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Originally published In Press as doi:10.1074/jbc.M003282200 on July 7, 2000

J. Biol. Chem., Vol. 275, Issue 38, 29308-29317, September 22, 2000
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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*

Marcin StopaDagger §, Dirk AnhufDagger §, Lara Terstegen, Petros Gatsios, Axel M. GressnerDagger , and Steven DooleyDagger ||

From the Dagger  Institut für Klinische Chemie und Pathobiochemie and  Institut für Biochemie, RWTH-Universitätsklinikum, 52074 Aachen, Germany

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.

* This study was financially supported by Deutsche Forschungsgemeinschaft Grant Do373/4-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF156727 (rat), AF156731 (human), and AF188834 (mouse).

§ Both authors contributed equally to this work.

|| To whom correspondence should be addressed: Inst. für Klinische Chemie und Pathobiochemie, Universitätsklinikum RWTH-Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. Tel.: 49-241-80-88-673; Fax: 49-241-80-512; E-mail: steven.dooley@post.rwth-aachen.de.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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