Tumor Necrosis Factor-α in Diabetic Plasma Increases the Activity of Core 2 GlcNAc-T and Adherence of Human Leukocytes to Retinal Endothelial Cells

Abstract

A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme core 2 β 1,6-N-acetylglucosaminyltransferase (GlcNAc-T) through protein kinase C (PKC)β2-dependent phosphorylation plays a fundamental role in increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. In the present study, we demonstrate that following exposure to plasma from diabetic patients, the human promonocytic cell line U937 exhibits a significant elevation in core 2 GlcNAc-T activity and increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by PKCβ2-dependent phosphorylation of the core 2 GlcNAc-T protein, were found to be triggered by increased plasma levels of tumor necrosis factor (TNF)-α. Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of diabetic retinopathy, with the highest values observed upon treatment with plasma of patients affected by proliferative retinopathy. Furthermore, we noted much higher correlation, as compared with control subjects, between increased values of core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-α in the development of diabetic retinopathy, these observations further validate the significance of core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.