Content area
Full Text
β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulationof freea-globin chains that form toxic aggregates1,2. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage3-6. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation7. Although erythroid transcription factor GATA-1, themaster transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase- 3 cleavage8. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation9. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free a-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.
To investigate the hypothesis that HSP70 can be sequestrated in the cytoplasm of mature b-TM erythroblasts by binding to free a-globin chains of haemoglobin or aggregates, we analysed its subcellular localization in bone marrow samples fromadult b-TM patients and healthy donors by confocal microscopy (n53). In contrast to normal erythroblasts, inb-TMmaturehaemoglobinized erythroblasts,HSP70wasmainly localized in the cytoplasm and GATA-1 was poorly expressed in the nucleus (Fig. 1).
To explore the role of HSP70 in b-TM ineffective erythropoiesis, we performed an in vitro two-phase liquid culture, allowing proliferation, survival and erythroid differentiation of b-TMor control CD341 progenitors towards the formation of acidophilic erythroblasts and reticulocytes. During the second phase of amplification (day 8), corresponding to erythroid terminal differentiation, differentiation in the b-TMgroup was accelerated. This was characterized by a higher percentage of polychromatophilic cells (26.268.4% versus 9.062.7%;P50.003) (Fig. 2a),...