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Erythropoiesis in β-thalassemia patients is ineffective, primarily because of death of the erythroid progenitor cells at the polychromatic normoblast stage. While it is known that autophagy plays a critical role during erythropoiesis by removing organelles from erythroid cells during terminal differentiation, its role in erythroid cells whose function is impaired remains to be explored. To investigate this, CD34+ erythroid progenitor cells from normal controls and β-thalassemia/Hb E patients were isolated from peripheral blood and cultured under conditions driving differentiation into an erythroid lineage, and levels of autophagy and apoptosis were analyzed both directly and after biochemical manipulation with L: -asparagine. A significantly higher level of autophagy was seen in β-thalassemia/Hb E erythroblasts as compared to normal control erythroblasts during erythropoiesis. Interestingly, this activation was mediated in part by the presence of high levels of Ca(2+) as modulation of Ca(2+) levels significantly reduced the level of autophagy in these cells. Inhibition of autophagic flux in normal erythroblasts significantly increased apoptosis in normal erythroblasts, but not in thalassemic erythroblasts, although sensitivity to autophagic flux inhibition was restored by reduction of Ca(2+) levels. These results suggest that high levels of autophagy in β-thalassemia/HbE erythroblasts may contribute to the increased levels of apoptosis that lead to ineffective erythropoiesis in β-thalassemia/Hb E erythroblasts.

Increased erythropoiesis of b-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activationThe 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 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 Ca 2+ 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 Ca 2+ . Modulation of either cAMP or Ca 2+ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly...

These results suggest the failure of thalassemic erythroblasts to cope with cellular stress caused by an impaired UPR function as a result of high Ca(2+) levels may exacerbate thalassemic cell death during erythropoiesis.

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