Efficient TGF-β Induction of the Smad7 Gene Requires Cooperation between AP-1, Sp1, and Smad Proteins on the Mouse Smad7 Promoter

Abstract

Sma-and Mad-related protein 7 (Smad7) is an antagonist of transforming growth factor-␤ (TGF-␤) signaling, which has been shown to be induced by TGF-␤ itself and also by other stimuli. In an effort to understand the molecular mechanisms underlying the transcriptional regulation of the Smad7 gene by TGF-␤, we cloned and functionally characterized a mouse genomic DNA fragment encompassing the mouse Smad7 proximal promoter. This region was found to contain a CpG island and to be devoid of a classical TATA box. Cloned upstream of a promoter-lacking luciferase reporter gene, this region conferred robust TGF-␤-induced transcription. Point mutations in a palindromic Smad binding element, abolished TGF-␤ inducibility completely. Through the use of electrophoretic mobility shift assays, we showed the presence of Smad2, Smad3, and Smad4 in complexes binding to the Smad binding element. Interestingly, we also found that point mutation and/or deletion of binding sites for the transcription factors activator protein-1 and Sp1 led to an attenuation of the basal promoter activity, as well as of the TGF-␤-mediated induction of Smad7. Taken together, our data imply that Smads, together with activator protein-1 and Sp1 transcription factors, are essential for efficient Smad7 promoter activity.

FLRG, an activin-binding protein, is a new target of TGFβ transcription activation through Smad proteins

“…This deletion signi®cantly reduced the response to TGFb or Smad proteins of the FLRG promoter reporter construct (Figure 3b,c). As previously reported for Smad7 gene (Brodin et al, 2000), our results suggest that Sp1 transcription factor is essential for basal transcription and for the optimal inducibility by TGFb of the FLRG promoter.…”

“…Several reports on di erent promoters have identi®ed Sp1 sites as major TGFb-responsive promoter elements (Brodin et al, 2000;Feng et al, 2000;Pardali et al, 2000). To analyse whether the Sp1 DNA binding sequences found on the FLRG promoter are involved in response to TGFb, we generated a reporter construct where the Sp1 sites were deleted.…”

Regulation of cell proliferation by Smad proteins

“…Multimers of SBE when placed in front of a minimal promoter reporter construct provide a strong enhancer function for TGF-b family members. SBE-like sequences have been shown to be critically important for TGF-binducibility of multiple TGF-b responsive genes (Dennler et al, 1998;Vindevoghel et al, 1998;Hanai et al, 1999;Nagarajan et al, 1999;Brodin et al, 2000;Chen et al, 2000;Taylor and Khachigian, 2000). The crystal structure of Smad3 MH1 domain with SBE showed that the b-hairpin loop in Smad3 is in contact region with the SBE (Shi et al, 1998).…”

Transient activation of NF-κB through a TAK1/IKK kinase pathway by TGF-β1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation

“…Smads and AP-1 complex have been shown to interact and synergistically promote TGF-b1-mediated transcription and apoptosis (Zhang et al, 1998;Engel et al, 1999;Brodin et al, 2000). To determine whether inhibition of NF-kB activity would enhance TGF-b1-dependent transcription through activation of JNK, we measured the effect of ectopic expression of 2N/3CIkB-a on transcription of the TGF-b1-responsive reporter construct p3TP-Lux, which contains the Smad-regulated PAI-1 promoter element and three AP-1 binding sites.…”

“…The rcDNA/Myc-DFosB (Yamamura et al, 2000) have been described previously. The SflI-XhoI fragment of the upstream regulatory sequence of the murine Smad7 promoter was cloned into the pGL-3 basic vector (Brodin et al, 2000). The 4XTRE-luc construct contains four repeats of the TPAresponsive element (TRE) driving a luciferase reporter gene (Williams et al, 2001).…”