Use of the ARPE-19 cell line as a model of RPE polarity: Basolateral secretion of FGF5

PURPOSE. To determine the polarity of fibroblast growth factor 5 (FGF5) sec retions from retinal pigment epithelium (RPE) cells and to examine the viab ility and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS. Influenza infection and adenovirus-mediated gene transfer were use d to deliver and express genes encoding influenza hemagglutinin (HA), p75-N TR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR) , and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA , p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned medi a. RESULTS. Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. in contrast, LDLR was as sociated preferentially with the basolateral membrane of ARPE-19 cells. Bio tinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS. ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling as says. This indicates that the ARPE-19 cell line is a valid model for studie s of RPE cell polarity. FGF5, a secreted protein normally produced by RPE c ells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.