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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

The two NFκB signaling pathways are of varying cell type-specific relevance for TWEAK-induced TRAF1 expression. (A) Cells were stimulated overnight with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml) and total cell lysates were analyzed by western blotting for NIK accumulation, p100 processing and expression of TRAF1, where indicated cells were pretreated for 30 min with 10 μM TPCA1. (B) Control vector and IκBα-SR transduced A172 cells were stimulated overnight with increasing concentrations of Flag-TWEAK and were assayed by western blotting of total cell extracts for TRAF1 production and activation of the alternative NFκB pathway (left panel). Efficacy of IκBα-SR-mediated repression of the classical NFκB pathway was proved by analyzing phosphorylation and degradation of IκBα in cells stimulated with TNF (100 ng/ml) (right panel). (C) The indicated cell lines were pretreated or not with 10 μM MLN4924 for 30 min and then stimulated with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml). The next day, total cell lysates were prepared and analyzed for the presence of the indicated proteins by western blotting.
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Figure 5: The two NFκB signaling pathways are of varying cell type-specific relevance for TWEAK-induced TRAF1 expression. (A) Cells were stimulated overnight with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml) and total cell lysates were analyzed by western blotting for NIK accumulation, p100 processing and expression of TRAF1, where indicated cells were pretreated for 30 min with 10 μM TPCA1. (B) Control vector and IκBα-SR transduced A172 cells were stimulated overnight with increasing concentrations of Flag-TWEAK and were assayed by western blotting of total cell extracts for TRAF1 production and activation of the alternative NFκB pathway (left panel). Efficacy of IκBα-SR-mediated repression of the classical NFκB pathway was proved by analyzing phosphorylation and degradation of IκBα in cells stimulated with TNF (100 ng/ml) (right panel). (C) The indicated cell lines were pretreated or not with 10 μM MLN4924 for 30 min and then stimulated with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml). The next day, total cell lysates were prepared and analyzed for the presence of the indicated proteins by western blotting.

Mentions: We have recently shown that TNF-induced IKK2-mediated activation of the classical NFκB pathway is strongly inhibited without a significant effect on TWEAK-induced IKK1-mediated activation of the alternative NFκB 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 (Figure 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 (Figure 5A). This emphasizes the efficacy of the inhibitory effect of TPCA1 and indicates that basal TRAF1 expression is at least partly maintained in some cell lines by weak constitutive classical NFκB signaling. The effect of TPCA1 on TWEAK-induced TRAF1 production, however, varied dependent on the cell line considered. TWEAK-induced TRAF1 expression was fully inhibited in TPCA1-treated A172 cells and likewise in IκBα-SR expressing A172 transfectants (Figures 5A,B). However, in the other cell lines investigated there was only partial inhibition of TWEAK-induced TRAF1 expression by TPCA1 (Figure 5A). This also argues for a capability of TWEAK to induce TRAF1 by classical NFκB pathway-independent mechanisms. Moreover, in all cell lines analyzed, including those where soluble TWEAK induces significant TRAF1 expression in the presence of TPCA1, the NEDD8-activating enzyme (NAE)-inhibitor MLN4924 (34), which interferes with IκBα degradation and p100 processing (33), inhibited upregulation of TRAF1 (Figure 5C). Although, MLN4924 also targets other signaling pathways and the cell cycle, this points to an important role of the alternative NFκB pathway in soluble TWEAK-induced TRAF1 expression.


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)

The two NFκB signaling pathways are of varying cell type-specific relevance for TWEAK-induced TRAF1 expression. (A) Cells were stimulated overnight with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml) and total cell lysates were analyzed by western blotting for NIK accumulation, p100 processing and expression of TRAF1, where indicated cells were pretreated for 30 min with 10 μM TPCA1. (B) Control vector and IκBα-SR transduced A172 cells were stimulated overnight with increasing concentrations of Flag-TWEAK and were assayed by western blotting of total cell extracts for TRAF1 production and activation of the alternative NFκB pathway (left panel). Efficacy of IκBα-SR-mediated repression of the classical NFκB pathway was proved by analyzing phosphorylation and degradation of IκBα in cells stimulated with TNF (100 ng/ml) (right panel). (C) The indicated cell lines were pretreated or not with 10 μM MLN4924 for 30 min and then stimulated with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml). The next day, total cell lysates were prepared and analyzed for the presence of the indicated proteins by western blotting.
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Figure 5: The two NFκB signaling pathways are of varying cell type-specific relevance for TWEAK-induced TRAF1 expression. (A) Cells were stimulated overnight with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml) and total cell lysates were analyzed by western blotting for NIK accumulation, p100 processing and expression of TRAF1, where indicated cells were pretreated for 30 min with 10 μM TPCA1. (B) Control vector and IκBα-SR transduced A172 cells were stimulated overnight with increasing concentrations of Flag-TWEAK and were assayed by western blotting of total cell extracts for TRAF1 production and activation of the alternative NFκB pathway (left panel). Efficacy of IκBα-SR-mediated repression of the classical NFκB pathway was proved by analyzing phosphorylation and degradation of IκBα in cells stimulated with TNF (100 ng/ml) (right panel). (C) The indicated cell lines were pretreated or not with 10 μM MLN4924 for 30 min and then stimulated with Flag-TWEAK (200 ng/ml) and Flag-TNF (100 ng/ml). The next day, total cell lysates were prepared and analyzed for the presence of the indicated proteins by western blotting.
Mentions: We have recently shown that TNF-induced IKK2-mediated activation of the classical NFκB pathway is strongly inhibited without a significant effect on TWEAK-induced IKK1-mediated activation of the alternative NFκB 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 (Figure 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 (Figure 5A). This emphasizes the efficacy of the inhibitory effect of TPCA1 and indicates that basal TRAF1 expression is at least partly maintained in some cell lines by weak constitutive classical NFκB signaling. The effect of TPCA1 on TWEAK-induced TRAF1 production, however, varied dependent on the cell line considered. TWEAK-induced TRAF1 expression was fully inhibited in TPCA1-treated A172 cells and likewise in IκBα-SR expressing A172 transfectants (Figures 5A,B). However, in the other cell lines investigated there was only partial inhibition of TWEAK-induced TRAF1 expression by TPCA1 (Figure 5A). This also argues for a capability of TWEAK to induce TRAF1 by classical NFκB pathway-independent mechanisms. Moreover, in all cell lines analyzed, including those where soluble TWEAK induces significant TRAF1 expression in the presence of TPCA1, the NEDD8-activating enzyme (NAE)-inhibitor MLN4924 (34), which interferes with IκBα degradation and p100 processing (33), inhibited upregulation of TRAF1 (Figure 5C). Although, MLN4924 also targets other signaling pathways and the cell cycle, this points to an important role of the alternative NFκB pathway in soluble TWEAK-induced TRAF1 expression.

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