Mpl ligand enhances the transcription of the cyclin D3 gene: A potential role for Sp1 transcription factor

Cyclin D3 plays a major role in the development of polyploidy in megakaryoc ytes. The expression of cyclin D3 gene and the level of cyclin D3 protein a re increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, a s indicated by Northern and Western blot analyses, and by nuclear run-on as says and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at -75 to -60 bp and at -134 to -92 bp, which display binding sites for the Sp famil y of transcription factors. Gel mobility shift assay and supershifts with s pecific antibodies indicate that Sp1 binds to these regions in the cyclin D 3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activ ity and eliminates the induction by Mpl ligand. We find that the nonphospho rylated form of SP1 has greater affinity for the cyclin D3 promoter and tha t the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatme nt results in increased levels of Sp1 protein, which also appears as nonpho sphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and s hifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl li gand to activate at once several Sp1-dependent genes during megakaryopoiesi s. (C) 1999 by The American Society of Hematology.