New Roles for Cyclin E in Megakaryocytic Polyploidization

Abstract

Megakaryocytes are platelet precursor cells that undergo endomitosis. During this process, repeated rounds of DNA synthesis are characterized by lack of late anaphase and cytokinesis. Physiologically, the majority of the polyploid megakaryocytes in the bone marrow are cell cycle arrested. As previously reported, cyclin E is essential for megakaryocyte polyploidy; however, it has remained unclear whether up-regulated cyclin E is an inducer of polyploidy in vivo. We found that cyclin E is up-regulated upon stimulation of primary megakaryocytes by thrombopoietin. Transgenic mice in which elevated cyclin E expression is targeted to megakaryocytes display an increased ploidy profile. Examination of S phase markers, specifically proliferating cell nuclear antigen, cyclin A, and 5-bromo-2-deoxyuridine reveals that cyclin E promotes progression to S phase and cell cycling. Interestingly, analysis of Cdc6 and Mcm2 indicates that cyclin E mediates its effect by promoting the expression of components of the pre-replication complex. Furthermore, we show that up-regulated cyclin E results in the up-regulation of cyclin B1 levels, suggesting an additional mechanism of cyclin E-mediated ploidy increase. These findings define a key role for cyclin E in promoting megakaryocyte entry into S phase and hence, increase in the number of cell cycling cells and in augmenting polyploidization. Megakaryocytes (MKs)2 are bone marrow precursor cells responsible for the production of platelets, which are renewed on a daily basis (1). In a process termed megakaryopoiesis, multipotent hematopoietic stem cells commit toward becoming megakaryocyte progenitors. This is followed by differentiation of progenitors into mature MKs while concomitantly undergoing polyploidization (reviewed in Ref. being 16N (6 -7). During this process, MKs increase the production of proteins necessary for platelet biogenesis and function (8). Mature MKs form proplatelet extensions that fragment and give rise to platelets (9).The mechanism by which MKs become polyploid is still not well understood. Following a series of normal cell divisions, MKs enter a cell cycle with a brief G1 phase, followed by a typical S phase and a very short G2 phase (6). Next, MKs undergo endoreplication, which represents a mitotic cell cycle that is terminated at the late anaphase stage. Repeated rounds of endoreplication eventually give rise to a polyploid megakaryocyte. The regulatory mechanisms that control polyploidization have been partially explored, with a major focus on the regulation of mitotic phase and cytokinesis (10 -13). As to mitotic regulation, it became clear that it is different in MK cell lines and primary MKs, as the former show decreased levels of cyclin B in polyploid MKs (14 -15), while primary MKs display cyclin B during endomitotis (16 -17). Other studies have focused on the G1 phase of the MK cell cycle as a regulatory phase of polyploidy. The first study in this area showed that cyclin D3, which is highly expressed at early G1 phase, is up-regulated in MKs (18). C...

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization

“…However, MKs are not subsequently blocked in 4N. Thus, abnormalities in the G1/S transition check point might also be present in the 4N MK 3,33,34 . In addition, it remains to understand why MYH9 does not accumulate in the cleavage furrow of MK in contrast to other cell types; in fact, myosin II isoform localization may depend on distinct upstream signalling pathways as shown in epithelial cells 31 .…”

Endoreplication and polyploidy: insights into development and disease

“…Cdk2 is the important kinase for endoreplication S phase in animal cells, although in the absence of Cdk2 in mammals, Cdk1 can act as a substitute (Ullah et al, 2009b). Cyclin E overexpression increases the ploidy of megakaryocytes (Eliades et al, 2010), suggesting that the Cyclin E/Cdk2 (Cdc2c -FlyBase) complex is the relevant kinase. Likewise, early work suggested that the Cyclin E/Cdk2 complex is the crucial, and perhaps only, CDK required for endoreplication in Drosophila (Lilly and Duronio, 2005).…”

Dynamin 2–dependent endocytosis is required for normal megakaryocyte development in mice

“…After washing twice, cells were incubated with 0.5 mg/mL of RNase (Sigma-Aldrich), incubated with 50 mg/mL of propidium iodide, and analyzed by flow cytometry. 30 Colony forming units, TGF-b1, and TPO levels and Dnm2 fl/fl Pf4-Cre were plated into MegaCult collagen-based medium (STEMCELL Technologies) and cultured for 7 days in the presence of 10 ng/mL of IL-3, 20 ng/mL of IL-6, and 50 ng/mL of TPO (R&D Systems). Cells were dehydrated and fixed in acetone and stained for acetylcholinesterase activity.…”