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

• Published in: Investigative ophthalmology & visual science Vol. 39; no. 13; pp. 2744 - 2749
• Main Authors: Dunn, KC, Marmorstein, AD, Bonilha, VL, Rodriguez-Boulan, E, Giordano, F, Hjelmeland, LM
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01.12.1998; Association for Research in Vision and Ophtalmology
• Subjects: Basement Membrane - metabolism; Biological and medical sciences; Blotting, Western; Cell Line; Cell Polarity; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Fibroblast Growth Factor 5; Fibroblast Growth Factors - genetics; Fibroblast Growth Factors - secretion; Hemagglutinin Glycoproteins, Influenza Virus - genetics; Hemagglutinin Glycoproteins, Influenza Virus - metabolism; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Microscopy, Confocal; Ophthalmology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Pigment Epithelium of Eye - metabolism; Pigment Epithelium of Eye - virology; Receptor, Nerve Growth Factor; Receptors, LDL - genetics; Receptors, LDL - metabolism; Receptors, Nerve Growth Factor - genetics; Receptors, Nerve Growth Factor - metabolism; Transfection; Human; Eye; Visual system; Fibroblast growth factor; Cell line; Secretion; Pigment epithelium; Exploration; Polarity; Retina
• ISSN: 0146-0404; 1552-5783

To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (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 media. Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation 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. ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, 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.