The Effect of High Glucose on PKC Isozyme Translocation and VEGF Expression in Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: To determine the effect of high glucose on protein kinase C (PKC) isozyme content and subcellular distribution, and vascular endothelial growth factor (VEGF) expression in the rat retinal pigment epithelial (RPE) cell type. Methods: Primary cultured RPE cells (T4-T8) were growth-arrested in normal (5.6 mM) and high (30 mM) glucose conditions. Western blot analysis of total cell lysates, cytosol, membrane and particulate fractions, and confocal immunofluorescent microscopy were used to analyze the total cell content and subcellular distribution of PKC isozymes, and total content of VEGF expression. ELISA was also used to analyze secreted VEGF. Results: Rat RPE cells expressed PKC-α, -ß₁, -δ, -ϵ, and -ζ isozymes, but not PKC-ß₂ or -γ. Phorbol ester (PMA) stimulation caused translocation of PKC-α, -ß₁, -δ, and -ϵ to the membrane fraction, and PKC-ζ did not translocate. Chronic PMA stimulation for 24h caused the disappearance of all PKC isozymes in all fractions, except PKC-ζ. Cells exposed to high glucose secreted VEGF as early as 3 h. VEGF secretion was inhibited by chronic PMA exposure. In high glucose, VEGF enhanced translocation of PKC-α, -ß₁, and -ζ to the membrane fraction and PKC-α, -ß₁, -δ, and -ϵ to the particulate fraction. Conclusions: In high glucose, DAG-sensitive PKC isozymes may be involved in VEGF expression in the RPE cell type. High glucose enhances the PKC isozyme translocation response to VEGF. Abnormal RPE cell phenotype in diabetes may involve high glucose-induced and VEGF-mediated signaling through PKC pathways and subsequent increases in VEGF expression.

Keywords: 388 diabetic retinopathy • 567 retinal pigment epithelium • 476 molecular biology