Abstract
Transforming growth factor beta (TGFbeta) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGFbeta-responsive region (-740 and -647) in the PAI-1 promoter. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGFbeta receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGFbeta responsive genes. Smad3 and Smad4 have been shown to bind to various sites in the PAI-1 promoter. To determine the number of Smad-binding sites within the 94-base pair major TGFbeta-responsive region and the mechanism of Smad-mediated transactivation, we systematically mapped the Smad-binding sites and show that Smad4 and Smad3 bind cooperatively to two adjacent DNA elements in this region. Both elements were required for TGFbeta-induced, Smad3- and Smad4-dependent activation of PAI-1 transcription. Contrary to previous reports, transactivation of the PAI-1 promoter was mediated by the amino- but not carboxyl-terminal domains of the Smads. Furthermore, oligomerization of Smad3 markedly enhanced its binding to the two binding sites. Finally, a Smad4 mutation identified in a human pancreatic carcinoma that inactivates Smad4 signaling abolished Smad4 DNA binding activity, hence preventing transactivation of TGFbeta-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis.
Highlights
Transforming growth factor  (TGF) activates transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene through a major TGF-responsive region (؊740 and ؊647) in the PAI-1 promoter
Smad3 and Smad4 Bind to Two Adjacent DNA Elements in the PAI-1 Promoter in a Cooperative Manner—Electrophoresis Mobility Shift Assays (EMSA) were used to determine whether Smad3 and Smad4 directly bind to the major TGF responsive region between Ϫ740 and Ϫ647 upstream of the initiation site in the PAI-1 promoter
The Two Smad-binding Sites in the PAI-1 Promoter Are Essential for TGF-induced, Smad3, and Smad4-dependent Transcriptional Activation—To determine whether the two Smad-binding sites we identified are necessary for TGF-induced activation of PAI-1 gene expression, luciferase reporter constructs that contained four copies of the 94-bp wild-type or mutant PAI-1 promoter sequences (Ϫ740/Ϫ647) inserted upstream of the TATA box in the SV40 early promoter were generated (Fig. 4A)
Summary
TGF, transforming growth factor ; PCR, polymerase chain reaction; bp, base pair; SBE, Smad-binding element; PAI-I, plasminogen activator inhibitor type-1; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay. By forming heteromeric complexes with various pathway restricted Smads, Smad participates in the activation of multiple signaling pathways initiated by TGF family members and may be directly involved in transcriptional activation of many downstream genes [14, 16] As such, it plays a key role in the control of cell growth and differentiation. The crystal structure of the MH1 domain of Smad bound to SBE determined a minimal Smad box (GTCT) required for binding to Smad3 [34], a high affinity interaction may require additional sequences These results suggest that direct DNA binding of Smad and Smad may play a role in transcriptional activation by TGF. We showed that oligomerization of the Smad proteins are not required for DNA binding
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