Distinct roles of the mTOR components Rictor and Raptor in MO7e megakaryocytic cells.

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.

Downregulation of signal transducer and activator of transcription 5 (STAT5) in CD34+ cells promotes megakaryocytic development, whereas activation of STAT5 drives erythropoiesis.

Although it has been proposed that the common myeloid progenitor gives rise to granulocyte/monocyte progenitors and megakaryocyte/erythroid progenitors (MEP), little is known about molecular switches that determine whether MEPs develop into either erythrocytes or megakaryocytes. We used the thrombopoietin receptor c-Mpl, as well as the megakaryocytic marker CD41, to optimize progenitor sorting procedures to further subfractionate the MEP (CD34(+)CD110(+)CD45RA(-)) into erythroid progenitors (CD34(+)CD110(+)CD45RA(-)CD41(-)) and megakaryocytic progenitors (CD34(+)CD110(+)CD45RA(-)CD41(+)) from peripheral blood. We have identified signal transducer and activator of transcription 5 (STAT5) as a critical denominator that determined lineage commitment between erythroid and megakaryocytic cell fates. Depletion of STAT5 from CD34(+) cells by a lentiviral RNAi approach in the presence of thrombopoietin and stem cell factor resulted in an increase in megakaryocytic progenitors (CFU-Mk), whereas erythroid progenitors (BFU-E) were decreased. Furthermore, an increase in cells expressing megakaryocytic markers CD41 and CD42b was observed in STAT5 RNAi cells, as was an increase in the percentage of polyploid cells. Reversely, overexpression of activated STAT5A(1*6) mutants severely impaired megakaryocyte development and induced a robust erythroid differentiation. Microarray and quantitative reverse transcription-polymerase chain reaction analysis revealed changes in expression of a number of genes, including GATA1, which was downmodulated by STAT5 RNAi and upregulated by activated STAT5.

Chemical characterisation of three haemolytic compounds from the microalgal species Fibrocapsa japonica (Raphidophyceae).

The molecular structures of the three main haemolytic compounds (Fj1, Fj2 and Fj3) isolated from the ichthyotoxic microalgal species Fibrocapsa japonica have been investigated by NMR, LC-ESI-MS, ESI-MS-MS, IR, GC-MS and GC-HRMS methods. They are polyunsaturated fatty acids which we identified as: 6,9,12,15-octadecatetraenoic acid (OTA, C18:4omega3), 5,8,11,14,17-eicosapentaenoic acid (EPA, C20:5omega3) and 5,8,11,14-eicosatetraenoic acid (arachidonic acid AA, C20:4omega6). The identity of the latter two was confirmed on the basis of commercial standards (C20:5omega3 and C20:4omega6). Both displayed the same spectroscopic and chromatographic characteristics as Fj2 and Fj3 and had a similar strong haemolytic effect. We propose that when F. japonica cells accumulate in fish gills during blooms these compounds could be the cause of icthyotoxicity.