PMA-induced c-mpl gene expression in CMK cells

Objectives: To further investgate the effect of phorbol 12-myristate 13-acetate(PMA) on c-mpl promoter activity, we examined the deletion constructs and site-directed mutagenesis on c-mpl promoter activity Methods: We examined the effect of phorbol 12-myristate 13-acetate(PMA) on c-mpl promoter activity by using transient transfection assay system. Besides pGL3-c-mpl(-310), we also examined the effect of the deletion constructs and site-directed mutagenesis on PMA-induced c-mpl promoter activity. Results: PMA dramatically, increased the c-mpl promoter activity, on the other hand, PKC inhibitors(GF109303X, H7 and CalphostinC) significantly inhibited the PMA-induced increase of c-mpl promoter activity. In case of the deletion constructs, the activity was clearly reduced from pGL3-c-mpl (-83). As to site-directed mutagenesis, we confirmed the positive regulatory elements involved in c-mpl gene expression induced by PMA. Conclusions: These data strongly suggest that PKC plays the key role in maintaining c-mpl transcription in megakaryocytic cells. It was made clear that pGL3-c-mpl (-121) is the most suitable construct for our experiment and also we elucidated the positive regulatory elements involved in PMA-induced c-mpl gene expression in CMK cells. *This study was supported by the Grant-in-Aid for Encouragement of Young Scientists (No.13771085) from Japan Society for the Promotion of Science(JSPS).