Characterization of 14-3-3β Expression during Differentiation of the Megakaryocytic Cell Line MEG-01

The 14-3-3 family of proteins is highly conserved among mammalian species which consists of seven homologous isoforms (β,ε, γ, η, 𝛉, σ and ζ). They are involved in the regulation of most cellular processes including several metabolic pathways, redox-regulation, transcription, RNA processing, protein synthesis, protein folding and degradation, cytoskeletal organization, apoptosis, intracellular trafficking/targeting, and cell cycle. In a previous study, we identified several novel genes associated with hematopoietic lineage commitment and/or differentiation using a hematopoietic liquid culture system and a differential display methodology. One of these, TP-3 (thrombopoietin-stimulated clone-3) is selectively expressed in normal human megakaryocytic lineage cells. cDNA sequences analysis revealed 100% identity with the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, beta polypeptide (YWHAB) gene that encodes for 14-3-3β protein. Accordingly, we set out to determine the role 14-3-3β may have in megakaryocyte proliferation and differentiation. For this purpose, we employed the megakaryocytic precursor cell line, MEG-01, that can be induce to differentiate by 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment. After TPA challenge, we detected an increase in the 14-3-3β mRNA expression in MEG-01 cells. Similarly, the phosphorylated form of 14-3-3β protein also was induced during MEG-01 cells differentiation by TPA treatment, while the total 14-3-3β protein levels did not reveal any differences between MEG-01 cells treated with or without TPA. To confirm the correlation between 14-3-3β expression and the megakaryocyte differentiation, we compared its expression patterns to that of the platelet-specific antigens GP IIb/IIIa and GP Ib which are known to be markers of maturation within the megakaryocyte lineage using flow cytometry and immunofluorescent staining methods. After TPA treatment, the percentage of MEG-01 cells expressing GP IIb/IIIa and GP Ib significantly increased from 19% to 95% and from 0% to 27% (p<0.05) over the course of 8 days, respectively. In contrast, 14-3-3β siRNA knockdown resulted in a reduction of platelet-specific antigens GP IIb/IIIa and GP Ib in TPA-treated population from 41.6 ± 7.01 to 12.8 ± 5.47 and from 24.1 ± 2.32 to 7.9 ± 0.21 (p<0.05), respectively. To better understand potential targets of 14-3-3β effects in megakaryocytic differentiation and proliferation, we investigated whether the changes of cell-cycle-related gene expressions were related to the MEG-01 cells differentiation. We used semi-quantitative real-time polymerase chain reaction (RT-PCR) to examine expression pattern of important known genes involved in cell-cycle regulation. We found enhanced expression of p21 and cyclin D1 in TPA-treated MEG-01 cells, while such treatment was associated with a suppression of Cdc25A and Cdc25C. By Western blot analysis, we confirmed this differential effect on gene expression was associated with corresponding changes in protein expression. Taken together, our data provide the new insights into the 14-3-3 β its role in the megakaryocyte differentiation via the signal transduction pathways of cyclin D1 and p21 expression. We are current investigating whether 14-3-3β dysfunction may contribute to the pathogenesis of certain acquired disorders of megakaryopoiesis.