Abstract
The availability of Erythropoietin (Epo) is essential for the survival of erythroid progenitors. Here we study the effects of Epo removal on primary human erythroblasts grown from peripheral blood CD34+ cells. The erythroblasts died rapidly from apoptosis, even in the presence of SCF, and within 24 hours of Epo withdrawal 60% of the cells were Annexin V positive. Other classical hallmarks of apoptosis were also observed, including cytochrome c release into the cytosol, loss of mitochondrial membrane potential, Bax translocation to the mitochondria and caspase activation. We adopted a 2D DIGE approach to compare the proteomes of erythroblasts maintained for 12 hours in the presence or absence of Epo. Proteomic comparisons demonstrated significant and reproducible alterations in the abundance of proteins between the two growth conditions, with 18 and 31 proteins exhibiting altered abundance in presence or absence of Epo, respectively. We observed that Epo withdrawal induced the proteolysis of the multi-functional proteins Hsp90 alpha, Hsp90 beta, SET, 14-3-3 beta, 14-3-3 gamma, 14-3-3 epsilon, and RPSA, thereby targeting multiple signaling pathways and cellular processes simultaneously. We also observed that 14 proteins were differentially phosphorylated and confirmed the phosphorylation of the Hsp90 alpha and Hsp90 beta proteolytic fragments in apoptotic cells using Nano LC mass spectrometry. Our analysis of the global changes occurring in the proteome of primary human erythroblasts in response to Epo removal has increased the repertoire of proteins affected by Epo withdrawal and identified proteins whose aberrant regulation may contribute to ineffective erythropoiesis.
Highlights
Red blood cell production in the bone marrow is maintained by a delicate balance between erythroid cell proliferation, differentiation and apoptosis
Primary human erythroblasts were cultured from CD34+ cells isolated from human peripheral blood in presence of Epo, Stem Cell Factor (SCF), Dexamethasone and lipids (ESDL) [21]
The blue square depicts the region of the gel shown in Figure 3B. (B) Typical example of a protein differentially represented in the 2 culture conditions, ESDL and SDL. 3D views of spot 5 in Figure 3A and identified by mass spectrometry as a proteolytic fragment of Hsp90 alpha
Summary
Red blood cell production in the bone marrow is maintained by a delicate balance between erythroid cell proliferation, differentiation and apoptosis. EpoR lacks kinase activity but Epo binding triggers the activation of the Janus family protein tyrosine kinase 2 (JAK2) [7], which in turn phosphorylates tyrosine residues in EpoR, creating docking sites for intracellular signalling proteins such as phosphatidylinositol 3-kinase [8], SHP1 [9] and STAT5 [10]. These events lead to the activation of multiple signal transduction pathways and specific gene expression that result in the survival, proliferation, and differentiation of erythroblasts [11]
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