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

Abstract

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.

Ischemia–Reperfusion Injury Induces Occludin Phosphorylation/Ubiquitination and Retinal Vascular Permeability in a VEGFR-2-Dependent Manner

“…19 Similar events were observed recently in vivo during VEGF-induced BRB breakdown. 14 Collectively, these data suggest that the posttranslational modifications to occludin contribute to alterations in tight junction organization and vascular permeability. Others have observed different occludin phosphorylation events associated with BBB permeability.…”

“…The cells were treated with 50 ng/mL of human recombinant VEGF (R&D systems, Minneapolis, MN, USA) for 15 minutes, then cells were washed with sterile phosphate-buffered saline twice and harvested in lysis buffer containing protease and phosphatase inhibitors as previously described. 14,29 Western Blotting Immediately after euthanization, retinas were excised, carefully cleaned of all vitreous and retinal pigmented epithelium and then placed in immunoprecipitation buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl, 50 mmol/L Tris (pH 7.5), 2 mmol/L N-ethylmaleimide, 1 mmol/L NaVO4, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 1 mmol/L benzamidine, and complete protease inhibitor cocktail) as previously described. 14 Retinas were gently sonicated followed by 20-minute incubation at 41C and a 10-minute centrifugation at 16,000 g and 41C to sediment insoluble material.…”

“…Endothelial Growth Factor Receptor-2 Activation Site Because occludin Ser490 phosphorylation and ubiquitination, as observed after retinal IR injury, are also observed after VEGF treatment both in vitro and in vivo, 14,19 we examined the possibility that IR leads to activation of the VEGF receptor VEGFR-2. Lysates from sham and IR retinas were immunoblotted with antibodies specific to VEGFR-2 phosphorylated at tyrosine 1175, (pTyr1175), a modification that is indicative of VEGFR-2 activation after VEGF binding.…”

Blood–brain barrier breakdown promotes macrophage infiltration and cognitive impairment in leptin receptor-deficient mice

“…On one hand, hyperglycemia increases retinal cerebrovascular permeability by activating PKCb. 27,45,46 This robust and widely replicated finding led to initiation of clinical trials to determine whether ruboxistaurin, a related PKCb inhibitor, might treat or delay the progression of diabetic retinopathy in humans. 47 On the other hand, nondiabetic obesity promotes cerebrovascular inflammation, leading to vascular adherence by circulating immune cells.…”

Association between cystoid spaces on indocyanine green hyperfluorescence and optical coherence tomography after vitrectomy for diabetic macular oedema

“…Trypsin digestion preparation showed that, in addition to pericyte loss, the endothelial cells also disappeared, the so-called acellular capillaries in patients with diabetic retinopathy; 9 experimentally, the intercellular junctions where the tight junction and adherens junction are highly integrated are disrupted in VEGF-treated or diabetic rodents, 10,11 suggesting the diversity of the pathogenesis in BRB breakdown. 12 In addition, histologic studies have reported that cystoid spaces in eyes with macular oedema are the primary structural changes in the retinal parenchyma.…”