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TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK.

Carmona Arana JA, Seher A, Neumann M, Lang I, Siegmund D, Wajant H - Front Immunol (2014)

Bottom Line: We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity.These data indicate 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg , Würzburg , Germany.

ABSTRACT
Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB 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 classical and alternative 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 similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate 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.

No MeSH data available.


Related in: MedlinePlus

Soluble TWEAK is superior to TNF in TRAF1 induction. (A,B) Cells were either stimulated overnight with the indicated concentrations of Flag-TWEAK and Flag-TNF (A) or were incubated with a constant amount of these cytokines (100 ng/ml Flag-TNF, 200 ng/ml Flag-TWEAK) for varying times (B). Total cell lysates were finally analyzed by western blotting with respect to the indicated proteins.
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Figure 2: Soluble TWEAK is superior to TNF in TRAF1 induction. (A,B) Cells were either stimulated overnight with the indicated concentrations of Flag-TWEAK and Flag-TNF (A) or were incubated with a constant amount of these cytokines (100 ng/ml Flag-TNF, 200 ng/ml Flag-TWEAK) for varying times (B). Total cell lysates were finally analyzed by western blotting with respect to the indicated proteins.

Mentions: To rule out that the observed limited effects of soluble TWEAK were related to a general poor TWEAK responsiveness of the cell lines investigated or low specific activity of the soluble TWEAK batch used for stimulation, we also assayed activation of the alternative NFκB pathway. Indeed, already at low concentrations soluble TWEAK triggered accumulation of the NFκB inducing kinase (NIK) and processing of p100, two events indicative for stimulation of the alternative NFκB pathway (23), while TNF had no effect in this regard (Figures 2A,B). We also analyzed induction of TRAF1, a well-recognized NFκB-regulated target of TNF (24–26). According to the literature, there was only weak expression of TRAF1 in unstimulated cells, but TRAF1 expression was readily induced by treatment with TNF. Surprisingly, however, soluble TWEAK was as efficient as, or even superior to TNF in TRAF1 induction (Figures 2A,B; Figure S1 in Supplementary Material). Although, soluble TWEAK-induced TRAF1 expression with somewhat slower kinetics than TNF, the maximal TRAF1 levels reached were regularly significantly higher (Figures 2A,B). Noteworthy, soluble TWEAK-induced TRAF1 production occurred with some delay with respect to NIK accumulation and p100 processing. (Figures 2A,B). NIK and IKK1 can also crosstalk into the classical NFκB pathway, e.g., at the level of IκBα phosphorylation (27). We thus re-analyzed phosphorylation of IκBα in the samples of, in comparison to Figure 1A, the extended time course used for evaluation of TRAF1 induction and activation of the alternative NFκB pathway. Indeed, there was now significant phosphorylation of IκBα in soluble TWEAK-treated cells (Figure 2B). However, analysis of total IκBα levels showed no major changes in the amount of this molecule and there was only a minor fraction of the slower migrating phosphorylated IκBα species. As the gene encoding IκBα itself is a bona fide target of the classical NFκB pathway, this argues for weak but persistent stimulation of the downstream steps of the classical NFκB pathway resulting in a balance between degradation and resynthesis of IκBα. However, as soluble TWEAK, in contrast to TNF, failed to trigger robust production of IL8 (Figure 1B), this hardly explains the superior induction of TRAF1 by soluble TWEAK.


TNF Receptor-Associated Factor 1 is a Major Target of Soluble TWEAK.

Carmona Arana JA, Seher A, Neumann M, Lang I, Siegmund D, Wajant H - Front Immunol (2014)

Soluble TWEAK is superior to TNF in TRAF1 induction. (A,B) Cells were either stimulated overnight with the indicated concentrations of Flag-TWEAK and Flag-TNF (A) or were incubated with a constant amount of these cytokines (100 ng/ml Flag-TNF, 200 ng/ml Flag-TWEAK) for varying times (B). Total cell lysates were finally analyzed by western blotting with respect to the indicated proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927163&req=5

Figure 2: Soluble TWEAK is superior to TNF in TRAF1 induction. (A,B) Cells were either stimulated overnight with the indicated concentrations of Flag-TWEAK and Flag-TNF (A) or were incubated with a constant amount of these cytokines (100 ng/ml Flag-TNF, 200 ng/ml Flag-TWEAK) for varying times (B). Total cell lysates were finally analyzed by western blotting with respect to the indicated proteins.
Mentions: To rule out that the observed limited effects of soluble TWEAK were related to a general poor TWEAK responsiveness of the cell lines investigated or low specific activity of the soluble TWEAK batch used for stimulation, we also assayed activation of the alternative NFκB pathway. Indeed, already at low concentrations soluble TWEAK triggered accumulation of the NFκB inducing kinase (NIK) and processing of p100, two events indicative for stimulation of the alternative NFκB pathway (23), while TNF had no effect in this regard (Figures 2A,B). We also analyzed induction of TRAF1, a well-recognized NFκB-regulated target of TNF (24–26). According to the literature, there was only weak expression of TRAF1 in unstimulated cells, but TRAF1 expression was readily induced by treatment with TNF. Surprisingly, however, soluble TWEAK was as efficient as, or even superior to TNF in TRAF1 induction (Figures 2A,B; Figure S1 in Supplementary Material). Although, soluble TWEAK-induced TRAF1 expression with somewhat slower kinetics than TNF, the maximal TRAF1 levels reached were regularly significantly higher (Figures 2A,B). Noteworthy, soluble TWEAK-induced TRAF1 production occurred with some delay with respect to NIK accumulation and p100 processing. (Figures 2A,B). NIK and IKK1 can also crosstalk into the classical NFκB pathway, e.g., at the level of IκBα phosphorylation (27). We thus re-analyzed phosphorylation of IκBα in the samples of, in comparison to Figure 1A, the extended time course used for evaluation of TRAF1 induction and activation of the alternative NFκB pathway. Indeed, there was now significant phosphorylation of IκBα in soluble TWEAK-treated cells (Figure 2B). However, analysis of total IκBα levels showed no major changes in the amount of this molecule and there was only a minor fraction of the slower migrating phosphorylated IκBα species. As the gene encoding IκBα itself is a bona fide target of the classical NFκB pathway, this argues for weak but persistent stimulation of the downstream steps of the classical NFκB pathway resulting in a balance between degradation and resynthesis of IκBα. However, as soluble TWEAK, in contrast to TNF, failed to trigger robust production of IL8 (Figure 1B), this hardly explains the superior induction of TRAF1 by soluble TWEAK.

Bottom Line: We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity.These data indicate 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg , Würzburg , Germany.

ABSTRACT
Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NFκB pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NFκB-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NFκB 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 classical and alternative 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 similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate 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.

No MeSH data available.


Related in: MedlinePlus