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MAD2B, a novel TCF4-binding protein, modulates TCF4-mediated epithelial-mesenchymal transdifferentiation.

ABSTRACT: T cell factor 4 (TCF4) interacts with beta-catenin in the WNT signaling pathway and transactivates downstream target genes involved in cancer progression. To identify proteins that regulate TCF4-mediated biological responses, we performed a yeast two-hybrid screen to search for a TCF4-binding protein(s) and found that MAD2B interacts with TCF4. We confirmed that MAD2B is a TCF4-binding protein by co-immunoprecipitation. Using the TOPFLASH reporter assay, we found that MAD2B blocks TCF4-mediated transactivation. The MAD2B binding regions of TCF4 were identified by TCF4 deletion mapping and electrophoretic mobility shift assay analysis. TCF4 and MAD2B interactions abolished the DNA binding ability of TCF4. Knockdown of MAD2B in SW480 colorectal cancer cells led to the conversion of epithelial cells to a mesenchymal fibroblastoid phenotype (epithelial-mesenchymal transdifferentiation). An E-cadherin promoter reporter analysis showed that MAD2B modulates TCF4-mediated E-cadherin expression. MAD2B knockdown blocked E-cadherin expression and significantly induced mesenchymal markers, such as N-cadherin and vimentin. Mesenchymal induction was accompanied by F-actin redistribution and the appearance of a fibroblastoid phenotype. MAD2B knockdown also increased both mRNA and protein levels of Slug, a known TCF4-induced E-cadherin transcriptional repressor. A chromatin immunoprecipitation assay showed that MAD2B silencing enhances the ability of TCF4 to bind the Slug promoter. Thus, MAD2B is a novel TCF4-interacting protein. This study provides the first evidence for the involvement of MAD2B in TCF4-mediated epithelial-mesenchymal transdifferentiation.

SUBMITTER: Hong CF

PROVIDER: S-EPMC2740587 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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MAD2B, a novel TCF4-binding protein, modulates TCF4-mediated epithelial-mesenchymal transdifferentiation.

Hong Chun-Fu CF Chou Yu-Ting YT Lin Young-Sun YS Wu Cheng-Wen CW

The Journal of biological chemistry 20090514 29

T cell factor 4 (TCF4) interacts with beta-catenin in the WNT signaling pathway and transactivates downstream target genes involved in cancer progression. To identify proteins that regulate TCF4-mediated biological responses, we performed a yeast two-hybrid screen to search for a TCF4-binding protein(s) and found that MAD2B interacts with TCF4. We confirmed that MAD2B is a TCF4-binding protein by co-immunoprecipitation. Using the TOPFLASH reporter assay, we found that MAD2B blocks TCF4-mediated ...[more]

PMID: 19443654

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Project description:Phenotype reprogramming during transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) is an extensive and dynamic process, orchestrated by the integration of biological signaling across multiple time scales. As part of the numerous transcriptional changes necessary for EMT, TGFβ-initiated Smad3 signaling results in remodeling of the redox environment and decreased nucleophilic tone. Because Smad3 itself is susceptible to attenuated activity through antioxidants, the possibility of a positive feedback loop exists, albeit the time scales on which these mechanisms operate are quite different. We hypothesized that the decreased nucleophilic tone acquired during EMT promotes Smad3 signaling, enhancing acquisition and stabilization of the mesenchymal phenotype. Previous findings supporting such a mechanism were characterized independent of each other; we sought to investigate these relationships within a singular experimental context. In this study, we characterized multivariate representations of phenotype as they evolved over time, specifically measuring expression of epithelial/mesenchymal differentiation, redox regulators, and Smad transcription factors. In-cell Western (ICW) assays were developed to evaluate multivariate phenotype states as they developed during EMT. Principal component analysis (PCA) extracted anticorrelations between phospho-Smad3 (pSmad3) and Smad2/Smad4, which reflected a compensatory up-regulation of Smad2 and Smad4 following cessation of TGFβ signaling. Measuring transcript expression following EMT, we identified down-regulation of numerous antioxidant genes concomitant with up-regulation of NADPH oxidase 4 (NOX4) and multiple mesenchymal phenotype markers. TGFβ treatment increased CM-H2DCF-DA oxidation, decreased H2O2 degradation rates, and increased glutathione redox potential. Our findings suggest that the decreased nucleophilic tone during EMT coincides with the acquisition of a mesenchymal phenotype over too long a time scale to enable enhanced Smad3 phosphorylation during initiation of EMT. We further challenged the mesenchymal phenotype following EMT through antioxidant and TGFβ inhibitor treatments, which failed to induce a mesenchymal-epithelial transition (MET). Our characterization of multivariate phenotype dynamics during EMT indicates that the decrease in nucleophilic tone occurs alongside EMT; however, maintenance of the mesenchymal phenotype following EMT is independent of both the nascent redox state and the continuous TGFβ signaling.

Dataset Information

MAD2B, a novel TCF4-binding protein, modulates TCF4-mediated epithelial-mesenchymal transdifferentiation.

Publications

MAD2B, a novel TCF4-binding protein, modulates TCF4-mediated epithelial-mesenchymal transdifferentiation.

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