Phosphatase inhibition promotes antiapoptotic but not proliferative signaling pathways in erythropoietin-dependent HCD57 cells

Erythropoietin (EPO) allows erythroid precursors to proliferate while prote cting them from apoptosis, Treatment of the EPO-dependent HCD57 murine cell line with 70 mu mol/L orthovanadate, a tyrosine phosphatase inhibitor, res ulted in both increased tyrosine protein phosphorylation and prevention of apoptosis in the absence of EPO without promoting proliferation. Orthovanad ate also delayed apoptosis in primary human erythroid progenitors, Thus, we investigated what survival signals were activated by orthovanadate treatme nt, Expression of Bcl-X-L and BAD phosphorylation are critical for the surv ival of erythroid cells, and orthovanadate in the absence of EPO both maint ained expression levels of antiapoptotic Bcl-X-L and induced BAD phosphoryl ation at serine 112, Orthovanadate activated JAK2, STAT1, STAT5, the phosph atidylinositol-3 kinase (PI-3 kinase) pathway, and other signals such as JN K and p38 without activating the EPO receptor, JAK1, Tyk2, Vav, STAT3, and SHC, Neither JNK nor p38 appeared to have a central role in either apoptosi s or survival induced by orthovanadate. Treatment with cells with LY294002, an inhibitor of PI-3 kinase activity, triggered apoptosis in orthovanadate -treated cells, suggesting a critical role of PI-3 kinase in orthovanadate- stimulated survival. Mitogen-activated protein kinase (MAPK) was poorly act ivated by orthovanadate, and inhibition of MAPK with PD98059 blocked prolif eration without inducing apoptosis. Thus, orthovanadate likely acts to grea tly increase JAK/STAT and PI-3 kinase basal activity in untreated cells by blocking tyrosine protein phosphatase activity, Activated JAK2/STAT5 then l ikely acts upstream of Bcl-X-L expression and PI-3 kinase likely promotes B AD phosphorylation to protect from apoptosis, In contrast, MAPK/ ERK activi ty correlates with only EPO-dependent proliferation but is not required for survival of HCD57 cells. (Blood, 2000;96: 2084-2092) (C) 2000 by The Ameri can Society of Hematology.