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S Proulx, S Landreville, SL Guérin, C Salesse; Effect of Confluency on Integrin Alpha5 Subunit Expression in Primary Cultures of Human Retinal Pigment Epthelial Cells and Arpe-19 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3649.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The retinal pigment epithelium (RPE) is differentiated and mitotically inactive in the normal eye, but several pathologies such as proliferative vitreoretinopathy (PVR) cause RPE cells to differentiate and proliferate. Integrins, a family of cell surface glycoproteins that mediate cell proliferation and differentiation, are thought to have fundamental roles in PVR. The aim of this study was to evaluate protein expression and gene regulation of integrin alpha5 subunit on proliferating and quiescent RPE cells. Methods: Cells at different confluency were used to evaluate expression of integrin alpha5 protein subunit on RPE cell surface by immunofluorescence and immunoprecipitation. Integrin alpha5 subunit expression was also assessed on differentiated RPE cells in situ by immunochemistry. Semi-quantitative RT-PCR and transient transfections were used to determine if increasing confluency affected alpha5 mRNA levels and promoter activity. These studies were performed with human primary RPE cell cultures as well as with the human RPE cell line ARPE-19. Results: We demonstrate that the integrin alpha5 subunit is present on human RPE cells both in vitro and in situ, and that protein level is influenced by confluency. Although mRNA transcripts are similar for non-confluent and confluent cells, there is a marked increase of promoter activity with increasing confluency. Alpha5 transcripts and promoter activity decrease when cells are confluent for a long period of time. We also show that major differences are present at the level of both the protein and gene regulation of integrin alpha5 subunit between primary human RPE and ARPE-19 cells. Conclusion: We demonstrate that confluency affects protein and gene expression of integrin alpha5 subunit. Proliferating RPE show a high level of expression of protein and mRNA transcripts as well as a high promoter activity. However, when cells become quiescent, gene expression is highly repressed and RPE cells have little integrin alpha5 subunit protein at the cell surface.
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