A New Role for Flavoproteins in the Control of Megakaryocyte Polyploidy.

Megakaryocytes (MKs) can reach a ploidy level of greater than 32N (with 2N representing diploid chromosomes), while polyploidization is important for generating an adequate number of functional platelets. The MK population in the marrow intriguingly consists of a large fraction of 8N and 16N cells that are cell cycle arrested. Here, we examined the expression of different NADPH (NOX) oxidases in MKs, using Western blotting and immunohistochemistry, because of their known role in upregulating regulators of the G1 phase of the cell cycle and in promoting cell cycle progression. It was found that in thrombopoietin-treated mouse bone marrow cultures, NOX1 is highly expressed in many, but not all MKs, while NOX4 is expressed at lower levels than NOX1. Treatment of mouse bone marrow or fetal liver cultures with a NOX inhibitor, diphenylene iodonium (DPI) lead to accumulation of viable MKs at diploid and tetraploid levels, and to a significant reduction in the fraction of high ploidy MKs, as measured by Flow cytometry analysis with a MK specific marker as well as by immunostaining. MKs isolated by an albumin gradient were subjected to Western blotting, which indicated a decrease in the level of the G1 phase cyclin, cyclin E, in DPI-treated cells. Taken together, this study shows for the first time the expression of NOX isoforms in MKs and highlights the important role of flavoproteins in promoting polyploidy in this lineage. It is suggested that the population of cell cycle-arrested intermediate-ploidy MKs is prevented from reaching higher ploidy, partly because of diminished levels of G1 phase regulators via NOX downregulation.