Abstract
Purpose: :
In diabetic retinopathy (RD) alteration of the outer retinal barrier, ensured by the tight-junctions of pigment epithelial cells (RPE), remain poorly understood. The PKCζ plays a key role in the formation and maintenance of TJ through its association with junctional protein such as PAR3 and PAR6 and the mechanisms that underline its role in retinal barriers integrity remain largely unknown. The aim of this study was to evaluate the role of PKCζ on RPE cell barrier disruption. PKCζ is activated in excess during early phase of diabetes. We studied the expression and localization of its endogenous inhibitor PAR4. This protein could be a candidate of therapeutic target preventing deleterious prolonged PKCζ activation and may offer new tools to inhibit early phase of RD.
Methods: :
To investigate in vivo the effects of diabetes on the hemato-retinal barrier: RPE, we used two diabetic rat models: the streptozotocin induced type 1 diabetes by one intraperitoneal injection of 60mg/kg and rats Goto-Kakizaki type 2. Structural and biochemical analysis were performed by immunohistochemical studies on RPE tissue and by western blotting studies on laser microdissected RPE tissue, using PKCζ, occludin, ZO1, PAR4 antibodies and Phalloidin.
Results: :
The breakdown of outer blood retinal barrier formed by the RPE cell tight junctions was observed in diabetic condition with double staining using anti PKCζ and anti occludin antibodies. These proteins colocalize, particularly in the loops depicted by occludin protrusion in control while the staining is discontinuous and punctiform in open junctions of the diabetic RPE. Interestingly in these RPE cellular junctions, Par-4 expression was clearly demonstrated and colocalize with PKCζ activated form. Furthermore, its subcellular localization and its expression was significantly modified in diabetic conditions associated with actin remodelling.
Conclusions: :
Our results point out a structural role of PKCζ, rather than its enzyme activity, in RPE cellular junction complexes. In control, expression and localization of PKCζ endogenous inhibitor Par-4 may prevent its deleterious excessive activation observed during early phase of diabetes. Exploring the mechanisms that are involved in turning on PKCζbeyond its normal levels or normal activity is proving to be a key area for new diabetes research to develope therapeutic strategy.
Keywords: cell adhesions/cell junctions • diabetic retinopathy • retinal pigment epithelium