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 members of the TGF-beta superfamily. To define the molecular mechanisms of negative control of TGF-beta signaling, we have isolated the human SMAD7 gene and characterized its promoter region. A -303 to +672 SMAD7 region contained a palindromic GTCTAGAC Smad binding element (SBE) between nucleotides -179 and -172 that was necessary for the induction of a Smad7 promoter luciferase reporter gene by TGF-beta. Electrophoretic mobility shift assays using oligonucleotide probes demonstrated that TGF-beta rapidly induced the binding of an endogenous SBE-binding complex (SBC) containing Smad2, Smad3, and Smad4. Transfection assays in mouse embryonic fibroblasts (MEFs), with targeted deletions of either Smad2 or Smad3, and the Smad4-deficient cell line MD-MBA-468 revealed that both Smad3 and Smad4, but not Smad2, were absolutely required for induction of the Smad7 promoter reporter gene by TGF-beta. Furthermore, the TGF-beta-inducible SBE-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 demonstrate that TGF-beta induces transcription of the human SMAD7 gene through activation of Smad3 and Smad4 transcription factor binding to its proximal promoter.
von Gersdorff
G
G
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Susztak
K
K
Rezvani
F
F
Bitzer
M
M
Liang
D
D
Böttinger
E P
EP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D004268:DNA-Binding Proteins; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D004247:DNA