Cooperative Binding of Smad Proteins to Two Adjacent DNA Elements in the Plasminogen Activator Inhibitor-1 Promoter Mediates Transforming Growth Factor β-induced Smad-dependent Transcriptional Activation

Abstract

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. This process requires the Smad family of signaling molecules. Upon phosphorylation by the TGF␤ receptors, Smad2 and Smad3 homoligomerize and heteroligomerize with Smad4, translocate to the nucleus and activate transcription of TGF␤ 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 TGF␤-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 TGF␤-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 TGF␤-responsive genes. These results underscore the importance of the Smad4 DNA binding activity in controlling cell growth and carcinogenesis. Transforming growth factor-␤ (TGF␤)1 is a multipotent cytokine that elicits many biological functions including inhibition of the growth of cells of epithelial, endothelial, and lymphoid origins, production of extracellular matrix components, and regulation of differentiation of many cell types (1). These activities are mediated by the cell surface types I and II TGF␤ receptors, T␤RI and T␤RII, which are receptor serine/threonine kinases (2-4). In the absence of ligand, while the T␤RI kinase is inactive, the T␤RII kinase is constitutively active and the receptor is autophosphorylated (2, 5). Binding of TGF␤1 to T␤RII results in the formation of a heteromeric complex containing T␤RI and T␤RII, followed by transphosphorylation of T␤RI by the T␤RII kinase (6). Phosphorylation of T␤RI by T␤RII is thought to activate the T␤RI kinase activity, allowing it to phosphorylate and activate downstream Smad2 (7-10) and Smad3 proteins (11, 12).The Smad family proteins are critical components of the TGF␤ signaling pathway. Depending on their mechanisms of action, the Smads can be divided into three classes: pathwayrestricted Smads, common-mediator Smads, and inhibitory Smads (13). All Smad proteins share considerable homology in their primary sequence and most contain two highly conserved Mad homology domains: MH1 in the amino-terminal half and MH2 in the carboxyl-terminal half separated by a diverse proline-rich linker. Upon stimulation by TGF␤1, the pathway restricted Smads, Smad2, and Smad3, interact with the TGF␤ receptor complex, and become phosphorylated on three ser...

Mutations of the Smad4 gene in acute myelogeneous leukemia and their functional implications in leukemogenesis

“…The probe was incubated with the isolated Smad4-Flag and then the complexes were electrophoresed. We observed a signi®cantly shifted band ( This band was not detected when we used a mutant probe in which SBE was changed to a sequence di erent from the consensus sequence (Stroschein et al, 1999) (data not shown). On the other hand, no band was detected when the probe was incubated and electrophoresed with the Flag-tagged form of P102L ( Figure 6, lane 4), indicating that P102L has lost the DNA-binding ability.…”

“…These results suggest that concomitant expression of P102L with wild-type Smad4 inhibits the DNA-binding ability of wild-type Smad4 to SBE. Because Smad4-induced transcription from the PAI-1 promoter is dependent on binding to SBE (Stroschein et al, 1999), these ®ndings account for the mechanism whereby the P102L mutant acts as a dominant negative inhibitor of wild-type Smad4 in the transcriptional response assays (Figure 3a).…”

“…Recently, it was shown that Smad4 binds to the region between 7688 and 7660 upstream of the initiation site in the PAI-1 promoter containing SBE with a single A to G change at position 5 (SBE, GTCTAGAC; PAI-1 7684/7677, GTCTGGAC) (Stroschein et al, 1999). Using a double-stranded oligonucleotide containing this region as a probe (Stroschein et al, 1999), we investigated the DNA-binding ability of Smad4P102L by the electrophoretic mobility shift assay (EMSA). We expressed Smad4-Flag in 293 cells and isolated Smad4-Flag by immunoprecipitation from the cell lysates of 293 cells using the anti-Flag antibody.…”

Inhibition of the TGF-Beta Tumor Suppression Pathway Through Repression of Smad Dependent Transcription

“…Flag-and/or HA-tagged Smads and SnoN were isolated from transfected 293T cells by immunoprecipitation with anti-Flag (Sigma) or anti-HA agarose, followed by elution with Flag or HA peptide as described previously (Zhou et al 1998a;Stroschein et al 1999a). To detect endogenous APC associated with the Smads, nuclear extracts were prepared from 293T cells transfected with Flag-tagged Smad as described previously (Lee et al 1987), and the Flag-tagged protein was isolated by immunoprecipitation with anti-Flag agarose (Sigma), followed by elution with the Flag peptide as described previously (Zhou et al 1998a;Stroschein et al 1999a).…”

“…To detect endogenous APC associated with the Smads, nuclear extracts were prepared from 293T cells transfected with Flag-tagged Smad as described previously (Lee et al 1987), and the Flag-tagged protein was isolated by immunoprecipitation with anti-Flag agarose (Sigma), followed by elution with the Flag peptide as described previously (Zhou et al 1998a;Stroschein et al 1999a). Associated Cdc27 and Cdcl6 were detected by Western blotting with anti-Cdc27 and anti-Cdcl6.…”