Soluble tumor necrosis factor (TNF)-like poor inducer of apoptosis 17-DMAG HCl (Alvespimycin) (TWEAK) in contrast to membrane TWEAK and TNF is only a poor activator of the classical NFκB pathway. pathway using the IKK2 inhibitor TPCA1. In contrast in some cell lines TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924 however which inhibits Tcf4 classical and alternate NFκB signaling blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NFκB signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction but potentiated the ability of soluble TWEAK to trigger production of the classical NFκB-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed comparable induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data show that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NFκB pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets which can be equally well induced by soluble and membrane TWEAK remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies. mRNA induction. (A B) TRAF1 expression was induced in the indicated cell lines by overnight activation with Flag-TWEAK (200?ng/ml) or Flag-TNF (100?ng/ml). Cells were then treated for additional … Soluble TWEAK induces TRAF1 by classical NFκB pathway-dependent and -impartial mechanisms The superior ability of soluble TWEAK compared to TNF to induce TRAF1 as well as the 17-DMAG HCl (Alvespimycin) kinetics of TWEAK-induced TRAF1 expression suggest that classical NFκB pathway-independent mechanisms play here a crucial role. Indeed oligomerization of soluble TWEAK a way to enhance the ability of soluble TWEAK to activate the classical NFκB pathway which however has practically no effect on the activation of the alternative NFκB pathway (15) showed no major enhancing effect on TRAF1 induction (Physique ?(Figure4A).4A). The ability of soluble TWEAK to induce the classical NFκB target IL8 however was strongly enhanced by oligomerization (Physique ?(Physique4B).4B). As on the one hand oligomerization enhances the ability of soluble TWEAK to trigger the classical NFκB pathway and as on the other hand oligomerization 17-DMAG HCl (Alvespimycin) has no effect on the dose response of TWEAK-induced TRAF1 induction the latter seems to be controlled to a significant extent by mechanisms independent from classical NFκB signaling. In line with our previous finding that 17-DMAG HCl (Alvespimycin) oligomerized soluble TWEAK mimics the activity of membrane TWEAK (15 32 we furthermore observed that cells expressing a non-cleavable mutant of membrane TWEAK efficiently trigger IL8 and TRAF1 production while soluble TWEAK generating cells showed strong TRAF1 induction but only very moderate IL8 induction (Figures ?(Figures44C D). Physique 4 Oligomerization of soluble TWEAK results in enhanced induction of IL8 but has no major effect on TRAF1 induction and option NFκB signaling. (A) Cells were stimulated overnight as indicated with Flag-TWEAK and 1?μg/ml of the … We have recently shown that TNF-induced IKK2-mediated activation of the classical NFκB pathway is usually strongly inhibited without a significant effect on TWEAK-induced IKK1-mediated activation of the alternative NFκB 17-DMAG HCl (Alvespimycin) pathway in cells treated with the IKK2-specific inhibitor TPCA1 (33). Under such conditions TNF-induced TRAF1 production was blocked in all cell lines investigated (Physique ?(Figure5A).5A). In some cell lines the minor levels of basal TRAF1 expression were also reduced by treatment with TPCA1. In these cases TPCA1 treatment reduced TNF-induced TRAF1 expression to the level or even below the level of basal expression of the untreated cells (Physique ?(Figure5A).5A). This emphasizes the efficacy of the.