744 Research Article Introduction Megakaryocytes (MKs) are characterized by high levels of ploidy during their normal development. During the MK life cycle, an initial proliferative stage (mitosis) is followed by an endomitotic process (successive rounds of DNA synthesis in the absence of cytokinesis), which results in a polyploid DNA content, before final maturation and platelet fragmentation (Zimmet and Ravid, 2000). The major regulator of megakaryocytic development, thrombopoietin (TPO), fully supports MK differentiation in vitro, including pro-platelet and platelet formation (Guerriero et al., 1995; Kaushansky et al., 1995; Cramer et al., 1997). As observed for other cytokines, its receptor binding might regulate specific cell functions activating several signaling pathways. An extensive cross-talk between these pathways might be crucial for controlling a wide array of biological processes (Rojnuckarin et al., 2001). However, in spite of the numerous studies carried out, the cellular and molecular mechanisms underlying MK polyploidization remain largely unknown. Several groups have reported that components of Janus kinase/signal transducer and activator of transcription (JAK/STAT) (Drachman et al., 1995; Wendling and Vainchenker, 1998) and Shc-Ras-MAPK pathways (Nagata and Todokoro, 1995; Yamada et al., 1995; Rouyez et al., 1997) are strongly activated in response to TPO. In particular, the importance of the mitogen-activated protein kinase (MAPK) pathway in megakaryocytopoiesis was corroborated by introduction of constitutively activated mutants of MAPK kinase (MEK) in several cell lines (Whalen et al., 1997; Racke et al., 1997; Melemed et al., 1997) and by treatment of cell lines and primary cells with pharmacological inhibitors of MEK such as PD98059 and UO126 (Rojnuckarin et al., 1999; Fichelson et al., 1999). Nevertheless, the conclusions from these studies on the role of TPO-induced MAPK activity were often controversial. These discrepancies may depend on the experimental conditions and/or on the source of cells used: cells lines, murine mature MKs, murine CD41-selected cells and human cord blood CD34 + cells. In addition to JAK/STAT and Ras-Raf-MAPK, the phosphoinositide 3-kinase (PI3K) pathway is important for mpl (TPO receptor) signaling (Sattler et al., 1997; Miyakawa et al., 2001). PI3K has been shown to be activated by many growth factors involved in hematopoiesis, and plays an important role in promoting cell survival (Songyang et al., 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 Summary Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes Raffaella Guerriero 1, *, Isabella Parolini 1 , Ugo Testa 1 , Paola Samoggia 2 , Eleonora Petrucci 1 , Massimo Sargiacomo 3 , Cristiana Chelucci 1 , Marco Gabbianelli 1 and Cesare Peschle 1,4 1 Department of Hematology, Oncology and Molecular Medicine, 2 Department of Cellular Biology and Neurosciences, and 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@iss.it) Accepted 7 November 2005 Journal of Cell Science 119, 744-752 Published by The Company of Biologists 2006 doi:10.1242/jcs.02784 Journal of Cell Science