Direct binding of Smad3 and Smad4 to critical TGFbeta -inducible elements in the promoter of human plasminogen activator inhibitor-type 1gene

Abstract

Smad proteins play a key role in the intracellular signalling of transforming growth factor β (TGFβ), which elicits a large variety of cellular responses. Upon TGFβ receptor activation, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. These complexes translocate to the nucleus where they control expression of target genes. However, the mechanism by which Smads mediate transcriptional regulation is largely unknown. Human plasminogen activator inhibitor-1 (PAI-1) is a gene that is potently induced by TGFβ. Here we report the identification of Smad3/Smad4 binding sequences, termed CAGA boxes, within the promoter of the human PAI-1 gene. The CAGA boxes confer TGFβ and activin, but not bone morphogenetic protein (BMP) stimulation to a heterologous promoter reporter construct. Importantly, mutation of the three CAGA boxes present in the PAI-1 promoter was found to abolish TGFβ responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGFβ. In addition, TGFβ induces the binding of a Smad3/Smad4-containing nuclear complex to CAGA boxes. Furthermore, bacterially expressed Smad3 and Smad4 proteins, but not Smad1 nor Smad2 protein, bind directly to this sequence in vitro. The presence of this box in TGFβ-responsive regions of several other genes suggests that this may be a widely used motif in TGFβ-regulated transcription.

ELAC2, a putative prostate cancer susceptibility gene product, potentiates TGF-β/Smad-induced growth arrest of prostate cells

“…We also confirmed that ELAC2 dosedependently potentiated TGF-b-induced (ARE) 2 -luc activity ( Figure 1c) although the effect of ELAC2 on TGF-b-induced (ARE) 2 -luc activity was lower than that on ALK5ca-induced (ARE) 2 -luc activity. (CAGA) 12 -luc is known to be activated by TGF-b-induced complex between Smad3 and Smad4 (Dennler et al, 1998). When we explored if ELAC2 can activate the TGF-b/Smad3-dependent promoter, ELAC2 could also potentiate the activity of (CAGA) 12 -luc upon ALK5 activation in dose-dependent manner ( Figure 1d).…”

“…Flag-Smad2 mutants were generously gifted by Dr T Imamura (Cancer Institute, Japan). The constructs for pcDEF3-Flag-Smad2, pcDEF3-myc-Smad2, pcDNA3-6xMyc-Smad2, pcDEF3-Flag-Smad3, pcDEF3-Flag-Smad4, 2xARE-luc, (CAGA) 12 -luc and GST-Smad2 have been described previously (Dennler et al, 1998;Kawabata et al, 1998;Itoh et al, 2000Itoh et al, , 2003. pGEX-4T-1 (GST alone) and CH110 were purchased from Amersham, Buckinghamshire, England.…”

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

“…After phosphorylation by the receptor, Smads form heteromeric complexes with Smad4, a constitutively phosphorylated common mediator of the two pathways. The Smad complexes then translocate to the nucleus where they control expression of diverse genes (Derynck and Feng, 1997;Dennler et al, 1998;Jonk et al, 1998). Smad4 is the unique member of the second class of Smad proteins: central ones.…”

“…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).…”

“…Smad2 and Smad3, the two TGFb pathwayrestricted members of the family, share an overall 92% identity at the amino-acid sequence level. We have previously described an eight nucleotide sequence called the CAGA box found in several promoters and responsible for TGFb responsiveness (Dennler et al, 1998;Jonk et al, 1998). Remarkably, the TGFbinduced nuclear protein complex that binds to this sequence contains Smad3 and Smad4 but not Smad2 (Dennler et al, 1998).…”