Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 10
Protein Kinase B (c-akt): a molecular switch in regulation of lineage choice decisions
during myelopoiesis
Miranda Buitenhuis, Liesbeth Verhagen, Christian Geest, Hanneke van Deutekom and
Paul J. Coffer.
Molecular Immunology Lab, Dept. of Immunology, University Medical Center Utrecht,
Lundlaan 6, 3584 EA Utrecht, The Netherlands.
Phosphatidylinositol 3-kinase (PI3K) has been reported to play a critical role in
proliferation and survival of a variety of cell types; however, a role in regulating blood cell
production is largely unknown. We have utilized an ex-vivo differentiation system, using
human umbilical cord blood derived CD34+ hematopoietic progenitor cells, to investigate the
role of PI3K and its downstream effectors in myeloid differentiation.
Inhibition of PI3K, with the pharmacological inhibitor LY294002, completely blocked
proliferation and differentiation during granulopoiesis. To pin-point the molecular
mechanisms responsible for this effect, we utilized specific pharmacological inhibitors of
PKB/c-akt or mTOR, both down-stream effectors of PI3K. Inhibition of PKB blocked
progenitor proliferation without affecting cell survival. Interestingly, inhibition of PKB,
abrogated neutrophil maturation, but conversely dramatically enhanced terminal
differentiation of eosinophils. Retroviral transductions were performed to ectopically express
a constitutively active PKB (myrPKB) in CD34+ cells. Transduction of cells with myrPKB
resulted in enhanced neutrophil differentiation and monocyte development compared to cells
transduced with empty vector alone. Conversely, ectopic expression of myrPKB resulted in an
almost complete block in eosinophil differentiation, surprisingly demonstrating neutrophil
differentiation even in the presence of IL-5. In order to investigate the role of PKB in
regulation of hematopoiesis in vivo, #2-microglobulin-/-NOD/SCID mice were transplanted
with CD34+ cells ectopically expressing active myrPKB or dominant-negative PKBcaax.
Transplantation of mice with CD34+ cells ectopically expressing myrPKB resulted in
enhanced neutrophil and monocyte development, whereas ectopic expression of PKBcaax
induced eosinophil development in vivo. To investigate which downstream signal
transduction pathways could be involved in PKB mediated regulation of myelopoiesis,
hematopoietic progenitors were either retrovirally transduced with myrPKB or treated with
pharmacological inhibitors, and western blot analysis was performed. Preliminary data
indicate that phosphorylation of C/EBPalpha, a transcription factor known to play an
important role in regulation of myelopoiesis, is inhibited upon PKB activation in
hematopoietic progenitors.
Taken together, these results demonstrate that PKB activity plays a critical role in the
regulation of cell fate choices during myeloid lineage commitment, most likely via regulation
of phosphorylation of C/EBPalpha. High PKB activity promotes neutrophil differentiation
and monocyte development, while reduction of PKB activity is required to induce eosinophil
differentiation.
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