Erythropoietin-induced phosphorylation/degradation of BIM contributes to survival of erythroid cells.
OBJECTIVE
A proapoptotic BH3-only protein BIM (BCL-2 interacting mediator of cell death) can link cytokine receptor signaling with the apoptotic machinery in hematopoietic cells. We investigated here the role of BIM in erythropoietin (EPO)-mediated survival in erythroid cells.


METHODS
We downregulated BIM in EPO-dependent HCD57 erythroid cells with short hairpin RNA (shRNA), and used real-time polymerase chain reaction, Western blots, and flow cytometry to characterize BIM expression and apoptosis. Hematologic analyses of BIM-deficient (Bim(-/-)) mice were conducted.


RESULTS
BIM expression increases in primary murine erythroid cells and HCD57 cells deprived of EPO. Whereas Bim mRNA increased less than twofold, BIM protein increased more than 10-fold after EPO withdrawal, suggesting posttranscriptional regulation of BIM. EPO treatment resulted in rapid phosphorylation of BIM at Serine 65 and phosphorylation correlated with degradation of BIM. Inhibition of extracellular signal-regulated kinase (ERK) by a MEK/ERK inhibitor, U0126, blocked both phosphorylation and degradation of BIM, resulting in apoptosis. Treatment with a proteasome inhibitor, MG-132, also blocked degradation of phosphorylated BIM. Downregulation of BIM with the shRNA resulted in HCD57 cells more resistant to apoptosis induced by either EPO withdrawal or ERK inhibition. Although we observed no significant changes in the number of erythrocytes or reticulocytes in the circulation of Bim(-/-) mice, erythroid progenitors from bone marrow in Bim(-/-) mice were reduced in number and more resistant to apoptosis induced by U0126 MEK/ERK inhibitor.


CONCLUSIONS
EPO protects erythroid cells from apoptosis in part through ERK-mediated phosphorylation followed by proteasomal degradation of BIM.
Abutin
Randolph M
RM
Department of Pharmacology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298-0035, USA.
Chen
Jingchun
J
Lung
Tina K
TK
Lloyd
Joyce A
JA
Sawyer
Stephen T
ST
Harada
Hisashi
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism*; D004921:Erythropoietin / Q000378:metabolism / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; C000606776:Bcl2l11 protein, mouse; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D004921:Erythropoietin; D048049:Extracellular Signal-Regulated MAP Kinases