title: Inhibition of protein kinase C β(2) prevents tumor necrosis factor-α-induced apoptosis and oxidative stress in endothelial cells: the role of NADPH oxidase subunits.
abstract: 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.
authors.affiliation: Department of Cardiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, PR China.
authors.forename: Bingqing
authors.initials: B
authors.lastname: Deng
authors.forename: Shuanglun
authors.initials: S
authors.lastname: Xie
authors.forename: Jingfeng
authors.initials: J
authors.lastname: Wang
authors.forename: Zhengyuan
authors.initials: Z
authors.lastname: Xia
authors.forename: Ruqiong
authors.initials: R
authors.lastname: Nie
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001578:Benzopyrans / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins / Q000502:physiology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000502:physiology; D018384:Oxidative Stress / Q000187:drug effects*; D010750:Phosphoproteins / Q000096:biosynthesis; D010797:Phthalimides / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors; D021122:Protein Subunits / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D010797:Phthalimides; D021122:Protein Subunits; D014409:Tumor Necrosis Factor-alpha; C079789:neutrophil cytosol factor 67K; C056165:acetovanillone; C085746:rottlerin; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051745:Protein Kinase C-delta; C098874:4,5-dianilinophthalimide