XPO1 regulates erythroid differentiation and is a new target for the treatment of ß-thalassemia.

ß-thalassemia major (ß-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of ß-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human ß-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of ß-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of ß-TM.

Defective nuclear localization of Hsp70 is associated with dyserythropoiesis and GATA-1 cleavage in myelodysplastic syndromes.

Normal human erythroid cell maturation requests the transcription factor GATA-1 and a transient activation of caspase-3, with GATA-1 being protected from caspase-3-mediated cleavage by interaction with the chaperone heat shock protein 70 (Hsp70) in the nucleus. Erythroid cell dysplasia observed in early myelodysplastic syndromes (MDS) involves impairment of differentiation and excess of apoptosis with a burst of caspase activation. Analysis of gene expression in MDS erythroblasts obtained by ex vivo cultures demonstrates the down-regulation of a set of GATA-1 transcriptional target genes, including GYPA that encodes glycophorin A (GPA), and the up-regulation of members of the HSP70 family. GATA-1 protein expression is decreased in MDS erythroblasts, but restores in the presence of a pan-caspase inhibitor. Expression of a mutated GATA-1 that cannot be cleaved by caspase-3 rescues the transcription of GATA-1 targets, and the erythroid differentiation, but does not improve survival. Hsp70 fails to protect GATA-1 from caspases because the protein does not accumulate in the nucleus with active caspase-3. Expression of a nucleus-targeted mutant of Hsp70 protects GATA-1 and rescues MDS erythroid cell differentiation. Alteration of Hsp70 cytosolic-nuclear shuttling is a major feature of MDS that favors GATA-1 cleavage and differentiation impairment, but not apoptosis, in dysplastic erythroblasts.

Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1.

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 differentiation by positively regulating promoters of erythroid and anti-apoptotic genes, is cleaved by caspases in erythroid precursors undergoing cell death upon erythropoietin starvation or engagement of the death receptor Fas. In contrast, by an unknown mechanism, GATA-1 remains uncleaved when these cells undergo terminal differentiation upon stimulation with Epo. 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-X(L), 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.

HSP27 controls GATA-1 protein level during erythroid cell differentiation.

Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34(+) human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.

Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia.

Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrations, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXΦ. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.