Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta

Smad7 is an inducible intracellular inhibitor of transforming growth factor -beta (TGF-beta) signaling that is regulated by diverse stimuli including m embers of the TGF-beta superfamily, To define the molecular mechanisms of n egative control of TGF-beta signaling, we have isolated the human SMAD7 gen e and characterized its promoter region. A -303 to +672 SMAD7 region contai ned a palindromic GTCTAGAC Smad binding element (SEE) between nucleotides - 179 and -172 that was necessary for the induction of a Smad7 promoter lucif erase reporter gene by TGF-beta, Electrophoretic mobility shift assays usin g oligonucleotide probes demonstrated that TGF-beta rapidly induced the bin ding of an endogenous SBE-binding complex (SBC) containing Smad2, Smad3, an d Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with ta rgeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell lin e MD-MBA-468 revealed that both Smad3 and Smad4, but not Smad2, were absolu tely required for induction of the Smad7 promoter reporter gene by TGF-beta . Furthermore, the TGF-beta-inducible SEE-binding complex was diminished in Smad2-deficient MEFs when compared with wild type MEFs and not detectable in Smad3-deficient MEFs and MD-MBA-468 cells. Taken together, our data demo nstrate that TGF-beta induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter.