TLR8-mediated NF-κB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappa B and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappa B and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappa B activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by I kappa B alpha phosphorylation and not I kappa B alpha degradation, whereas interleukin (IL)-1 stimulation causes both I kappa B alpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 ( including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of I kappa B alpha phosphorylation and NF-kappa B and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappa B activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappa B and JNK activation are IRAK- dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated I kappa B alpha phosphorylation, NF-kappa B, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappa B activation are also reflected at the level of I kappa B kinase (IKK) complex. TLR8 ligands induced IKK gamma phosphorylation, whereas IKK alpha/beta phosphorylation and IKK gamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKK gamma phosphorylation might play an important role in the activation of IKK complex, leading to I kappa B alpha phosphorylation.