Cyclin D3 and megakaryocyte development: Exploration of a transgenic phenotype

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Abstract
The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity, p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.