measurement of 59Fe in haem and non-haem fractions was carried out as described previously using an acid precipitation method [28,29]. Cells where collected by centrifugation (9500 g for 1 min), washed in ice-cold PBS, lysed in water and boiled in 1 ml of 0.2 M HCl. The samples were then transferred to an ice-bath and 59Fe-labelled haem-containing proteins were precipitated with ice-cold 7% (v/v) TCA (trichloroacetic acid) solution and collected by centrifugation. Precipitated proteins (containing 59Fe in haem) were washed with 7% (v/v) TCA post-centrifugation; supernatants contained non-haem 59Fe. Following collection of supernatants into separate tubes, radiolabelled iron measurements of 59Fe radioactivities in haem and non-haem fractions were carried out on a Packard Cobra γ-counter (PerkinElmer).

Transcriptional reporter assays

A reporter construct PN38 comprising the γ-globin promoter and HS2 enhancer, linked to the firefly luciferase gene (a gift from Dr Paul Ney, Department of Biochemistry, St. Jude Children's Hospital, Memphis, TN, U.S.A.), was transfected into MEL cells using Amaxa Nucleofector technology. At 1 h following transfection, the cells were split equally into four wells. The cells were treated or not with 4 mM HMBA and additionally with 5 μM U0126 or 3 μM SB202190. The MAPK inhibitors were added 1 h prior to HMBA. Luciferase activity was determined 48 h following transfection using a Berthold luminometer.

Quantification and statistical analysis

Quantification of Northern blot experiments was performed using a phosphoimager (Molecular Dynamics) and Image Quant software (version 5.2). All results are means±S.E.M., and were analysed for significance (*P<0.05, **P<0.01 and ***P<0.001) using a two-tailed Student's t test (Graph Pad Prism 4 software).

RESULTS

Modulation of haemoglobin levels by inhibition of MAPK signalling pathways

To gain insights into the role of MAPKs in erythroid differentiation, we determined haemoglobin levels in chemically induced differentiating MEL cells in the absence and presence of MAPK inhibitors. We exposed the cells to HMBA, a potent inducer of erythroid differentiation [3]. At 1 h before HMBA induction, we treated the cells with the MEK1/2 inhibitor U0126 to block the ERK1/2 MAPK signalling pathway and SB202190 as a specific inhibitor of the p38α and p38β MAPKs [15,30,31]. We used U0126 and SB202190 at low concentrations of 5 and 3 μM respectively to minimize non-specific inhibition of other kinases [30,31]. First, we verified the effect of U0126 on ERK1/2 phosphorylation in an immunoblot assay. A one-time U0126 treatment led to complete inhibition of ERK1/2 phosphorylation at 24 h following HMBA induction and significant inhibition at later time points (Figure 1). We then determined the haemoglobin concentration in the cells (Figure 2A) using a spectrophotometric assay [24]. Haemoglobin levels in cells treated with HMBA strongly increased when compared with uninduced control cells. Addition of U0126 resulted in an even higher haemoglobin concentration in MEL cells, at both 24 and 48 h following HMBA treatment. In a parallel series of experiments, haemoglobin levels in the presence of HMBA were significantly reduced when cells were treated with SB202190. In addition, we verified the expression of globin transcripts and proteins by Northern blot and immunoblot experiments respectively (Figures 2B and 2C). We found an increase in β-globin mRNA and protein levels in the presence of U0126 and a decrease in the presence of SB202190 in HMBA-treated MEL cells. These findings correlate well with the results of the haemoglobin assay and suggest that the ERK1/2 pathway negatively, and the p38 MAPK pathway positively, regulate haemoglobin production.

MAPK inhibitors and ERK1/2 phosphorylation

Figure 1
MAPK inhibitors and ERK1/2 phosphorylation

MEL cells were treated with 4 mM HMBA and cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190 for the times indicated. Immunoblot analysis using antibodies specific for total and phospho-ERK1/2 was performed. The results shown are representative of three independent experiments.

Figure 1
MAPK inhibitors and ERK1/2 phosphorylation

MEL cells were treated with 4 mM HMBA and cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190 for the times indicated. Immunoblot analysis using antibodies specific for total and phospho-ERK1/2 was performed. The results shown are representative of three independent experiments.

MAPK pathway inhibition modulates haemoglobin and globin synthesis

Figure 2
MAPK pathway inhibition modulates haemoglobin and globin synthesis

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190. (A) The haemoglobin content of cells was assessed by spectrophotometry 24 and 48 h following HMBA treatment. The results shown are the means±S.E.M. for five independent experiments at 24 h and four independent experiments at 48 h performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. (B) Northern blot analysis of MEL cells using probes specific for Hbb-b1 and 18S. The quantification shown are the means±S.E.M. for five independent experiments at 24 h and four independent experiments at 48 and 72 h. **P<0.01 and ***P<0.001. (C) Immunoblot analysis of MEL cells using an antibody specific for haemoglobin. HSP70 levels are shown as a loading control. For the 24 h samples, 2.4 times more protein was loaded. The blot is representative of three independent experiments.

Figure 2
MAPK pathway inhibition modulates haemoglobin and globin synthesis

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190. (A) The haemoglobin content of cells was assessed by spectrophotometry 24 and 48 h following HMBA treatment. The results shown are the means±S.E.M. for five independent experiments at 24 h and four independent experiments at 48 h performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. (B) Northern blot analysis of MEL cells using probes specific for Hbb-b1 and 18S. The quantification shown are the means±S.E.M. for five independent experiments at 24 h and four independent experiments at 48 and 72 h. **P<0.01 and ***P<0.001. (C) Immunoblot analysis of MEL cells using an antibody specific for haemoglobin. HSP70 levels are shown as a loading control. For the 24 h samples, 2.4 times more protein was loaded. The blot is representative of three independent experiments.

Haem biosynthesis is affected by MAPK inhibitors

To assess the role of MAPK signalling in haem biosynthesis, we measured the amount of intracellular haem following inhibition of ERK1/2 and p38α/β MAPK in HMBA-induced MEL cells using a fluorimetric assay (Figure 3A). Haem levels at the 24 h time point were below the sensitivity of the assay (results not shown). Following 48 and 72 h of HMBA induction, we found that U0126 treatment resulted in an additional increase in haem levels in MEL cells. In contrast, exposure of MEL cells to SB202190 led to a significant reduction in intracellular haem. We conclude that the production of haem in the presence of MAPK inhibitors correlates well with our haemoglobin data (Figure 2). To examine this regulation of haem in more detail, we examined the expression of the gene coding for ALAS2, a rate-limiting enzyme of haem biosynthesis [32]. Previous studies have shown that induction of MEL cell differentiation by DMSO or HMBA led to increased Alas2 transcript levels [8,33]. We thus examined whether the inhibition of specific MAPK pathways had an effect on Alas2 mRNA levels (Figure 3B). In untreated cells, Alas2 mRNA levels were very low. In the presence of HMBA, Alas2 transcript levels increased and were elevated further upon U0126 treatment. In contrast, inhibition by SB202190 resulted in a significant decrease in Alas2 transcript levels at 72 h following HMBA induction. Our results provide a new link between MAPK signalling and the regulation of haem biosynthesis.

Haem biosynthesis is affected by MAPK pathway inhibitors

Figure 3
Haem biosynthesis is affected by MAPK pathway inhibitors

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190 for the times indicated. (A) Haem levels were assessed at 48 and 72 h of culture using a fluorimetric analysis. The results are the means±S.E.M. for five independent experiments performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. (B) Northern blot analysis using probes specific for Alas2 and 18S. The quantification shown are the means±S.E.M. for five independent experiments at 24 h and six independent experiments at 48 h and 72h. **P<0.01 and ***P<0.001.

Figure 3
Haem biosynthesis is affected by MAPK pathway inhibitors

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM U0126 or 5 μM SB202190 for the times indicated. (A) Haem levels were assessed at 48 and 72 h of culture using a fluorimetric analysis. The results are the means±S.E.M. for five independent experiments performed in triplicate. *P<0.05, **P<0.01 and ***P<0.001. (B) Northern blot analysis using probes specific for Alas2 and 18S. The quantification shown are the means±S.E.M. for five independent experiments at 24 h and six independent experiments at 48 h and 72h. **P<0.01 and ***P<0.001.

Regulation of iron metabolism by MAPK signalling pathways

To examine whether iron uptake is under the control of MAPK signalling pathways, we measured total iron and haem iron uptake in MEL cells using 59Fe-labelled Tf [34]. We found that blocking ERK1/2 led to higher total iron uptake as well as intracellular haem iron levels (Figure 4A). Inhibition of the p38α/β MAPKs had the opposite effect. We thus investigated a series of genes known to be involved in cellular iron metabolism. First, we determined whether the Tfr1 protein, essential for uptake of iron from the extracellular environment, is modulated through MAPK signalling pathways. Treatment of HMBA-induced MEL cells with UO126 led to increased Tfr1 protein levels, whereas exposure to SB202190 resulted in lower levels (Figure 4B). We found a similar regulation of the transcript levels of Mfrn1 that codes for the principal mitochondrial iron transporter, a protein that is also essential for haem synthesis (Figure 4C) [26]. In contrast, transcript levels of Glrx5, coding for a mitochondrial enzyme required for the synthesis of iron–sulfur clusters [35], did not appear to be regulated by ERK1/2 and p38α/β MAPKs (Figure 4D), although mRNA levels were induced following 48 and 72 h of HMBA treatment. These experiments clearly demonstrate a link between the regulation of iron metabolism and MAPK signalling. In summary, our findings suggest a co-ordinated regulation of globin production, haem biosynthesis and iron uptake through ERK1/2 and p38α/β MAPKs.

Regulation of iron metabolism by MAPK signalling pathways

Figure 4
Regulation of iron metabolism by MAPK signalling pathways

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM or 5 μM SB202190. (A) Total iron uptake (upper panel) and haem iron incorporation (lower panel) were assessed. The results shown are the means±S.E.M. for five independent experiments. **P<0.01. (B) Immunoblot analysis using an antibody specific for Tfr1. HSP70 levels are shown as loading control. The blot is representative of three independent experiments. (C and D) Northern blot analysis using specific probes for Mfrn1 (C) and Glrx5 (D) was performed. The small arrows indicate larger Mfrn1 transcripts. 18S was used as a control. The quantification shown are the means±S.E.M. for four independent experiments at 24 and 72 h and three independent experiments at 48 h. *P<0.05, **P<0.01 and ***P<0.001.

Figure 4
Regulation of iron metabolism by MAPK signalling pathways

MEL cells were treated or not with 4 mM HMBA for the times indicated. HMBA-exposed cells were cultured in the presence (+) or absence (−) of 3 μM or 5 μM SB202190. (A) Total iron uptake (upper panel) and haem iron incorporation (lower panel) were assessed. The results shown are the means±S.E.M. for five independent experiments. **P<0.01. (B) Immunoblot analysis using an antibody specific for Tfr1. HSP70 levels are shown as loading control. The blot is representative of three independent experiments. (C and D) Northern blot analysis using specific probes for Mfrn1 (C) and Glrx5 (D) was performed. The small arrows indicate larger Mfrn1 transcripts. 18S was used as a control. The quantification shown are the means±S.E.M. for four independent experiments at 24 and 72 h and three independent experiments at 48 h. *P<0.05, **P<0.01 and ***P<0.001.

Regulation of transcriptional activity during erythroid differentiation by MAPKs

To determine whether MAPK signalling controls transcriptional activity in differentiating erythroid cells, we used a luciferase reporter under the control of a globin promoter and the HS2 enhancer, an LCR (locus control region), as a model. We measured luciferase activity following transfection of MEL cells and subsequent treatment with HMBA and MAPK inhibitors. Globin promoter and enhancer activity increased considerably in the presence of the differentiation inducer HMBA (Figure 5). Treatment with the ERK1/2 inhibitor UO126 augmented further the transcriptional activity of the reporter, whereas exposure to the p38α/β MAPK inhibitor SB202190 led to reduced luciferase activity. Our findings suggest that MAPK signalling pathways act, at least in part, at the transcriptional level.

Regulation of transcriptional activity during erythroid differentiation by MAPKs

Figure 5
Regulation of transcriptional activity during erythroid differentiation by MAPKs

MEL cells were treated or not with 4 mM HMBA for 48 h. HMBA-exposed cells were cultured in the presence or absence of 3 μM U0126 or 5 μM SB202190. Activity of a reporter construct comprising a globin promoter and enhancer (HS2) linked to luciferase transfected into MEL cells was assessed. The results shown are the means±S.E.M. for five independent experiments. *P<0.05 and **P<0.01.

Figure 5
Regulation of transcriptional activity during erythroid differentiation by MAPKs

MEL cells were treated or not with 4 mM HMBA for 48 h. HMBA-exposed cells were cultured in the presence or absence of 3 μM U0126 or 5 μM SB202190. Activity of a reporter construct comprising a globin promoter and enhancer (HS2) linked to luciferase transfected into MEL cells was assessed. The results shown are the means±S.E.M. for five independent experiments. *P<0.05 and **P<0.01.

DISCUSSION

A series of previous reports have suggested a role for MAPKs in the control of erythroid differentiation [1220]. These studies mainly focused on the response of erythroid cells to inducing agents such as EPO and butyrate, with particular emphasis on the regulation of globin gene regulation by MAPKs. In the present study, we investigated MAPK function in HMBA-induced erythroid differentiation in MEL cells, analysing their role in globin synthesis as well as haem production and iron metabolism.

MEL cells have served as a valuable model to study the molecular mechanisms involved in erythroid differentiation. The control of globin gene expression, haem biosynthesis and iron metabolism in MEL cells has been studied extensively [36,37]. However, many aspects of the regulation of these pathways remain to be elucidated. In the present study, we investigated the functions of the ERK and p38 MAPK signalling pathways in HMBA-treated MEL cells. The ERK pathway MEK1/2 inhibitor UO126 further promoted induced erythroid differentiation, as shown by the increase in haemoglobin protein and β-globin transcript levels. These results strongly suggest that the ERK1/2 MAPK signalling pathway represses globin chain synthesis in MEL cells. In contrast, treatment with SB202190 led to a decrease in haemoglobin protein and globin transcript levels, suggesting that the p38α/β MAPK pathway signalling acts as positive regulator of globin production. Our present results correlate well with previous studies that have analysed MAPK signalling in EPO-induced murine and human erythroid cells [1214,18,38,39]. Down-regulation of p38 MAPK expression by antisense technology or p38 MAPK activity using the inhibitor SB203580 blocked erythroid differentiation in murine SKT6 cells induced by EPO [12,13]. A study investigating the murine EPO-responsive cell line ELM-I-1 showed that β-globin transcript levels were elevated in the presence of UO126 and reduced by SB203580 [18]. Additional reports using hydroxyurea or butyrate as inducing agents [15,19,40] supported further the notion that ERKs and p38 MAPKs play critical roles in erythroid differentiation.

Importantly, our present results also provide novel evidence that MAPKs control the haem biosynthesis pathway in erythroid cells. We observed an increase in haem levels and in the transcript levels of Alas2 upon exposure to UO126, whereas treatment with SB202190 had the opposite effect. ALAS2 plays a key role as the first enzyme of the pathway, catalysing a rate-limiting step in the synthesis of haem [32,35]. Intriguingly, iron uptake is regulated in a similar manner, as UO126 augments total iron uptake and its incorporation into haem as well as Mfrn1 transcript and Tfr1 protein levels, and exposure to SB202190 leads to a decrease in these parameters. We conclude that ERK and p38 MAPK pathways act antagonistically in HMBA-induced erythroid differentiation, as shown by their regulation of haemoglobin production, haem biosynthesis and iron metabolism. This does not appear to be a pleiotropic effect of the MAPKs, as Glrx5, encoding a protein involved in the synthesis of iron–sulfur clusters, is not significantly modulated upon exposure of the cells to UO126 or SB202190, but shows inducibility by HMBA at later time points. An interesting question is whether the processes of haemoglobin and haem synthesis as well as iron uptake, identified as effectors of MAPK signalling in our present study, are co-regulated or whether MAPKs act directly and independently on each of these. With respect to the latter, MAPKs may act on one pathway, which then leads to the co-ordinated up-regulation of the others. For example, it has been shown that addition of the end product of the haem pathway, haem, leads to the rapid induction of globin mRNA induction in MEL cells [4]. This may also be the case for iron uptake through, for example, the up- or down-regulation of Tfr1 expression, since adequate iron levels are essential for functional haem and globin synthesis [41]. Experiments supplying different doses of haem or iron to the cells in combination with MAPK inhibitors may help to gain further insights into the regulation of these processes.

Of note, in our reporter assays, we found that MAPK inhibitors exert their effect at the transcriptional level. This is in line with previous reports that have shown that MAPKs can function by controlling the activity of specific transcription factors through phosphorylation [42]. Potential targets for MAPKs in the regulation in erythroid cells are transcription factor, such as GATA1 (GATA-binding protein 1), EKLF (erythroid Krüppel-like factor), PU.1 (PU box-binding protein 1), NF-E2 (nuclear factor-erythroid 2), Bach1 (BTB and CNC homology 1) and/or their cofactors, that have been previously implicated in the regulation of globin synthesis, haem production and possibly iron uptake [5,4346]. Future studies will be aimed at clarifying whether MAPKs control the expression, DNA-binding and/or transcriptional activity of these transcription factors, which in turn control the expression of genes required for erythroid differentiation.

In conclusion, our present results suggest that the ERK and p38 MAPK signalling pathways perform antagonistic functions in HMBA-induced erythroid differentiation of MEL cells, with ERK1/2 suppressing and p38α/β MAPK promoting this process. Importantly, we have shown that, besides the regulation of globin production, MAPKs also control haem biosynthesis and iron uptake in erythroid cells. Our functional studies suggest that these signalling molecules act, at least in part, via modulation of transcriptional activity. MEL cells will provide a valuable model to pursue the identification of the missing links in the MAPK signalling pathway in erythroid differentiation.

Abbreviations

     
  • ALAS2

    erythroid-specific 5-aminolaevulinic acid synthase 2

  •  
  • EPO

    erythropoietin

  •  
  • ERK

    extracellular signal-regulated kinase

  •  
  • Glrx5

    glutaredoxin 5

  •  
  • Hbb-b1

    βmajor-globin

  •  
  • HMBA

    hexamethylene bisacetamide

  •  
  • HSP70

    heat-shock protein 70

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MEL

    cell, mouse erythroleukaemia cell

  •  
  • MEK

    MAPK/ERK kinase

  •  
  • Mfrn1

    mitoferrin 1

  •  
  • TCA

    trichloracetic acid

  •  
  • Tf

    transferrin

  •  
  • Tfr

    Tf receptor

AUTHOR CONTRIBUTION

Louay Mardini carried out the majority of the experiments, analysed the data and helped in manuscript preparation. Jadwiga Gasiorek performed the reporter experiments. Anna Derjuga assisted with the expression, haem biosynthesis and iron uptake studies. Lucie Carrière helped with the expression and iron uptake experiments. Barry Paw provided mitoferrin constructs and provided support in the preparation of the manuscript. Matthias Schranzhofer and Prem Ponka provided reagents as well as assistance with iron uptake experiments and manuscript preparation. Volker Blank designed the project, analysed the data and wrote the manuscript.

We thank Zaynab Nouhi, Grégory Chevillard and Benoît Chénais for helpful discussions and/or reading of the manuscript. We also thank Leonard Zon, Peter Curtis and Paul Ney for providing plasmids, and Andrea LeBlanc for reagents. We acknowledge the help of Amy Moore, Mansouria Merad Boudia and Damien Lehalle in the early stages of this project.

FUNDING

This research was supported by the Canadian Institutes of Health Research [grant number MOP-79361] to V.B. L.M was the recipient of a Leukemia Research Fund of Canada Studentship, a Judith Ann Wright Litvack Bursary and McGill University Faculty of Medicine Research bursaries. J.G. is currently a recipient of a studentship from the Thalassemia Foundation of Canada/Canadian Institutes of Health Research. M.S. was supported by a fellowship from the Canadian Institutes of Health Research and the Canadian Blood Services. P. P. was supported by grants from the Canadian Institutes of Health Research. V.B. acknowledges the receipt of studentship/fellowship support from the Thalassemia Foundation of Canada.

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Author notes

1

This paper is dedicated to the memory of Damien Lehalle, who lost his life while on a humanitarian mission with Médecins sans Frontières.