The formation of a ␤-catenin⅐TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the ␤-catenin⅐TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks ␤-catenin⅐TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interacts with ␤-catenin and TCF4. Interestingly, whereas the interaction of p15RS with ␤-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, overexpression of p15RS reduces the interaction of ␤-catenin with TCF4, whereas the depletion of p15RS enhances their interaction. We further demonstrate that overexpression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, whereas depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of CYCLIN D1 and c-MYC, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting ␤-catenin⅐TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters.

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The Raf kinase inhibitory protein (RKIP) is down-regulated in multiple types of human cancers. Decreased RKIP transcription activity may be one of the major mechanisms responsible for the downregulation of RKIP expression in human diseases. To test this hypothesis, we need to gain basic knowledge of the transcriptional regulation of RKIP. To achieve this objective, we made a systematic effort to identify cis-acting elements and trans-acting factors that control RKIP promoter activity. We found that full RKIP promoter activity requires the region −56 to +261 relative to the transcription start site. Within the full promoter region, there are two motifs rich in G/C that responded to transcription factor Sp1, one cAMP-responsive element that responded to the transcription factor CREB, and one docking site for the histone acetylase p300. In human melanoma A375 cells and human cervical cancer HeLa cells, mutation or deletion of each of these cis-acting elements decreased promoter activity. In A375 cells, knockdown of the corresponding transcription factors Sp1, CREB, or p300 decreased RKIP promoter activity, whereas overexpression of CREB and p300 increased RKIP promoter activity. The results obtained with HeLa cells also supported the idea that Sp1 and CREB play positive roles in the regulation of RKIP transcription. These findings suggest that regulators of the expression or activity of Sp1, CREB, and p300 are involved in regulating RKIP transcription.

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