CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 74 AFRICA Role of nuclear factor kappa-B in TNF-induced cytoprotection Roisin Kelly-Laubscher, Sarin Somers, Lydia Lacerda, Sandrine Lecour Abstract Ischaemic heart disease (IHD) is a leading cause of death worldwide. Understanding prosurvival signalling pathways that protect against ischaemia–reperfusion injury (IRI) may assist in the development of novel cardioprotective strategies against IHD. In this regard, the transcription factor, nuclear factor kappa-B (NFκB) is activated by tumour necrosis factor (TNF), but its role in TNF-induced cytoprotection is unknown. Therefore, to investigate the role of NFκB in TNF-induced cytoprotection, C2C12 cells were pretreated with TNF (0.5 ng/ml) in the presence and absence of an NFκB inhibitor, pyrrolidine derivative of dithiocarbamate (PDTC; 100 µM). Cells were subjected to simulated IRI and treated with PDTC, either during TNF exposure or at reperfusion. Phosphorylation of IkB was measured after the TNF stimulus. Cytoprotection by TNF in cells subjected to IRI (cell viability: 43.7 ± 8.1% in control vs 70.6 ± 6.1% with TNF, p < 0.001) was abrogated by co-administration of PDTC (40.6 ± 1.9%, p < 0.001 vs TNF) but not by exposure to PDTC at reperfusion (70.7 ± 1.7%). Cytosolic IkB phosphorylation [1.5 ± 0.2 arbitrary units (AU) for TNF vs 1.0 ± 0.0 for untreated, p < 0.01]) was increased after TNF exposure and this increase was abolished by co-administration with PDTC (0.8 ± 0.3 AU, p < 0 01 vs TNF). Our data suggest that NFκB acts as a key component in TNF-induced cytoprotection. These findings may pave the way for the development of novel therapeutic drugs that target TNF/NFκB signalling to protect against IHD. Keywords: TNF, NFκB, ischaemia–reperfusion, preconditioning, cardioprotection Submitted 18/8/20, accepted 10/5/22 Published online 8/6/22 Cardiovasc J Afr 2023; 34: 74–80 www.cvja.co.za DOI: 10.5830/CVJA-2022-023 Ischaemic heart disease (IHD) is the leading cause of death worldwide and its global prevalence rate is on a constant rise, expected to reach more than 1 845 per 100 000 population by the year 2030.1 Although various possible cardioprotective strategies have been pinpointed as promising therapies to protect against ischaemia–reperfusion injury (IRI) in the preclinical setting, very few have translated into the clinical setting in the past 30 years.2 A better understanding of the signalling pathways that may promote cell survival against IRI are key to the successful development of future therapies that may limit the cellular damage associated with the disease. In this regard, the pro-inflammatory cytokine, tumour necrosis factor (TNF), is implicated as a mediator in cardiovascular disease, including acute myocardial infarction,3,4 atherosclerosis,5 chronic heart failure6,7 and ischaemia–reperfusion.8-10. TNF can induce cardiomyocyte apoptosis via signalling through the TNF receptorassociated death domain (TRADD).11 In contrast, TNF is also a key signalling component whose activation is required to promote cell survival in ischaemic preconditioning and postconditioning.12-14. Most importantly, TNF can mimic the protective effect of ischaemic conditioning in a dose- and time-dependent manner.13,15 TNF is traditionally known as a potent activator of nuclear factor kappa-B (NFκB). This transcription factor mediates the protective effects of various forms of ischaemic and pharmacological preconditioning, including classic ischaemic preconditioning,16,17 late ischaemic preconditioning,18,19, remote ischaemic preconditioning20 and erythropoietininduced cardioprotection.20 Despite a clear role for NFκB in cytoprotection induced by other treatments, the role of NFκB in TNF-induced cytoprotection has not been investigated. Inactive NFκB resides in the cytoplasm bound to inhibitory IkB proteins.21 NFκB activation can be initiated through receptor-mediated events such as ligand binding (e.g. TNF, interleukin-1 or lipopolysaccharide) or non-receptor-mediated pathways such as oxidative stress or ultraviolet radiation.22 This causes phosphorylation and activation of the IkB kinases (IKKs) by upstream protein kinases such as Akt. The IKKs, in turn, phosphorylate IkB at its amino terminus, leading to its disassociation from NFκB and subsequent proteasomal degradation by ubiquination.23 NFκB, now in its active form, translocates to the nucleus where it binds to specific recognition sequences and regulates transcription.24 TNF initiates a prosurvival signalling cascade termed the survivor activating-factor enhancement (SAFE) pathway.25 This pathway includes activation of TNF receptor 2 (TNFR2) by TNF, which leads to activation of the signal transducer and activator of transcription 3 (STAT-3) pathways and ultimately inhibits opening of the mitochondrial permeability transition pore to promote cell survival.13,15 Recently, the mitochondrial effects of TNFR2 activation have been suggested to be mediated by activation of the transcription factor, NFκB,26 but the role of NFκB in TNF-induced cytoprotection against IRI is unknown. Hatter Institute for Cardiovascular Research in Africa, Faculty of Health Sciences, University of Cape Town, South Africa; and Department of Pharmacology and Therapeutics, College of Medicine and Health, University College Cork, Ireland Roisin Kelly-Laubscher, PhD, Roisinkelly@ucc.ie Hatter Institute for Cardiovascular Research in Africa, Faculty of Health Sciences, University of Cape Town, South Africa Sarin Somers, PhD, sarin.somers@gmail.com Lydia Lacerda, PhD Sandrine Lecour, PhD
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