Abstract
Purpose: :
RPE are unusual among epithelial cells in expressing high N– (rather than E–) cadherin and using N–cadherin to form a zonular adherens junction typical of epithelial cells. Here an MDCK subclone (clone–YH), selected for having high endogenous N–cadherin and very low E–cadherin (similar to RPE), was used to analyze cell type differences/similarities in epithelial phenotype development.
Methods: :
Clone–YH and RPE cell lines ARPE–19 and hTERT–RPE1 were analyzed for cadherin and beta1–integrin protein expression and glycosylation state by western blotting, protein distribution by immunostaining, and cell phenotype by phase contrast, epifluorescence and confocal microscopy. N–cadherin expression was knocked down by siRNA transfection.
Results: :
RPE cells are variably epithelioid and become more so as culture density increases and N–cadherin becomes zonular. Clone–YH cells are initially highly fusiform with N–cadherin at multiple sites. E–cadherin contributes little to clone–YH junctions; protein levels are low and only after N–cadherin knockdown does junctional E–cadherin become detectable. With culture time, N–cadherin in clone–YH (but not RPE) becomes de–glycosylated and coincidentally the cadherin and actin cytoskeleton develop an epithelial zonular pattern. These events can be induced in early clone–YH cultures by treatment with the N–glycosylation inhibitor tunicamycin, or by treatment with medium conditioned by confluent cells (with or without serum), suggesting that time–dependent protein de–glycosylation is triggered by a secreted product. Beta1–integrin, which like cadherins is an N–glycoprotein, remains glycosylated in clone–YH with time in culture indicating that protein de–glycosylation is not universal and that integrin glycosylation state is likely not related to clone–YH phenotype. However, beta1–integrin, which is both a cell–cell and cell–substrate adhesion protein, differs in both protein amounts and in distribution patterns in clone–YH and RPE cells.
Conclusions: :
Both RPE cells and the MDCK subclone clone–YH are E–cadherin deficient but can acquire an epithelial shape using N–cadherin to generate a zonular, epithelial–type junction. However, the mechanisms of epithelialization may differ in the two cell types with a differential role for beta1–integrin, and a role for protein de–glycosylation in clone–YH but not RPE.
Keywords: retinal pigment epithelium • cell adhesions/cell junctions