Proteomic analysis identifies nuclear protein effectors in PKC-δ signaling under high glucose-induced apoptosis in human umbilical vein endothelial cells

Abstract

Although experimental animal and clinical trials have suggested that additional mechanisms other than protein kinase C (PKC)-β activation are involved in the vascular pathology of diabetic complications, current knowledge on the role of PKC-delta is incomplete and inconclusive. Human umbilical vein endothelial cells (HUVECs) were cultured in both high and normal glucose conditions and infected with recombinant adenovirus to overexpress PKC-delta. PKC-delta expression was also down-regulated using the PKC-delta inhibitor, rottlerin. Using flow cytometric analysis, we showed that PKC-delta is activated and translocates to the nucleus under high glucose conditions. Augmented cell apoptosis and cell cycle arrest were observed in a PKC-delta-dependent manner in the HUVECs. Furthermore, proteomic analyses identified 51 high glucose-induced and PKC-delta-associated proteins, and subsequent matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis uncovered a total of 37 unique proteins. The majority of identified proteins were previously unknown targets of PKC-delta signaling and were involved in the regulation of the cell cycle and apoptosis, tumor suppression, transcription, stress and signal transduction within the nucleus. Our data show that PKC-delta is an important mediator of cell apoptosis and cell cycle arrest in HUVECs under high glucose stress.