Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.
Smad proteins play a key role in the intracellular signalling of transforming growth factor beta (TGF beta), which elicits a large variety of cellular responses. Upon TGF beta 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 TGF beta. 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 TGF beta 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 TGF beta responsiveness. Thus, CAGA elements are essential and sufficient for the induction by TGF beta. In addition, TGFbeta 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 TGF beta-responsive regions of several other genes suggests that this may be a widely used motif in TGF beta-regulated transcription.
Dennler
S
S
Laboratoire Glaxo Wellcome, 25 avenue de Québec, 91951 Les Ulis Cedex, France.
Itoh
S
S
Vivien
D
D
ten Dijke
P
P
Huet
S
S
Gauthier
J M
JM
D028341:Activins; D000818:Animals; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007265:Inhibins / Q000494:pharmacology; D008907:Mink; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D017395:Plasminogen Activator Inhibitor 1; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D028341:Activins; D007265:Inhibins