Cell Biology/Signaling

Both Internalization and AIP1 Association Are Required for Tumor Necrosis Factor Receptor 2-Mediated JNK Signaling

Weidong Ji, Yonghao Li, Ting Wan, Jing Wang, Haifeng Zhang, Hong Chen, Wang Min
https://doi.org/10.1161/ATVBAHA.112.253666
Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2271-2279
Originally published August 15, 2012

Jump to

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.

Conclusion—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.

Introduction

Vascular endothelial cells (EC) are among the principal physiological targets of prototypic inflammatory cytokine tumor necrosis factor (TNF).1,2 In EC, as in other cell types, TNF elicits a broad spectrum of biological effects including proliferation, differentiation, and apoptosis.3 The nature of the TNF effects depends on TNF concentration, and the type and growth state of the target cells.4 These differences in TNF-induced response are also due, in part, to the presence of 2 distinct TNF-specific plasma membrane-localized receptors, type I 55 kDa TNFR (TNFR1) and type II 75 kDa TNFR (TNFR2).5 Mice with genetic deletion of TNFR1 or TNFR2 (TNFR1-knockout [KO] and TNFR2-KO) are viable and do not show any overt phenotypic abnormalities. Data from studies using these mice suggest that TNFR1 is primarily responsible for TNF-mediated host defense and inflammatory responses.6 We have previously characterized the vascular phenotypes of TNFR1-KO and TNFR2-KO mice.7,8 These studies have demonstrated that TNFR1-KO and TNFR2-KO mice are normal in vascular development. However, TNFR1-KO and TNFR2-KO mice have distinct phenotype in ischemia-induced models. TNFR2, but not TNFR1, is highly upregulated in vascular endothelium in response to ischemia. TNFR1-KO mice have enhanced, whereas TNFR2-KO have reduced capacity in ischemia-induced angiogenesis and tissue recovery.7,8

TNFR1-mediated signaling pathways have been well characterized.9 The intracellular portion of TNFR1 can be subdivided into a membrane proximal and a membrane distal part. The latter contains a death domain which can also be found in other receptors involved in cell death (eg, Fas). A current model postulates that TNF-binding triggers trimerization of TNFR1, which recruits adaptor proteins and signaling molecules by their intracellular domains to form a receptor-signaling complex.9 The first to be recruited is the TNFR-associated death domain protein (TRADD).10 TRADD functions as a platform adaptor that recruits TNFR-associated factor-2 (TRAF2), and receptor-interacting protein kinase-1 (RIPK1).11,12 It has been proposed that TNF-induced nuclear factor-κB (NF-κB), c-Jun N-terminal kinase (JNK), and apoptosis pathways are sequentially activated, and early NF-κB signaling can inhibit JNK and apoptosis in EC. Specifically, the initial plasma membrane-bound complex (complex I, comprising TNFR1, TRADD, RIPK1, and TRAF2) for NF-κB activation, and a cytoplasmic complex (complex II or the death-inducing signaling complex), in which the internalized TRADD, TRAF2, and RIPK1, recruits Fas-associated death domain protein and procaspase-8 for apoptotic signaling.13 Because the NF-κB-induced protein synthesis of cellular FLICE inhibitory protein normally prevents the recruitment of procaspase-8/10 to Fas-associated death domain protein and therefore the formation of complex II, complex II formation can only be detected at 2 to 8 hours posttreatment with TNF in cells overexpressing the NF-κB inhibitor IκBα. Data from Schutze’s laboratory has also demonstrated that TNFR1 endocytosis is required for the formation of death-inducing signaling complex.14 Different from the findings in a study by Tschopp’s group, TNFR1 is a component of the intracellular death-inducing signaling complex complex. Our previous data support that an intermediate complex between complex I and complex II (likely an internalized complex I) is responsible for TNF-induced JNK activation.15–17 Because ASK1-interacting protein-1 (AIP1) is a different component than complex I, we name it the AIP1 complex. We have demonstrated that AIP1 complex is structurally and functionally different from complex I and complex II. AIP1 complex activates JNK, whereas it inhibits NF-κB signaling. Moreover, AIP1-apoptosis signal-regulating kinase-1-JNK activation induces apoptotic signaling, which is dependent on intrinsic (mitochondria-dependent) pathways.15–17 In EC, NF-κB is associated with cell survival, whereas the JNK pathway is involved in apoptosis.1,2

In contrast, the function and signaling of the second TNF receptor, TNFR2, is less understood. This is because of several reasons. First, TNFR2 expression is limited to specific cell types, including EC, cardiac myocytes and hematopoietic cells.6 Moreover, TNFR2 is often induced in response to certain stimuli, for instance, in the activation of macrophage and T lymphocytes that results in a marked increase in TNFR2 levels.6,18 TNFR2 is also highly induced in vascular EC in ischemia hind limb, as we recently demonstrated.7,8 Second, the intracellular domains of TNFR2 and interacting proteins are not well defined. Although TRAF2 was initially identified as a TNFR2-binding protein,19 subsequent studies have been focused on its role in TNFR1 signaling. Trimerization of TNFR2 in response to TNF leads to the recruitment of TRAF2 and TRAF2-associated TRAF1, cellular inhibitor of apoptosis-1, and cellular inhibitor of apoptosis-2. We have previously identified a Bmx-binding sequence at the C terminus of TNFR2, which constitutively binds to nonreceptor tyrosine kinase Bmx, contributing to TNFR2-mediated Akt activation, EC migration, and proliferation.7,8,20,21 It has been shown that the Bmx-binding sequence also recruits TRAF2, regulating TNFR2-mediated NF-κB signaling.22,23 Recent studies suggest that TNFR2, independent of TNFR1, activates both canonical and noncanonical NF-κB pathways.18 Specifically, TNFR2 through PI3K/Akt induces phosphorylation of IκBα kinase-β and subsequent activation of the canonical NF-κB pathway.24 Only membrane-bound TNF triggers TNFR2-mediated NF-κB-inducing kinase/IKKα-dependent noncanonical pathway.25 However, the mechanism by which TNFR2 induces JNK activation has not been investigated.

In the present study, we show that TNFR2 significantly contributes to TNF-induced NF-κB and JNK activation in vascular EC. We identify 2 sequences other than the TRAF2-binding site that are required for TNFR2-dependent JNK activation. Moreover, these sequences of TNFR2 are critical for its internalization and its association with AIP1, an adaptor molecule specific for JNK signaling. This is the first study, to our knowledge, to dissect the TNFR2-mediated JNK-signaling pathway.

Methods

Detailed materials and methods are provided in the online-only Data Supplement. Most of the methods have been previously published, including plasmids, cell culture, transfection, reporter gene assay, immunoprecipitation, immunoblotting, indirect immunofluorescence micropscopy, and quantification of apoptosis.7

Results

TNFR2 Contributes to TNF-Induced NF-κB and JNK-Signaling Pathways in Vascular EC

To determine the role of TNFR2 in TNF signaling, we first examined the TNF responses in mouse lung EC (MLEC) isolated from wild-type (WT) and TNFR2-KO mice. MLEC were treated with murine recombinant TNF for the indicated times (0, 15, 30, and 60 minutes), and activation of the NF-κB and JNK pathways were determined by immunoblotting with phospho-specific antibodies. Consistent with previous observations in other EC types (human umbilical vein EC and bovine aortic endothelial cells),15–17 TNF-induced phosphorylation of IκBα (an indication of NF-κB activation) and JNK, peaking at 15 to 30 minutes in WT cells. TNFR2 deletion in EC reduced TNF-induced NF-κB and JNK signaling by 50 to 60% compared with WT EC (Figure 1A with quantifications in Figure 1B and 1C). Further deletion of TNFR1 (TNFR1/2-double knockout) in EC (Figure IA in the online-only Data Supplement for TNFR1 and TNFR2 expression) completely abolished the TNF responses (Figure 1A with quantifications in Figure 1B and 1C). Knockdown of TNFR2 in human EC (human umbilical vein EC) by small interfering RNA also reduced human TNF-induced NF-κB and JNK activation (Figure IB in the online-only Data Supplement). These data suggest that TNFR2 contributes to TNF-induced NF-κB and JNK activation in EC.

Figure 1.
Figure 1.

Tumor necrosis factor receptor 2 (TNFR2) contributes to TNF-induced nuclear factor-κB (NF-κB) and JNK-signaling pathways in endothelial cell (EC). Wild-type (WT), TNFR2-knockout (KO), and TNFR1/2-KO mouse lung EC (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 3 independent experiments. *P<0.05 indicating statistic significance compared with the untreated vector control.

TRAF2-Binding Sites Are Required for TNFR2-Mediated NF-κB but Not JNK Activation

It has been shown that TNFR2 contains 2 TRAF2-binding sites at the C terminus, the first being the SKEE motif and the second is the last 16 aa sequence22,23 that overlaps with a Bmx-binding sequence.7,8⇓,20 Each TRAF2 site mediates TNFR2-transduced NF-κB activation independently.22,23 Besides the TRAF2-binding consensus sequences, sequence alignment of TNFR2 intracellular regions from different species revealed 3 additional conserved domains—domain I (aa266–289), domain II (aa289–338), and domain III (aa338–380) (Figure 2A). To determine the TNFR2 domain critical for JNK activation, we constructed a series of TNFR2 truncations with deletions of these various domains (Figure 2A and 2B). Activities of TNFR2 mutants in NF-κB and JNK signaling were determined by NF-κB and JNK-reporter gene assays in TNFR2-null MLEC (Figure 2B). Overexpression of TNFR2-WT, similar to overexpression of TNFR1 or TRAF2,19 activated both NF-κB and JNK-reporter genes in a ligand-independent manner. Consistent with our previous findings,7 deletion of the TRAF2-binding sites (TNFR2–59) abolished TNFR2-mediated NF-κB activation (Figure 2C). To our surprise, TNFR2–59 retained the ability to activate the JNK-reporter gene, suggesting that TRAF2-binding sites are not required for TNFR2-mediated JNK signaling. However, a partial (TNFR2–84) or a complete deletion of the domain III (TNFR2–112) blunted TNFR2-dependent JNK activation. Further deletions of domain II and domain I (TNFR2–150 and TNFR2-TM) had similar effects on the JNK reporter gene (Figure 2D). These data suggest that TNFR2 utilizes different regions to transduce NF-κB and JNK signaling, and the sequence between TNFR2–84 and TNFR2–59 could be important for TNFR2-mediated JNK activation.

Figure 2.
Figure 2.

The tumor necrosis factor receptor associated factor-2 (TRAF2)-binding sites are required for TNFR2-mediated nuclear factor-κB (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). 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 and 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-knockout (KO) mouse lung endothelial cell (MLEC) in the presence of vector control (–), TNFR2-WT or mutants. Cells were harvested for luciferase/renilla assays at 48 hours posttransfection. Data are presented as mean±SEM of duplicate samples from 4 independent experiments. *P<0.05 indicating statistical significance compared with the vector control (normalized to 1.0). Arrows indicate that TNFR2–59 cannot activate NF-κB, but retains JNK activation. TM indicates transmembrane.

Domain II and Domain III Are Required for TNFR2-Mediated JNK Activation

The results indicated above using the C-terminal truncations supported a role of domain III in TNFR2-mediated JNK activation. However, the role of domain I and domain II could not be determined. To this end, each domain was then internally deleted (Δ266–289 for domain I, Δ289–338 for domain II, Δ338–355 for the first half of domain III, and Δ355–380 for the second half of domain III—the sequence located between TNFR2–84 to –59 that is critical for JNK activation). Domain II contains a di-leucine motif, which has recently been shown to be important for TNFR2 internalization.26 Therefore, we made a site-specific mutation at the di-leucine motif (L319/320A) (Figure 3A). These internal deletions had no effects on TNFR2 expression (not shown; Figure 4). Effects of these deletions/mutations on TNFR2-mediated signaling were assessed in the reporter gene assays. TNFR2 with a deletion of domain I, domain II, or domain III retained the ability to activate NF-κB reporter gene. Similar to the deletion of domain II, mutations at the di-leucine motif had no effects on TNFR2-mediated NF-κB activation (Figure 3B), consistent with a recent report.26 In contrast, a deletion of domain II or a mutation at the di-leucine motif was sufficient to abolish TNFR2 activity in JNK activation (Figure 3C). A deletion at the first half of domain III (Δ338–355) had no effects on JNK activation. However, deletion at the second half (Δ355–380), similar to TNFR2–84, diminished TNFR2-mediated JNK activation (Figure 3C). These data suggest that both di-leucine motif within domain II and the second half of domain III are required for TNFR2-mediated JNK activation.

Figure 3.
Figure 3.

Both domain II and domain III are required for tumor necrosis factor receptor 2 (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). B and C, Effects of TNFR2 internal deletions on nuclear factor-κB (NF-κB) and JNK activation in reporter gene assays. A NF-κB–reporter gene or JNK-reporter gene was transfected into TNFR2-knockout (KO) mouse lung endothelial cell (MLEC) in the presence of vector control (–), TNFR2-wild-type (WT) or mutants. Cells were harvested for luciferase/renilla assays at 48 hours posttransfection. Data are presented as mean±SEM of duplicate samples from 4 independent experiments. *P<0.05 indicating statistical significance compared with the TNFR2-WT. Arrows indicate that a deletion of domain III or domain II or mutation at the di-leucine motif abolished the JNK activity. LL indicates a di-luecine motif.

Figure 4.
Figure 4.

Both domain II and domain III of tumor necrosis factor receptor 2 (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-knockout (KO) mouse lung endothelial cell (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.

Domain II and Domain III of TNFR2 Regulate TNFR2 Intracellular Localization

The cellular localization of TNFR1 determines its activity in the NF-κB and JNK-signaling pathways and in apoptosis.13,14,16 The results that mutations of the di-leucine motif diminished TNFR2-dependent JNK, but not NF-κB activity prompted us to examine TNFR2 localization. TNFR2-WT, up expression into TNFR2-KO MLEC, exhibited both cytoplasmic membrane and intracellular localizations. Deletions of the TRAF2/Bmx motifs (−59) enhanced TNFR2 intracellular localization (Figure 4A and 4B), indicating that the TRAF2 (and Bmx)-binding sites are required to maintain TNFR2 on the membrane. Internalized TNFR2 was detected in intracellular vesicles, where it colocalized with the endocytic marker green fluorescent protein-FYVE-1, containing 2 tandem-arranged FYVE domains of the late endosomal protein Hrs27 (Figure II in the online-only Data Supplement). To our surprise, a further deletion of domain III (TNFR2–84) was sufficient to block TNFR2 intracellular vesicle staining, even in the presence of the di-leucine motif (Figure 4B). This result was confirmed using the internal deletion mutants (Figure 4C and 4D). An internal deletion of aa355–380 drastically reduced TNFR2 intracellular staining. In contrast, deletion of domain I (aa266–289) or the first half of domain III (aa324–355) did not block TNFR2 internalization. As controls, a deletion or a mutation at the di-leucine motif (Δ289–338 and L319/320A, respectively) caused a loss of intracellular vesicle localization of the TNFR2 protein. These data suggest that both the di-leucine motif of domain II and aa355–380 within domain III are required for TNFR2 internalization.

Domain III of TNFR2 Is Critical for the Association With AIP1, an Adaptor Molecule for JNK Activation

As aa355–380 of domain III specifically mediates TNFR2-induced JNK but not NF-κB activation, we reasoned that it may be required for its association with JNK-specific intracellular signaling molecules. AIP1 is an adaptor molecule mediating JNK, but not NF-κB activation in EC,15–17 and therefore we examined whether TNFR2 via its domain III associated with AIP1. To this end, AIP1-WT, TNFR2–59, and TNFR2–84 (Figure 5A) were expressed in TNFR2-KO MLEC, and association of TNFR2 with endogenous AIP1 was determined by immunoprecipitation with anti-AIP1 followed by Western blot with anti-TNFR2. Association of TNFR2 with TRAF2 was used as a control. As expected, TNFR2-WT, but not TNFR2–59 or TNFR2–84, strongly associated with TRAF2. In contrast, both TNFR2-WT and TNFR2–59 associated with AIP1, and TNFR2–59 exhibited much stronger AIP1 binding than TNFR2-WT (Figure 5B), correlating with its prominent intracellular localization and higher activity in JNK signaling. A deletion of domain III (TNFR2–84) abolished the AIP1 binding. Similar results were observed for association of TNFR2 with the AIP1 effector apoptosis signal-regulating kinase-1 (Figure 5B), an upstream MAP kinase kinase kinase in JNK activation.

Figure 5.
Figure 5.

Domain III of tumor necrosis factor receptor 2 (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-knockout (KO) mouse lung endothelial cell (MLEC) were transfected with TNFR2 mutants. Association of TNFR2 with AIP1, apoptosis signal-regulating kinase-1 (ASK1), and TRAF2 was determined by coimmunoprecipitation with the respective antibodies followed by immunoblotting with anti-TNFR2. C, Association of endogenous TNFR2 and AIP1 in response to TNF. Human umbilical vein endothelial cells (HUVEC) were untreated or treated with human TNF (10 ng/mL) for 15 minutes. TNFR2 and AIP1 in the lysates were detected by immunoblotting with respective antibodies. Association of endogenous TNFR2 with AIP1 was determined by coimmunoprecipitation with anti-TNFR2 (αR2) followed by immunoblotting with anti-AIP1. Immunoprecipitation with a goat IgG isotype was used as a control.

We then determined whether endogenous TNFR2 and AIP1 proteins form a complex in EC. human umbilical vein EC were untreated or treated with TNF (10 ng/mL for 15 minutes), and association of TNFR2 with AIP1 was determined in a coimmunoprecipitation assay. TNFR2 associated with AIP1 in resting EC and this association was increased in response to TNF (Figure 5C). We also examined interactions of TNFR2 and AIP1 in EC by a colocalization assay. Colocalization of TNFR2 and AIP1 was detected in untreated cells, primarily on the cytoplasmic membrane. However, TNF treatment enhanced their colocalization in intracellular vesicles (Figure III in the online-only Data Supplement). Taken together, these data suggest that TNFR2 via its domain III binds to AIP1 (and its effector apoptosis signal-regulating kinase-1) to activate the JNK signaling.

TNFR2-WT Inhibits, Whereas TNFR2–59 Augments, TNF-Induced Cell Apoptosis

JNK signaling has been implicated in cell apoptosis by activating caspase-dependent pathways.28,29 As TNFR2–59 specifically activates JNK but not the NF-κB survival pathway, we used it to determine the cellular function of TNFR2-dependent JNK signaling and JNK-dependent apoptosis in EC. EC are normally resistant to cytotoxic actions of TNF because of NF-κB–induced gene expression of antiapoptotic proteins that inhibit cell death by both the caspase and the JNK pathways. To potentiate TNF-mediated EC death, TNF is provided in combination with either a global inhibitor of new gene transcription (such as actinomycin D) or of protein synthesis (such as cycloheximide or shiga-like toxin) or in combination with a selective inhibitor of NF-κB activation.1,2 To this end, TNFR2-KO MLEC were infected with lentivirus to express TNFR2-WT or TNFR2–59, and cells were treated with an apoptotic stimulus (TNF plus a protein synthesis inhibitor cycloheximide). Cell apoptosis was determined by annexin V/propidium iodide staining followed by flow cytometry analysis. Expression of TNFR2-WT significantly blocked TNF+ cycloheximide-induced apoptosis, consistent with its activity in NF-κB survival signaling. In contrast, expression of TNFR2–59 strongly induced EC apoptosis, even in the absence of the apoptotic stimulus. The TNFR-59-induced responses were further augmented by TNF+ cycloheximide treatment (Figure 6A, with quantification in Figure 6B). Similar results were obtained by detection of caspase-3 activation as measured for caspase-3 cleavage (Figure 6C). These data suggest that TNFR2-dependent JNK activity contributes to EC apoptosis.

Figure 6.
Figure 6.

Tumor necrosis factor receptor 2 (TNFR2)-wild-type (WT) inhibits, whereas TNFR2–59 augments, TNF-induced cell apoptosis. A and B, TNFR2-knockout (KO) mouse lung endothelial cell (MLEC) were infected with lentivirus expressing a control gene LacZ, TNFR2-WT, or TNFR2–59. Forty-eight hours postinfection, cells were treated with TNF (10 ng/mL) plus cycloheximide (CHX, 10 μg/mL) for 6 hours. Apoptosis was determined by fluorescence-activated cell sorter (FACS) analysis with fluorescein isothiocyanate (FITC)-conjugated annexin V (X-axis) and propidium iodide (PI, Y-axis). The numbers indicate percentage of cell population in each quadruplet. Bottom left: normal cells; top left: necrotic cells; bottom right: apoptotic cells; top right: late apoptotic cells. Quantification of apoptotic (annexin V-positive, both top and bottom right quadruplets in FACS) are shown in B. Data presented are means (±SEM) of 3 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 nuclear factor-κB (NF-κB) survival signaling. Domain II and domain III are required for TNFR2 internalization and AIP1-ASK1 association, leading to activation JNK and apoptosis.

Discussion

In the present study, we have made several important findings. First, TNFR2 significantly contributes to TNF-induced NF-κB and JNK signaling in vascular EC. This is based on the studies using primary vascular EC with TNFR2 deletion or knockout. Second, by reexpressing various TNFR2 mutants into TNFR2-KO EC, we have identified the critical domains of TNFR2 involved in TNFR2-mediated NF-κB and JNK signaling (Figure 6D). Specifically, the C terminal 59 residues (aa380–439) containing the TRAF2- and Bmx-binding sites are essential for NF-κB activation; the adjacent domain II (aa289–338) and domain III (aa338–380) are critical for JNK activation; and the membrane proximal domain I is dispensable for both NF-κB and JNK signaling. Third, TNFR2 cellular localization correlates with its activity in NF-κB and JNK signaling. While the TRAF2/Bmx-binding sites are required for its localization on the membrane and its ability to activate NF-κB, the di-leucine motif within domain II and aa355–380 within domain III of TNFR2 are required for its internalization and its ability to activate JNK. We further show that the TNFR2 domain III is required for its association with the JNK-specific signaling molecule AIP1, providing the molecular basis for its role in JNK activation. Finally, we demonstrate that TNFR2-mediated NF-κB activity is associated with cell survival, whereas the JNK signaling is involved in EC apoptosis.

One surprising finding in our study is that the TRAF2-binding sites are not required for, and in fact inhibits, TNFR2-mediated JNK activation. The C-terminal TNFR2 contains 2 TRAF2-binding sites, 1 is the canonical TRAF2 motif (SKEE), which has been shown to be critical for TRAF2 binding and NF-κB activation. The second site, overlapping with the Bmx-binding sequence, is located at the C-terminal 16 aa residues. The role of this second TRAF2-binding site in TNFR2-mediated NF-κB signaling has also been characterized.22,23 However, the mechanism by which TNFR2 induces JNK activation has not been investigated. Our present study suggests that a deletion of both TRAF2-binding sites (TNFR2–59) diminishes TNFR2-mediated NF-κB but augments TNFR2-mediated JNK signaling. On the basis of previous and current findings, we propose the following possible models by which the TRAF2-binding sites inhibit TNFR2-mediated JNK signaling. (1) The TRAF2-binding sites regulate TNFR2 intracellular localization. The TRAF2/Bmx-binding sites appear to be important in maintaining TNFR2 on the plasma membrane, leading to NF-κB but not JNK activation. However, deletion of this sequence (TNFR2–59) causes TNFR2 intracellular localization and specific JNK activation. Previous work has demonstrated that TRAF2 regulates TNFR2 membrane localization.30 It has also been shown that Bmx is localized on the plasma membrane.20,31,32 It will be interesting to determine whether TRAF2 and Bmx proteins cooperatively regulate TNFR2 membrane localization and internalization. (2) NF-κB inhibits JNK signaling. In TNFR1 signaling, NF-κB induces gene expression of antiapoptotic proteins that inhibit both the JNK and the caspase pathways. The best characterized factor is FLICE inhibitory protein (cellular FLICE inhibitory protein)33 and A20.34,35 Cellular FLICE inhibitory protein competes with procaspase 8 for binding to Fas-associated death domain protein and prevents the autocatlytic activation of caspase 8 (and 10) by the TRADD/RIPK1/TRAF2/Fas-associated death domain protein complex. A20 exhibits both deubiquinating and E3 ubiquitin ligase activities, and is thought to remove the signaling scaffold of lysine 48-linked ubiquitin polymer that forms on RIPK1 as part of the NF-κB and JNK activation pathways, replacing them with a lysine 68-linked ubiquitin polymer that targets RIPK1 for proteosome-mediated degradation.34,36 As a result, both JNK and caspase activation are inhibited. The importance of NF-κB–induced protective proteins is demonstrated by the potentiation of TNF-mediated EC death when TNF is provided in combination with either a global inhibitor of new gene transcription (such as actinomycin D) or of protein synthesis (such as cycloheximide or shiga-like toxin) or in combination with a selective inhibitor of NF-κB activation. (3) Our recent data suggest that TRAF2/RIPK1 in TNFR1 complex I recruits IKK, which could prevent TRAF2/RIPK1 from association with AIP1, therefore inhibiting AIP1-mediated JNK activation (W. Min, unpublished data, 2012). It is conceivable that in TNFR2 signaling pathway TRAF2-dependent NF-κB inhibits TNFR2-mediated JNK signaling by similar mechanisms.

TNFR1 endocytosis has been extensively investigated.37 A TNFR1-internalization domain, comprising YxxW internalization motif in the cytoplasmic tail of TNFR1, has been identified and implicated in mediating endocytosis of TNFR1.14,37 Caveolae also regulate TNFR1 endocytosis and signaling.38,39 In the present study, we have identified 2 sequences (the di-leucine motif within the domain II and aa355–380 within domain III) on TNFR2 that regulate both TNFR2 internalization and TNFR2-dependent JNK activation. TNFR2 contains a di-leucine motif, a typical sequence associated with clathrin-dependent endocytosis. A site-specific mutation at this motif (L319/320A) blocks TNFR2 internalization, but has no effect on TNFR2-mediated NF-κB signaling. However, TNFR2-L319/320A abolishes TNFR2-induced JNK activation. These data suggest that TNFR2, like TNFR1,13 initiates NF-κB signaling on the plasma membrane whereas it activates JNK on internalization. It is intriguing that aa355–380 within domain III, containing no known consensus sequence(s) associated with endocytosis, is also required for TNFR2 intracellular localization as well as TNFR2-dependent JNK activation. We have uncovered that aa355–380 within the domain III of TNFR2 is required for its association with AIP1, an intracellular adaptor molecule specific for JNK signaling. Of note, JNK inhibition had no effects on the cellular localization of TNFR2-WT or TNFR2–59, a mutant TNFR2 exhibits strong internalization and JNK activation (Figure IV in the online-only Data Supplement). These data suggest that JNK activation is likely a consequence of TNFR2 internalization. Interestingly, the domain III of TNFR2 contains serine/threonine-rich sequences that are potentially phosphorylated.23 We have previously shown that AIP1 via one of the poly-lysine clusters binds to a serine-rich sequence on apoptosis signal-regulating kinase-1.15 It is conceivable that internalized TNFR2 via serine/threonine-rich sequences forms a complex with AIP1, leading to JNK activation.

What is the in vivo relevance of TNFR2-mediated JNK signaling? Dr DiCorleto’s laboratory has shown that TNFR2 is essential for TNF-induced E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 expression as well as for TNF-induced leukocyte-EC interaction in vivo. More importantly, TNFR2 is also required for progression of atherosclerosis in mouse models.40,41 It is conceivable that TNFR2-mediated NF-κB and JNK synergistically regulate expression of the adhesion molecules as we demonstrated previously.42 TNFR2-mediated JNK activation could be detected in ischemic hind limb at day 3 postsurgery, correlating with ischemia-induced apoptosis. This transient JNK activation under ischemia is likely because of decreased expression of TRAF2 in response to ischemia.7,8 The JNK activation and cellular apoptosis may be an early event during the adaptive response, which is required for revascularization and ischemic tissue regeneration.7,8 Therefore, modulation of TNFR2-mediated JNK signaling in EC may provide a novel strategy for the treatment of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral arterial disease.

Acknowledgments

We thank Dr Jordan S. Pober for discussion.

Sources of Funding

This work was supported by grants from National Institutes of Health grants R01HL65978 and R01HL109420.

Disclosures

None.

Footnotes

  • Received May 19, 2012.
  • Accepted June 13, 2012.

References

  1. 1.↵
    1. Pober JS,
    2. Min W
    . Endothelial cell dysfunction, injury and death. Handb Exp Pharmacol. 2006;176:135–156.
  2. 2.↵
    1. Pober JS,
    2. Min W,
    3. Bradley JR
    . Mechanisms of endothelial dysfunction, injury, and death. Annu Rev Pathol. 2009;4:71–95.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Locksley RM,
    2. Killeen N,
    3. Lenardo MJ
    . The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001;104:487–501.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Fiers W,
    2. Beyaert R,
    3. Boone E,
    4. et al.
    TNF-induced intracellular signaling leading to gene induction or to cytotoxicity by necrosis or by apoptosis. J Inflamm. 1995;47:67–75.
    OpenUrlPubMed
  5. 5.↵
    1. Wallach D,
    2. Varfolomeev EE,
    3. Malinin NL,
    4. Goltsev YV,
    5. Kovalenko AV,
    6. Boldin MP
    . Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol. 1999;17:331–367.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Carpentier I,
    2. Coornaert B,
    3. Beyaert R
    . Function and regulation of tumor necrosis factor receptor type 2. Curr Med Chem. 2004;11:2205–2212.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Luo D,
    2. Luo Y,
    3. He Y,
    4. Zhang H,
    5. Zhang R,
    6. Li X,
    7. Dobrucki WL,
    8. Sinusas AJ,
    9. Sessa WC,
    10. Min W
    . Differential functions of tumor necrosis factor receptor 1 and 2 signaling in ischemia-mediated arteriogenesis and angiogenesis. Am J Pathol. 2006;169:1886–1898.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Luo Y,
    2. Xu Z,
    3. Wan T,
    4. He Y,
    5. Jones D,
    6. Zhang H,
    7. Min W
    . Endothelial-specific transgenesis of TNFR2 promotes adaptive arteriogenesis and angiogenesis. Arterioscler Thromb Vasc Biol. 2010;30:1307–1314.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Wajant H,
    2. Scheurich P
    . TNFR1-induced activation of the classical NF-κB pathway. FEBS J. 2011;278:862–876.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Hsu H,
    2. Shu HB,
    3. Pan MG,
    4. Goeddel DV
    . TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell. 1996;84:299–308.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Rothe M,
    2. Wong SC,
    3. Henzel WJ,
    4. Goeddel DV
    . A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell. 1994;78:681–692.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Stanger BZ,
    2. Leder P,
    3. Lee TH,
    4. Kim E,
    5. Seed B
    . RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death. Cell. 1995;81:513–523.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Micheau O,
    2. Tschopp J
    . Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell. 2003;114:181–190.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Schneider-Brachert W,
    2. Tchikov V,
    3. Neumeyer J,
    4. Jakob M,
    5. Winoto-Morbach S,
    6. Held-Feindt J,
    7. Heinrich M,
    8. Merkel O,
    9. Ehrenschwender M,
    10. Adam D,
    11. Mentlein R,
    12. Kabelitz D,
    13. Schütze S
    . Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity. 2004;21:415–428.
    OpenUrlPubMed
  15. 15.↵
    1. Zhang R,
    2. He X,
    3. Liu W,
    4. Lu M,
    5. Hsieh JT,
    6. Min W
    . AIP1 mediates TNF-alpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. J Clin Invest. 2003;111:1933–1943.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Zhang H,
    2. Zhang R,
    3. Luo Y,
    4. D’Alessio A,
    5. Pober JS,
    6. Min W
    . AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation. J Biol Chem. 2004;279:44955–44965.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Zhang H,
    2. Zhang R,
    3. Zhang H,
    4. Lin Y,
    5. Li J,
    6. Pober JS,
    7. Min W
    . RIP1-mediated AIP1 phosphorylation at a 14-3-3-binding site is critical for tumor necrosis factor-induced ASK1-JNK/p38 activation. J Biol Chem. 2007;282:14788–14796.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Naudé PJ,
    2. den Boer JA,
    3. Luiten PG,
    4. Eisel UL
    . Tumor necrosis factor receptor cross-talk. FEBS J. 2011;278:888–898.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Rothe M,
    2. Pan MG,
    3. Henzel WJ,
    4. Ayres TM,
    5. Goeddel DV
    . The TNFR2-TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell. 1995;83:1243–1252.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Pan S,
    2. An P,
    3. Zhang R,
    4. He X,
    5. Yin G,
    6. Min W
    . Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis. Mol Cell Biol. 2002;22:7512–7523.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Al-Lamki RS,
    2. Wang J,
    3. Vandenabeele P,
    4. Bradley JA,
    5. Thiru S,
    6. Luo D,
    7. Min W,
    8. Pober JS,
    9. Bradley JR
    . TNFR1- and TNFR2-mediated signaling pathways in human kidney are cell type-specific and differentially contribute to renal injury. FASEB J. 2005;19:1637–1645.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Grech AP,
    2. Gardam S,
    3. Chan T,
    4. Quinn R,
    5. Gonzales R,
    6. Basten A,
    7. Brink R
    . Tumor necrosis factor receptor 2 (TNFR2) signaling is negatively regulated by a novel, carboxyl-terminal TNFR-associated factor 2 (TRAF2)-binding site. J Biol Chem. 2005;280:31572–31581.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Rodríguez M,
    2. Cabal-Hierro L,
    3. Carcedo MT,
    4. Iglesias JM,
    5. Artime N,
    6. Darnay BG,
    7. Lazo PS
    . NF-kappaB signal triggering and termination by tumor necrosis factor receptor 2. J Biol Chem. 2011;286:22814–22824.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Marchetti L,
    2. Klein M,
    3. Schlett K,
    4. Pfizenmaier K,
    5. Eisel UL
    . Tumor necrosis factor (TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by N-methyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated phosphatidylinositol 3-kinase-dependent NF-kappaB pathway. J Biol Chem. 2004;279:32869–32881.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Rauert H,
    2. Wicovsky A,
    3. Müller N,
    4. Siegmund D,
    5. Spindler V,
    6. Waschke J,
    7. Kneitz C,
    8. Wajant H
    . Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2). J Biol Chem. 2010;285:7394–7404.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Fischer R,
    2. Maier O,
    3. Naumer M,
    4. Krippner-Heidenreich A,
    5. Scheurich P,
    6. Pfizenmaier K
    . Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NF-κB pathway. Cell Signal. 2011;23:161–170.
    OpenUrlCrossRefPubMed
  27. 27.↵
    1. Chen H,
    2. De Camilli P
    . The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin. Proc Natl Acad Sci USA. 2005;102:2766–2771.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Tournier C,
    2. Hess P,
    3. Yang DD,
    4. Xu J,
    5. Turner TK,
    6. Nimnual A,
    7. Bar-Sagi D,
    8. Jones SN,
    9. Flavell RA,
    10. Davis RJ
    . Requirement of JNK for stress-induced activation of the cytochrome c-mediated death pathway. Science. 2000;288:870–874.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Zhang R,
    2. Al-Lamki R,
    3. Bai L,
    4. Streb JW,
    5. Miano JM,
    6. Bradley J,
    7. Min W
    . Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a JNK-independent manner. Circ Res. 2004;94:1483–1491.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Feng X,
    2. Gaeta ML,
    3. Madge LA,
    4. Yang JH,
    5. Bradley JR,
    6. Pober JS
    . Caveolin-1 associates with TRAF2 to form a complex that is recruited to tumor necrosis factor receptors. J Biol Chem. 2001;276:8341–8349.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Vargas L,
    2. Nore BF,
    3. Berglof A,
    4. Heinonen JE,
    5. Mattsson PT,
    6. Smith CI,
    7. Mohamed AJ
    . Functional interaction of caveolin-1 with Bruton’s tyrosine kinase and Bmx. J Biol Chem. 2002;277:9351–9357.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Jones D,
    2. Xu Z,
    3. Zhang H,
    4. He Y,
    5. Kluger MS,
    6. Chen H,
    7. Min W
    . Functional analyses of the nonreceptor kinase bone marrow kinase on the X chromosome in vascular endothelial growth factor-induced lymphangiogenesis. Arterioscler Thromb Vasc Biol. 2010;30:2553–2561.
  33. 33.↵
    1. Kreuz S,
    2. Siegmund D,
    3. Scheurich P,
    4. Wajant H
    . NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol. 2001;21:3964–3973.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    1. Wertz IE,
    2. O’Rourke KM,
    3. Zhou H,
    4. Eby M,
    5. Aravind L,
    6. Seshagiri S,
    7. Wu P,
    8. Wiesmann C,
    9. Baker R,
    10. Boone DL,
    11. Ma A,
    12. Koonin EV,
    13. Dixit VM
    . De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature. 2004;430:694–699.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Slowik MR,
    2. Min W,
    3. Ardito T,
    4. Karsan A,
    5. Kashgarian M,
    6. Pober JS
    . Evidence that tumor necrosis factor triggers apoptosis in human endothelial cells by interleukin-1-converting enzyme-like protease-dependent and -independent pathways. Lab Invest. 1997;77:257–267.
    OpenUrlPubMed
  36. 36.↵
    1. Heyninck K,
    2. Beyaert R
    . A20 inhibits NF-kappaB activation by dual ubiquitin-editing functions. Trends Biochem Sci. 2005;30:1–4.
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Schütze S,
    2. Tchikov V,
    3. Schneider-Brachert W
    . Regulation of TNFR1 and CD95 signalling by receptor compartmentalization. Nat Rev Mol Cell Biol. 2008;9:655–662.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. D’Alessio A,
    2. Al-Lamki RS,
    3. Bradley JR,
    4. Pober JS
    . Caveolae participate in tumor necrosis factor receptor 1 signaling and internalization in a human endothelial cell line. Am J Pathol. 2005;166:1273–1282.
    OpenUrlCrossRefPubMed
  39. 39.↵
    1. D’Alessio A,
    2. Kluger MS,
    3. Li JH,
    4. Al-Lamki R,
    5. Bradley JR,
    6. Pober JS
    . Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells. J Biol Chem. 2010;285:23868–23879.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Chandrasekharan UM,
    2. Mavrakis L,
    3. Bonfield TL,
    4. Smith JD,
    5. DiCorleto PE
    . Decreased atherosclerosis in mice deficient in tumor necrosis factor-alpha receptor-II (p75). Arterioscler Thromb Vasc Biol. 2007;27:e16–e17.
    OpenUrlCrossRefPubMed
  41. 41.↵
    1. Chandrasekharan UM,
    2. Siemionow M,
    3. Unsal M,
    4. Yang L,
    5. Poptic E,
    6. Bohn J,
    7. Ozer K,
    8. Zhou Z,
    9. Howe PH,
    10. Penn M,
    11. DiCorleto PE
    . Tumor necrosis factor alpha (TNF-alpha) receptor-II is required for TNF-alpha-induced leukocyte-endothelial interaction in vivo. Blood. 2007; 109:1938–1944.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Min W,
    2. Pober JS
    . TNF initiates E-selectin transcription in human endothelial cells through parallel TRAF-NF-kappaB and TRAF-RAC/CDC42-JNK-c-Jun/ATF2 pathways. J Immunol. 1997;159:3508–3518.
    OpenUrlAbstract
View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • Both Internalization and AIP1 Association Are Required for Tumor Necrosis Factor Receptor 2-Mediated JNK Signaling
    Weidong Ji, Yonghao Li, Ting Wan, Jing Wang, Haifeng Zhang, Hong Chen and Wang Min
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2012;32:2271-2279, originally published August 15, 2012
    https://doi.org/10.1161/ATVBAHA.112.253666
    Permalink:
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Subjects