Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug

Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.