Objective: During megakaryopoiesis, hematopoietic progenitor cells in the bone marrow proliferate and ultimately differentiate in mature megakaryocytes (MK). We and others have recently described a role for the mammalian target of Rapamycin (mTOR) in proliferation and differentiation of MK cells. Two non-redundant complexes of mTOR have been described; mTORC1 containing rapamycin-associated TOR protein (Raptor) and mTORC2 containing Rapamycin-insensitive companion of mTOR (Rictor). The individual roles of these complexes in MK development have so far not been elucidated, and were investigated in this study.
Methods: We have used an siRNA approach to selectively knock down either Rictor or Raptor expression in MO7e megakaryoblastic cells. Using flow cytometry, nuclear ploidity, and cell cycling as assessed by BrdU incorporation were investigated. Electron microscopy and cotransductions with GFP-LC3 were used to quantify autophagy. Activation of intracellular signal transduction pathways was studied by Western blot analysis.
Results: We observed a reduced cell cycling upon Rictor siRNA transduction, resulting in decreased numbers of polypoid cells. Knocking down Raptor expression resulted in a reduced expansion and a reduced cell size. In addition, increased autophagy was observed in Raptor siRNA-transduced cells, in correspondence with an attenuation of activation of the p70S6K/S6, and 4E-BP pathways.
Conclusions: The current study shows that the mTORC1 and mTORC2 complexes have distinct, non-redundant functions in MO7e MK cell proliferation, and development. The mTOR/Rictor complex affects megakaryopoiesis by regulating nuclear division and subsequent cell cycle progression, whereas Raptor signaling protects MK cells from autophagic cell death, enabling normal megakaryopoiesis to take place.