The molecular mechanisms mediating endomitosis in megakaryocytes are incompletely understood. Studies investigating endomitosis have been hampered by the rarity of megakaryocytes, difficulty separating direct effects from general perturbations of cell maturation, complications associated with synchronizing the cell cycle, use of transformed cell lines, and potential differences between rodent and human megakaryocytes.

Cyclins and Cyclin-Dependent Kinases

 Two classes of proteins control the cell cycle in mammalian cells. These are the cyclins, so named for their cyclical synthesis and degradation during the cell cycle, and cell division kinases (Cdks, also known as cyclin-dependent kinases). Together, these two families of proteins form a protein-kinase complex in which the regulatory unit is the cyclin and the catalytic unit is the Cdk. The role of these kinase complexes in cell cycle control is complex. At least seven members of the cyclin gene family and seven distinct Cdk genes have been identified.

Given the importance of cyclins and Cdks in controlling the cell cycle, they have been the focus of considerable attention in investigations of the mechanisms underlying megakaryocyte endomitosis. The most compelling evidence probably exists for the role of the D-type cyclins in megakaryocyte endomitosis. The D-type cyclins are unique in that their activity can be modulated by extracellular mitogens. Megakaryocytes express cyclin D3 and, to a lesser extent, cyclin D1. Levels of both of these factors increase after treatment with TPO. Overexpression of cyclin D3 results in increased mega karyocyte ploidy in transgenic mouse models. Complexes of cyclin D3 and its major kinase subunit, Cdk2, show high kinase activity in polyploid cells. Antisense knockdown of cyclin D3 levels suppresses endomitosis and abrogates normal development of primary mouse megakaryocytes.

Cyclin D1 is a direct target gene of GATA1, a transcription factor required for megakaryocyte polyploidization and maturation. Overexpression of cyclin D1 in transgenic mice increases mega karyocyte modal ploidy compared with nontransgenic littermates, and the combination of cyclin D1 and Cdk4 kinase activity restores polyploidization of GATA1-deficient murine megakaryocytes. Conversely, enforced expression of p16^ink4a, a cell cycle inhibitor of Cdk4/6, blocks polyploidization in murine megakaryocytes. p16ink4a is also potently repressed by GATA1.

Cyclin E−/− mice have impaired megakaryocytopoiesis with reduced modal ploidy. These mice also have defective trophoblast development, another tissue characterized by atypical mitosis. Cyclin B1/CDC2 is a mitotic cyclin complex. Yeast strains deficient in cyclin B1 or CDC2 undergo an additional round of DNA replication without cytokinesis. Several studies have shown that low levels of cyclin B1/CDC2 are required for the progression of endomitosis in megakaryocytic cell lines. However, studies of primary megakaryocytes have shown normal cyclin B1 and CDC2 levels and functional mitotic activity during endomitosis.

Other Mitotic Kinases

Aurora-B kinase (also called AIM-1 kinase) is involved in late ana phase and cytokinesis, and mRNA transcript levels of Aurora-B kinase have been reported to decrease during polyploidization of primary megakaryocytes and megakaryocytic cell lines. This suggests that Aurora-B kinase may play a mechanistic role in megakaryocyte endomitosis. However, functional activity of Aurora-B kinase appears normal in late anaphase of endomitotic primary megakaryocytes, indicating that a simple deficiency of Aurora-B kinase activation is an unlikely mechanism to explain endomitosis. A polo-like kinase (PLK-1) is a serine-threonine kinase required for assembly of the mitotic spindle, separation of chromosomes during anaphase, and exit from mitosis. PLK-1 mRNA and protein levels decrease during polyploidization of murine megakaryocytes, and enforced expression of PLK-1 in primary murine megakaryocytes impairs endomitosis. However, the effects of overexpression are modest, preferentially affect lower-ploidy megakaryocytes, and are complicated by alterations in cell cycle kinetics.

Microtubules and the Mitotic Spindle

Microtubules play key roles in mitosis by enabling the formation of the mitotic spindle. Therefore, factors that regulate their assembly have also been investigated as candidates involved in megakaryocyte endomitosis. The protein regulator of cytokinesis 1 (PRC-1) is involved in mitotic spindle elongation and cytokinesis. However, no differences in PRC-1 levels were detected in primary murine megakaryocytes undergoing polyploidization compared with non endomitotic precursors. Stathmin is a microtubule-depolymerizing factor that plays an important role in the regulation of the mitotic spindle. Levels of stathmin are inversely related to the level of ploidy of megakaryocytic cell lines and primary megakaryocytes. Inhibition of stathmin in K562 cells increases their propensity to undergo endomitosis when induced to differentiate into megakaryocytes, and overexpression of stathmin prevents the transition from mitotic to endomitotic cell cycles. Conversely, lentiviral-mediated overexpression of stathmin in primary megakaryocytes delayed cytoplasmic maturation and impaired their ability to achieve high levels of ploidy. Together, these findings support a possible role of stathmin in modulating endomitosis.

The Spindle Checkpoint. During mitosis of normal diploid cells, a spindle assembly checkpoint prevents the progression of anaphase until all of the chromosomes are aligned with the mitotic spindle and each sister chromatid is properly attached to spindle microtubules originating from the opposing spindle pole. This ensures that each daughter cell receives the proper complement of chromosomes. The anaphase-promoting complex (APC) is a multisubunit protein complex with ubiquitin ligase activity that regulates chromosome segregation and anaphase progression by targeting key factors for degradation. Since some chromosomal missegregation occurs during megakaryocyte endomitosis, several groups have examined the expression levels and/or activity of certain APC components and associated factors. These studies have shown no significant difference in protein levels of the core APC protein CDC27 or the kinetochore-associated signaling protein hsMAD2 in primary murine megakaryocytes undergoing polyploidization compared with nonendomitotic precursors. Haploinsufficiency of BUBR1, a key component of the spindle checkpoint, perturbs megakaryocyte development and polyploidization in mice but does not cause alterations in circulating platelet counts.

Contractile Ring Activity

Cytokinesis requires the assembly and activity of an actin-based con tractile ring which enables cell separation. This process is regulated by RhoA involving actin polymerization, myosin II accumulation, and activation which provide the contractile forces for abscission. The failure to complete cytokinesis during endomitotic cell cycles in megakaryocytes involves functional and quantitative defects in the RhoA, Rock, F actin, and myosin II. Silencing of nonmuscle myosin heavy chain IIB (Myh10) by the transcription factor RUNX1 is also required for efficient megakaryocyte polyploidization. Further studies will be required to fully dissect the molecular pathways involved in megakaryocyte endomitosis.

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مواضيع ذات صلة

Cellular and Molecular Mechanisms in Development: Morphogens and Cell-to-Cell Signaling

Thrombopoietin Signaling

Megakaryocytopoiesis

Ontogeny of Erythropoiesis

Cellular Dynamics in Erythropoiesis: Steady State and Stress Erythropoiesis

Erythropoiesis and Iron Regulation

Hematopoietic Microenvironment

Intrinsic Control of Erythropoiesis:Erythropoietin

Nuclear Organization

Unfolded Protein Response

Autophagy

Nonapoptotic forms of cell death