Effect of the deletion constructs on PMA-induced c-mpl promoter activity

Objectives: To further examine the roles of PKC in c-mpl gene expression, we examined the effect of phorbol 12-myristate 13-acetate(PMA) and the deletion constructs 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 on PMA-induced c-mpl promoter activity at the same time. To examine the effect of PMA and PKC inhibitors on c-mpl promoter activity, CMK was treated with these reagents and the inducibility of c-mpl promoter activities were measured. Results: PMA dramatically, increased the c-mpl promoter activity 24hr after pretreatment with PMA. On the other hand, PKC inhibitors(GF109303X, H7 and CalphostinC) reduced the basal level of the c-mpl promoter activity. In case of the deletion constructs, the activity was clearly reduced from pGL3-c-mpl (-83). Conclusions: These data strongly suggest that PKC plays the key role in maintaining c-mpl transcription in megakaryocytic cells and it was made clear that pGL3-c-mpl (-121) is the most suitable construct for the following our experiment. 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).