Background: Erythropoietin (Epo) is required for correct execution of the erythroid differentiation program. Erythropoiesis requires Bcl-X_L , a major late target of Epo-receptor signaling. Mice lacking Bcl-X_L die around embryonic age E12.5, forming normal erythroid progenitors but lacking functional red cells. Recently, serum-free culture conditions for expansion of murine red cell progenitors were developed, yielding cells capable of in vivo-like terminal differentiation into enucleated erythrocytes, in response to Epo/insulin. Here we address whether Epo function during terminal maturation involves a cytokine-independent "default program," requiring only apoptosis inhibition through Epo-dependent upregulation of Bcl-X_L.

Results: Exogenous expression of Bcl-X_L or Bcl-2 in primary murine erythroblasts or clonal erythroblast lines derived from p53^−/− mice allowed these cells to undergo terminal erythroid maturation, in the complete absence of cytokines. A potential autocrine Epo loop was ruled out by respective neutralizing antibodies. Importantly, sustained proliferation of Bcl-X_L-expressing immature erythroblasts still required respective factors (Epo, stem cell factor [SCF], and the glucocorticoid receptor ligand dexamethasone [Dex]). Epo-independent differentiation in these Bcl-X_L- or Bcl-2-expressing, primary erythroblasts was thus triggered by removal of the renewal factors SCF and Dex. This initiated the maturation-specific expression cascade of erythroid transcription factors, followed by differentiation divisions (characterized by a short G1 phase and decrease in cell size), hemoglobin accumulation, and enucleation.

Conclusions: During erythroid maturation, Epo regulates red cell numbers via apoptosis inhibition, caused by Epo-dependent upregulation of the antiapoptotic protein Bcl-X_L. This allows "default" terminal differentiation of apoptosis-protected, committed erythroblasts, independent of any exogenous signals.