IL-1β stimulates a novel, IKKα-dependent, NIK-independent activation of non-canonical NFκB signalling

In this study, we examined the activation of non-canonical nuclear factor Kappa B (NF kappa B) signalling in U2OS cells, a cellular metastatic bone cancer model. Whilst Lymphotoxin alpha 1 beta 2 (LTa1 beta(2)) stimulated the expected slow, delayed, sustained activation of serine 866/870 p100 phosphorylation and increased cellular expression of p52 NF kappa B, we found that canonical agonists, Interleukin-1 beta (IL-1 beta) and also Tumour necrosis factor-alpha (TNF alpha) generated a rapid transient increase in pp100, which was maximal by 15-30 min. This rapid phosphorylation was also observed in other cells types, such as DU145 and HCAECs suggesting the phenomenon is universal. IKKa deletion using CRISPR/Cas9 revealed an IKKa-dependent mechanism for serine 866/870 and additionally serine 872 p100 phosphorylation for both IL-1 beta and LT alpha(1)beta(2). In contrast, knockdown of IKK beta using siRNA or pharmacological inhibition of IKK beta activity was without effect on p100 phosphorylation. Pre-incubation of cells with the NF kappa B inducing-kinase (NIK) inhibitor, CW15337, had no effect on IL-1 beta induced phosphorylation of p100 however, the response to LT alpha(1)beta(2) was virtually abolished. Surprisingly IL-1 beta also stimulated p52 nuclear translocation as early as 60 min, this response and the concomitant p65 translocation was partially reduced by IKK alpha deletion. Furthermore, p52 nuclear translocation was unaffected by CW15337. In contrast, the response to LT alpha(1)beta(2) was essentially abolished by both IKKa deletion and CW15337. Taken together, these finding reveal novel forms of NF kappa B non-canonical signalling stimulated by ligands that activate the canonical NF kappa B pathway strongly such as IL-1 beta.