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

ABSTRACT

In this study, we examined the activation of non-canonical nuclear factor Kappa B (NFκB) signalling in U2OS cells, a cellular metastatic bone cancer model. Whilst Lymphotoxin α1ß2 (LTα1ß2) stimulated the expected slow, delayed, sustained activation of serine 866/870 p100 phosphorylation and increased cellular expression of p52 NFκB, we found that canonical agonists, Interleukin-1ß (IL-1ß) and also Tumour necrosis factor-α (TNFα) 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. IKKα deletion using CRISPR/Cas9 revealed an IKKα-dependent mechanism for serine 866/870 and additionally serine 872 p100 phosphorylation for both IL-1ß and LTα1ß2. In contrast, knockdown of IKKß using siRNA or pharmacological inhibition of IKKß activity was without effect on p100 phosphorylation. Pre-incubation of cells with the NFκB inducing-kinase (NIK) inhibitor, CW15337, had no effect on IL-1ß induced phosphorylation of p100 however, the response to LTα1ß2 was virtually abolished. Surprisingly IL-1ß also stimulated p52 nuclear translocation as early as 60 min, this response and the concomitant p65 translocation was partially reduced by IKKα deletion. Furthermore, p52 nuclear translocation was unaffected by CW15337. In contrast, the response to LTα1ß2 was essentially abolished by both IKKα deletion and CW15337. Taken together, these finding reveal novel forms of NFκB non-canonical signalling stimulated by ligands that activate the canonical NFκB pathway strongly such as IL-1ß.