Abstract
Purpose: :
Our objective is to better understand the mechanisms which modulate vascular permeability in health and disease. Recent research has begun to define the molecular mechanisms in diabetic retinopathy which lead to enhanced vascular permeability. VEGF is elevated in the eye in diabetes and acts through protein kinase C to cause the phosphorylation of a transmembrane tight junction protein, occludin, on multiple sites. Preventing this phosphorylation with PKC inhibitors reduces VEGF induced vascular permeability. Therefore, mapping these phosphorylation sites and identifying their functional relevance is a critical step in understanding the pathophysiology of increased vascular permeability in diabetic retinopathy.
Methods: :
Bovine retinal endothelial cells (BREC) were treated with VEGF for 15min, occludin was immunoprecipitated and separated by SDS–PAGE. Occludin bands were excised, digested with trypsin, and analyzed by MALDI TOF mass spectrometry to identify sites of occludin phosphorylation. Phosphopeptides were identified based on mass accuracy, repeat observations, identification in multiple overlapping fragments, presence in VEGF treated and gel shifted (hyperphosphorylated) occludin bands.
Results: :
Six putative phosphopeptides were identified within occludin. This data led to the successful creation of a phospho–specific antibody to phospho–Ser490. Immunoblotting experiments have confirmed that Ser490 is phosphorylated in vivo and that VEGF stimulation of BREC increases Ser490 phosphorylation. Moreover, substitution of aspartate for serine at residue 490 alters occludin–ZO–1 binding as determined by pull–down assay.
Conclusions: :
To our knowledge, this is the first demonstration of site specific occludin phosphorylation within cells. Ser490 is located within the cytoplasmic coiled–coil domain of occludin known to interact with ZO–1 and phosphorylation mimics of Ser490 showed altered binding to ZO–1. These studies provide a possible mechanism by which VEGF regulates tight junction architecture and vascular permeability. Future experiments, including mutational analysis, will identify the functional significance of occludin phosphorlation and altered interaction with ZO–1.
Keywords: diabetic retinopathy • vascular cells • signal transduction