Interleukin-1b stimulates a novel early activation of non-canonical NF Kappa B signalling pathway linked to CXCL12 production.

ID:1557
Introduction
The non-canonical nuclear factor kappa B (NFB) pathway plays an important role in inflammatory and immune responses. Ligands such as lymphotoxin 12 (LT12) mediate an Inhibitory Kappa B kinase α (IKK dependent phosphorylation and processing of NFκB2 p100 which results in nuclear translocation of p52 NFB. The process is further regulated by NF kappa B- inducing kinase (NIK) [1]. The non-canonical pathway has become a target for new drug design directed again certain cancers [2] and we have used novel pharmacological tools to examine the potential of co-regulation by IL-1
Methods
U2OS cells were used throughout this study. Western blotting assessed intermediates of the non-canonical signalling pathway. Induction of CXCL12 was measured by luciferase reporter assay and mRNA levels using qRT-PCR. Statistical analysis was performed by One-way ANOVA with Dunnett’s Post-test (*p<0.05, **p<0.01, ***p<0.001).
Results
LT12 stimulated a delayed activation of p100 phosphorylation (fold stimulation 4 h = 2.96 ± 0.32, n=5) and nuclear accumulation of p52 NFB. Unexpectedly we found that IL-1 generated a more rapid increase in pp100 (fold stimulation 30 minutes: 4.56 ± 0.86, n=5). IKK deletion abolished pp100 phosphorylation in response to IL-1 and LT12. Pre-treatment with SU1261, a novel first-in-class selective inhibitor of IKK, developed in-house, reduced p100 phosphorylation in response to both IL-1and LT12. In contrast, pre-incubation with a NIK inhibitor, CW15337, had no effect however, the response to LT12 was significantly reduced. IL-1 also stimulated a rapid induction of CXCL12 reporter activity. IKK knockdown reduced IL-1 induced reporter activity by approx. 50% (IL-1β + IKKα = 50.74 % ± 5.94 %, n=3). Pre-treatment with SU1261 resulted in a concentration-dependent inhibition of CXCL12 with an IC50 value of 0.42 µM (n=5). Induction of CXCL12 mRNA levels were also studied (fold stimulation: IL-1β 8 h = 5.33 ± 0.83, IL-1β + siRNA IKKα = 2.07 ± 0.40, P<0.05, IL-1β + SU1261 = 1.62 ± 0.53, P<0.001; n=3).
Conclusion (s)
Taken together these results point to a novel kinetically distinct mode of regulation of non-canonical NFB signalling linked to CXCL12 induction which is IKK dependent but NIK independent and mediated by traditional canonical activating ligands such as IL-1.
References (s)
1.Sun, S.C., The non-canonical NF-kappaB pathway in immunity and inflammation. Nat Rev Immunol, 2017. 17(9): p. 545-558.
2.Gamble, C., et al., Inhibitory kappa B Kinases as targets for pharmacological regulation. Br J Pharmacol, 2012. 165(4): p. 802-19.