Nuclear Factor of Activated T Cells Regulates Down Syndrome Critical Region 1 Gene Expression in Megakaryocytes.

The calcineurin-dependent NFAT (Nuclear Factors of Activated T cells) transcription factors were initially characterized as central mediators of inducible gene expression in activated T cells, but recently have also been implicated in regulating differentiation and function of a number of cell types outside the immune system. We have previously found that one member of the NFAT family, NFATc2, is strongly expressed in bone marrow megakaryocytes. The function of NFAT in this cell type, however, is unclear. The Down Syndrome Critical Region 1 (DSCR1) gene, located within the Down syndrome critical region of human chromosome 21, is overexpressed about 1,5-fold in patients with Down syndrome (DS) and has been implicated in the pathology of the disease. DSCR1 is a member of the calcipressin family of calcineurin inhibitors, and thereby serves as an endogenous suppressor of NFAT signalling. Furthermore, the expression of a specific isoform of the DSCR1 gene (exons 4–7) is thought to be regulated by NFAT, thus creating a potential regulatory feedback mechanism. Given the strong expression of NFATc2 in megakaryocytes, the complex interaction between DSCR1 and NFAT proteins, and the fact that DS children have a ~500-fold increased incidence of acute megakaryoblastic leukemia, we set out to analyze the expression and regulation of NFAT and DSCR1 in megakaryopoiesis. Pure populations of culture-derived (CD) megakaryocytes were obtained by culturing leukapheresis CD34⁺ cells in the presence of thrombopoietin. Using the calcium ionophor ionomycin and the calcineurin inhibitor cyclosporin A (CsA) in CD megakaryocytes as well as in various megakaryocytic cell lines, NFAT activation was found to be regulated in the same calcineurin-dependent and CsA-sensitive manner as is known from T lymphocytes. During the differentiation of CD34⁺ cells into megakaryocytes, the expression of NFATc2 protein was maintained at a high level, and progressive dephosphorylation of the protein in the course of the culture indicated activation of NFATc2 during the differentiation process. DSCR1 mRNA expression in CD34⁺ cells was low, but markedly upregulated during megakaryopoiesis. This upregulation was inhibited when differentiation was performed in the presence of CsA, an inhibitor of NFAT activation. In mature CD megakaryocytes as well as in all megakaryocytic cell lines tested, the expression of DSCR1 was further inducable by stimulating the cells with ionomycin and inhibited by treatment with CsA. To test the involvement of NFAT in the transcriptional regulation of the DSCR1 gene in megakaryocytes, CMK cell lines stably overexpressing either NFATc2 or the specific peptide inhibitor of NFAT activation, VIVIT, were generated by retroviral transduction. Overexpression of NFATc2 potently augmented, while VIVIT suppressed DSCR1 promoter transcriptional activity, confirming transcriptional regulation of DSCR1 expression by NFAT. These results establish DSCR1 as a transcriptional target gene for NFAT in megakaryocytes. The upregulation of DSCR1 expression during megakaryopoiesis suggests a possible implication of DSCR1 in megakaryocytic differentiation and should encourage further investigation into the reciprocal roles of DSCR1 and NFAT in the development of megakaryoblastic leukemia in DS.