The role of erythropoietin signal transduction in BCL-2 protein family-mediated survival of erythroid and erythroleukemic cells

Erythropoietin (EPO) is the requisite hormone for the survival of erythroid progenitors. The BCL-2 family of proteins, which include the proapoptotic proteins BIM (B&barbelow;CL-2 I&barbelow;nteracting M&barbelow;ediator of Cell Death) and BAD (B&barbelow;CL-XL/B&barbelow;CL-2 A&barbelow;ssociated D&barbelow;eath Promoter), are major determinants of cell fate in growth factor-dependent hematopoietic cells. Survival factors, such as EPO, or mutations in cancerous cells may activate protein kinases that suppress the activities of proapoptotic molecules leading to phosphorylation, abrogation of expression, and/or sequestration of proteins to promote survival. Withdrawal of EPO from primary and apoptotis-sensitive HCD57 erythroid cells resulted in significant expression of BIM which correlated with apoptotic cell death. EPO treatment of erythroid cells resulted in apparent phosphorylation and disappearance of BIM protein. Inhibition of EPO-induced MAP kinase activation, with U0126 MEK1 inhibitor, effectively blocked phosphorylation and disappearance of BIM resulting in an overall increase of protein correlative with apoptosis. Whereas others have implicated the PI3 kinase/Akt and AP1/c-Jun pathways in regulating BIM expression, experiments with PI3 kinase inhibition, transfections of constitutively active Akt, and AP1/c-Jun dominant negative protein rule out these pathways in erythroid cells. Treatment with the ubiquitin-proteasome inhibitor, MG-132, resulted in the persistence of BIM following EPO-dependent phosphorylation suggesting that degradation of BIM is a key regulatory mechanism of EPO-mediated survival. Knockdown of endogenous BIM expression through transfection of apoptosis-sensitive HCD57 cells with small interfering RNA resulted in cells which were resistant to EPO withdrawal and U0126-induced apoptosis. In contrast to BIM phosphorylation through MAP kinase alone, kinase inhibition studies suggest that EPO-dependent PI3 kinase, MAP kinase, and/or protein kinase C pathways serve to phosphorylate BAD in erythroid cells. BAD kinase inhibition resulted in increased association with prosurvival BCL-XL which is known to trigger the mitochondrial gateway inducing apoptosis. However, EPO-dependent BAD kinase inhibition did not correlate with apoptosis in the HCD57R cell line. This and assessment of BAD in primary and early passage HCD57 cells suggest that BAD is not a major regulator of apoptosis in erythroid cells. Therefore, in contrast to BAD, the phosphorylation and subsequent degradation of BIM through a MAP kinase pathway may have a significant role in EPO-dependent survival of erythroid cells.