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Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase

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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.

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Acknowledgments

This work was supported by a grant from SingHealth Research Foundation (SHF/FG436P/2010), Singapore to KTM.

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No conflict of interest to disclose for any of the authors.

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Authors and Affiliations

  1. Research and Development Unit, National Heart Centre Singapore, 7 Hospital Drive SingHealth Research Facilities Block A, #03-05, Singapore, 169611, Singapore

    Kyaw Thu Moe, Katwadi Khairunnisa, Nwe Oo Yin & Philip Wong

  2. Baker IDI Heart and Diabetes Institute, Melbourne, Australia

    Jaye Chin-Dusting

  3. Department of Pharmacology, National University of Singapore, Singapore, Singapore

    Meng Cheong Wong

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  1. Kyaw Thu Moe
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Moe, K.T., Khairunnisa, K., Yin, N.O. et al. Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase. J Physiol Biochem 70, 769–779 (2014). https://doi.org/10.1007/s13105-014-0345-0

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