Abstract
Abstract: :
Purpose: Increased occludin phosphorylation in response to VEGF and diabetes is closely linked to increased retinal vascular permeability. The purpose of the current study was to determine if protein kinase C (PKC) is involved in the pathway leading to occludin hyper–phospohorylation in order to understand the molecular mechanism by which PKC regulates vascular permeability. Methods: Primary bovine retinal endothelial cells (BREC) in culture were transfected with expression plasmids for active wild type PKC ß and a dominant negative kinase dead S217A mutant PKC ß. Western blots of whole cell lystaes were used to determine the relative amounts of phosphorylated occludin in cells transfected with PKC expression plasmid vs. mock transfected cells. Phosphorylation of MAPK was also determined because it is a known endpoint of VEGF stimulation in these cells. Results: Treatment of BREC with 50 ng/ml VEGF caused an increase in occludin phosphorylation and induced an increase in permeability of a BREC monolayer to 70 kD RITC–dextran. Overexpression of wt PKC ß in BREC using transient transfection increased occludin phosphorylation by 60% in response to VEGF. The dominant negative PKC ß S217A blocked the increase in occludin phosphorylation in response to VEGF and, in fact, was no different from untreated control cells. Phosphorylation of MAPK, known to be stimulated by VEGF in endothelial cells, showed a similar pattern of response in transfected cells. The results of these transfection experiments support data from previous studies in which a synthetic PKC inhibitor decreased the response of BREC to VEGF stimulation with regard to occludin phosphorylation and permeability. Conclusions: We conclude that PKC is involved in the pathway leading to occludin phoshporylation in response to VEGF in retinal endothelial cells. Furthermore, PKC ß specifically can regulate occludin phosphorylation. These data support a role for PKC in the VEGF regulation of vascular permeability.
Keywords: diabetic retinopathy • signal transduction • phosphorylation