Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.     

Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity

Transforming growth factor-beta1 (TGF-beta1) mediates expression of collagen 1A2 (Col 1A2) gene via a synergistic cooperation between Smad2/Smad3 and Sp1, both act on the Col 1A2 gene promoter. In our previous study, we reported that a retinoic acid derivative obtained from Phellinus linteus (designated PL) antagonizes TGF-beta-induced liver fibrosis through regulation of ROS and calcium influx. In this continuing study we seek further the effect of PL on the Smad signaling pathway. We used a Col 1A2 promoter-luciferase construct to study the action of PL on Smad through TGF-beta. We found that PL decreases the promoter activity of Col 1A2, hinders the translocalization of phosphorylated Smad2/3-Smad 4 complex from cytosol into nucleus and inhibits Sp1 binding activity. These results suggest that PL inhibits TGF-beta1-induced Col 1A2 promoter activity through blocking ROS and calcium influx as well as impeding Sp1 binding and translocalization of pSmad 2/3-Smad4 complex into nucleus.