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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

TRAF1 expression interferes with CD40-induced signaling. (A) U2OS and 786-O cells with and without TWEAK priming (200 ng/ml, 6 h) and U2OS-TRAF1 and 786-O-TRAF1 transfectants were stimulated in 96-well plates in triplicates with the indicated concentrations of Fc–Flag-CD40L. Next day, supernatants were assayed for production of IL8 or IL6 by ELISA. Prior stimulation cell culture medium was replaced to reduce the background caused by constitutive cytokine production. (B) U2-OS and 786-O cells with and without TWEAK priming and TRAF1 expressing U2-OS and 786-O transfectants were stimulated with Fc–Flag-CD40L for 5 and 15 min and were finally analyzed by western blotting to detect the indicated molecules. Please note, in the case of the TRAF1 western blots a relatively short exposure time is shown to ensure reasonable visibility of overexpressed and TWEAK-induced TRAF1. (C) 786-O and U2-OS cells and their corresponding TRAF1 transfectants were primed overnight with soluble TWEAK or remained untreated and were then analyzed by FACS for CD40 cell surface expression. (D) Cells (2 × 105 cells/well) were seeded in 24-well plates. Next day, half of the samples of each cell type were pretreated for 1 h at 37°C with 2 μg/ml of Fc–Flag-CD40L. Next, untreated and Fc–Flag-CD40L pretreated cells were incubated pairwise with increasing concentrations of GpL–Fc–Flag-CD40L (1 h, 37°C), a fusion protein of Fc–Flag-CD40L with the luciferase from Gaussia princeps. After removal of unbound CD40L molecules, cells were scratched in 50 μl medium to measure cell-associated luciferase activity. Specific binding values were obtained by subtraction of the non-specific binding values derived of Fc–Flag-CD40L pretreated cells from the corresponding total binding values. Specific binding values were fitted by non-linear regression using the GraphPad Prism5 software. Specific binding values were normalized according to the maximum binding value obtained from the linear regression.
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Figure 6: TRAF1 expression interferes with CD40-induced signaling. (A) U2OS and 786-O cells with and without TWEAK priming (200 ng/ml, 6 h) and U2OS-TRAF1 and 786-O-TRAF1 transfectants were stimulated in 96-well plates in triplicates with the indicated concentrations of Fc–Flag-CD40L. Next day, supernatants were assayed for production of IL8 or IL6 by ELISA. Prior stimulation cell culture medium was replaced to reduce the background caused by constitutive cytokine production. (B) U2-OS and 786-O cells with and without TWEAK priming and TRAF1 expressing U2-OS and 786-O transfectants were stimulated with Fc–Flag-CD40L for 5 and 15 min and were finally analyzed by western blotting to detect the indicated molecules. Please note, in the case of the TRAF1 western blots a relatively short exposure time is shown to ensure reasonable visibility of overexpressed and TWEAK-induced TRAF1. (C) 786-O and U2-OS cells and their corresponding TRAF1 transfectants were primed overnight with soluble TWEAK or remained untreated and were then analyzed by FACS for CD40 cell surface expression. (D) Cells (2 × 105 cells/well) were seeded in 24-well plates. Next day, half of the samples of each cell type were pretreated for 1 h at 37°C with 2 μg/ml of Fc–Flag-CD40L. Next, untreated and Fc–Flag-CD40L pretreated cells were incubated pairwise with increasing concentrations of GpL–Fc–Flag-CD40L (1 h, 37°C), a fusion protein of Fc–Flag-CD40L with the luciferase from Gaussia princeps. After removal of unbound CD40L molecules, cells were scratched in 50 μl medium to measure cell-associated luciferase activity. Specific binding values were obtained by subtraction of the non-specific binding values derived of Fc–Flag-CD40L pretreated cells from the corresponding total binding values. Specific binding values were fitted by non-linear regression using the GraphPad Prism5 software. Specific binding values were normalized according to the maximum binding value obtained from the linear regression.

Mentions: We observed recently that priming of cells with soluble TWEAK for a few hours strongly inhibits CD40 signaling and traced this back to impaired formation of TRAF2–cIAP1/2 containing CD40 signaling complexes (20). Noteworthy, TRAF1 forms with high efficiency heterotrimers with TRAF2 and this heteromeres interact much stronger with cIAP2 than homotrimeric TRAF2 (21). On the other side, however, there is evidence that TRAF1-TRAF2 heteromers bind weaker to CD40 than TRAF2 homotrimers (22). To evaluate a possible role of TRAF1 induction in the crosstalk of TWEAK and CD40, we took advantage of 786-O and U2-OS cells that we have stably transfected for another project with a caspase cleavage-resistant TRAF1 variant with unchanged protein–protein interaction properties. In accordance with our previous findings (20), priming with soluble TWEAK diminished CD40L-induced upregulation of the classical NFκB-controlled cytokine IL8 in U2-OS cells as well as induction of the likewise classical NFκB-regulated cytokine IL6 in 786-O cells (Figure 6A).


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)

TRAF1 expression interferes with CD40-induced signaling. (A) U2OS and 786-O cells with and without TWEAK priming (200 ng/ml, 6 h) and U2OS-TRAF1 and 786-O-TRAF1 transfectants were stimulated in 96-well plates in triplicates with the indicated concentrations of Fc–Flag-CD40L. Next day, supernatants were assayed for production of IL8 or IL6 by ELISA. Prior stimulation cell culture medium was replaced to reduce the background caused by constitutive cytokine production. (B) U2-OS and 786-O cells with and without TWEAK priming and TRAF1 expressing U2-OS and 786-O transfectants were stimulated with Fc–Flag-CD40L for 5 and 15 min and were finally analyzed by western blotting to detect the indicated molecules. Please note, in the case of the TRAF1 western blots a relatively short exposure time is shown to ensure reasonable visibility of overexpressed and TWEAK-induced TRAF1. (C) 786-O and U2-OS cells and their corresponding TRAF1 transfectants were primed overnight with soluble TWEAK or remained untreated and were then analyzed by FACS for CD40 cell surface expression. (D) Cells (2 × 105 cells/well) were seeded in 24-well plates. Next day, half of the samples of each cell type were pretreated for 1 h at 37°C with 2 μg/ml of Fc–Flag-CD40L. Next, untreated and Fc–Flag-CD40L pretreated cells were incubated pairwise with increasing concentrations of GpL–Fc–Flag-CD40L (1 h, 37°C), a fusion protein of Fc–Flag-CD40L with the luciferase from Gaussia princeps. After removal of unbound CD40L molecules, cells were scratched in 50 μl medium to measure cell-associated luciferase activity. Specific binding values were obtained by subtraction of the non-specific binding values derived of Fc–Flag-CD40L pretreated cells from the corresponding total binding values. Specific binding values were fitted by non-linear regression using the GraphPad Prism5 software. Specific binding values were normalized according to the maximum binding value obtained from the linear regression.
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Figure 6: TRAF1 expression interferes with CD40-induced signaling. (A) U2OS and 786-O cells with and without TWEAK priming (200 ng/ml, 6 h) and U2OS-TRAF1 and 786-O-TRAF1 transfectants were stimulated in 96-well plates in triplicates with the indicated concentrations of Fc–Flag-CD40L. Next day, supernatants were assayed for production of IL8 or IL6 by ELISA. Prior stimulation cell culture medium was replaced to reduce the background caused by constitutive cytokine production. (B) U2-OS and 786-O cells with and without TWEAK priming and TRAF1 expressing U2-OS and 786-O transfectants were stimulated with Fc–Flag-CD40L for 5 and 15 min and were finally analyzed by western blotting to detect the indicated molecules. Please note, in the case of the TRAF1 western blots a relatively short exposure time is shown to ensure reasonable visibility of overexpressed and TWEAK-induced TRAF1. (C) 786-O and U2-OS cells and their corresponding TRAF1 transfectants were primed overnight with soluble TWEAK or remained untreated and were then analyzed by FACS for CD40 cell surface expression. (D) Cells (2 × 105 cells/well) were seeded in 24-well plates. Next day, half of the samples of each cell type were pretreated for 1 h at 37°C with 2 μg/ml of Fc–Flag-CD40L. Next, untreated and Fc–Flag-CD40L pretreated cells were incubated pairwise with increasing concentrations of GpL–Fc–Flag-CD40L (1 h, 37°C), a fusion protein of Fc–Flag-CD40L with the luciferase from Gaussia princeps. After removal of unbound CD40L molecules, cells were scratched in 50 μl medium to measure cell-associated luciferase activity. Specific binding values were obtained by subtraction of the non-specific binding values derived of Fc–Flag-CD40L pretreated cells from the corresponding total binding values. Specific binding values were fitted by non-linear regression using the GraphPad Prism5 software. Specific binding values were normalized according to the maximum binding value obtained from the linear regression.
Mentions: We observed recently that priming of cells with soluble TWEAK for a few hours strongly inhibits CD40 signaling and traced this back to impaired formation of TRAF2–cIAP1/2 containing CD40 signaling complexes (20). Noteworthy, TRAF1 forms with high efficiency heterotrimers with TRAF2 and this heteromeres interact much stronger with cIAP2 than homotrimeric TRAF2 (21). On the other side, however, there is evidence that TRAF1-TRAF2 heteromers bind weaker to CD40 than TRAF2 homotrimers (22). To evaluate a possible role of TRAF1 induction in the crosstalk of TWEAK and CD40, we took advantage of 786-O and U2-OS cells that we have stably transfected for another project with a caspase cleavage-resistant TRAF1 variant with unchanged protein–protein interaction properties. In accordance with our previous findings (20), priming with soluble TWEAK diminished CD40L-induced upregulation of the classical NFκB-controlled cytokine IL8 in U2-OS cells as well as induction of the likewise classical NFκB-regulated cytokine IL6 in 786-O cells (Figure 6A).

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