Apoptosis protection by the Epo target Bcl-X(L) allows factor-independent differentiation of primary erythroblasts.
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.
Division of Molecular Biology, Institute of Medical Biochemistry, 1030 Vienna, Austria.
Helmut
H
Dolznig
Bianca
B
Habermann
Katharina
K
Stangl
Eva Maria
EM
Deiner
Richard
R
Moriggl
Hartmut
H
Beug
Ernst W
EW
Müllner
D000818:Animals; D017209:Apoptosis; D002454:Cell Differentiation; D003907:Dexamethasone / Q000378:metabolism; D004900:Erythroblasts / Q000166:cytology* / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D008810:Mice, Inbred C57BL; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D019089:Stem Cell Factor / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D019089:Stem Cell Factor; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; D004921:Erythropoietin; D003907:Dexamethasone