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M. Economopoulou, T. Chavakis, S. Miller; Localization and Function of Junctional Adhesion Molecule (JAM)-C in the Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2007;48(13):4925.
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Tight and adherence junctions are crucial for RPE function and help maintain the chemical composition of the subretinal and choroidal extracellular spaces. Tight junctions consist of three types of transmembrane proteins, claudins, occludin and JAMs. The purpose of this study was to localize JAM-C in RPE and begin to characterize its functions.
The localization of JAM-C was studied by immunofluorescence confocal microscopy in 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 ± 283Ω·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, 20 hours after Ca reconstitution (n = 3; p=0.01). During junction reformation JAM-C was recruited to the initial cell-cell contacts. It has been shown in other systems that JAM-C may act as a ligand for the ß2-integrin Mac-1 on leukocytes. We studied the Mac-1-dependent basal to apical transmigration of human monocytes through the hfRPE monolayer. Both Mac-1 and JAM-C inhibition significantly decreased the chemokine induced transmigration of monocytes through the hfRPE monolayer compared to control (p=0.02 and 0.03, respectively; 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. By interacting with leukocyte integrin Mac-1, JAM-C promotes the basal to apical transmigration of monocytes through the hfRPE monolayer
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