Abstract
Purpose: :
Vascular endothelial growth factor (VEGF) increases in diabetic retinopathy (DR) and contributes to vascular permeability. VEGF phosphorylation of the tight juction (TJ) protein occludin is associated with endothelial permeability and previous work has mapped 5 occludin phosphorylation sites using MALDI-TOF mass spectrometry. Serine 490, one of the identified phosphosites, is located on the occludin carboxy terminus within a coiled-coil domain and conforms to a PKCζ phosphorylation consensus motif. Phospho-Ser490 specific antibodies reveal a site specific phosphorylation in response to VEGF. We hypothesize that occludin serine 490 phosphorylation regulates endothelial permeability.
Methods: :
Site directed mutagenesis was used to mutate Ser 490 to alanine (S490A) to block phosphorylation or to aspartic acid (S490D) to mimic phosphorylation in the expression vector pUB6A. Primary bovine retinal endothelial cells (BREC) in culture were cotransfected with PKCζ and occludin plasmids and were plated onto transwell filters. After 24 hours of confluence samples were treated with 50 ng/ml VEGF for 15 min before permeability measures using 70kDa RITC-dextran and compared to untreated controls. In separate experiments, cells were harvested for Western blotting.
Results: :
Transfection of PKCζ had no effect on basal permeability but augmented VEGF induced permeability. Overexpression of PKCζ led to a rapid loss of occludin content in the samples treated with VEGF (15min). Transfection of the occludin S490A mutant blocked the VEGF induced increase in permeability while the S490D mutant increased basal permeability similar to the effect of VEGF. VEGF had no additional effect on S490D transfected BREC.
Conclusions: :
These results demonstrate that phosphorylation of occludin at S490 contributes to VEGF induced endothelial permeability. The ability of S490D to increase permeability suggests that phosphorylation of occludin directly effects barrier properties. The mechanisms by which single occludin point mutations control permeability are currently being investigated. These data also point to PKCζ as a potential therapeutic target to treat endothelial permeability for diseases such as DR.
Keywords: diabetic retinopathy • cell adhesions/cell junctions • protein modifications-post translational