Caspase-3 is activated during both terminal differentiation and erythropoietin-starvation-induced apoptosis of human erythroid precursors. The transcription factor GATA-1, which performs an essential function in erythroid differentiation1,2 by positively regulating promoters of erythroid and anti-apoptotic genes3,4,5,6, is cleaved by caspases in erythroid precursors undergoing cell death upon erythropoietin starvation or engagement of the death receptor Fas7,8. In contrast, by an unknown mechanism, GATA-1 remains uncleaved when these cells undergo terminal differentiation upon stimulation with Epo9,10,11. Here we show that during differentiation, but not during apoptosis, the chaperone protein Hsp70 protects GATA-1 from caspase-mediated proteolysis. At the onset of caspase activation, Hsp70 co-localizes and interacts with GATA-1 in the nucleus of erythroid precursors undergoing terminal differentiation. In contrast, erythropoietin starvation induces the nuclear export of Hsp70 and the cleavage of GATA-1. In an in vitro assay, Hsp70 protects GATA-1 from caspase-3-mediated proteolysis through its peptide-binding domain. The use of RNA-mediated interference to decrease the Hsp70 content of erythroid precursors cultured in the presence of erythropoietin leads to GATA-1 cleavage, a decrease in haemoglobin content, downregulation of the expression of the anti-apoptotic protein Bcl-XL, and cell death by apoptosis. These effects are abrogated by the transduction of a caspase-resistant GATA-1 mutant. Thus, in erythroid precursors undergoing terminal differentiation, Hsp70 prevents active caspase-3 from cleaving GATA-1 and inducing apoptosis.
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We thank C. Pouzet for her assistance in confocal analysis, F. Valensi and V. Asnafi for their assistance in cytological analysis, Y. Dumez, A. Benachi and F. Audat for providing us with cord blood samples; U. Testa for the cDNAs of GATA-1 and poly(ADP-ribose) polymerase subcloned in PET21; and A. Benmerah for providing us with leptomycin B. This work was supported by grants from the Ligue nationale contre le cancer (LNC), the Fondation pour la recherche médicale (FRM), the Association pour la recherche sur le cancer (ARC), Cancéropole d’Île de France, Fondation de France, Ministère de la recherche and AMGEN.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
The file contains a Supplementary Note on Hsp70 and Hsc70, a Supplementary Methods section describing all reagents used as well as detailed protocols, Supplementary Figures 1-8 with legends and a Supplementary References. (PDF 935 kb)
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Ribeil, J., Zermati, Y., Vandekerckhove, J. et al. Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1. Nature 445, 102–105 (2007) doi:10.1038/nature05378
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