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First published online 31 January 2006
doi: 10.1242/jcs.02784

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Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes

¹ Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
² Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
³ Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy
⁴ T. Jefferson University, Kimmel Cancer Institute, 233 South 10th St, Philadelphia, PA 19107-5541, USA

^* Author for correspondence (e-mail: r.guerriero{at}iss.it)

Accepted 7 November 2005

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.

Key words: Signal transduction, Megakaryocytopoiesis, Polyploidization, MAPK, PI3K, mTOR

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