The murine gastrin promoter is synergistically activated by transforming growth factor-beta/Smad and Wnt signaling pathways.
The transforming growth factor-beta (TGF-beta) and Wnt/wingless pathways play critical roles in the specification of cell fate during development and also contribute to cancer formation and progression. Whereas Wnt signaling is clearly pro-oncogenic, TGF-beta signaling is cell- and context-dependent, manifesting both inhibitory and proliferative effects. The growth factor, gastrin, has previously been shown to be a downstream target of the Wnt pathway and a promoter of gastrointestinal cancer. In this study, we show that the mouse gastrin promoter is regulated synergistically by TGF-beta/Smads and beta-catenin/T-cell factor (TCF). Co-transfection of Smad3/Smad4 and beta-catenin expression constructs synergistically activated mouse gastrin promoter activity 30-60-fold in AGS cells with minimal effect seen with either construct alone. This activation was further potentiated by TGF-beta1 treatment. Mutating either the TCF binding site or the Smad-binding element (SBE) diminished the activation of gastrin expression by Smad3/Smad4 and beta-catenin and led to a loss of gastrin promoter responsiveness to TGF-beta1 treatment. Wnt and TGF-beta regulated endogenous gastrin mRNA levels in AGS cells in a similar fashion, as revealed by small interference RNA studies or overexpression of Smads and TCF4/beta-catenin. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that the putative SBE and T-cell factor (TCF) sites were able to bind a complex containing Smads and beta-catenin/TCF4. In addition, the synergy between Smads and beta-catenin/TCF4 was dependent on CREB-binding protein (CBP)/P300, as demonstrated by overexpression of CBP or E1A. Moreover, by using a heterogeneous promoter reporter system, we showed that this complex containing Smads/TCF4/beta-catenin complex was able to up-regulate transcription at isolated SBE or TCF sites. Thus, the Wnt signaling pathway is able to activate some target genes through its actions as a co-activator at non-TCF sites and has the potential to profoundly alter transcriptional responses to TGF-beta signaling.
Division of Gastroenterology, Department of Medicine, Columbia College of Physicians and Surgeons, New York, New York 10032, USA.
Shi
S
Lei
Alexander
A
Dubeykovskiy
Abhijit
A
Chakladar
Lindsay
L
Wojtukiewicz
Timothy C
TC
Wang
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005755:Gastrins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D015854:Up-Regulation; D051153:Wnt Proteins; D051176:beta Catenin
C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D005755:Gastrins; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins; D051176:beta Catenin; D004247:DNA; D008156:Luciferases