TAK1 is an upstream MAP 3 K regulator of a novel non-canonical NFkB pathway stimulated by IL-1b

There are two well-established NFκB signalling pathways known as the canonical and non-canonical NFκB signalling cascades. The non-canonical NFκB pathway, stimulated by TNFα superfamily members, involves a NIK- and IKKα-dependent phosphorylation of p100 and subsequent proteasomal degradation. Previous research within the laboratory has identified a novel axis within the non-canonical pathway which involves IKKα-dependent and NIK-independent phosphorylation of p100 without subsequent degradation. To further identify the intermediates upstream of this axis, we have examined the role of the MAP 3 K TAK1. SDS-PAGE and western blotting was applied in two cell types, a human Osteosarcoma cell line, U2OS, and human umbilical vein endothelial cells (HUVECs) using phospho- and total antibodies. Results demonstrated that IL-1β stimulated an increase in p100 phosphorylation which was maximal at 30–60 minutes (fold stim at 60 minutes = 12.1 ± 0.9, ****p < .0001, n = 3). In IKKα CRISPR knockdown U2OS cells, IL-1β-mediated p100 phosphorylation was significantly reduced (fold stim at 60 minutes = wildtype 10.07 ± 0.48, knockdown 2.49 ± 1.73, **p < .01, n = 3). To assess if Transforming growth factor β-activated kinase 1 (TAK1), a member of the MAP 3 K family, is an upstream kinase to IKKα in response to IL-1β stimulation, U2OS cells were pre-treated with increasing concentrations (1-20 μM) of a selective TAK1 inhibitor, 5Z-7-oxozeaenol. IL-1β-mediated p100 phosphorylation was significantly reduced (fold stim: IL-1β = 6.9 ± 0.95, IL-1β + 10 μM = 1.28 ± 0.3, ***p < .001, n = 4). Further studies demonstrated IL-1β stimulated IKKα phosphorylation by using a mixed phospho-IKKα/ IKKβ antibody and identifying the 85 kDa IKKα band. Pre-treatment with 5Z-7-oxozeaenol (1-20 μM) strongly inhibited both IKKα and IKKβ phosphorylation (fold stim: IL-1β-mediated pIKKα/pIKKβ = 23.76 ± 5.31/94.54 ± 6.33, pIKKα/pIKKβ at IL-1β + 10 μM = 1.37 ± 0.23, ***p < .001/2.4 ± 0.81, ****p < .0001, n = 3). Similar effects of TAK1 inhibition were observed in HUVECs. Finally, we also demonstrate 5Z- 7-oxozeaenol pre-treatment substantially reduced IL-1β stimulation of p52 and p65 accumulation in the nucleus as assessed by nuclear extracts (fold stim: IL-1β-mediated p52/p65 = 1.52 ± 0.42/1.45 ± 0.13, p52/p65 at IL-1β + 10 μM = 0.73 ± 0.16, **p < .01/0.22 ± 0.03, ****p < .0001, n = 3). We have demonstrated for the first time that TAK1 is an equivalent MAP 3 K that can mediate IKKα activity and phosphorylation of p100 in response to IL-1β. Furthermore, this study gives merit into pharmacological targeting of this pathway in several inflammatory-based diseases and cancers.