Continuous expression of Bcl-xL protein during megakaryopoiesis is post-translationally regulated by thrombopoietin-mediated Akt activation, which prevents the cleavage of Bcl-xL.
BACKGROUND
One of the important biological activities of thrombopoietin (TPO) is to prevent the apoptosis of megakaryocytes. As the antiapoptotic protein Bcl-xL, which has been proven to be indispensable for erythroid differentiation, is also abundantly expressed in megakaryocytes, it is assumed that Bcl-xL plays an important role in megakaryopoiesis.


OBJECTIVE
We investigated the expression of Bcl-xL during megakaryopoiesis and the underlying regulatory mechanism.


RESULTS
In stem cell-derived megakaryocytes, expression of Bcl-xL increased in the early and mid-stages of the differentiation. Both in vitro in cell culture and in vivo in an animal model, expression of Bcl-xL protein was maintained until the platelet-producing stage. TPO depletion caused significant decrease in Bcl-xL protein level without affecting its mRNA in both megakaryocytes and TPO-dependent megakaryocytic UT-7/TPO cells, suggesting that Bcl-xL protein level in TPO-dependent cells is post-translationally regulated. In agreement with this finding, we recognized the appearance of a 12-kD fragment of Bcl-xL upon TPO depletion. This cleavage of Bcl-xL was inhibited by a caspase-3-specific inhibitor. Furthermore, pretreatment of UT-7/TPO with a phosphatidylinositol 3-kinase (PI3 K) inhibitor resulted in the cleavage of Bcl-xL even in the presence of TPO. We thus hypothesized that PI3 K inhibits the activation of caspase-3 and consequent cleavage of Bcl-xL. To prove this, we prepared UT-7/TPO cells transfected with constitutively active Akt-1. When TPO was depleted, the transfectant was significantly less liable to caspase-3 activation and Bcl-xL cleavage.


CONCLUSIONS
Bcl-xL protein is expressed throughout megakaryopoiesis until platelets are produced, and its expression level is at least in part post-translationally regulated through TPO-mediated Akt activation.
Kozuma
Y
Y
Division of Hematology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Kojima
H
H
Yuki
S
S
Suzuki
H
H
Nagasawa
T
T
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005260:Female; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D034061:Thrombopoiesis / Q000502:physiology*; D013926:Thrombopoietin / Q000378:metabolism*; D014162:Transfection; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
C494811:BCL2L1 protein, human; C494812:Bcl2l1 protein, mouse; D018076:DNA, Complementary; D034741:RNA, Small Interfering; D051020:bcl-X Protein; D013926:Thrombopoietin; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt