Protein Kinase Cβ Phosphorylates Occludin Regulating Tight Junction Trafficking in Vascular Endothelial Growth Factor―Induced Permeability In Vivo

• Published in: Diabetes (New York, N.Y.) Vol. 61; no. 6; pp. 1573 - 1583
• Main Authors: MURAKAMI, Tomoaki, FREY, Tiffany, CHENGMAO LIN, ANTONETTI, David A
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01.06.2012
• Subjects: Animals; Biological and medical sciences; Blood-Retinal Barrier - metabolism; Capillary Permeability - drug effects; Cattle; Complications; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Male; Medical sciences; Membrane Proteins - metabolism; Occludin; Phosphorylation - drug effects; Protein Kinase C - metabolism; Protein Kinase C beta; Protein Kinase Inhibitors - pharmacology; Rats; Rats, Sprague-Dawley; Retina - cytology; Retina - drug effects; Retina - metabolism; Tight Junctions - drug effects; Tight Junctions - metabolism; Vascular Endothelial Growth Factor A - pharmacology; Endocrinopathy; In vivo; Vascular endothelium growth factor; Enzyme; Transferases; Diabetes mellitus; Non-specific serine/threonine protein kinase; Cell junction; Tight junction; Permeability
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/db11-1367

Vascular endothelial growth factor (VEGF)-induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.