Increased erythropoiesis of b-thalassaemia/Hb ... - Wiley Online Library

research paper

Increased erythropoiesis of b-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation

Tirawat Wannatung,1 Pathrapol Lithanatudom,1 Amporn Leecharoenkiat,1 Saovaros Svasti,2 Suthat Fucharoen2 and Duncan R. Smith1 1

Molecular Pathology Laboratory, and

2

Thalassaemia Research Centre, Institute of

Molecular Biosciences, Mahidol University, Nakorn Pathom, Thailand

Received 30 March 2009; accepted for publication 2 June 2009 Correspondence: Dr Duncan R. Smith, Molecular Pathology Laboratory, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthon Sai 4, Salaya, Nakorn Pathom, Thailand 73170. E-mail: [email protected]

Summary b-thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of b-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34+ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signalregulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca2+ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in b-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca2+. Modulation of either cAMP or Ca2+ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of b-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca2+. Keywords: b-thalassaemia/Hb E, erythropoiesis, cAMP, Ca2+, signal transduction.

The thalassaemias are a group of disorders characterized by the under production of globin chains as a consequence of a wide range of underlying genetic abnormalities (Weatherall & Clegg, 2001). The a and b chains of haemoglobin A are the most commonly affected genes and both a- and b-thalassaemias represent a worldwide clinical problem (Weatherall & Clegg, 2001). The underproduction of one chain leads to a resultant excess of the other chain in the red cell that is toxic to the cell and leads to a reduced red cell life span, increased haemolysis and ineffective erythropoiesis, which is believed to arise through accelerated cell death by apoptosis of the immature erythroid cell (Yuan et al, 1993), either as a direct or indirect consequence of the unpaired globin chain deposition within the cell (Mathias et al, 2000). In b-thalassaemia apoptosis is

believed to occur primarily at the polychromatophilic normoblast stage (Mathias et al, 2000). The combination of ineffective erythropoiesis, increased haemolysis and reduced life span of the mature red cell lead to the anaemic condition, which stimulates the production of erythropoietin (EPO) leading to erythroid hyperplasia in the bone marrow (Chen et al, 1992; Centis et al, 2000; Mathias et al, 2000; Pootrakul et al, 2000; Kittikalayawong et al, 2005; Mai et al, 2007) and markedly increased levels of circulating EPO have been documented in thalassaemic patients (Nisli et al, 1997; Chaisiripoomkere et al, 1999; Paritpokee et al, 2002). However, despite the increase in erythroid precursors, the overall response to the anaemia is limited due to the ineffective erythropoiesis leading to the accelerated death of immature erythroid cells. In severe cases of

ª 2009 Blackwell Publishing Ltd, British Journal of Haematology, 146, 557–568

First published online 6 July 2009 doi:10.1111/j.1365-2141.2009.07794.x

T. Wannatung et al thalassaemia, this ineffective feedback loop can result in massive expansion of the bone marrow mass leading to characteristic skeletal problems including overgrowth of the maxilla and frontal skull bossing as well as frequent fracture (Mohamed & Jackson, 1998). While erythroid expansion can be either nullified through allogeneic haemopoietic cell transplantation or abrogated by regular transfusions, these treatment options are often not available in developing countries (Mohamed & Jackson, 1998). EPO acts as an erythropoiesis-regulating cytokine through several well-known signal transduction cascades collectively called the EPO pathway (Klingmuller, 1997; Cheung & Miller, 2001) and includes the Janus Kinase 2/Signal Transducer and Activator of Transcription 5 (JAK2/STAT5) (Klingmuller, 1997; Oda et al, 1998), Phosphatidylinositol-3-kinase (PI3K)/ AKT (Klingmuller, 1997; Haseyama et al, 1999; Myklebust et al, 2002) and Mitogen-activated protein kinase/Extracellular regulated protein kinase (MAPK/ERKs) pathways (Klingmuller, 1997; Sui et al, 1998; Schmidt et al, 2004). In particular, the MAPK/ERKs pathway is a well-characterized cascade known to be important for the growth, proliferation and differentiation response in many cell types including erythroid cells in which EPO is the primary regulatory cytokine for this pathway. In erythroid cells EPO activates the pathway by inducing EPO receptor (EPO-R) phosphorylation (Sui et al, 1998), stimulating the activation of the adapter proteins Shc and Grb2, which in turn activate the downstream adaptor protein, Ras (Cutler et al, 1993; Damen et al, 1993). Activated Ras can then induce activation of its effector proteins, B-Raf, and c-Raf leading to subsequent activation of MAPK/ERK kinases (MEKs) and ERKs (Schmidt et al, 2004). In addition to this classic signal transduction pathway, Ca2+-dependent isoforms of Protein kinase C (PKC), which can be directly activated by EPO stimulation (von Lindern et al, 2000; Myklebust et al, 2000) in a dose-dependent manner (Miller & Cheung, 1994) have also been reported to be alternative signalling modulators activating MEKs and ERKs either dependent on (von Lindern et al, 2000), or independently of, Raf proteins (Schmidt et al, 2004). Another signalling cascade that has been shown to modulate the MAPK/ERKs cascade is the cyclic adenosine monophosphate (cAMP)/Protein kinase A (PKA) pathway. PKA is major target protein for cAMP and has been reported to stimulate B-Raf while inhibiting c-Raf. Therefore, the activity of downstream signalling proteins, such as MEKs and ERKs could be either enhanced or inhibited depending on the balance of c-Raf and B-Raf activation (Houslay & Kolch, 2000; Kolch, 2000; Boer et al, 2003). While the EPO-regulated signalling cascade has been extensively studied in normal erythroid cells, this pathway remains unexplored in thalassaemic erythroid cells. Given the importance of erythroid hyperplasia to the presentation of thalassaemia, the present study sought to address this question. In particular, this study investigated EPO-mediated activation of the MAPK/ERKs cascade in proerythroblasts from b-thalassaemia/Hb E patients, a compound heterozygous 558

genotype that is very common in parts of Southeast Asia, as well as being associated with a remarkable spectrum of disease severity (Fucharoen et al, 2000).

Patients, materials and methods Patients Peripheral blood samples were taken from Thai/Chinese b-thalassaemia/Hb E patients and healthy normal subjects after study approval by the Committee on Human Rights and Related to Human Experimentation, Mahidol University, Thailand. All patients had been diagnosed as b-thalassaemia/ Hb E, as described previously (Sripichai et al, 2008) and all controls were screened to be normal for red blood cell indices and haemoglobin (Langlois et al, 2008). No patient had received a blood transfusion for at least 1 month prior to the study and had not taken any medication for at least 2 weeks prior to blood collection. Informed consent was obtained from all subjects. Clinical data of the patients is shown in Table I.

CD34+ haemopoietic progenitor cells (HPCs) isolation, primary culture, and treatment of erythroid progenitors and precursors Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml (b-thalassaemia/Hb E patients) or 50 ml (healthy controls) venous blood by layering over LymphoprepTM (AXIS-SHIELD PoC AS, Oslo, Norway) according to the manufacturer’s protocol. Cells were then enriched for CD34+ HPCs by using a direct CD34 progenitor cell isolation kit with MACSTM isolation system (Miltenyi Biotech, Auburn, CA, USA) according to manufacturer’s protocol. The purity of Table I. Clinical characteristics of patients.

Patient

Sex

Hb (g/l)

Serum EPO (iu per l)

Genotype(mutation)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

F F F F F F M M M M F F F F F M M M

65 84 50 62 84 61 75 73 62 68 66 91 68 66 68 57 76 66

137Æ4 204Æ3 960Æ0 74Æ8 – – 1058Æ6 590Æ0 – – 114Æ7 61Æ1 110Æ5 225Æ5 157Æ3 1126Æ9 331Æ2 –

bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b bE/b

(codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon (codon

41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT) 41/42; -TTCT 41/42; -TTCT) 17; A->T) 17; A->T) 17; A->T) 17; A->T) 17; A->T) 17; A->T) 17; A->T) 17; A->T)


ERK1/2 in b-thalassaemia Proerythroblasts selected CD34+ HPCs was more than 85% as evaluated by flow cytometry using a phycoerythrin (PE)-conjugated mouse anti-human CD34 monoclonal antibody and a fluorescein isothiocyanate (FITC)-conjugated mouse anti human CD45 monoclonal antibody (BD Bioscience, San Jose, CA, USA). Isolated CD34+ HPCs were cultured essentially as described elsewhere (Choi et al, 2000). Briefly, CD34+ HPCs were grown at a density of 105/ml in Iscove’s modified Dulbecco medium (IMDM; GIBCO BRL, Grand Island, NY, USA) containing 15% heat-inactivated fetal calf serum (FCS; GIBCO BRL, Grand Island, NY, USA), 15% human AB serum, 2 U/ml recombinant human (rHu) EPO (CILAG GmbH, Zug, Switzerland), 20 ng/ml rHu stem cell factor (SCF; Promokine, Heidelberg, Germany), and 10 ng/ml rHu interleukin-3 (IL-3; Promokine) at 37C in a high-humidity, 5% CO2 incubator for first 3 d. On day 3, the culture medium was replaced with fresh complete medium without rHuIL-3 and the cells were incubated for a further 4 d. The proportion of cells at this stage with proerythroblast morphology was more than 95% as evaluated by cytospin preparation followed by WrightGiemsa’s staining. Numbers of erythroid cells and viability were determined by haemocytometery (BOECO, Hamburg, Germany) after trypan blue staining. For serum and cytokines deprivation studies, proerythroblasts were incubated in serumfree liquid medium containing half mixture of IMDM (GIBCO BRL, Grand Island, NY, USA) and F-12 medium (SigmaAldrich, St Louis, MO, USA) with 1% detoxified bovine serum albumin (BSA; Stem Cell Technologies Inc, Vancouver, BC, USA), 300 lg/ml iron-saturated transferrin (Roche Diagnostics, Indianapolis, IN, USA), lipid suspensions (2Æ8 lg/ml oleic acid, 4Æ0 lg/ml L-a-phosphatidylcholine; 3Æ9 lg/ml cholesterol; all from Sigma-Aldrich, St Louis, MO, USA) prepared as described elsewhere (Choi et al, 2000) at 37C in a highhumidity, 5% CO2 incubator for 4 h. For EPO stimulation, cytokines-deprived proerythroblasts were stimulated by the addition of 25 U/ml rHuEPO in serum-free liquid medium. After 10 min or 3 h of stimulation cells were harvested and proteins extracted. For cAMP and calcium inhibition studies, proerythroblasts from b-thalassaemia/Hb E patients were incubated in serum-free liquid medium with addition of ethyleneglycoltetraacetic acid (EGTA; Sigma-Aldrich, St Louis, MO, USA), 2¢,5¢-dideoxyadenosine (DDA; Sigma-Aldrich, St Louis, MO, USA), or U0126 (Cell Signaling Technologies, Beverly, MA, USA) for 4 h at 37C, 5% CO2 after which cells were harvested and proteins extracted. Alternatively, b-thalassaemia/Hb E erythroid precursors at day 3 post CD34+ isolation were grown in complete medium with or without the addition of EGTA, DDA, or U0126 for a further 4 d at 37C, 5% CO2 after which time proteins were extracted.

Western blot analysis Harvested proerythroblasts were lysed using phosphorylation lysis buffer (50 mmol/l Tris (tris(hydroxymethyl)aminomethane)–HCl pH 7Æ5, 15 mol/l NaCl, 50 mmol/l NaF, 1 mmol/l

EDTA (ethylenediaminetetraacetic acid), 1 mmol/l EGTA, 1% Triton-X 100, 0Æ5 mmol/l activated sodium orthovanadate (Na3VO4), 5 mmol/l sodium pyrophosphate, and 10 mmol/l b-glycerophosphate) containing protease inhibitors cocktail (Bio Basic, Markham, Ontario, Canada). Cells were ruptured by vortexing and sonication for 5 s, repeated 4 times. The lysate was clarified by centrifugation at 16 000 rpm for 10 min at 4C. Protein concentration in the clear supernatant was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA) and a total of 40 lg of proteins were separated by electrophoresis through denaturing 8Æ5% polyacrylamide gels. Separated proteins were then electrophoretically transferred onto polyvinylidene difluoride membranes (PVDF; Millipore Corporation, Bedford, MA, USA) by using Mini-PROTEAN II Cell (Bio-Rad, Hercules, CA, USA). Efficiency of transfer was evaluated after staining membranes with Ponceau S. Membranes were blocked by incubating with 5% non-fat dry milk in Tris–buffered saline (TBS) containing 0Æ05% Tween 20 (TBS-T) for 1 h at room temperature before overnight incubation with 1:1000 dilutions of primary antibodies in TBS-T containing 5% BSA at 4C. Primary antibodies used were directed against human phospho-B-Raf (Ser445), phospho-c-Raf (Ser338), phospho-MEK (Ser217/221), phosphoERK1/2 (Thr202/Tyr204), c-Raf, ERK1/2, MAPK phosphatase 3 (MKP3) (all from Cell Signaling Technologies, Beverly, MA, USA), erythropoietin receptor (EPO-R; R&D Systems, Minneapolis, MN, USA) and B-Raf and GAPDH (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with the specific primary antibody, membranes were incubated with either a 1:4000 dilution of a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or a 1:8000 dilution of a HRP-conjugated rabbit anti-mouse IgG antibody. Signal was developed using the Enhanced Chemiluminescense Plus system (ECL plus; GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction.

Measurement of intracellular cAMP and calcium levels Intracellular cAMP levels in 1Æ5 · 106 proerythroblasts were determined using a cAMP enzyme immunoassay (EIA) kit (Cayman Laboratories, Ann Arbor, MI, USA) according to the manufacturers’ protocol. The absorbance was measured using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). To determine intracellular calcium levels, 3 · 105 proerythroblasts were loaded with the calciumsensitive indicator dye calcium green-1-AM (Molecular Probes, Eugene, OR, USA) at final concentration of 5 lmol/l in IMDM containing 5% FCS for 45 min at 37C in the dark. Subsequently, cells were washed three times and resuspended with sample buffer (phosphate-buffered saline pH 7Æ4 containing 5 mmol/l glucose, 1 mmol/l MgCl2, and 5% FCS) for sample reading (Fsample), or sample buffer supplemented with 10 mmol/l EGTA for minimum signal (Fmin), or sample buffer supplemented with 50 lmol/l ionomycin (Sigma-Aldrich, St Louis, MO, USA) for maximum signal (Fmax). Fluorescence


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T. Wannatung et al signal was measured in a JASCO FP-6300 spectrofluorometer (JASCO Corporation, Hachioji-shi, Tokyo, Japan) using 531 nm fluorescence emission and 506 nm fluorescence excitation at 1 h following addition of buffer. The intracellular calcium concentration was determined in nmol/l using the equation

(A)

[Ca2þ i ¼ K d ðF sample F min Þ=ðF max F sample Þ; K d ¼ 190 nmol/l:

Statistical analysis (B) Band intensities from Western blot analysis were quantified using the ImageJ 1.41 programme (National Institutes of Health, USA). Data are reported as mean values plus or minus standard error of the mean (SEM). Statistical analyses were performed using the graphpad prism software (GraphPad Software, La Jolla, CA, USA). The statistical significance of differences between mean values was calculated using the nonparametric t-test; a P value of less than 0Æ05 was considered significant.

Results In vitro erythroid expansion The expansion of erythroid precursors of 12 b-thalassaemia/ Hb E patients and 12 non-thalassaemic individuals was investigated by isolating CD34+ haemopoietic progenitor cells from peripheral blood by magnetic bead separation, which gave 85% CD34+ cells (Fig 1A) and culturing under standard conditions (Choi et al, 2000). Morphological examination of erythroid cells on day 7 post-culture by Wright-Giemsa’s staining showed more than 90% proerythroblasts in both groups (Fig 1B). Expansion ratios, calculated from quantitating isolated CD34+ progenitors on day 0 as compared with proerythroblasts on day 7 of culture, showed approximately 2Æ5-fold greater expansion for b-thalassaemia/Hb E erythroid cells than normal controls (P = 0Æ001) (Fig 1C). Given that culture conditions with respect to cytokine concentration were identical, this would suggest either a greater response to cytokine stimulation, or that erythroid precursor cells from b-thalassaemia/Hb E patients have an inherently greater proliferative capacity than those from controls. Given the well characterized role of EPO in erythroid proliferation, activation of MAPK/ERKs cascade was examined.

Activation of the MAPK/ERKs signalling pathway in proerythroblasts Western blotting was used to examine the degree of phosphorylation of B-Raf, c-Raf, MEK and ERK1/2 in proerythroblasts under steady state growth conditions, after serum and cytokine deprivation and post-EPO stimulation. In addition, the level of the EPO-R, total ERK1/2, B-Raf, and MAPK phosphatase3 (MKP3) were also evaluated under the same 560

(C)

Fig 1. In vitro expansion of erythroid progenitor cells. (A) Purity of isolated HPCs from a normal subject as determined by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) using PE-conjugated anti CD34 and FITC-conjugated anti CD45 antibodies. (B) Normal and b-thalassaemia/Hb E CD34+ haemopoietic progenitor cells were cultured for 7 d to allow differentiation into proerythroblasts and morphology examined by Wright-Giemsa’s staining. Images (magnification 1000·) were captured on an Olympus CH40 microscope with a connected digital DP12-2 camera (Olympus, Tokyo, Japan). (C) Expansion of normal (n = 12) and b-thalassaemia/Hb E (n = 12) progenitor cells into proerythroblasts was determined by haemocytometry after trypan blue staining on day 0 and 7 of culture. The mean (± SEM) values of erythroid expansion of 12 control subjects and b-thalassaemia/Hb E patients are shown. An asterisk (*) indicates P = 0Æ001.

conditions. Steady state levels of MEK and ERK1/2 phosphorylation were found to be higher in b-thalassaemia/Hb E proerythroblasts than in normal controls, while phospho-c-Raf was marginally higher and B-Raf was significantly lower than


ERK1/2 in b-thalassaemia Proerythroblasts normal controls (Figs 2 and 3). In response to serum and cytokine deprivation, normal control proerythroblasts showed a significant reduction in phosphorylation of B-Raf, MEK and ERK1/2 while c-Raf showed a slight reduction in phosphorylation. For b-thalassaemia/Hb E proerythroblasts, there was a marginal reduction in phospho-B-Raf and phospho-c-Raf, a significant reduction in phospho-MEK, and in marked contrast to normal proerythroblasts, only a marginal reduction in levels of phospho-ERK1/2, and a significant level of ERK1/2 phosphorylation after 4 h deprivation was clearly seen in Western blot analysis (Fig 2). In response to stimulation with EPO for 10 min, control proerythroblasts showed an increase in the levels of phosphoB-Raf, phospho-c-Raf, phospho-MEK and phospho-ERK1/2. Under similar stimulation, b-thalassaemia/Hb E proerythroblasts showed a significant increase in levels of phospho-B-Raf, and phospho-ERK1/2, a marginal increase in levels of

phospho-MEK, and essentially unchanged levels of phosphoc-Raf. Only marginal changes in the phosphorylation levels were observed after 3 h stimulation in either normal control or b-thalassaemia/Hb E proerythroblasts, although it is notable that levels of phospho-ERK1/2 were significantly higher than steady state levels in b-thalassaemia/Hb E proerythroblasts (Figs 2 and 3). The significantly higher level of phosphorylation of ERK1/2 in cytokine-deprived proerythroblasts from b-thalassaemia/Hb E patients, and the relatively normal response of c-Raf, B-Raf, and MEK expression suggested that modulation of ERK1/2 phosphorylation was occurring at this level of the signal transduction pathway. High levels of ERK1/2 phosphorylation could result from defects in proteins regulating the dephosphorylation of ERK1/2. To investigate this possibility the expression levels of MKP3, which specifically inactivates ERK1/ 2 by dephosphorylation (Muda et al, 1996), was investigated.

Fig 2. Western blot analysis of EPO-mediated MAPK/ERKs signal transduction. Western analysis of MAPK/ERKs signalling proteins in steady state proerythroblasts (Day 7) and after cytokine deprivation and subsequent EPO stimulation. Proerythroblasts were deprived of cytokines for 4 h and then stimulated with 25 U/ml rHuEPO for 10 min or 3 h. Total proteins were extracted and subjected to immunoblotting to detect EPO-R, B-Raf, ERK1/2, and GAPDH as well as levels of phosphorylated (p) c-Raf, B-Raf, MEK and ERK1/2.


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T. Wannatung et al

Fig 3. Quantitative analysis of EPO-mediated MAPK/ERKs signal transduction. Band intensities for phosphorylated (p) c-Raf, B-Raf, MEK and ERK1/2 were quantified and normalized against GAPDH. The mean (± SEM) values of from six control subjects and 11 b-thalassaemia/Hb E patients for c-Raf and ERK1/2 and five control subjects and nine b-thalassaemia/Hb E patients for B-Raf and MEK are shown. An asterisk (*) or hashmark (#) indicates P < 0Æ05 between values. Dpv, deprivation; Sti, stimulation.

Essentially identical levels of MKP3 were shown in both steady state cells, cytokine/serum deprived cells and in 3-h EPOstimulated cells from both patients and controls (Fig 4). Interestingly, a slight but significant increase in MKP3 levels were seen in b-thalassaemia/Hb E proerythroblasts stimulated for 10 min with EPO as compared to control proerythroblasts (Fig 4), suggesting that a significant deficit in MKP3 was not the cause of the high levels of ERK1/2 phosphorylation seen in b-thalassaemia/Hb E proerythroblasts.

Levels of Ca

2+

and cAMP in proerythroblasts

The essentially normal response of upstream signal transduction proteins and the essentially normal expression of MKP3 suggest that alternative signalling pathways modulate the level of phosphorylation of ERK1/2. As it is known that ERKs activation can be modulated by cAMP/PKA signalling either in a c-Raf independent (Lee, 1999) or c-Raf/B-Raf dependent manner (Erhardt et al, 1995; Kolch, 2000) and moreover, that certain PKC isotypes activated by Ca2+ have also been implicated in c-Raf (Kolch et al, 1993), MEK and ERK1/2 activation (Kolch et al, 1993; Schonwasser et al, 1998; Schmidt et al, 2004) intracellular levels of both Ca2+ and cAMP were determined in normal control and b-thalassaemia/Hb E proerythroblasts. Results showed that cAMP levels in 562

b-thalassaemia/Hb E proerythroblasts were significantly increased as compared to normal controls (P = 0Æ003) (Table II). This result is similar to that found in a previous study, which examined cAMP levels in b-thalassaemia nucleated erythroblasts (Bailey et al, 2007). Similarly, levels of Ca2+ were also significantly elevated in b-thalassaemia/Hb E erythroblasts (P < 0Æ05) (Table II) when compared with normal controls.

Intracellular Ca2+ and cAMP modulate ERK1/2 phosphorylation and erythroid expansion in b-thalassaemia/Hb E proerythroblasts To determine whether there was a causal association between the elevated levels of Ca2+ and cAMP and the high level of basal ERK1/2 phosphorylation seen in b-thalassaemia/Hb E proerythroblasts, erythroid progenitor cells from one patient were cultured for 7 d and deprived of cytokines as above, but in this case the cytokine deprivation medium was supplemented with either the adenylyl cyclase inhibitor 2¢-5¢dideoxyadenosine (DDA) to reduce cAMP levels, EGTA to reduce intracellular Ca2+ levels or with U0126, a direct inhibitor of MEK as a control, following which the level of phosphorylation of ERK1/2 was determined by Western blotting as above. There was a strong, dose-dependent


ERK1/2 in b-thalassaemia Proerythroblasts

(A)

(B)

Fig 4. Expression of MKP3 in b-thalassaemia/Hb E proerythroblasts. (A) Western analysis of MKP3 expression levels in proerythroblasts. (B) Band intensities for MKP3 were quantified and normalized against GAPDH. The mean (± SEM) values of from five control subjects and six b-thalassaemia/ Hb E patients are shown. An asterisk (*) indicates P < 0Æ05 between values. Dpv, deprivation; Sti, stimulation.

Table II. Levels of intracellular cAMP and Ca2+ in control and b–thalassaemia/Hb E proerythroblasts.

Control b-thal/Hb E

cAMP levels

Ca2+ levels

n

n

(nmol/l)

7 18

57Æ17 ± 2Æ68 79Æ49 ± 9Æ89**

5 17

(pmol/l) 160Æ90 ± 16Æ69 259Æ40 ± 24Æ47*

*P < 0Æ05. **P < 0Æ005.

reduction in the basal level of ERK1/2 phosphorylation with U0126, while EGTA reduced the level of ERK1/2 phosphorylation by some 90% at concentrations of 1 mmol/l or above (Fig 5). At all concentrations tested, DDA reduced ERK1/2 phosphorylation by approximately 70% (Fig 5A). To determine whether or not the high basal levels of ERK1/2 phosphorylation were driving the increased proliferation of b-thalassaemia/Hb E progenitor cells, day 3 b-thalassaemia/Hb E erythroid progenitor cells were incubated in complete growth medium supplemented with DDA, EGTA or U0126 for a further 4 d. On day 7 post-culture, proerythroblasts were

assessed for both steady state levels of ERK1/2 phosphorylation as well as the degree of erythroid expansion as compared with untreated b-thalassaemia/Hb E proerythroblasts. Steady state levels of ERK1/2 phosphorylation were shown to be reduced in an apparently dose-dependent manner by EGTA, DDA, and U0126 (Fig 5B). Inhibition of MEK with 10 lmol/l U1026 significantly reduced b–thalassaemia/Hb E erythroid expansion by approximately 50%, while 1 mmol/l EGTA and 200 lmol/l DDA also significantly reduced erythroid expansion by 30–40% (Fig 5C). No difference was observed in cell viability between treated and untreated b-thalassaemia/Hb E cells (data not shown).

Discussion Increased erythropoiesis in b-thalassaemia/Hb E patients has been proposed to occur as compensation for the anaemic condition resulting from the loss of erythroid cells (Olivieri, 1999) as a consequence of ineffective erythropoiesis (Mathias et al, 2000). The anaemic state is believed to result in increased production of EPO, which drives the increased erythropoiesis, and increased levels of circulating EPO in thalassaemic patients have been reported in several studies (Nisli et al, 1997;


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(A)

(B)

(C)

Fig 5. Modulation of intracellular Ca2+ and cAMP in b-thalassaemia/Hb E proerythroblasts. (A) Proerythroblasts of one b-thalassaemia/Hb E patient were treated with MEK inhibitor; U0126, Ca2+ Chelator; EGTA, or the adenylyl cyclase Inhibitor; DDA at the concentration shown, in serum-free medium for 4 h. Total proteins were extracted and subjected to immunoblotting to detect levels of phospho-ERK1/2, ERK1/2 and GAPDH. Levels of ERK1/2 phosphorylation were determined by densitometry and normalized against GAPDH and calculated as percentage ERK1/2 phosphorylation relative to untreated b-thalassaemia/Hb E control. (B) Day 3 erythroid precursors from one b-thalassaemia/Hb E patient were cultured in complete medium in the presence of U0126, EGTA, and DDA at the concentrations indicated for 4 d until reaching 7 d of culture and the phosphorylation levels of ERK1/2 was compared relative to untreated b-thalassaemia/Hb E control. (C) Day 3 erythroid precursors form four b-thalassaemia/Hb E patients were cultured in complete medium in the presence of U0126, EGTA, and DDA at the concentrations indicated for 4 d until reaching 7 d and the degree of expansion to proerythroblasts was determined by hemocytometry after trypan blue staining. Percent expansion was calculated relative to untreated b-thalassaemia/Hb E control cells. The mean (± SEM) values of percent expansion are shown. *P < 0Æ05, P < 0Æ01 compared to control (untreated) b-thalassaemia/Hb E erythroid cells.

Chaisiripoomkere et al, 1999; Paritpokee et al, 2002) and are consistent with the levels found in the patients in this study, which showed a 3- to 60-fold elevation of EPO levels, with no observable relationship between EPO levels and either total haemoglobin or haemoglobin F levels (data not shown). As such, the central belief, that increased EPO (either directly or in synergy with other cytokines) results in increased erythropoiesis, in thalassaemic patients, remains largely unsupported and untested. 564

An increased expansion in cultured erythroid precursor cells of b-thalassaemia patients as compared to normal controls has been previously reported (Chen et al, 1992; Yaowaree et al, 2005). In the present study, in which early erythroid precursor cells were isolated from peripheral CD34+ haemopoietic progenitor cells in culture, we observed a significantly increased expansion of the earliest stage of erythroid precursors; proerythroblasts, with thalassaemic erythroid cells expanding some 35- to 40-fold in liquid culture compared to


ERK1/2 in b-thalassaemia Proerythroblasts 10- to 15-fold expansion for normal erythroid cells. Given that the culture conditions were identical and that levels of EPO were the same for cells from patients and controls, it is difficult to reconcile with a model in which increased levels of EPO drives increased proliferation in thalassaemic erythroid precursor cells, and would rather point to either an increased sensitivity to the action of EPO or to an inherent mechanism driving the increased proliferative capacity. While EPO acts through several signal transduction pathways (Klingmuller, 1997) the MAPK/ERKs pathway is perhaps the best characterized in erythroid precursor cells (Sui et al, 1998; Bugarski et al, 2007). Investigation of this pathway in proerythroblasts from b-thalassaemia/Hb E patients revealed a surprisingly high basal level of phosphorylation of ERK1/2. A central role for ERKs in erythroid expansion has previously been demonstrated. Inhibition of ERKs phosphorylation with the MEK inhibitor PD98059 in normal primary erythroid cells resulted in a dose-dependent inhibition of erythroid colony formation (Sui et al, 1998) as well as a reduction of proerythroblast expansion (Arcasoy & Jiang, 2005). Additionally, expression of a constitutively active Ras, Raf or MEKs in primary erythroid cells not only constitutively activated ERKs phosphorylation, but also highly enhanced erythroid expansion in an EPO-independent manner (Zhang & Lodish, 2004). In this study, while proerythroblasts from normal controls showed a significant reduction in the level of ERK1/2 phosphorylation in response to serum and cytokine deprivation, b-thalassaemia/Hb E proerythroblasts underwent only modest reductions in the level of phosphorylation, and levels were significantly above those seen in normal control cells. In

contrast, MEK, c-Raf and B-Raf responses were approximately similar between normal and b-thalassaemia/Hb E proerythroblasts, suggesting that alternative signalling pathways may be responsible for the increased phosphorylation of ERK1/2 seen in b-thalassaemia/Hb E proerythroblasts. While such a result could arise from defects in the appropriate dephosphorylation of ERK1/2, examination of the expression of MKP3, a protein that specifically dephosphorylates ERK1/2, showed essentially normal levels under most conditions examined. Interestingly, a slight increase in expression of MKP3 was observed in EPOstimulated b-thalassaemia/Hb E proerythroblasts, which would be consistent with the increased phosphorylation levels of ERK1/2 seen in these cells. However, from the results observed, it would seem that a failure to appropriately dephosphorylate ERK1/2 is not the major cause of the high levels of ERK1/2 activation observed, and suggests that other regulators of ERK1/2 phosphorylation are the principal cause of the high levels of ERK1/2 activation seen in b-thalassaemia/Hb E proerythroblasts. This was supported by the abnormally raised levels of both Ca2+ and cAMP observed in b-thalassaemia/Hb E proerythroblasts as it has been reported that MAPK/ERKs signalling cascade can be activated by the Ca2+/PKC (Ueda et al, 1996; von Lindern et al, 2000; Agell et al, 2002) and cAMP/PKA cascades (Boer et al, 2003), as well the fact that modulation of Ca2+ and cAMP levels reduced the basal level of phosphorylation of ERK1/2. Collectively, these results support a model in which MAPK/ERKs signal transduction in proerythroblasts from b-thalassaemia/Hb E patients is different to the pathway normally observed in proerythroblasts (Fig 6).

Fig 6. Schematic representation of activation of the MAPK/ERKs pathway. In normal proerythroblasts, EPO-mediated signal transduction is primarily transmitted through the classical Raf/MEK/ERK cascade. In b-thalassaemia/Hb E proerythroblasts, elevated levels of Ca2+ and cAMP lead to elevated basal levels of ERK1/2 phosphorylation, probably through PKA and PKC activation, which drive the enhanced expansion of b-thalassaemia/ Hb E erythroid progenitor cells.


565

T. Wannatung et al Ca2+ is an abundant intracellular signalling molecule that is known to regulate several cellular processes including cell proliferation and differentiation. Changes in the levels of intracellular Ca2+ have been previously reported to modulate the MAPK/ERK pathway (Cullen & Lockyer, 2002), which support our findings in b-thalassaemia/Hb E proerythroblasts of significantly increased levels of intracellular free Ca2+ being associated with markedly increased phosphorylation of ERK1/ 2. While elevation of levels of intracellular Ca2+ in mature erythrocytes of b-thalassaemia patients has long been known (Shalev et al, 1984; Bookchin et al, 1988), there is, as yet, no generally accepted explanation for the elevated levels. One possible cause is defects of the erythroid cell membranes, which may affect certain gates or channels involved in Ca2+ homeostasis regulation (Shalev et al, 1984) and as a consequence, lead to cell shrinkage (Lang et al, 2004). The present study reports, for the first time, that increased levels of Ca2+ occur in b-thalassaemia/Hb E precursors as early as the proerythroblast stage. While this does not identify the primary cause of Ca2+ deregulation, it suggests that Ca2+ ion regulation requires further detailed study in b-thalassaemia erythropoiesis. Similarly, cAMP is a well-characterized second messenger that has been shown to control several signalling cascades. The formation of cAMP is principally regulated through the activation of certain G-coupled protein receptors, which leads to the conversion of ATP to cAMP by the enzyme adenylyl cyclase, and the significant elevation of intracellular cAMP observed in b-thalassaemia/Hb E proerythroblasts suggest a possible deregulation of the G-protein cascade. Significantly, a role for cAMP in inducing c-globin expression in b-thalassaemia erythroblasts has been recently reported (Bailey et al, 2007) and the marked elevation of intracellular cAMP levels in both peripheral erythrocytes and cultured erythroblasts reported from b-thalassaemia intermedia patients in that study is consistent with the results reported here. Whilst Bailey et al (2007) reported a direct correlation between the levels of cAMP in late-stage nucleated erythroblasts (polychromatic and orthochromatic erythroblasts) and blood Hb F levels from eight patients, our data in early stage nucleated erythroblasts (proerythroblasts) showed no significant correlation between cAMP levels and blood Hb F levels (n = 17; data not shown). However, given that our study was undertaken on early stage erythroid progenitor cells the lack of a correlation is of uncertain significance, but perhaps highlights the multifunctional role of second messenger molecules. Apart from the role of MAPK/ERK1/2 in signal transduction in erythroid proliferation, increased activation of ERK1/2 has also been shown to mediate apoptosis through the up-regulation of the anti-apoptotic protein Bcl-XL and in late stage haemoglobinized precursors the anti-apoptotic effect of Bcl-XL is dominant (Motoyama et al, 1999; Rhodes et al, 2005), and it has been proposed that in case of b-thalassaemia, Bcl-XL is up-regulated to ameliorate 566

apoptosis primarily caused by accumulation of excess a globin chains. Interestingly, a second proliferative cascade mediated by EPO, the JAK/STAT5 pathway, has been shown in erythroid precursor cells of b-thalassaemia major mice to have significantly elevated levels of phosphorylated JAK2, which resulted in the increased expression of Bcl-XL (Libani et al, 2008). However, a study by Bailey et al (2007) in human nucleated erythroblasts from b-thalassaemia patients showed an extremely low level of STAT5 phosphorylation. As STAT5 is downstream of JAK2, the significance of these two studies is unclear. It does however provide evidence that interactions between EPO-mediated signalling pathways and the apoptotic cascade may be critical in mediating the inefficient erythropoiesis that is a hallmark of b-thalassaemia erythropoiesis. It is possible that the high levels of ERK1/2 phosphorylation seen in this study are a compensationary mechanism of the cell to protect against even higher levels of apoptosis in b)thalassaemia erythroblasts. However, there is a possibility that phosphorylated ERK1/2 interacts with other members of the Bax/Bcl2 family. The Bax/Bcl2 family contains a number of proteins with significant homology domains, and includes proteins that are both pro-apoptotic as well as anti-apoptotic (Yang & Korsmeyer, 1996). It is feasible that as yet uncharacterized interactions of highly activated ERK1/2 with members of the Bax/Bcl2 family could be the driving force behind the increased apoptosis of b-thalassaemia erythroblasts, and overall the increased levels of cAMP and Ca2+ could drive both increased proliferation of the b-thalassaemia erythroid precursors and increased apoptosis at the polychromatophilic normoblast stage. Perhaps most significantly, modulation of Ca2+ or cAMP levels significantly reduced the degree of expansion of b-thalassaemia/Hb E erythroid precursor cells, without any observable effect on cell viability, suggesting that erythroid expansion in these cells is an inherent characteristic driven by disordered Ca2+ and cAMP levels acting through ERK1/2. This suggests that current models in which erythroid expansion is driven by increased levels of EPO, acting directly, or in synergy with other cytokines, such as transforming growth factor-beta or SCF, are incorrect, and leads to the possibility of clinically being able to directly intervene to correct erythroid hyperplasia in thalassaemic patients without dramatically affecting levels of anaemia. Such an approach may serve to ameliorate the more severe consequences of thalassaemia, such as skeletal and facial malformation.

Acknowledgements This work and T.W. were supported by a Research Strengthening Grant from the National Centre for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA). P.L. is supported by a Thai Royal Golden Jubilee Fund Scholarship and A.L. is supported by the Thai Commission on Higher Education.


ERK1/2 in b-thalassaemia Proerythroblasts

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