Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7 - The TGF-beta response element ofthe promotor requires functional Smad binding element and E-box sequences for transcriptional regulation

Smad7 has recently been identified as a player that antagonizes transformin g growth factor beta (TGF-beta) signals by acting downstream of TGF-beta re ceptors, TGF-beta rapidly induces expression of Smad7 mRNA in a variety of cell types, suggesting participation in a negative feedback loop to control TGF-beta beta responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain r eaction cloning of the corresponding promoter regions of human and murine S mad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-b eta response element within a strongly conserved region, containing a perfe ct Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobilit y shift assay and cotransfection experiments, we were able to delineate DNA -binding complexes and identified Smad3, Smad4, and Smad2. Mutation of the SBE completely abolished TGF-beta inducibility of Smad7 in HepG2 cells, ind icating that this sequence is necessary for TGF-beta-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TG F-beta-dependent promoter activation and an overlapping AP1 site is also in volved. We conclude that regulation of Smad7 transcription by TGF-beta is m ediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.