title: Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. THE TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.
abstract: Smad7 has recently been identified as a player that antagonizes transforming growth factor beta (TGF-beta) signals by acting downstream of TGF-beta receptors. 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 responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain reaction cloning of the corresponding promoter regions of human and murine Smad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-beta response element within a strongly conserved region, containing a perfect Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobility 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, indicating that this sequence is necessary for TGF-beta-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TGF-beta-dependent promoter activation and an overlapping AP1 site is also involved. We conclude that regulation of Smad7 transcription by TGF-beta is mediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, RWTH-Universitätsklinikum, 52074 Aachen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Stopa
authors.forename: D
authors.initials: D
authors.lastname: Anhuf
authors.forename: L
authors.initials: L
authors.lastname: Terstegen
authors.forename: P
authors.initials: P
authors.lastname: Gatsios
authors.forename: A M
authors.initials: AM
authors.lastname: Gressner
authors.forename: S
authors.initials: S
authors.lastname: Dooley
mesh_terms: D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; C497784:Smad2 protein, rat; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D051906:Smad7 Protein; C497808:Smad7 protein, rat; D015534:Trans-Activators