title: Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase.
abstract: An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes.
authors: Research and Development Unit, National Heart Centre Singapore, 7 Hospital Drive SingHealth Research Facilities Block A, #03-05, Singapore, 169611, Singapore, moe.kyaw.thu@nhcs.com.sg.
authors: Kyaw Thu
authors: KT
authors: Moe
authors: Katwadi
authors: K
authors: Khairunnisa
authors: Nwe Oo
authors: NO
authors: Yin
authors: Jaye
authors: J
authors: Chin-Dusting
authors: Philip
authors: P
authors: Wong
authors: Meng Cheong
authors: MC
authors: Wong
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D053583:Interleukin-1beta / Q000235:genetics; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D015854:Up-Regulation; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics
chemical_list: D000098:Acetophenones; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D016328:NF-kappa B; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C007517:diphenyleneiodonium; C056165:acetovanillone; D019255:NADPH Oxidases; D051550:I-kappa B Kinase