RESUMO
UNLABELLED: The WW domain containing oxidoreductase (WWOX) has recently been shown to inhibit of the Wnt/ß-catenin pathway by preventing the nuclear import of disheveled 2 (DVL2) in human breast cancer cells. Here, it is revealed that WWOX also interacts with the BCL9-2, a cofactor of the Wnt/ß-catenin pathway, to enhance the activity of the ß-catenin-TCF/LEF (T-cell factor/lymphoid enhancer factors family) transcription factor complexes. By using both a luciferase assay in MCF-7 cells and a Xenopus secondary axis induction assay, it was demonstrated that WWOX inhibits the BCL9-2 function in Wnt/ß-catenin signaling. WWOX does not affect the BCL9-2-ß-catenin association and colocalizes with BCL9-2 and ß-catenin in the nucleus of the MCF-7 cells. Moreover, WWOX inhibits the ß-catenin-TCF1 interaction. Further examination found that HDAC3 associates with BCL9-2, enhances the inhibitory effect of WWOX on BCL9-2 transcriptional activity, and promotes the WWOX-BCL9-2 interaction, independent of its deacetylase activity. However, WWOX does not influence the HDAC3-BCL9-2 interaction. Altogether, these results strongly indicate that nuclear WWOX interacts with BCL9-2 associated with ß-catenin only when BCL9-2 is in complex with HDAC3 and inhibits its transcriptional activity, in part, by inhibiting the ß-catenin-TCF1 interaction. The promotion of the WWOX-BCL9-2 interaction by HDAC3, independent of its deacetylase activity, represents a new mechanism by which this HDAC inhibits transcription. IMPLICATIONS: The inhibition of the transcriptional activity of BCL9-2 by WWOX and HDAC3 constitutes a new molecular mechanism and provides new insight for a broad range of cancers.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Histona Desacetilases/genética , Oxirredutases/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HEK293 , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Camundongos , Oxirredutases/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW , Xenopus , beta Catenina/metabolismoRESUMO
Signaling by transforming growth factor-ß (TGF-ß) is critical for various developmental processes and culminates in the activation of the transcription factors Smad2 and Smad3. MAN1, an integral protein of the inner nuclear membrane, inhibits TGF-ß signaling by binding to Smad2 and Smad3. Depletion of the gene LEMD3 encoding MAN1 leads to developmental anomalies in mice, and heterozygous loss-of-function mutations in LEMD3 in humans cause sclerosing bone dysplasia. We modeled the three-dimensional structure of the MAN1-Smad2 complex from nuclear magnetic resonance and small-angle x-ray scattering data. As predicted by this model, we found that MAN1 competed in vitro and in cells with the transcription factor FAST1 (forkhead activin signal transducer 1) for binding to Smad2. The model further predicted that MAN1 bound to activated Smad2-Smad4 or Smad3-Smad4 complexes, which was confirmed by in vitro experiments; however, in cells, MAN1 bound only to Smad2 and Smad3 and not to the Smad4-containing complexes. Overexpression of MAN1 led to dephosphorylation of Smad2 and Smad3, thus hindering their recognition by Smad4, and MAN1 bound directly in vitro to the phosphatase PPM1A, which catalyzes the dephosphorylation of Smad2/3. These results demonstrate a nuclear envelope-localized mechanism of inactivating TGF-ß signaling in which MAN1 competes with transcription factors for binding to Smad2 and Smad3 and facilitates their dephosphorylation by PPM1A.
