Both internalization and AIP1 association are required for tumor necrosis factor receptor 2-mediated JNK signaling

Abstract

Objective: The proinflammtory cytokine tumor necrosis factor (TNF), primarily via TNF receptor 1 (TNFR1), induces nuclear factor-κB (NF-κB)-dependent cell survival, and c-Jun N-terminal kinase (JNK) and caspase-dependent cell death, regulating vascular endothelial cell (EC) activation and apoptosis. However, signaling by the second receptor, TNFR2, is poorly understood. The goal of this study was to dissect how TNFR2 mediates NF-κB and JNK signaling in vascular EC, and its relevance to in vivo EC function.

Methods and results: We show that TNFR2 contributes to TNF-induced NF-κB and JNK signaling in EC as TNFR2 deletion or knockdown reduces the TNF responses. To dissect the critical domains of TNFR2 that mediate the TNF responses, we examine the activity of TNFR2 mutant with a specific deletion of the TNFR2 intracellular region, which contains conserved domain I, domain II, domain III, and 2 TNFR-associated factor-2-binding sites. Deletion analyses indicate that different sequences on TNFR2 have distinct roles in NF-κB and JNK activation. Specifically, deletion of the TNFR-associated factor-2-binding sites (TNFR2-59) diminishes the TNFR2-mediated NF-κB, but not JNK activation; whereas, deletion of domain II or domain III blunts TNFR2-mediated JNK but not NF-κB activation. Interestingly, we find that the TNFR-associated factor-2-binding sites ensure TNFR2 on the plasma membrane, but the di-leucine LL motif within the domain II and aa338-355 within the domain III are required for TNFR2 internalization as well as TNFR2-dependent JNK signaling. Moreover, domain III of TNFR2 is responsible for association with ASK1-interacting protein-1, a signaling adaptor critical for TNF-induced JNK signaling. While TNFR2 containing the TNFR-associated factor-2-binding sites prevents EC cell death, a specific activation of JNK without NF-κB activation by TNFR2-59 strongly induces caspase activation and EC apoptosis.

Conclusions: Our data reveal that both internalization and ASK1-interacting protein-1 association are required for TNFR2-dependent JNK and apoptotic signaling. Controlling TNFR2-mediated JNK and apoptotic signaling in EC may provide a novel strategy for the treatment of vascular diseases.

Fig.1. TNFR2 contributes to TNF-induced NF-κB and JNK signaling pathways in EC

WT, TNFR2-KO and TNFR1/2-KO MLEC (1×10⁶) were treated with murine recombinant TNF (10 ng/ml) for the indicated times. Phospho- and total IκBα and JNK were determined by immunoblotting with the respective antibodies (A). The quantification of the ratios of p-IκBα/ IκBα and p-JNK/JNK are presented in B and C, respectively. Data are the mean ± SEM from three independent experiments. *, p<0.05 indicating statistic significance compared to the untreated VC.

Fig.2. The TRAF2-binding sites are required for TNFR2-mediated NF-κB but not JNK activation

A. Schematic structure of TNFR2 and the truncated deletions at the C terminus. The numbers refer to the amino acid number, indicating the boundary of the extracellular and intracellular domains (I–III). TM: transmembrane; A TRAF2-binding motif (SKEE) and Bmx-binding sequence are also indicated. B. Expression of TNFR2 truncates as detected by immunoblotting with anti-TNFR2. C–D. Effects of TNFR2 truncation on NF-κB and JNK activation in reporter gene assays. A NF-κB-reporter gene or JNK-reporter gene was transfected into TNFR2-KO MLEC in the presence of vector control (−), TNFR2-WT or mutants. Cells were harvested for luciferase/renilla assays at 48 h post-transfection. Data are presented as mean ± SEM of duplicate samples from four independent experiments. *, p<0.05 indicating statistic significance compared to the vector control (normalized to 1.0). Arrows indicated that TNFR2-59 cannot activate NF-κB, but retains JNK activation.

Fig.3. Both domain II and domain III are required for TNFR2-mediated JNK activation

A. Schematic structure of TNFR2 and the internal deletion mutants. The numbers refer to the amino acid number, indicating the boundary of the extracellular and intracellular domains (I–III). LL: a di-luecine motif. B–C. Effects of TNFR2 internal deletions on NF-κB and JNK activation in reporter gene assays. A NF-κB-reporter gene or JNK-reporter gene was transfected into TNFR2-KO MLEC in the presence of vector control (−), TNFR2-WT or mutants. Cells were harvested for luciferase/renilla assays at 48 h post-transfection. Data are presented as mean ± SEM of duplicate samples from four independent experiments. *, p<0.05 indicating statistic significance compared to the TNFR2-WT. Arrows indicated that a deletion of domain III or domain II or mutation at the di-leucine motif abolished the JNK activity.

Fig.4. Both domain II and domain III of TNFR2 regulate TNFR2 intracellular localization

A and C. Schematic structure of TNFR2 truncation and internal deletion are shown. B and D. Effects of truncations and deletions on TNFR2 cellular localization. TNFR2-KO MLEC were transfected with various TNFR2 mutants, and the localization of the TNFR2 protein was determined by indirect fluorescence microscopy with anti-TNFR2 followed by Alexa Fluor-488 donkey anti-goat IgG. TNFR2-KO cells showed no staining (not shown). Representative images from 5 cells for each truncate are shown.

Fig.5. Domain III of TNFR2 is critical for the association with AIP1, an adaptor molecule for JNK activation

A. Schematic structure of TNFR2-WT, −59 and −84 are shown. B. TNFR2-KO MLEC were transfected with TNFR2 mutants. Association of TNFR2 with AIP1, ASK1 and TRAF2 was determined by co-immunoprecipitation with the respective antibodies followed by immunoblotting with anti-TNFR2. C. Association of endogenous TNFR2 and AIP1 in response to TNF. HUVEC were untreated or treated with human TNF (10 ng/ml) for 15 min. TNFR2 and AIP1 in the lysates were detected by immunoblotting with respective antibodies. Association of endogenous TNFR2 with AIP1 was determined by co-immunoprecipitation with anti-TNFR2 (αR2) followed by immunoblotting with anti-AIP1. Immunoprecipitation with a goat IgG isotype was used as a control.

Fig.6. TNFR2-WT inhibits, whereas TNFR2-59 augments, TNF-induced cell apoptosis

A–B. TNFR2-KO MLEC were infected with lentivirus expressing a control gene LacZ, TNFR2-WT or TNFR2-59. 48 h post-infection, cells were treated with TNF (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for 6h. Apoptosis was determined by FACS analysis with FITC-conjugated annexin V (X-axis) and propidium iodide (PI, Y-axis). The numbers indicate % of cell population in each quadruplet. Lower left: normal cells; upper left: necrotic cells; lower right: apoptotic cells; upper right: late apoptotic cells. Quantification of apoptotic (annexin V-positive, both upper and lower right quadruplets in FACS) are shown in B. Data presented are means (±SEM) of three independent experiments. *, p<0.05. C. Caspase-3 activation was measured by immunoblotting with an antibody that recognizes a cleaved form of caspase-3. Densitometry quantifications were indicated, with untreated control as 1.0. Expression of TNFR2-WT and TNFR2-59 was determined by immunoblotting with anti-TNFR2. D. A summary for TNFR2-mediated signaling. TNFR2 via the TRAF2-binding sites recruits TRAF2 to activate NF-κB survival signaling. Domain II and domain III are required for TNFR2 internalization and AIP1-ASK1 association, leading to activation JNK and apoptosis.