Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes.
The megakaryocyte is a paradigm for mammalian polyploid cells. However, the mechanisms underlying megakaryocytic polyploidization have not been elucidated. In this study, we investigated the role of Shc-Ras-MAPK and PI3K-AKT-mTOR pathways in promoting megakaryocytic differentiation, maturation and polyploidization. CD34+ cells, purified from human peripheral blood, were induced in serum-free liquid suspension culture supplemented with thrombopoietin (TPO) to differentiate into a virtually pure megakaryocytic progeny (97-99% CD61+/CD41+ cells). The early and repeated addition to cell cultures of low concentrations of PD98059, an inhibitor of MEK1/2 activation, gave rise to a population of large megakaryocytes showing an increase in DNA content and polylobated nuclei (from 45% to 70% in control and treated cultures, respectively). Conversely, treatment with the mTOR inhibitor rapamycin strongly inhibited cell polyploidization, as compared with control cultures. Western blot analysis of PD98059-treated progenitor cells compared with the control showed a downmodulation of phospho-ERK 1 and phospho-ERK 2 and a minimal influence on p70S6K activation; by contrast, p70S6K activation was completely inhibited in rapamycin-treated cells. Interestingly, the cyclin D3 localization was nuclear in PD98059-induced polyploid megakaryocytes, whereas it was completely cytoplasmic in those treated with rapamycin. Altogether, our results are in line with a model in which binding of TPO to the TPO receptor (mpl) could activate the rapamycin-sensitive PI3K-AKT-mTOR-p70S6K pathway and its downstream targets in promoting megakaryocytic cell polyploidization.
Guerriero
Raffaella
R
Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. guerriero@iss.it
Parolini
Isabella
I
Testa
Ugo
U
Samoggia
Paola
P
Petrucci
Eleonora
E
Sargiacomo
Massimo
M
Chelucci
Cristiana
C
Gabbianelli
Marco
M
Peschle
Cesare
C
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors*; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008533:Megakaryocytes / Q000166:cytology* / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011003:Ploidies; D011494:Protein Kinases / Q000187:drug effects*; D020123:Sirolimus / Q000494:pharmacology*; D058570:TOR Serine-Threonine Kinases; D013926:Thrombopoietin / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D005419:Flavonoids; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D013926:Thrombopoietin; C546842:MTOR protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one