IL-1β stimulates a novel axis within the NFκB pathway in endothelial cells regulated by IKKα and TAK-1

In this study we examined the activation of the non-canonical NF kappa B signalling pathway in endothelial cells. In HUVECs, LIGHT stimulated a delayed induction of serine 866/870 p100 phosphorylation linked to p52 NF kappa B formation. Surprisingly, the canonical ligand, IL-1 beta, stimulated a rapid phosphorylation or p100 which was not associated with p52 formation. Inhibition of IKK alpha activity, using DN-IKK alpha adenovirus, IKK alpha siRNA or a novel first-in-class selective IKK alpha inhibitor, SU1261, revealed IL-1 beta induced p100 phosphorylation to be dependent on IKK alpha. In contrast, IKK beta inhibition was found to be without effect. The NIK inhibitor, CW15337, did not affect IL1 beta induced p100 phosphorylation however, both p100 and pIKK alpha/ beta phosphorylation was substantially reduced by inhibition of the upstream kinase TAK-1, suggesting phosphorylation of p100 is mediated by IKK alpha from within the canonical NEMO/IKK beta /IKK alpha complex. IL-1 beta also stimulated a rapid increase in nuclear translocation of p52, which was not affected by NIK inhibition, suggesting a source of p52 independent of p100 processing. Inhibition of TAK-1 abolished p52 and p65 nuclear translocation in response to IL-1 beta. SiRNA deletion or inhibition with dominant-negative virus of IKK alpha activity partially reduced p52 translocation, however pharmacological inhibition of IKK alpha was without effect. Inhibition of IKK beta abolished both p52 and p65 translocation. Taken together these results show that IL-1 beta stimulates a novel IKK alpha -dependent axis within the non-canonical NF kappa B pathway in endothelial cells which is NIK-independent and regulated by TAK-1. However, this pathway is not primarily responsible for the early nuclear translocation of p52, which is dependent on IKK beta. Elucidation of both these new pathways may be significant for NF kappa B biology within the endothelium.