Hsp70 Is a New Major Regulator of Erythropoiesis by Preventing Caspase-3-Mediated Cleavage of GATA-1.

Red blood cell production depends on apoptosis rate of erythroid progenitors and precursors. This process is mainly regulated by the glycoprotein Erythropoietin (Epo), which positively regulates through the JAK/STAT5 pathway, in synergy with the transcription factor GATA-1, the expression of the antiapoptotic protein Bcl-XL. Thus, Epo starvation results in caspase-3 activation through the intrinsic mitochondrial pathway, and as a consequence GATA-1 is cleaved and apoptosis occurs. Recently we have shown that caspase-3 activation is also absolutely required for the morphological changes that occur during normal human terminal erythroid differentiation, including chromatin condensation as well as nucleus and cell size reduction. In this context, although activated caspase-3 colocalizes in the nucleus with GATA-1, apoptosis and GATA-1 cleavage do not occur (

Heat Shock Proteins (Hsp) are chaperons that play a major role as a modulator of apoptosis. Here, we report by western blot and confocal analysis that inducible Hsp70 is constituvely expressed during human terminal eythroid differentiation, in both nuclear and cytoplasmic erythroblast compartments. Hsp70 co-localizes and co-immunoprecipitates with GATA-1 in the nucleus at the onset of caspases activation during terminal erythroid differentiation. Rapidly (<6h) after Epo starvation, while Hsp70 nuclear localization is lost, GATA-1 is cleaved. In contrast, in the presence of the nuclear export inhibitor Leptomycin B, Hsp70 remains in the nucleus and GATA-1 is not cleaved. In order to demonstrate the direct role of Hsp70 in GATA-1 protection, in an in vitro assay, we have shown that in a dose dependant manner recombinant Hsp70, but not Hsp90, inhibits recombinant caspase-3-mediated proteolysis of GATA-1. In human erythroid CD36+GPA+ progenitors, transfection of siRNA Hsp70, but not of a scramble siRNA, reduces significantly (80% vs 0%of inhibition) Hsp 70 nuclear and cytoplasmic expression, and is associated with GATA-1 cleavage at the onset of caspase-3 activation occuring during erythroid differentiation. Hsp70 inhibition leads to significant decrease of GATA-1 regulated genes expression (hemoglobinization (8% vs 50% of benzidine positive cells) ; Bcl-XL expression ( 80% inhibition) and apoptosis (8% vs 40%trypan blue positive cells). Interestingly, only mature cells are affected by siRNA Hsp70 and exhibit apoptotic features as assessed by topro3 stain and nuclear fragmentation (acidophils (90%) vs basophilic erythroblasts (10%)).

Therefore, Hsp70 exerts a new critical antiapoptotic role during terminal erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1. We propose a model in which, Epo determines the fate of erythroblasts (apoptotsis vs differentiation) downstream of caspase-3 activation by regulating the localization of Hsp70 (cytoplasmic vs nuclear and cytoplasmic).