The Effect of Claudin-5 Overexpression on the Interactions of Claudin-1 and-2 and Barrier Function in Retinal Cells

摘要

Claudin-5, one of the dominant tight junctions (TJs) proteins, plays an important role in maintaining the barrier function in the blood brain and retinal barrier. This study aimed to investigate the effect of claudin-5 overexpression on the interactions of claudin-1 and -2 and barrier functions in primary cultured human retinal pigment epithelium cells (HRPECs) and human retina endothelial cells (HRECs). Lentivirus was used to mediate the overexpression of claudin-5 in retinal cells. Significantly increased mRNA and protein levels of claudin-5 were detected in the transfection group. After the transfected cells grew on the transwell membrane for three weeks, a stable monolayer cell barrier model was established in vitro. The claudins expressions analysis showed that overexpressed claudin-5 significantly increased the expression of claudin-1, while it decreased the expression of claudin-2 in both mRNA and protein level. Co-IP experiments and barrier function assay revealed that claudin-5 overexpression promoted the interactions of claudin-1 and claudin-2 and enhanced the barrier function of retinal cells. Intriguingly, the exogenous expression of claudin-5 induced new interaction pattern between claudin-5 and claudin-1 or -2 in HRPECs, which do not have endogenous claudin-5 expression. In addition, claudin-5 overexpression decreased cell mobility and the sprouting capability of vessel tube formation in vitro. This study demonstrated that claudin-5 has a positive regulation in the formation of retinal barrier. Claudin elements and their interactions can be modulated and that such dynamic properties are important for the functions of TJs, ranging from the regulation of retinal barrier integrity to junction-associated signaling mechanisms.