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


METHODS
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.


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 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.


CONCLUSIONS
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.
Dunn
K C
KC
Section of Molecular and Cellular Biology, University of California-Davis, USA.
Marmorstein
A D
AD
Bonilha
V L
VL
Rodriguez-Boulan
E
E
Giordano
F
F
Hjelmeland
L M
LM
D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D016764:Cell Polarity; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000821:virology; D020800:Receptor, Nerve Growth Factor; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C496449:FGF5 protein, human; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D020800:Receptor, Nerve Growth Factor; D011973:Receptors, LDL; D017475:Receptors, Nerve Growth Factor; D051521:Fibroblast Growth Factor 5; D005346:Fibroblast Growth Factors