The role of the Smad3 protein in phorbol ester-induced promoter expression

The Spl transcription factor plays an important role in mediating the p53-i ndependent activation of the p21(WAF1) (WAF1) promoter by phorbol 12-myrist ate13-acetate (PMA) in hematopoietic cells. Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional act ivity of Sp1 independent of the WAF1 promoter. This activation does not req uire the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids ( 152-193) of Sp1 that are important for the interaction with human TAF130, B ecause transforming growth factor-beta enhances WAF1 promoter activity thro ugh both Sp1 and Smad proteins, the role of Smads in PMA transcriptional ac tivation was examined. PMA addition to hematopoietic cells was found to act ivate a GAL4/Smad-dependent promoter and the transforming growth factor-bet a-responsive promoter, p3TP-lux. Immunofluorescence data demonstrate that P MA addition to hematopoietic cells induces the translocation of Smads to th e nucleus. However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this ups tream region. Additionally, transfection of Smad3 did not enhance the activ ation of GAL4/Sp1 by PMA. These results demonstrate that, while PMA can act ivate Smad-mediated transcription Smad proteins do not appear to play a maj or role in the PMA induction of the WAF1 promoter.