Inhibition of Protein Kinase C β2 Prevents Tumor Necrosis Factor-α-Induced Apoptosis and Oxidative Stress in Endothelial Cells: The Role of NADPH Oxidase Subunits

• Published in: Journal of vascular research Vol. 49; no. 2; pp. 144 - 159
• Main Authors: Deng, Bingqing, Xie, Shuanglun, Wang, Jingfeng, Xia, Zhengyuan, Nie, Ruqiong
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland Karger 01.04.2012
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Research Paper; Vertebrates: cardiovascular system; Human umbilical vein endothelial cells; Reactive oxygen species; Protein kinase C β2; NADPH oxidase; Apoptosis; Human; NADPH; Oxidative stress; Endothelial cell; Oxygen; Protein kinase C; Enzyme; Transferases; Cytokine; Oxidase; Subunit; Vertebrata; Mammalia; Oxidoreductases; Circulatory system; Inhibition; Protein kinase C β; Reactive oxygen specie; Umbilical vein; Tumor necrosis factor α
• ISSN: 1018-1172; 1423-0135
• DOI: 10.1159/000332337

We investigate the cell signal transduction pathway protein kinase C (PKC) and the role of NADPH subunits in the process of TNF-α-induced endothelial apoptosis. Human umbilical vein endothelial cells (HUVEC) were treated with one of these: 1 mM PKC β 2 inhibitor CGP53353, 10 mM PKC δ inhibitor rottlerin, combination CGP53353 with rottlerin, 3 ×10 –4 M NADPH oxidase inhibitor apocynin, 5 × 10 –6 M NADPH oxidase peptide inhibitor gp91ds-tat. The apoptosis process was assessed by Hoechst 33342 stain, flow cytometry and Western blot analysis, while intracellular reactive oxygen species (ROS) production was detected by 2,7’-dichlorodihydrofluorescein diacetate (DCFH-DA). The NADPH oxidase subunit gene and protein expression were assessed by quantitative real-time PCR and Western blot analysis, respectively. TNF-α significantly induced HUVEC apoptosis and ROS production, accompanying with dramatic upregulation of NADPH oxidase subunits: NOX2/gp91 phox , NOX4, p47 phox and p67 phox , whereas these enhancements were abolished by the treatment with PKC inhibitors. High TNF-α level exposure induces HUVEC apoptosis, as well as a ROS generation increase via the PKC β 2 -dependent activation of NADPH oxidase. Although the PKC δ pathway may enhance TNF-α-induced HUVEC apoptosis, it does not involve the ROS pathway. Upregulation of expression of NADPH subunits is important in this process, which leads to a new target in antioxidative therapy for vascular disease prevention.