Abstract
Abstract: :
Purpose:The retinal pigment epithelium (RPE) plays a vital role in maintaining homeostasis between the neuroretina and the choriocapillaris. This homeostatic balance is disrupted in diseases such as age–related macular degeneration (ARMD) and diabetic retinopathy. RPE cells are highly differentiated, but can undergo phenotypic modulation in response to tissue damage. In this study, the expression of differentiation markers characteristic of RPE cells was evaluated in an ARPE–19 cell culture model. Methods:ARPE–19 cells at passage 25–35 were grown in DMEM/F12 with 10% FBS and fed weekly. Semiquantitive PCR was performed on proliferating and confluent cells over a 27 day time course. DNase I hypersensitivity assays were performed with non–differentiated (proliferating) and differentiated cells. Micrococcal nuclease studies were performed using passage 29 cells over a 19 day time course. Results:Proliferating and non–confluent cultures showed little expression of genes associated with the vision cycle or with pigmentation. However, within two to four weeks of continuous culture, transcription of these genes was initiated. In our cell culture model, the expression of the visual cycle genes, RPE65 and CRALBP, become activated after 15 days of culture. Interestingly, the genes associated with pigmentation, including tyrosinase and the tyrosinase–related proteins, TYRP–1 and Dct (TYRP–2), are up–regulated starting at 5, 8, and 15 days, respectively. Changes in promoter architecture, including the presence of DNase I hypersensitive sites and the positioning of nucleosomes, were evaluated for several of these genes. In addition, the ability of promoter:reporter constructs to mimic the differentiation–associated transcription of these genes was evaluated. Conclusions:This represents a unique model to study the induction of RPE cell markers from minimal transcription to robust expression in response to discrete steps in RPE cell differentiation.
Keywords: transcription • retinal pigment epithelium • gene/expression