Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2) - PubMed (original) (raw)

Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2)

Hilka Rauert et al. J Biol Chem. 2010.

Abstract

Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFkappaB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFkappaB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1- and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFkappaB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFkappaB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFkappaB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF.

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Figures

FIGURE 1.

FIGURE 1.

TNF receptor specificity of TNC-scTNF(143N/145R) and Flag-TNF(32W/86T). A, domain architecture of the soluble TNF variants used in this study. Flag indicates the epitope DYKDDDDK. TNC represents the compact disulfide-bonded trimerization domain of chicken tenascin-C comprising amino acids 110–139 of the molecule. The numbering of amino acids of TNF refers to the sequence of the mature soluble TNF protein. B, Flag-TNF(32W/86T) and TNC-scTNF(143N/145R) were transiently produced in HEK293 cells and purified by affinity chromatography on anti-Flag-agarose. Purified proteins were analyzed by SDS-PAGE and silver staining under reducing and non-reducing conditions. C, TNF variants were subjected to gel filtration on a BioSep-Sec-S3000 column, and molecular masses were calculated based on the elution volume of the standards thyroglobulin (670 kDa), immunoglobulin G (150 kDa), ovalbumin (44 kDa), and myoglobulin (17 kDa). D, HeLa, HeLa-TNFR2-vector, and HeLa-TNFR2-TRAF1 cells were seeded in 96-well plates (20 × 103 cells/well). The following day, cells were stimulated in duplicates for 18 h with 200 ng/ml of Flag-TNF(32W/86T), Flag-TNF(143N/145R), or TNC-scTNF(143N/145R) and IL-8 production was measured by ELISA analysis of the supernatants. To reduce the background related to constitutive IL-8 synthesis cell culture medium was changed prior stimulation. E, HeLa-TNFR2-TRAF1 cells were again seeded in 96-well plates and challenged with 20 ng/ml of TNC-scTNF(143N/145R) or Flag-TNF(32W/86T) in the presence and absence of 20 μg/ml TNFR2-Fc or 10 μg/ml TNFR1-Fc. Finally, IL-8 production was determined as in D.

FIGURE 2.

FIGURE 2.

TNFR2 stimulation induces p100 processing, whereas TNFR1 activation results in p100 up-regulation. A, HeLa-TNFR2, Jurkat-TNFR2, Kym1, and EM-2 cells were stimulated with the indicated mixtures of the TNFR2-specific TNF mutant TNC-scTNF(143N/145R) (200 ng/ml) and the TNFR1-specific TNF mutant Flag-TNF(32W/86T) (200 ng/ml) for 18 h. As both TNF receptors induce apoptosis in Kym1 cells, these cells were analyzed in the presence of the pan-caspase inhibitor z-VAD-fmk (40 μ

m

) to block apoptosis induction. Total cell lysates were analyzed by Western blotting with primary antibodies recognizing the indicated proteins and IRDye-conjugated secondary antibodies to allow quantification with the Odyssey® Infrared Imaging System. Relative p100 and p52 intensities were indicated in arbitrary units. B, for analysis of the activation of the classical NFκB pathway HeLa-TNFR2 cells and Jurkat-TNFR2 were challenged with 200 ng/ml Flag-TNF(32W/86T) or 200 ng/ml TNC-scTNF(143N/145R) for the indicated times. Whole cell lysates were subjected to Western blot analysis using antibodies specific for phospho-IκBα, IκBα, and tubulin as a loading control. C, Jurkat-TNFR2 cells were stimulated with 200 ng/ml of the indicated TNF variants for 18 h, and total cell lysates were again analyzed by Western blotting for p100 processing. D and E, indicated cells were stimulated for 2, 6, and 18 h via TNFR2 with 200 ng/ml TNC-scTNF(143N/145R). Cells were directly dissolved in Laemmli sample buffer and analyzed by Western blotting for the presence of TRAF2 (D) and NIK (E). F, samples of the various cell lines were pretreated or not with 10 μ

m

of the IKK2 inhibitor TPCA-1 (1 h, 37 °C) and were than either stimulated with 200 ng/ml Flag-TNF(32W/86T) for 3 and 20 min for the analysis of IκBα phosphorylation or for 18 h with TNC-scTNF(143N/145R) to analyze p100 processing. G, HeLa-TNFR2-vector and HeLa-TNFR2-TRAF1 cells were stimulated for 18 h with the indicated concentrations of TNC-scTNF(143N/145R) and p100 processing was analyzed by Western blotting.

FIGURE 2.

FIGURE 2.

TNFR2 stimulation induces p100 processing, whereas TNFR1 activation results in p100 up-regulation. A, HeLa-TNFR2, Jurkat-TNFR2, Kym1, and EM-2 cells were stimulated with the indicated mixtures of the TNFR2-specific TNF mutant TNC-scTNF(143N/145R) (200 ng/ml) and the TNFR1-specific TNF mutant Flag-TNF(32W/86T) (200 ng/ml) for 18 h. As both TNF receptors induce apoptosis in Kym1 cells, these cells were analyzed in the presence of the pan-caspase inhibitor z-VAD-fmk (40 μ

m

) to block apoptosis induction. Total cell lysates were analyzed by Western blotting with primary antibodies recognizing the indicated proteins and IRDye-conjugated secondary antibodies to allow quantification with the Odyssey® Infrared Imaging System. Relative p100 and p52 intensities were indicated in arbitrary units. B, for analysis of the activation of the classical NFκB pathway HeLa-TNFR2 cells and Jurkat-TNFR2 were challenged with 200 ng/ml Flag-TNF(32W/86T) or 200 ng/ml TNC-scTNF(143N/145R) for the indicated times. Whole cell lysates were subjected to Western blot analysis using antibodies specific for phospho-IκBα, IκBα, and tubulin as a loading control. C, Jurkat-TNFR2 cells were stimulated with 200 ng/ml of the indicated TNF variants for 18 h, and total cell lysates were again analyzed by Western blotting for p100 processing. D and E, indicated cells were stimulated for 2, 6, and 18 h via TNFR2 with 200 ng/ml TNC-scTNF(143N/145R). Cells were directly dissolved in Laemmli sample buffer and analyzed by Western blotting for the presence of TRAF2 (D) and NIK (E). F, samples of the various cell lines were pretreated or not with 10 μ

m

of the IKK2 inhibitor TPCA-1 (1 h, 37 °C) and were than either stimulated with 200 ng/ml Flag-TNF(32W/86T) for 3 and 20 min for the analysis of IκBα phosphorylation or for 18 h with TNC-scTNF(143N/145R) to analyze p100 processing. G, HeLa-TNFR2-vector and HeLa-TNFR2-TRAF1 cells were stimulated for 18 h with the indicated concentrations of TNC-scTNF(143N/145R) and p100 processing was analyzed by Western blotting.

FIGURE 3.

FIGURE 3.

TNFR2 stimulation induces nuclear translocation of p52 and RelB. A and B, HeLa-TNFR2 (A) and Jurkat-TNFR2 (B) cells were stimulated for 45 min with 200 ng/ml of Flag-TNF(32W/86T) or 18 h with 200 ng/ml of TNC-scTNF(143N/145R). Cytoplasmic and nuclear fractions were isolated and probed for the presence of the indicated NFκB proteins by Western blotting. As a control nuclear and cytoplasmic fractions were analyzed with respect to the marker proteins lamin B (nuclear) and tubulin (cytoplasmic). C, primary T-cells of donor no. 3 were cultivated in the presence of 20 units/ml IL2 and 5 μg/ml PHA. After 7 days, cells were stimulated with 200 ng/ml of TNC-scTNF(143N/145R), 200 ng/ml Flag-TNF(32W/86T), or a mixture of both for 18 h. Total cell lysates were then analyzed by Western blotting with antibodies recognizing the indicated proteins.

FIGURE 4.

FIGURE 4.

Analysis of TNFR2-induced nuclear translocation of p52 and RelB by immunolocalization. A and B, HeLa-TNFR2 cells were grown on glass coverslips and challenged for 45 min with 200 ng/ml Flag-TNF(32W/86T) or for 18 h with 200 ng/ml of TNC-scTNF(143N/145R). Cells were stained with antibodies specific for p65, RelB, and p100/p52. Shown are representative images (A) and the ratio of nuclear to cytoplasmic fluorescence intensity (B). To calculate the ratio of nuclear to cytoplasmic fluorescence intensity, measured values obtained from three independent experiments with at least 120 analyzed cells were used. Asterisks indicate p values <0.05.

FIGURE 5.

FIGURE 5.

Characterization of TNFR1- and TNFR2-specific membrane TNF mutants. A, domain architecture of the membrane TNF variants used in this study. THD, TNF homology domain; TM, transmembrane domain; PM, plasma membrane; TACE, TNF-α-converting enzyme. B, HEK293 cells transiently transfected with expression plasmids encoding the indicated membrane TNF variants were incubated with TNFR1-Fc and TNFR2-Fc or TRAILR3-Fc as a control. After three washes with PBS, bound proteins were detected with PE-labeled anti-human-Fc by FACS. Conventional FACS staining with anti-TNF-PE and a corresponding isotype control were included as a control. C, left panel, HeLa, HeLa-TNFR2-vector, and HeLa-TNFR2-TRAF1 cells were challenged in 96-well plates in triplicate with NCTC transfectants expressing the indicated membrane TNF variant, and after 18 h supernatants were removed to determine their IL-8 content by ELISA. Right panel, HeLa-TNFR2-TRAF1 cells were cocultured with the indicated NCTC transfectants in the presence or absence of 10 μg/ml TNFR1-Fc and IL-8 induction was analyzed by ELISA after 18 h.

FIGURE 6.

FIGURE 6.

Membrane TNF induces p100 processing via TNFR2. A, Hela and Hela-TNFR2-TRAF1 cells were cocultivated with NCTC transfectants expressing the indicated proteins. IL-8 production was measured after 18 h by ELISA. B, Jurkat-TNFR2 cells were cocultivated with the indicated NCTC transfectants and were analyzed after 18 h by Western blotting with respect to p100 processing.

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