Resumen de Tumor necrosis factor-?-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase
Kyaw Thu Moe, Katwadi Khairunnisa, Nwe Oo Yin, Jaye Chin-Dusting, Philip Wong, Meng Cheong Wong
An elevated level of tumor necrosis factor (TNF)-? is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-? 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-? and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-?-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-? with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-?-induced ROS production was measured using 5-(and-6)-chloromethyl-2�, 7�-dichlorodihydrofluorescein diacetate assay. TNF-?-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-?-induced upregulation of interleukin (IL)-1? and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-?-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-?/? and p65, degradation of IkappaB?, binding of NF-kappaB to its binding motif, and upregulation of IL-1? and VCAM-1 induced by TNF-? were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-?-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1? and VCAM-1, in human cardiomyocytes.
