Proteome Survey of Proliferating and Differentiating Rat RPE-J Cells KAREN A. WEST, LIN YAN, MASARU MIYAGI, JOHN S. CRABB, ALAN D. MARMORSTEIN, LIHUA MARMORSTEIN AND JOHN W. CRABB * Cole Eye Institute, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195, U.S.A. (Received St. Louis 16 April 2001 and accepted in revised form 14 June 2001) The suitability of the rat derived SV-40T immortalized RPE-J cell line for identifying proteome changes associated with RPE differentiation was evaluated by surveying changes in protein expression levels. Rat RPE-J cells were induced to undergo differentiation in culture by growth at the nonpermissive temperature of 408C in the presence of retinoic acid. Total proteins were extracted from cells grown under proliferating or differentiating conditions and separated by 1D and 2D gel electrophoresis. Gel spots were excised, digested in situ with trypsin, and analysed by mass spectrometry to identify proteins. Computer assisted image analysis was used to align gel patterns and quantify spot intensities. Neither proliferating nor differentiating RPE-J cell cultures exhibited detectable levels of cellular retinaldehyde- binding protein, RPE65, 11-cis-retinol dehydrogenase or lecithin retinol acyl transferase, suggesting that RPE-J cells are not appropriate for visual cycle studies. About 18 % of the 61 identified proteins appear to change expression levels with the cell growth conditions. Seven proteins appeared to be up-regulated and four proteins down-regulated when the cells were changed from proliferating to differentiating culture conditions. The majority of the apparent changes in protein expression levels were associated with stress response genes. Significant changes in the apparent mass and charge properties of proteins were also observed and for select proteins, the modifications appeared to be correlated with cell growth conditions. The results demonstrate that proteome differences in RPE-J cells associated with growth conditions can be identified and support the suitability of RPE-J cells for more targeted and/or more global proteome analysis of RPE differentiation. # 2001 Academic Press Key words: proteomics; retinal pigment epithelium; mass spectrometry; 2D gel electrophoresis. 1. Introduction The retinal pigment epithelium (RPE) serves many vital functions in the vertebrate visual process. In mammals, this single layer of polarized cells between the photoreceptors and vascular choroid transports nutrients, growth factors and hormones to the rods and cones, phagocytizes shed photoreceptor outer segments, helps remove waste products to the blood, absorbs scattered light and participates in the regeneration of bleached visual pigment (Bok, 1993). Visual pigment regeneration may be the most special- ized function of the RPE and involves enzymatic isomerization of all-trans- to 11-cis-retinol following photoisomerization of 11-cis-retinol to all-trans-retinal in the adjacent photoreceptor cells. Disruption and/or malfunction of the RPE is commonly observed in retinal degenerative diseases such as retinitis pigmen- tosa and age-related macular degeneration (Lowen- stein, Bressler and Bressler, 1998; Phelan and Bok, 2000). Because of its critical role in retinal cell biology, several immortalized RPE cell lines have been devel- oped for studying RPE physiology such as the human cell lines D407 (Davis et al., 1995) and ARPE 19 (Dunn et al., 1996) and rat cell lines RPE-J (Nabi et al., 1993) and BPEI-1 (McLaren et al., 1993). Such in vitro model systems offer advantages for morpho- logical, biochemical and molecular biological studies because they allow the investigation of a pure, single cell type under controlled experimental conditions and can be manipulated in ways not possible with tissues in situ. However, the RPE cell lines differ from each other in various properties and unfortunately, none are ideal models of in vivo RPE. Here we explore the suitability of the RPE-J cell line from rat for character- izing proteome changes associated with differentiation and the differentiated RPE-J cell phenotype. RPE-J cells have been used for studying rod outer segment phagocytosis (Finnemann et al., 1997), RPE protein trafficking (Marmorstein et al., 1998a), and epithelial morphogenesis (Marmorstein et al., 1998b, Bonilha, Finnemann and Rodriguez-Boulan, 1999). The cell line was created by immortalization with SV40-T and is temperature sensitive for growth. For differentiation, RPE-J cells are grown on Matrigel at 328C in media containing all-trans-retinoic acid for 6 days, then switched to 408C for an additional 36–48 hr. Under these conditions the cells develop a polarized epithelial phenotype and form tight junctions (Nabi et al., 1993). The aim of this study was to test the feasibility of using RPE-J cells for Exp. Eye Res. (2001) 73, 479–491 doi:10.1006/exer.2001.1058, available online at http://www.idealibrary.com on 0014-4835/01/10047913 $35.00/0 # 2001 Academic Press * Author for correspondence. E-mail: crabbj@ccf.org