PI-3k-Akt axis inhibits apoptosis in normal human megakaryoblasts and is efficiently activated by thrombopoietin

While optimizing a clinical protocol for ex vivo expansion of human megakaryocytic progenitor cells (CFU-Meg) we found that megakaryocytic cells gradually undergo apoptosis upon prolonged culture or decrease in optimal concentrations of growth factors. Hence, we become interested in the anti-apoptotic influence of several megakaryopoietic stimulators (TPO, IL-6, IL-11 and SDF-1). Megakaryoblasts were expanded ex vivo in serum free liquid cultures from CD34 + cells. When necessary cells were further purified by immunoselection (final purity >95% of α IIb /β 3 + cells), and then exposed to TPO, IL-6, IL-11 or SDF-1. We focused on i) influence of these factors on survival of megakaryocytic cells and ii) correlation with activation of different intracellular signaling pathways (phosphorylation of AKT, MAPK p42/44 and JAK-STAT proteins) with antiapoptotic effects. We found that TPO only, and not IL-6, IL-11 or SDF-1 i) protected human megakaryoblasts from undergoing apoptosis (Annexin-V binding, PARP and MTT assays and activation of caspase-3) and ii) activated the JAK-STAT, MAPK p42/44 and PI-3K-AKT pathway (Table). We also found that blocking PI-3K activity with wortmanin or Ly 294002 induced apoptosis in human megakaryoblasts. In contrast inhibiting MAPK p42/44 activity by PD980092 did not induce apoptosis in these cells. This suggests that the PI-3K-AKT axis plays an important role in inhibiting apoptosis in megakaryocytic cells. Interestingly, while the PI-3K-AKT axis was stimulated in normal human megakaryoblasts by both TPO and SDF-1, only TPO inhibited apoptosis. We conclude that i) PI-3K-AKT axis plays an important anti-apoptotic role in normal human megakaryoblasts, and ii) TPO in contrast to SDF-1 efficiently activates pathways downstream from AKT or parallel to the PI-3K-AKT pathway. Activation of this cascade appears to prevent normal human megakaryocytic cells from undergoing apoptosis.