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

Z Wang, P Wade, K J Mandell, A Akyildiz, C A Parkos, R J Mrsny, A Nusrat

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Abstract

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.
Original languageEnglish
Pages (from-to)1222-1230
Number of pages9
JournalOncogene
Volume26
Issue number8
DOIs
Publication statusPublished - 2007

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Occludin
Tight Junction Proteins
Zinc Fingers
Gastropoda
Epithelial-Mesenchymal Transition
Snail Family Transcription Factors
Chromatin Immunoprecipitation
Site-Directed Mutagenesis
Small Interfering RNA
Up-Regulation
Down-Regulation
Epithelial Cells
Phenotype

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Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug. / Wang, Z; Wade, P; Mandell, K J; Akyildiz, A; Parkos, C A; Mrsny, R J; Nusrat, A.

In: Oncogene, Vol. 26, No. 8, 2007, p. 1222-1230.

Research output: Contribution to journalArticle

Wang, Z ; Wade, P ; Mandell, K J ; Akyildiz, A ; Parkos, C A ; Mrsny, R J ; Nusrat, A. / Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug. In: Oncogene. 2007 ; Vol. 26, No. 8. pp. 1222-1230.
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T1 - Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug

AU - Wang, Z

AU - Wade, P

AU - Mandell, K J

AU - Akyildiz, A

AU - Parkos, C A

AU - Mrsny, R J

AU - Nusrat, A

PY - 2007

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N2 - 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.

AB - 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.

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