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D. C. Dean, Y. Liu, H. J. Kaplan, Q. Lu; The Epithelial-Mesenchymal Transcription Factor, Zeb1, and Cell-Cell Contacts Regulate Retinal Pigment Epithelial Cell Differentiation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2439.
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When RPE cells are placed in culture with serum growth factors, the normally quiescent cells begin proliferating and undergo dedifferentiation with hallmarks of an epithelial-mesenchymal transition (EMT), which is characterized by hypopigmentation and fibroblastic morphology. Our purpose was to investigate this dedifferentiation pathway. We hypothesized that classic EMT-promoting transcriptional repressors such as Zeb1 and Snail might be involved in the process.
Immunostaining of eyes from wild-type and Zeb1 mutant mice, real time PCR analysis of RPE mRNA levels, cell proliferation assays, and Chromatin immunoprecipitation assays were used to analyze the effects of Zeb1 mutation on RPE gene expression and dedifferentiation.
The classic epithelial specification gene, E-cadherin, was ectopically expressed on the RPE when Zeb1 was mutated. And Zeb1 and Snail were overexpressed when RPE cells were exposed to serum growth factors in culture, which coincided with dedifferentiation changes: onset of proliferation, repression of the RPE specification transcription factor MITF, hypopigmentation, and transition to a fibroblastic morphology. Heterozygous mutation of Zeb1 prevented these dedifferentiation changes. Zeb1 represses pigment cell specification transcription factor, MITF, leading to downregulation of pigment cell genes and cyclin dependent kinase (cdk) inhibitors, which normally restrict proliferation. A RPE phenotype can be reprogrammed following EMT by imposing cell-cell contacts via sphere formation. This leads to downregulation of Zeb1, induction of MITF and re-expression of pigment cell genes and cdk inhibitors.
The EMT transcription factor, Zeb1, is required to repress E-cadherin expression on the RPE in vivo. Additionally, Zeb1 is overexpressed as RPE proliferate and undergo dedifferentiation in culture. Decreasing the gene dosage of Zeb1 via heterozygous mutation prevents this proliferation and dedifferentiation. The role of Zeb1 overexpression in dedifferentiation appears linked to its binding to the MITF A promoter and repression of the gene. This repression of MITF in turn is associated with downregulation of MITF target genes involved in pigment synthesis and maintaining cell cycle arrest. Expression of Zeb1 and thus RPE dedifferentiation is linked to cell-cell contacts.
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