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Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of ?-catenin.

ABSTRACT: Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of ?-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor ? and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of ?-catenin messenger RNA (mRNA) and Wnt signaling.We used microarray analysis to associate levels of Smad4 and ?-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(?1638/+)) and (APC(?1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection.In human CRC samples, reduced levels of Smad4 correlated with increased levels of ?-catenin mRNA. In Smad4-depleted cell lines, levels of ?-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the ?-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of ?-catenin mRNA. In mice with heterozygous disruption of Apc(APC(?1638/+)), Smad4-deficient intestinal adenomas had increased levels of ?-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(?1638/+) mice that expressed Smad4.Transcription of ?-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor ?, BMP, and Wnt signaling pathways in progression of CRC.

SUBMITTER: Freeman TJ

PROVIDER: S-EPMC3343368 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of β-catenin.

Freeman Tanner J TJ Smith J Joshua JJ Chen Xi X Washington M Kay MK Roland Joseph T JT Means Anna L AL Eschrich Steven A SA Yeatman Timothy J TJ Deane Natasha G NG Beauchamp R Daniel RD

Gastroenterology 20111122 3

<h4>Background & aims</h4>Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of β-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor β and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss o ...[more]

PMID: 22115830

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Project description:Oxidants and canonical Wnt/?-catenin signaling have been shown to decrease cardiac Na(+) channel activity by suppressing NaV1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/?-catenin signaling and promotes ?-catenin nuclear activity, leading to suppression of NaV1.5 expression and if this suppression requires the interaction of ?-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased ?-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total ?-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on NaV1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of ?-catenin or stabilizing ?-catenin by GSK-3? inhibitors, LiCl and Bio, suppressed NaV1.5 expression in HL-1 cells. In contrast, destabilization of ?-catenin by a constitutively active GSK-3? mutant (S9A) upregulated NaV1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na(+) channel activity in these cells. Using immunoprecipitation (IP), we showed that ?-catenin interacted with TCF4 indicating that ?-catenin as a co-transfactor, regulates NaV1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both ?-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that ?-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting ?-catenin significantly increased SCN5a promoter activity, leading to enhanced NaV1.5 expression. As expected, ?-catenin SiRNA prevents H2O2 suppressive effects on both SCN5a promoter activity and NaV1.5 expression. Our findings indicate that H2O2 inhibits NaV1.5 expression by activating the Wnt/?-catenin signaling and ?-catenin interacts with TCF4 to transcriptionally suppress cardiac NaV1.5 expression.

Dataset Information

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of ?-catenin.

Publications

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of β-catenin.

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