Tumor necrosis factor (TNF) receptor-associated factor 7 is required for TNFα-induced Jun NH2-terminal kinase activation and promotes cell death by regulating polyubiquitination and lysosomal degradation of c-FLIP protein

Abstract

The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.

FIGURE 1.

TRAF7 is required for JNK activation following TNFα stimulation. A, HEK293 cells were transfected with vectors encoding for an shRNA targeting human TRAF7 (shTRAF7) (30) or a control sequence (scrambled), along with an AP1-luciferase reporter plasmid. 16 h later, cells were treated with TNFα (30 ng/ml) for 6 h, and luciferase activity was determined. Data shown represent relative normalized luciferase activity and are representative of six independent experiments done in triplicate. B, HEK293 cells were transfected with an expression vector empty or encoding TRAF7. 24 h later, cells were stimulated with TNFα, and the activation state of JNK was monitored by in vitro kinase assay. Cells exposed to anisomycin served as a positive control for JNK activation. C, HEK293 cells were transfected as indicated and stimulated with TNFα. The activation state of JNK and p38 was monitored by immunoblot assay. D, HEK293 cells were infected with lentiviral vectors encoding for shTRAF7 or a scramble control sequence. Cells were stimulated with TNFα, and the activation state of JNK was monitored by in vitro kinase assay.

FIGURE 2.

TRAF7 promotes polyubiquitination of c-FLIP_L. A, HEK293 cells were transfected with an expression vector encoding for HA-ubiquitin along with a vector empty or expressing a FLAG-tagged version of TRAF7. 24 h later, cell lysates were immunoprecipitated with anti c-FLIP antibody, separated by SDS-PAGE, and transferred onto membranes subsequently probed with anti-HA. B, HEK293 cells transfected with an expression vector encoding for TRAF7 were monitored for c-FLIP expression by immunoblot assay. c-FLIP_L expression levels were quantitated by ImageJ (lower panel). C and D, HEK293 cells were cotransfected with HA-tagged ubiquitin mutants and FLAG-tagged TRAF7 (C) or TRAF6 (D). The numbers indicate the only lysine residue remaining in the ubiquitin molecule. Immunoprecipitates with anti-c-FLIP antibody were resolved by SDS-PAGE and blotted onto a membrane subsequently probed with anti-HA. E, HEK293 cells were transfected as indicated, and the ubiquitination state of JNK was monitored by immunoblot assay probed with an anti-ubiquitin antibody (P4D1).

FIGURE 3.

Lysosome inhibitors restore c-FLIP_L expression in TRAF2^−/− EF. A, TRAF2^−/− EFs were treated for 12 h with the indicated inhibitors (2.5 μm MG132, 1.25 nm leupeptin, 2.5 μm NH₄Cl), and normalized lysates were examined for c-FLIP_L expression by immunoblot analysis. Lysates from NIH-3T3 cells served as positive control c-FLIP_L expression. B, TRAF2^−/− EFs were pretreated for 1 h with the indicated inhibitors, and TNFα was added at the indicated concentration. 16 h later, cell viability was determined with the ATPLite^TM assay. Each data point represents the mean ± S.D. cell survival expressed as a percentage of untreated cells in six replicates. C, TRAF2^−/− EFs pretreated for 1 h with the indicated inhibitors were stimulated with TNFα for the indicated time periods, and caspase-8 activation was monitored by immunoblot assay. D, HEK293 cells were infected with retroviral vectors encoding for a shRNA targeting human TRAF2 or a control scramble sequence. Cells were pretreated for 1 h with lysosomes inhibitors, and then TNFα was added at the indicated concentration. 16 h later, cell viability was determined with the ATPLite^TM assay. Each data point represents the mean ± S.D. cell survival expressed as a percentage of untreated cells in four replicates. E, NIH-3T3 cells were left untreated or treated with lysosomes inhibitors for indicated time periods. c-FLIP_L expression levels were monitored by Western blot analysis and quantitated by ImageJ. Each data point shown in the graph represents relative normalized optical density percentage.

FIGURE 4.

TRAF7 depletion restores c-FLIP_L expression in TRAF2^−/− EF. A, TRAF2^−/− EFs were infected with retroviral vectors encoding for two different shRNAs targeting murine TRAF7 (shTRAF7#5 and shTRAF7#2) or a control sequence (scramble). After selection, cells were left untreated or stimulated with TNFα (10 ng/ml) for 6 h, and then normalized lysates were examined for c-FLIP_L expression by immunoblot analysis. B, TRAF2^−/− EFs retrovirally infected as indicated were treated with TNFα at the indicated concentrations for 16 h, and then cell viability was determined with the ATPLite^TM assay. Lysates from NIH-3T3 cells served as positive control for c-FLIP_L expression. Each data point represents the mean ± S.D. cell survival expressed as a percentage of untreated cells in six replicates. Statistical analysis was by the one-tailed unpaired Student's test. C, p65^−/− EFs retrovirally infected as indicated were treated as in B.

FIGURE 5.

TRAF7 depletion protects cells independently of JNK function. JNK^−/− DKO EFs retrovirally infected as indicated were treated with TNFα plus cycloheximide (1 μg/ml) at the indicated concentration for 16 h, and then cell viability was determined with the ATPLite^TM assay. Each data point represents the mean ± S.D. cell survival expressed as a percentage of untreated cells in six replicates. Statistical analysis was by the one-tailed unpaired Student's test.

FIGURE 6.

TRAF7 depletion in Jurkat and HeLa cells. A, Jurkat cells were infected with retroviral vectors encoding either for an shRNA targeting human TRAF7 or for a scrambled sequence. Cells were treated with either anti-Fas (upper panel) or TNFα (lower panel) at the indicated concentrations plus cycloheximide (1 μg/ml). 16 h later, cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Each data point represents the mean ± S.D. cell survival expressed as percentage of untreated cells in four replicates. Statistical analysis was by the one-tailed unpaired Student's test. B, Jurkat cells infected and treated with TNFα as in A were monitored for c-FLIP_L expression levels by Western blot and quantitated by ImageJ. Data shown represent relative normalized optical density percentage and are representative of five independent experiments. C, HeLa cells infected and treated with TNFα as indicated were monitored for c-FLIP_L and c-FLIP_S expression levels.