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J.S. Vogel, E.C. Bullen, C.L. Teygong, E.W. Howard; Transcriptional Regulation of Pigmentation During Retinal Pigment Epithelial Cell Differentiation . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3159.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Retinal pigment epithelial (RPE) cells are highly differentiated, but can undergo phenotypic modulation in response to tissue damage. We previously established a cell culture model to evaluate ARPE–19 cells in proliferating versus differentiated phenotypes. Our goal is to investigate the mechanisms involved in this differentiation process. To do this, we are determining how several marker genes associated with pigmentation become transcriptionally activated during differentiation. Methods: ARPE–19 cells were stably transfected with increasing lengths of promoter fragments attached to a luciferase reporter. The cells were grown in DMEM/F12 with 10% FBS and fed weekly. RNA was collected at weekly intervals for 5 weeks, and semi–quantitative RT–PCR was performed on proliferating (less than 3 days) and confluent (greater than 3 days) cells over a 5 week time course with primers to tyrosinase, and tyrosinase–related proteins 1 and 2. Results:Over the course of several weeks, ARPE–19 cells in culture differentiate to the extent that they express both pigmentation and vision cycle genes. They become pigmented, and are capable of engulfing rod outer segments. In addition, cells stably transfected with pigmentation gene promoter fragments also undergo this differentiation. Importantly, several of these constructs demonstrate a pattern of upregulation versus control that recapitulates the expression of the endogenous gene, in that increased expression occurs at a similar time point over the differentiation time course. Further studies are underway to pinpoint critical promoter elements needed for expression of pigment–related genes during differentiation. Conclusions:We have demonstrated that ARPE–19 cells represent a unique model capable of undergoing at least some of the differentiation steps associated with RPE cell development. We will now utilize this model to study the transcriptional regulation of genes involved in the vision cycle and pigmentation during this differentiation process.
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