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

The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21(WAF1) (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells. Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter. This activation does not require 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. Because transforming growth factor-beta enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined. PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-beta-responsive promoter, p3TP-lux. Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus. However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region. Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA. These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter.