Human Smad3 and Smad4 Are Sequence-Specific Transcription Activators

Abstract

Mounting evidence indicates that Smad proteins are required for TGF beta signaling, but the way(s) in which Smad proteins propagate this signal is unclear. We found that two human Smad proteins (Smad3 and Smad4) could specifically recognize an identical 8 bp palindromic sequences (GTCTAGAC). Tandem repeats of this palindrome conferred striking TGF beta responsiveness to a minimal promoter. This responsiveness was abrogated by targeted deletion of the cellular Smad4 gene. These results define a novel biochemical property of Smad proteins that is likely to play a direct role in the biologic responses to TGF beta and related ligands.

Aryl hydrocarbon receptor inhibition downregulates the TGF-β/Smad pathway in human glioblastoma cells

“…The ARNT-CMV4 construct (Mason et al, 1994) was a kind gift of L Poellinger (Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden). Transcriptional activity mediated by TGF-b1 and TGF-b2 signaling were assessed using the GL3-SBE-4 Luc plasmid, containing four copies of an 8 bp palindrome Smad binding element discovered through an unbiased approach (Zawel et al, 1998), a reporter plasmid kindly provided by B Vogelstein (Baltimore, MD, USA).…”

SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

“…HepG2 cells were transfected with either the Smad/TGF-b-responsive SBE4-luc reporter construct, which contains four tandem repeats of SBE (Smad-binding element) (Zawel et al, 1998), or the 3TP-lux reporter construct. After transfection, cells were subjected to serum-starved conditions for 24 h and then incubated with 20% serum in the presence or absence of TGF-b1.…”

“…Briefly, HepG2 cells were transiently transfected with SBE4-luc (Zawel et al, 1998), 3TP-Lux (Carcamo et al, 1995) and the internal control pCMV-b-gal in six-well plates using Lipofectin (Invitrogen Corp., Rockville, MD, USA) according to the manufacturer's instructions. After a 24 h transfection, cells were treated with 5 ng/ml TGF-b1 for 24 h in media.…”

A short amino-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3

“…The N-terminus MH1 domain of Drosophila Mad (Kim et al, 1997), Smad3 and Smad4 (Yingling et al, 1997;Dennler et al, 1998;Zawel et al, 1998) binds DNA and shows no homology with other known DNA binding domains. The C-terminus MH2 domain interacts with the receptor and displays transcriptional activity when fused to a heterologous Gal4 DNA-binding domain (Liu et al, 1996).…”