Abstract
Purpose :
The ARPE19 cell line gradually loses RPE properties with cell passage. One potential cause is that ARPE19 fails to uniformly express the major claudins found in native RPE tight junctions. As tight junctions regulate cell shape, morphology and gene expression, we explored whether exogenous expression of claudin-3 or claudin-19 might restore native RPE properties.
Methods :
ARPE19 were transduced with adenoviral vectors that expressed claudin-3, claudin-19, or as a control, green fluorescent protein (GFP). We used the transepithelial electrical resistance (TER) to assess barrier properties; RT-PCR arrays of RPE markers or junctional proteins to assess gene expression; and immunoblotting and immunofluorescence to assess protein expression. Comparisons were made with human fetal RPE (hfRPE). A scratch assay and inhibitors of EGF-stimulated pathways were used to evaluate wound-healing.
Results :
PCR arrays revealed substantial quantitative differences between hfRPE and ARPE19 with respect to RPE signature/maturation genes. Exogenous claudin-3 and claudin-19 increased the TER and the expression of ~50% of the genes that were under-expressed in ARPE19. The claudins had little effect on the expression of other tight, adherens, and gap junction proteins. Expression of the EGF receptor was decreased, which prompted us to examine wound-healing. In ARPE19, EGF stimulated cell proliferation and wound-healing using the AKT arm of the EGF-signaling pathway. In contrast, wound-healing was retarded in claudin-expressing ARPE19 and was insensitive to EGF. Instead, AKT was constitutively activated. Wound-healing was more epithelial-like in that wounds were slowly closed by a combination of cell spreading and cell proliferation.
Conclusions :
Directly or indirectly, claudin-3 and -19 effected changes in gene expression that partially restored a native RPE phenotype. The results suggest claudins and AKT signaling might be therapeutic targets for treating proliferative vitreoretinopathy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.