The localization of JAM-C, ZO-1, N-cadherin and ezrin was studied by immunofluorescence confocal microscopy in confluent and subconfluent cultures of human fetal RPE (hfRPE) and in adult native RPE wholemounts. Western blot was used to analyze JAM-C protein expression. The transepithelial resistance was measured by EVOM. A calcium switch assay was used to determine the importance of JAM-C in junction formation by monitoring changes in transepithelial resistance (TER). The steady-state resistance of the hfRPE cultures was 935+/- 283 ohm.cm2 (mean SD; n=9). A transepithelial migration assay (basal to apical) was used to study the role of JAM-C in the migration of leukocytes through the hfRPE monolayer.? JAM-C was found in the tight junctions of both cultured hfRPE cells and adult native RPE where it colocalized with ZO-1. In addition, only partial colocalization of JAM-C with E-cadherin or desmoplakin was found. JAM-C localization or expression was not altered by stimulation of the cells with proinflammatory cytokines. The inhibition of JAM-C resulted in a significant delay in the reassembly of the hfRPE junctions after calcium depletion-induced reduction in TER. In control experiments, this recovery was 90.7 +/- 3.9% of the initial TER while in the presence of the JAM-C inhibitor the recovery was only 67.9+/-9.8% of the initial TER in the same time-frame, 24 hours after Ca reconstitution (n = 3; p=0.01). During junction reformation JAM-C was recruited to the initial cell-cell contacts and after JAM-C knockdown, the recruitment of N-cadherin and ZO-1 at the site of cell-cell contact was reduced. Furthermore, JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by the reduced apical staining of ezrin at selected time points. It has been shown in other systems that JAM-C may act as a ligand for the beta2-integrin Mac-1 on leukocytes. We studied the basal to apical transmigration of human monocytes and neutrophils through the hfRPE monolayer. JAM-C inhibition significantly decreased the chemokine induced transmigration of leukocytes through the hfRPE monolayer compared to control (p= 0.03; n =3).? JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. JAM-C is important in the initial stages of tight junction formation in hfRPE possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts and has a role in the polarization of hfRPE cells.Finally, JAM-C promotes the basal to apical transmigration of leukocytes through the hfRPE monolayer.
