JBJS | Transcriptional Regulation by Smads: Crosstalk between the TGF-β and Wnt Pathways

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 1

Scientific Article | March 01, 2001

Transcriptional Regulation by Smads: Crosstalk between the TGF-β and Wnt Pathways

Ainhoa Letamendia, PhD; Etienne Labbé, PhD; Liliana Attisano, PhD

Department of Anatomy and Cell Biology and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
Ainhoa Letamendia, PhD Liliana Attisano, PhD Department of Anatomy and Cell Biology Etienne Labbé, PhD Department of Medical Biophysics Medical Sciences Building, Room 6336, 1 King's College Circle, University of Toronto, Toronto, ON M5S 1A8, Canada. E-mail address for L. Attisano: liliana.attisano@utoronto.ca
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Canadian Institute for Health Research and National Cancer Institute of Canada. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:S31-S39

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Abstract

Background: Several studies have shown that cooperation between transforming growth factor beta (TGF-ß) and Wnt/wingless signaling pathways plays a role in controlling certain developmental events. These factors elicit their biological effects through distinct pathways in which TGF-ß and Wnt signaling induce activation of the transcriptional regulators Smads and lymphoid enhancer binding factor/T-cell-specific factor (LEF/TCF), respectively. To understand the mechanism for cooperativity between these pathways, we have investigated the molecular mechanism for this synergistic effect.

Methods: Transcriptional assays were conducted by transient transfection of HepG2 cells with use of luciferase reporter constructs. Protein/protein interaction studies were conducted in vitro with the use of glutathione-S-transferase pull-down assays and in intact cells by immunoprecipitation and immunoblotting.

Results: We show that Smads physically interact with LEF1/TCF transcription factors and that specific DNA binding sites in the Xenopus twin promoter are required for synergistic activation by TGF-ß and Wnt pathways. In addition, we demonstrate that TGF-ß-dependent activation of LEF1/TCF target genes can occur independently of ß-catenin, an essential component of the Wnt signaling pathway.

Conclusions: TGF-ß and Wnt signaling pathways can independently or cooperatively regulate LEF1/TCF target genes. This suggests that the cooperation between these pathways may be important for the specification of cell fates during development.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

The members of the transforming growth factor beta (TGF-ß) superfamily elicit their biological effects through heteromeric complexes of type I and type II transmembrane serine/threonine kinase receptors ^{1,9,13,24,36,37,40} . A heteromeric receptor complex is formed when ligand binds to receptor II and receptor I is recruited into the complex. Receptor II then phosphorylates receptor I on serine and threonine residues in the highly conserved juxtamembrane "GS domain." Once phosphorylated, receptor I is activated to signal to downstream targets, members of the Smad family of signaling transducers ( Fig. 1 ).

The Smad family of signal transduction molecules is composed of intracellular proteins that are critical for transmitting TGF- b signals from the cell surface into the nucleus1^{,9,13,24,36,37,40} . Numerous studies in Drosophila, Xenopus, C. elegans , and mammals have defined three major classes of Smads. The receptor-regulated Smads (R-Smads) are direct targets of the type I receptor and are phosphorylated on the last two serines at the carboxy-terminus at a conserved SSXS motif. The R-Smads function in specific pathways such that Smad1, 5, and 8 are all targeted by the bone morphogenetic protein (BMP) receptors, whereas Smad2 and 3 are targeted by TGF-ß and activin receptors. Interestingly, a novel FYVE domain protein, called SARA (Smad Anchor for Receptor Activation) appears to play an important role in recruiting Smad2 to the TGF-ß receptor complex, and it is postulated that a related protein may serve a similar function in the BMP pathway ³¹ . The second class of Smad proteins, the common mediator Smads, comprise Smad4 and in Xenopus also include Smad4b. Phosphorylation of R-Smads induces heteromeric complexes with Smad4, and this R-Smad/Smad4 complex then translocates to the nucleus to target specific gene responses. In addition to these positively acting Smads, the inhibitory Smads, represented by Smad6 and Smad7, block TGF-ß superfamily signals.

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Fig. 1:: The transforming growth factor beta (TGF-ß) and Wnt signaling pathways. TGF-ß (left) and Wnt (right) activate distinct signaling pathways (see text for details). Here, we investigated whether the two pathways cooperate through the association of Smads with lymphoid enhancer binding factor/T-cell-specific factor (LEF1/TCF) transcription factors.

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Fig. 2-A:: Fig. 2 Transforming growth factor beta (TGF-ß) and Wnt signaling pathways synergistically activate Xtwn transcription. A : TGF-ß and lymphoid enhancer binding factor (LEF1)-dependent activation of Xtwn-Lux in HepG2 cells. Cells were transiently transfected with Xtwn-Lux reporter alone or together with various combinations of LEF1 or Smads. The cells were incubated overnight with or without TGF-ß (left) or were cotransfected with the constitutively active bone morphogenetic protein (BMP) type I receptor, ALK6 (right). Luciferase activity was normalized to ß-galactosidase activity and is expressed as the mean SD.

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Fig. 2-B:: Fig. 2 Transforming growth factor beta (TGF-ß) and Wnt signaling pathways synergistically activate Xtwn transcription. B : The Smad binding elements (SBE) and the LEF1/T-cell-specific factor (TCF) binding sites are required for synergistic activation of Xtwn by TGF-ß and Wnt signaling pathways. A schematic representation of the Xtwn promoter, the deletion constructs (left), and a summary of results obtained with these promoters are shown (right). The location of the SBEs and the triple LEF1/TCF binding sites (LEF) are indicated. Smad3 and LEF1-DNA binding was assessed by gel shift assays using bacterially expressed GST-LEF1 or GST-Smad3 MH1 domain. TGF-ß and LEF-dependent signaling was determined by transfection of HepG2 cells with LEF1 and the indicated Xtwn-Lux reporter constructs. Figure modified from Labbé et al. ¹⁹ with permission from National Academy of Sciences, U.S.A. Copyright (2000).

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Fig. 3-A:: Fig. 3 Lymphoid enhancer binding factor/T-cell-specific factor (LEF1/TCF) transcription factors associate with Smads. A : Interaction of LEF1 with Smad2 and Smad3 in mammalian cells. COS-l cells were transiently transfected with Flag-tagged Smad2 or Smad3 and HA-tagged LEF1 in the presence of a constitutively activated activin type I receptor, ActRIB. Cell lysates were subjected to anti-Flag antibody immunoprecipitation (IP) and were analyzed by immunoblotting with anti-HA antibodies. Protein levels were determined by immunoblotting of total cell lysates with use of antiFlag or anti-HA antibodies.

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Fig. 3-B:: Fig. 3 Lymphoid enhancer binding factor/T-cell-specific factor (LEF1/TCF) transcription factors associate with Smads. B : Interaction of LEF1 with bacterially expressed full-length MH1, MH2, or non-conserved domains of Smad3. A schematic representation of Smad3 and locations of the MH1, MH2, and non-conserved (NC) domains are shown. A summary of the association of LEF1 with Smads is shown at the right.

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Fig. 3-C:: Fig. 3 Lymphoid enhancer binding factor/T-cell-specific factor (LEF1/TCF) transcription factors associate with Smads. C : Determination of the domains in LEF1 that mediate association with Smad3. A schematic representation of mutant versions of LEF1 is shown. The ß-catenin binding domain (ß-cat BD) and the HMG box are marked. The location of the three helices within the HMG box (overline) and the MH1 and MH2 domain binding domains (BD; underline) are indicated. A summary of the interaction of LEF1 expressed in mammalian cells with bacterially expressed full-length (FL) MH1 and MH2 domains of Smad3 is shown (right). Figure modified from Labbé et al. ¹⁸ with permission from National Academy of Sciences, U.S.A. Copyright (2000).

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Fig. 4-A:: Fig. 4 Introduction of Smad binding elements into Topflash confers transforming growth factor beta (TGF-ß)-responsiveness to the promoter. A : HepG2 cells were transiently transfected with Xtwn-Lux, Topflash, or Twntop reporters alone or together with various combinations of lymphoid enhancer binding factor (LEF1), Smad3, and Smad4 as in Fig. 2-A .

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Fig. 4-B:: Fig. 4 Introduction of Smad binding elements into Topflash confers transforming growth factor beta (TGF-ß)-responsiveness to the promoter. B : Smad enhances LEF1-dependent signaling in the absence of ß-catenin. HepG2 cells were transiently transfected with Xtwn-Lux reporter alone or together with various combinations of Smad3 and Smad4, wild type LEF1, or LEF1 lacking the ß-catenin binding domain, LEF1 D20. Figure modified from Labbé et al. ¹⁹ with permission from National Academy of Sciences, U.S.A. Copyright (2000).

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Fig. 5:: A model for activation of specific target genes by transforming growth factor beta (TGF-ß) and Wnt pathways. In the presence of TGF-ß signaling alone, promoters with Smad binding elements (SBE) adjacent to the lymphoid enhancer binding factor/T-cell-specific factor (LEF1/TCF) binding sites can be activated by the TGF-ß pathway in the absence of ß-catenin (top panel). TGF-ß-dependent activation requires interaction of Smads with the HMG box of LEF1. In the presence of Wnt signaling alone, promoters with LEF1/TCF binding sites are activated by association of LEF1/TCF with ß-catenin (middle panel). In promoters containing SBEs and LEF1/TCF binding sites, TGF-ß and Wnt signals can cooperate to enhance transcriptional activation of LEF1 target genes (bottom panel). Figure modified from Labbé et al. ¹⁹ with permission from National Academy of Sciences, U.S.A. Copyright (2000).

Nuclear Functions of Smads

Once in the nucleus, Smads function to target specific gene promoters. Smads can directly bind DNA; however, they rely on interactions with DNA binding partners to target specific genes ^1,9,36,37 . The first Smad nuclear partner to be identified was Xenopus FAST ( forkhead activin signal transducer ), a forkhead DNA binding protein ⁴ . Related proteins in the mouse and human have also been described ^18,21,32,39 . FAST proteins (now known as FoxH) bind to specific elements in the promoters of genes including Xenopus Mix.2 and mouse goosecoid and nodal-related genes but cannot activate transcription ^4,18 . However, stimulation of activin or TGF-ß pathways results in the nuclear translocation of heteromeric complexes of Smad2-Smad4, which bind to FAST and activate transcription. Several Smad2 and Smad3 DNA binding partners that function in TGF-ß signaling have now been identified and include fos/jun, ATF2, TFE3, Runx, and Vitamin D receptor ^1,9,36,37 . Once recruited to specific elements, Smads can activate transcription by recruiting coactivators, such as CBP/p300 or MSG1, or corepressors such as TGIF or Ski family members. In addition to these more extensive studies on TGF-ß/activin targets, a number of Smad1 nuclear partners that function in the BMP pathway have been described. Analysis of a BMP-responsive element in the Xenopus Vent2 promoter led to the identification of OAZ (Olf-1/EBF associated zinc finger) as a DNA binding factor that can mediate BMP-dependent activation of Xvent2 ¹² . Smad1 has also been shown to interact with the runt-related gene 2 (Runx2). Cleidocranial dysplasia (CCD), a human bone disease, is thought to be caused by mutations in the runt-related gene 2, and one of these mutations prevents Smad1 interaction with CCD, thereby implicating the BMP pathway in this disease ³⁸ . Interestingly, Hoxc-8, a homeodomain-containing transcription protein can regulate BMP-dependent activation of the osteopontin promoter in what appears to be a unique mechanism for Smads. Hoxc-8 DNA binding represses the osteopontin promoter and activation of BMP signaling results in Smad1 binding to Hoxc-8, which dislodges the inhibitory Hoxc-8 from the promoter, thereby activating osteopontin gene expression ³⁰ . Smad1 can also cooperate with STAT3 to promote neural progenitors to differentiate into astrocytes. However, in this case the interaction between Smads and the DNA-binding partner is indirect and is bridged by the association of both proteins with p300 ²⁵ .

TGF-ß and Wingless Signaling in Early Development

TGF-ß-like factors are important in numerous developmental events and can specify cell fates and tissue types ^16,34 . For example, these factors play an important role in establishment of the basic body plan during gastrulation in Xenopus and mammals and in dorsal-ventral patterning in Drosophila. Interestingly, several studies have shown that cooperation between TGF-ß and Wnt/wingless signaling pathways plays a significant role in controlling certain developmental events. In Xenopus, activin and Wnt both contribute to the spatial restriction of early gene transcription in the Spemann organizer ⁶ . In Drosophila, the vestigial and ultrabithorax genes coordinately receive inputs from both dpp (a BMP homolog) and wingless ^15,29 .

The Wnt pathway is quite distinct from that of TGF-ß ( Fig. 1 ) ^5,10,35 . Briefly, in the absence of Wnt signaling, glycogen-synthase kinase-3 (GSK-3) phosphorylates ß-catenin, thereby inducing the degradation of ß-catenin by the ubiquitin-proteasome pathway. At activation of Wnt signaling, GSK-3 activity is inhibited. This prevents ß-catenin degradation, resulting in its accumulation, nuclear translocation, and association with members of the lymphoid enhancer binding factor/T-cell-specific factor (LEF/TCF) transcription factor family. The LEF1/TCF factors are a family of DNA-binding proteins that associate with ß-catenin and thereby the activation of Wnt pathway gene targets. The molecular details that underlie the cooperation between the TGF-ß and Wnt signaling cascades are currently unclear. Thus, in this study, we focussed on investigating the molecular mechanism that might mediate this effect ( Fig. 1 ).

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Transcriptional Response Assays

A 322 base pair fragment of the Xtwn promoter was obtained by polymerase chain reaction (PCR) from Xenopus laevis genomic DNA and was subcloned upstream of a luciferase reporter gene in the pGL2-promoter vector (Promega, Madison, WI, U.S.A.). The pGL3-Topflash promoter (pOT) was kindly provided by B. Vogelstein. For luciferase assays, HepG2 cells were transiently transfected by the calcium phosphate precipitation method as previously described ^18,19 .

Protein/Protein Interaction and Protein/DNA Gel Shift Assays

For immunoprecipitations, COS-1 or 293T cells were transfected with diethylaminoethyl (cellulose)-dextran or calcium phosphate, respectively, cell lysates were subjected to immunoprecipitation with monoclonal anti-Flag M2 antibody (Sigma Chemical, St. Louis, MO, U.S.A.), and associated proteins were detected by immunoblotting with anti-HA 12CA5 antibodies by chemiluminescence ¹⁹ . For GST-pull downs, cell lysates from transfected cells were incubated with glutathione-sepharose bound GST-fusion proteins and associated proteins were visualized by immunoblotting. Electrophoretic mobility shift assays (EMSA) were conducted exactly as described previously ^18,19 .

Results

Introduction | Materials and Methods | Results | Discussion | References

Identification of Xtwn as a Target of TGF-ß and Wnt Signaling Pathways

Siamois is a Xenopus homeobox gene closely related to Xtwn. Both genes share a similar expression pattern in early development. Investigations on the regulation of Siamois in Xenopus embryos have pointed to an important developmental role for synergism between the TGF-ß and Wnt signaling pathways. In Xenopus, Siamois expression can be induced by Wnt signaling by way of activation of ß-catenin ^6,11 or to a lesser degree through signaling by Smads ^2,7 . However, when Smad2 was combined with Wnt signaling, Siamois was induced much more strongly, and this cooperativity is thought to limit the spatial domain of Siamois expression to the dorsal mesendoderm ⁶ . To understand the mechanism of the cooperativity between these two pathways, we focussed on Twin (Xtwn), a homeobox gene that is closely related to Siamois and displays comparable expression patterns during Xenopus development ²⁰ . Previously, Laurent and colleagues used Xenopus assays to demonstrate that Xtwn expression is induced by Wnt and requires LEF1/TCF binding sites in the promoter ²⁰ . Thus, to investigate LEF1 regulation of the Xtwn promoter in mammalian cells, we isolated a fragment of the Xtwn promoter that includes these LEF1/TCF binding sites. We subcloned this fragment upstream of a luciferase reporter gene to generate Xtwn-Lux. We tested for LEF1 and TGF-ß responsiveness using HepG2 cells, which harbor an activated form of ß-catenin ⁸ and which contain endogenous TGF-ß receptors and Smads ^18,23 . We observed that coexpression of LEF1 mediated induction of the Xtwn-lux reporter ( Fig. 2-A , Fig. 2-B ). In the absence of LEF1 expression, TGF-ß had minimal effects; however, in the presence of LEF1, activation of the Xtwn promoter was stimulated by TGF-ß treatment of the cells and coexpression of Smads greatly enhanced this activity. In contrast, the Xtwn promoter was unaffected by activation of the BMP signaling pathway even in the presence of Smad1 ( Fig. 2-A , Fig. 2-B . Thus, we conclude that activation of the Xtwn promoter is specific for the TGF-ß/activin pathway and that TGF-ß/activin-dependent activation of the promoter requires LEF1.

Binding of Smads and LEF1 to the Xtwn Promoter Mediates Synergistic Activation by TNF-ß and Wnt Signaling Pathways

To gain insights into the mechanism for the synergistic activation of Xtwn by TGF-ß and Wnt, we next determined whether Smads and LEF1 could simultaneously bind to the Xtwn promoter. EMSAs using nuclear extracts from COS-1 cells transiently expressing full-length Smads and LEF1 demonstrated the presence of a DNA binding complex containing LEF1, Smad3, and Smad4 only in the presence of the activated receptor (data not shown ¹⁹ ). Thus, these results indicate that Smad3, Smad4, and LEF1 can simultaneously bind to the Xtwn promoter on activation of the TGF-ß signaling pathway.

We next focussed on determining the promoter elements that were required for Smad and LEF DNA binding and synergy. For this, we constructed a series of deletions and point mutations of the promoter and then tested the effect of these alterations on TGF-ß and Wnt signaling as well as on Smad and LEF/TCF DNA binding activity. Previous work has established that Smad3 and Smad4, through their MH1 domains, can bind GC-rich regions of DNA ^1,9,36,37 and that LEF1/TCF transcription factors bind to the consensus sequence CTTTG(A/T)(A/T) ¹⁴ . Examination of the Xtwn promoter sequence revealed the presence of two regions of putative Smad binding elements (SBE) at positions -218 to -203 and -160 to -155 and three LEF1 binding sites ( Fig. 2-B ). We observed that deletion of the first SBE and deletion or mutation of the second SBE reduced or blocked, respectively, Smad-dependent activation of the promoter. Consistent with this, these deleted promoter constructs caused a strong reduction in Smad3 MH1 domain DNA binding activity as assessed by EMSA with use of bacterially expressed Smad3 MH1 domain fusion proteins. Importantly, LEF1-dependent signaling was still observed in these SBE mutant promoter constructs. We also examined the requirement for LEF1/TCF binding. Introduction of point mutations into the LEF1 binding motifs prevented LEF1 DNA binding as determined by EMSA analyses and abolished the activity of the Xtwn promoter ( Fig. 2-B ). Together, these results indicate that the two SBEs and the LEF1 binding motif are all required for TGF-ß-dependent activation of the Xtwn promoter.

LEF1/TCF Transcription Factors Associate with Smad2 and Smad3

Previous work has shown that Smads associate with DNA binding proteins to regulate target genes. Thus, we wondered whether the synergistic activation of Xtwn might be the result of a physical association between Smads and LEF/TCF. We first tested for this putative interaction using transiently transfected mammalian cells. HA-tagged LEF1 was expressed in COS-1 cells together with Flag-tagged Smads in the presence of the constitutively active, activin type I receptor, ActRIB. In anti-Flag immunoprecipitates of cells expressing Smad2 and Smad3, coprecipitation of LEF1-HA was observed ( Fig. 3-A ). Examination of the association of endogenous Smad3 with endogenous TCFs using TGF-ß-treated 293T cells demonstrated that the Smad3/TCF association was enhanced by TGF-ß ¹⁹ . This interaction is direct, as bacterially expressed Smad3 associated with in vitro transcribed and translated LEF1 (data not shown ¹⁹ ).

Smad proteins contain conserved amino and carboxy terminal MH1 and MH2 domains, respectively, which are separated by a non-conserved linker region ^1,9,13,37 . Both the MH1 and MH2 domains can associate with DNA binding proteins. To determine the domains in Smad3 that mediate its association with LEF1, we expressed HA-tagged LEF1 in COS-1 cells and examined the ability of full-length LEF1 to interact with the MH1, MH2, or non-conserved linker regions of Smad3 expressed as bacterial fusion proteins. We observed that LEF1 interacted with both the MH1 and MH2 domains but not with the non-conserved linker region of Smad3 ( Fig. 3-B ). A similar association of XTcf3, a TCF family member that is coexpressed with Xtwin in Xenopus, with bacterially expressed full-length MH1 and MH2 domains of Smad3 was observed (data not shown). The same results were obtained when the association of Smad2 with LEF1 was examined ( Fig. 3-B ). Interestingly, we also detected an interaction between Smad4 and LEF1 in this assay, as previously described ²⁶ . However, in this case, Smad4 associated with LEF1 only through its MH1 domain ( Fig. 3-B ).

To identify the Smad-interacting domains in LEF1, we first constructed HA-tagged versions of LEF1 that lack either the amino-terminal ß-catenin binding domain (LEF1 ?ßC) or the carboxy terminal HMG box (LEF1 ?H) ( Fig. 3-C ). Although LEF1 ?ßC interacted normally with Smad3, LEF1 DH was unable to associate with the full-length MH1 or MH2 domains of Smad3 ( Fig. 3-C ). To further define the Smad-interaction domain, we created a series of LEF1 constructs containing varying deletions of the carboxy-terminus ( Fig. 3-C ). This analysis revealed that amino acids 324 to 334 mediate binding to the Smad3 MH2 domain whereas a lysine and arginine-rich region between amino acids 370 and 383 is required for association with the MH1 domain. Analysis of the structure of the HMG box of LEF1 reveals that the region that is required for binding to the Smad3 MH2 domain forms part of a-helix 2 ²² . Structural studies of HMG boxes show that helix 2 is required to maintain the structural integrity of the HMG box but is not involved in mediating specific DNA contacts ^22,28,33 . Thus, this a-helix would be free to interact with the Smad3 MH2 domain. In contrast, the MH1 domain of Smad3 binds to a basic region that makes extensive contacts with the DNA ^22,28 and that may also serve as a nuclear localization signal ²⁷ . Thus, it is possible that these properties prevent the association of the MH1 domain with LEF1 at the Xtwn promoter. It is intriguing to speculate that a-helix 2 might also mediate interaction of LEF1 with other transcriptional regulators.

Wnt-dependent Activation of LEF1 Target Genes can Occur Independently of TGF-ß

We were next interested in determining whether all LEF1/TCF target genes can be synergistically activated by TGF-ß and Wnt pathways. Association of ß-catenin with LEF1 in response to Wnt signaling has been shown to activate transcription from the Topflash reporter that contains three multimerized LEF1/TCF binding sites ¹⁷ . Thus, we examined whether this reporter could also be activated in the presence of Smads. Unlike the Xtwn promoter, coexpression of Smad3 and Smad4 had no effect on LEF1-dependent induction of luciferase activity from the Topflash reporter ( Fig. 4-A ). Since the Topflash promoter lacks SBEs, we postulated that this results in the inability to respond to TGF-ß signaling. To test this, we generated a chimeric promoter (TwnTop) that contained the SBEs from the Xtwn promoter upstream of the LEF/TCF binding sites in Topflash. We observed that activation of the Twntop promoter was stimulated by TGF-ß treatment of LEF1-expressing cells ( Fig. 4-B ). Furthermore, coexpression of Smad3 and Smad4 resulted in strong enhancement of LEF1-dependent transcriptional activity. Thus, these results demonstrate that the presence of SBEs in the promoters of LEF-dependent target genes is required to mediate TGF-ß-dependent enhancement of transcriptional activity.

TGF-ß-dependent Activation of LEF1 Target Genes Occurs Independently of ß-Catenin

Our analysis of the Topflash reporter demonstrated that activation of LEF/TCF target genes can occur independently of the TGF-ß signaling pathway. We were next interested in investigating whether TGF-ß signaling could activate the Xtwn promoter in the absence of ß-catenin function. To examine this, we constructed a version of LEF1 that lacked the first 20 amino acids (LEF1 ?20) and thus was missing a functional ß-catenin binding domain. We observed that HepG2 cells transfected with wild type or mutant LEF1 (?20) both yielded TGF-ß-dependent activation of the Xtwn reporter gene that was increased by coexpression of Smad3 and Smad4 ( Fig. 4-B ). Thus, in the absence of ß-catenin binding, Smads can stimulate transcriptional activation of the Xtwn promoter in an LEF1-dependent manner. Currently, we are directing efforts toward establishing whether these results are also observed with use of endogenously expressed proteins. Nevertheless, the results show that activation of both TGF-ß and Wnt pathways can independently or cooperatively activate the Xtwn promoter.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

TGF-ß and Wnt proteins are members of two distinct groups of secreted proteins that are required during numerous developmental stages in animals ^3,34 . Although each of these factors signals through unique downstream effectors, a cooperative interaction between these pathways has been described in the regulation of developmental events in Drosophila and Xenopus. In this study, we investigated the molecular mechanism for this cooperative effect. We show that synergistic activation of Xtwn, a Wnt and TGF-ß target gene, is mediated by a physical association between intracellular components of these two pathways, namely, Smad3 and LEF1/TCFs . We also demonstrate that specific Smad and LEF1/TCF DNA binding sites are required in the Xtwn promoter for this cooperative effect. On Topflash, another LEF1-regulated element that does not contain Smad binding sites, TGF-ß signaling had no effect on the activity of the promoter. However, TGF-ß-responsiveness was conferred by introducing SBEs into the Topflash promoter. In addition to their cooperative activity, TGF-ß and Wnt pathways can also independently regulate LEF1 target genes ( Fig. 5 ). Wnt-dependent activation of LEF1 through association with ß-catenin has been well described ^5,10,35 . Furthermore, we show that TGF-ß can stimulate activation of LEF1 target genes in the absence of ß-catenin binding to LEF1. Thus, our findings suggest that TGF-ß and Wnt-dependent induction of LEF1 target genes occurs in a context-dependent manner ( Fig. 5 ) and that this differential regulation may play a role in early developmental processes.

Activation of the TGF-ß signaling pathway stimulates the assembly of a heteromeric complex between the receptor-regulated Smads, Smad2 or Smad3, and the co-Smad, Smad4, and induces the nuclear accumulation of this complex ^1,9,13,37 . Analysis of numerous TGF-ß target genes has demonstrated that once in the nucleus Smads interact with a host of specific DNA binding proteins and are thereby recruited to specific regulatory elements. This promotes binding of Smads to DNA at adjacent sites. Our study of TGF-ß-dependent activation of the Xtwn promoter is consistent with this model. Thus, we have shown that Smads interact with the DNA binding transcription factor LEF1 to activate transcription of an element from the Xtwn promoter. Deletion of the SBEs that are located adjacent to the LEF1/TCF binding sites also abrogated Smad-dependent transcriptional activation of the promoter. Consistent with this, the Topflash promoter was not responsive to TGF-ß but the introduction of SBEs adjacent to the LEF1/TCF binding sites converted it into a TGF-ß and Smad-responsive promoter. Thus, we propose a model ( Fig. 5 ) in which Smad regulation of LEF1 target elements is dependent not only on the physical association of the two proteins but also on the presence of a SBE adjacent to the LEF1 site. This mechanism may provide the basis for promoter specificity in TGF-ß-dependent regulation of LEF1 target genes, since LEF1 binding sites that are not adjacent to a SBE will not be regulated by the Smad pathway.

Activation of LEF1 target genes through the Wnt signaling pathway is mediated by association of ß-catenin with LEF1; however, the exact mechanism by which ß-catenin activates transcription is not known. Intriguingly, a recent report suggests that the common Smad, Smad4, plays an important role in Wnt-dependent activation of LEF1 target genes. In this study, it was shown that Smad4 interacts directly with LEF1/TCF and indirectly with ß-catenin. Activation of the Wnt pathway alone enhanced the Smad4/ß-catenin interaction and in Xenopus animal caps resulted in a concomitant nuclear accumulation of both proteins. The effect of activating TGF-ß/activin signaling pathways together with the Wnt signal was not investigated; however, the results of our study indicate that a cooperative enhancement of transcriptional activation would be observed. The examination of the role of Smad4 as a common mediator of both Wnt and TGF-ß pathways represents an intriguing area for future work.

Interaction between components of the TGF-ß and Wnt signaling pathways is likely to play an important role during development. Since disruption of mediators of TGF-ß and Wnt signaling has been shown to be associated with human cancers, it will be interesting to determine whether these interactions also play a role in tumorigenesis.

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