Inhibition of TPO-induced MEK or mTOR activity induces opposite effects on the ploidy of human differentiating megakaryocytes
Authors: Raffaella Guerriero
Eleonora Petrucci
Ugo Testa
Cesare Peschle
Isabella Parolini
Massimo Sargiacomo
Cristiana Chelucci
Paola Samoggia
Marco Gabbianelli
Journal: Journal of Cell Science (2006)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: megakaryocyte; polyploidization; differentiation; maturation; Shc-Ras-MAPK pathway
Affiliations: Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. , Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy, Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy, Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy, T. Jefferson University, Kimmel Cancer Institute, 233 South 10th St, Philadelphia, PA 19107-5541, USA

Abstract:  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. 

Retracted: No

PubPeer comments:
 How to explain the duplications, including a mirror image? , 
null, 
null, 
 2019 expression of concern. http: jcs.biologists.org content 132 11 jcs233684 Journal of Cell Science was informed that several bands in each of the ERK1 2, P-AKT, AKT and p70S6K blots in Fig. 5 look very similar. After discussions with the first author, Raffaella Guerriero, who was unable to locate the original data, the journal referred this matter to the Istituto Superiore di Sanitu00e0. As the institute does not have a suitable body to investigate this matter, the journal is publishing this Expression of Concern to alert readers to issues with the western blots shown in Fig. 5. Without the original full blots and an impartial investigation by the institute, the journal cannot determine whether the results and conclusions reported in the paper are compromised. The authors apologise to readers for any inconvenience caused. , 
 https: retractionwatch.com 2019 06 17 an-author-told-a-journal-their-institution-had-no-one-who-handled-allegations-turns-out-that-wasnt-true , 
 https: retractionwatch.com 2019 09 23 based-on-the-literature-we-have-no-reason-not-to-believe-to-the-authors , 
 How naive can you be?! Serious journals that cares about the science should show more responsibility when research institutions fail. , 
 This Expression of Concern relates to J. Cell Sci. (2006) 119, 744-752 (doi:10.1242 jcs.02784). Journal of Cell Science was informed that several bands in each of the ERK1 2, P-AKT, AKT and p70S6K blots in Fig. 5 look very similar. After discussions with the first author, Raffaella Guerriero, who was unable to locate the original data, the journal referred this matter to the Istituto Superiore di Sanitu00e0. As the institute does not have a suitable body to investigate this matter, the journal is publishing this Expression of Concern to alert readers to issues with the western blots shown in Fig. 5. Without the original full blots and an impartial investigation by the institute, the journal cannot determine whether the results and conclusions reported in the paper are compromised. The authors apologise to readers for any inconvenience caused. Update: After this note was published, Journal of Cell Science was contacted by the Istituto Superiore di Sanitu00e0 to clarify that they do in fact have a committee to investigate research integrity matters, and that they would endeavour to make this more obvious. They investigated this case and issued the following statement: The Bioethics Unit and the Research Ethics Committee (REC) of the Istituto Superiore di Sanitu00e0 (ISS: Italian National Institute of Health) are in charge of dealing with ethical issues and research integrity matters. The ISS Bioethics Unit and the ISS REC carried out an independent investigation following the Expression of Concern published by the Journal of Cell Science. The corresponding author, Dr Raffaella Guerriero, provided relevant and pertinent documentation and information. Following the investigation, the ISS Research Ethics Committee and the ISS Bioethics Unit confirm the results and conclusions of the paper by Guerriero et al. (2006). The Head of the ISS Research Coordination and Support Service was informed about the results of this investigation. The President of the ISS, Prof. Silvio Brusaferro, approved the results of the investigation and the present note. Via: https: jcs.biologists.org content 132 16 jcs237495 , 
 After the u201cinvestigationu201d above was approved by Prof. Silvio Brusaferro, readers might find his possible involvement in an Italian WHO matter of interest: u201cThe head of Italyu2019s national health institute [Brusaferro] distanced himself Friday from a scandal over a spiked World Health Organization report into Italyu2019s coronavirus response, saying he had no means to interfere with the U.N. agency and had never censored even inconvenient information from the public.u201d https: apnews.com article pandemics-italy-coronavirus-pandemic-united-nations-fdd018c21a19f9bc36a523657ebb39cf ,