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Molecular and Cellular Biology, March 2005, p. 2130-2137, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2130-2137.2005

TRAF2 Plays a Key, Nonredundant Role in LIGHT-Lymphotoxin ß Receptor Signaling

You-Sun Kim,1 Sergei A. Nedospasov,2 and Zheng-gang Liu1*

Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,1 Basic Research Program, SAIC-Frederick, Inc., and Laboratory of Molecular Immunoregulation, NCI-Frederick, Frederick, Maryland2

Received 20 August 2004/ Returned for modification 17 September 2004/ Accepted 17 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LIGHT is a member of the tumor necrosis factor (TNF) superfamily, and its function is mediated by at least two receptors, including lymphotoxin ß receptor (LTßR) and herpes simplex virus entry mediator. However, the molecular mechanism of LIGHT signaling mediated by LTßR has not been clearly defined. In this report, we demonstrate that TRAF2 is critical for LIGHT- and LTßR-mediated activation of both the transcription factor NF-{kappa}B and the mitogen-activated protein kinase JNK. In HeLa cells, LIGHT induces NF-{kappa}B and JNK activation, which can be blocked by the dominant negative mutant of TRAF2. In these cells, LIGHT causes the recruitment of TRAF2, TRAF3, and I{kappa}B kinase into the LTßR complex. Importantly, while both NF-{kappa}B and JNK are activated by LIGHT in wild-type mouse embryonic fibroblasts, no activation of either of these two pathways is observed in TRAF2 null fibroblasts. However, LIGHT-induced NF-{kappa}B and JNK activation can be restored by ectopic expression of TRAF2 in TRAF2–/– cells. Interestingly, in contrast to TNF signaling, the activation of both NF-{kappa}B and JNK by LIGHT was normal in RIP–/– and TRAF5–/– cells. Taken together, our data demonstrate that TRAF2, an important effector molecule of TNF signaling, plays a critical, nonredundant role in LIGHT-LTßR signaling.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIGHT is a cell surface-bound member of the tumor necrosis factor (TNF) superfamily implicated in costimulation and T-cell homeostasis (19, 43). LIGHT forms a membrane-anchored homotrimeric complex that is capable of binding to both lymphotoxin ß receptor (LTßR) and herpes simplex virus entry mediator (HVEM) (41, 56). LTßR is a member of the TNF receptor superfamily, playing a critical role in the development, organization, and differentiation of lymphoid tissue (37, 51). The expression of LTßR can be detected in most types of cells, including fibroblast, epithelial, and myeloid lineage cells, but not on T or B lymphocytes (14). Similar to other members of the TNF receptor (TNFR) family, the engagement of ligands, such as LIGHT, to LTßR induces receptor aggregation and subsequent activation of multiple signaling pathways. Signaling of LTßR activated with LT{alpha}1ß2 or agonist anti-LTßR antibody leads to the induction of cell death of certain tumor cells (4, 21, 44, 56) and the expression of genes encoding integrins, chemokines, and cytokines, such as B-cell activating factor (BAFF) and beta interferon (8, 20). Recently, studies in mice expressing recombinant LIGHT or after administration of soluble HVEM proteins to block LIGHT activity revealed that LIGHT is required for activation and expansion of T cells and plays an important role in T-cell homeostasis (46, 53).

At the mechanistic level, LTßR ligation can lead to activation of the transcription factor NF-{kappa}B and the mitogen-activated protein (MAP) kinase JNK (6, 38, 52). Importantly, this receptor activates the so-called alternative NF-{kappa}B pathway (3) which, along with the JNK pathway, is thought to play a critical role in mediating LTßR function. The transcription factor NF-{kappa}B family regulates the expression of genes crucial to innate and adaptive immune responses, cell growth, and apoptosis (18, 26, 48). In mammalian cells, the NF-{kappa}B family is composed of five members, RelA, RelB, c-Rel, p50/NF-{kappa}B1, and p52/NF-{kappa}B2 (47). p50 and p52 are the proteolytic processing products of the p105/NF-{kappa}B1 and p100/NF-{kappa}B2 proteins, respectively. In most cells, the NF-{kappa}B dimer is sequestered in the cytosol and its nuclear translocation can be induced by a wide variety of stimuli (18, 47). These stimuli trigger activation of the I{kappa}B kinase (IKK) complex, which consists of two catalytic subunits, IKK{alpha} and IKKß, and one regulatory subunit, IKK{gamma}, causing phosphorylation and ubiquitin-dependent degradation of the I{kappa}Bs (27). The NF-{kappa}B processing not only serves to generate p50 and p52 but also plays a role in liberating specific NF-{kappa}B complexes. Most studies on NF-{kappa}B processing implicate the role of p105, but in the alternative signaling pathway NF-{kappa}B activity is induced via the processing of p100 protein. This alternative mechanism for inducing NF-{kappa}B activity has emerged based on the observation that inducible IKK{alpha}-dependent p100 processing allows the resultant p52 to function as a transcriptional activator. In most cell types only a small amount of p52 is produced relative to its precursor p100 (2), and the regulated expression of p52 might be important for generating NF-{kappa}B dimers with specific functions (5, 16, 24). Recently, overexpression of NF-{kappa}B-inducing kinase has been shown to trigger the processing of p100 to p52 by site-specific p100 phosphorylation and subsequent ubiquitination (54). Thus, the processing of p100 to generate p52 is an important alternative step in NF-{kappa}B regulation and is different from the classical pathway in that it presumably activates a distinct set of downstream genes (3).

JNK (c-Jun N-terminal kinase), also known as stress-activated kinase (7, 25), is activated by many apoptosis-inducing stimuli, and it is thought to be an important apoptotic mediator (33, 36). In addition to apoptosis, JNK activation is also involved in many other biological processes, such as cell proliferation, embryogenesis, and immunological responses (7, 12, 25, 49). It is believed that AP-1 is the major target of the JNK pathway and that most biological functions of JNK are achieved by regulating AP-1 activity, although the mechanism of JNK-mediated apoptosis remains unknown (7, 25, 33). Most members of the TNF superfamily are potent inducers of JNK activation (9). For instance, JNK activity is dramatically elevated by TNF treatment (7, 25). While it has been reported that overexpression of HVEM leads to the activation of JNK, it is largely unknown whether LIGHT-LTßR can activate JNK (22, 39).

Like other members of the TNFR superfamily, the cytoplasmic region of LTßR lacks domains with enzymatic activity, such as protein kinases. LTßR signaling is achieved through the formation of ligand-induced receptor signaling complex (13, 31). Previous reports demonstrated that several TRAF proteins, including TRAF2, -3, -4, and -5, can bind to the cytoplasmic domain of LTßR (39, 52). However, the functional significance of each of these TRAFs in LTßR signaling has not been fully understood. In this study, we demonstrate that TRAF2 is critical for the activation of NF-{kappa}B and JNK mediated by LIGHT-LTßR interaction. We show that in HeLa cells LIGHT induces NF-{kappa}B and JNK activation and that TRAF2 and TRAF3 are recruited to the LTßR complex following LIGHT binding. Additionally, we show that while NF-{kappa}B is activated by LIGHT in wild-type (WT) mouse embryonic fibroblasts (MEF), this activation is diminished in TRAF2 null fibroblasts (TRAF2–/– cells) but can be restored by ectopic expression of TRAF2 in TRAF2–/– cells. Collectively, our data identify TRAF2, an important effector molecule of TNF signaling, as a critical, nonredundant component in LIGHT-LTßR signaling.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Murine recombinant TNF-{alpha}, human recombinant LIGHT, anti-TNF-R1 antibody, and anti-LTßR antibody were purchased from R&D Systems. Agonistic monoclonal anti-murine LTßR antibodies were kindly provided by J. Browning (Biogene Inc.). Anti-TRAF2, anti-TRAF3, anti-TRAF5, anti-IKK{alpha}, anti-IKKß, anti-Sp1, antihemagglutinin (anti-HA), anti-JNK1, and anti-NF-{kappa}B p52 antibodies were purchased from Santa Cruz Biotechnology. Anti-RIP antibody was purchased from Transduction Laboratories. Antiactin and anti-FLAG antibodies were purchased from Sigma.

Cell culture. WT, RIP–/–, TRAF2–/–, and TRAF5–/– MEF cells were previously described (28, 50, 55). TNFR1–/– and LTßR–/– MEF cells were generated from TNFR1 and LTßR knockout mice, respectively. MEF and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. Previously established stable cell lines TRAF2–/–/TRAF2 and TRAF2–/–/TRAF2 (87-501) (10) were cultured in this medium plus 300 µg of hygromycin/ml.

Western blot analysis and coimmunoprecipitation. After treatments as described in the figure legends, cells were collected and lysed in M2 buffer (20 mM Tris at pH 7, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM ß-glycerol phosphate, 1 mM sodium vanadate, 1 µg of leupeptin/ml). Fifty micrograms of the cell lysates was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotted. The proteins were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham). For immunoprecipitation assays, HeLa cells were treated with LIGHT (50 ng/ml) as indicated below in the legend of Fig. 3 and then collected in lysis buffer (50 mM HEPES at pH 7.6, 250 mM NaCl, 0.1% NP-40, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg of leupeptin/ml, 1 µg of aprotinin/ml, 1 µg of pepstatin/ml). The lysates were mixed and precipitated with anti-LTßR antibody (R&D) and protein G-agarose beads by incubation at 4°C overnight. The beads were washed four times with 1 ml of lysis buffer, and the bound proteins were resolved in 4 to 20% SDS-polyacrylamide gels and detected by Western blot analysis.



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  		FIG. 3. Recruitment of TRAF2 and IKK{alpha} and -ß to LTßR following LIGHT treatment. Cell extracts were prepared from HeLa cells after treatment with LIGHT for various times as indicated. After normalization of the protein content according to the protein assay results, cell extracts were immunoprecipitated with the anti-LTßR antibody overnight. Immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blotting with anti-TRAF2, anti-TRAF3, anti-RIP, anti-IKK{alpha}, and anti-IKKß antibodies. One percent of cell extracts from each sample was used as a control of protein input.

 
EMSA. Nuclear extracts were prepared from WT, RIP–/–, TRAF2–/–, TRAF5–/–, TNFR1–/–, LTßR–/–, TRAF2–/–/TRAF2, TRAF2–/–/TRAF2 (87-501), and HeLa cells as previously described (23). For binding reactions, 5 µg of nuclear extract was incubated at room temperature for 20 min with reaction buffer containing 20 mM HEPES (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 200 µg of bovine serum albumin/ml, and 2 µg of poly(dI-dC) · poly(dI-dC). Then, the 32P-labeled double-stranded oligonucleotide (1 ng; ≥105 cpm) containing the NF-{kappa}B binding consensus sequence (5'-GGCAACTGGGGACTCTCCCTTT-3') was added to the reaction mixture for an additional 10 min at room temperature. The reaction products were fractionated on a nondenaturing 5% polyacrylamide gel, which was then dried and subjected to autoradiography. As controls of nuclear protein content, Oct1 oligonucleotides (5'-GGTGTCGAATGCAAATCACTAGAA-3') and anti-Sp-1 antibody were used in an electrophoretic mobility shift assay (EMSA) and Western blotting, respectively.

Transfection, kinase assay, and luciferase assay. Transfection experiments in HeLa cells were performed with Lipofectamine Plus reagent by following the instructions provided by the manufacturer (GIBCO/BRL). HeLa cells were cotransfected with the dominant negative TRAF2 (87-501), TRAF6 (289-522) construct (1), or empty vector, as indicated below in Fig. 4. JNK1 or HA-JNK1 was immunoprecipitated with anti-JNK1 or anti-HA antibody, and JNK kinase activities were determined by in vitro kinase assay using glutathione S-transferase-c-Jun (1-79) as the substrate. For luciferase assay, cells were cotransfected with p2XNF-{kappa}B-Luc, pRSV-ß-galactosidase, and the dominant negative TRAF2 (87-501) or empty vector. At 24 h after transfection, the cells were treated with LIGHT (50 ng/ml) for an additional 8 h, and luciferase activity of each sample was measured using a luciferase assay kit (Promega). Luciferase activity was normalized relative to the ß-galactosidase activity of each sample.



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  		FIG. 4. The dominant negative mutant of TRAF2, TRAF2 (87-501), inhibits LIGHT-induced NF-{kappa}B and JNK activation. (A) HeLa cells were transfected with HA-I{kappa}B{alpha} and pcDNA, mutant FLAG-TRAF2 (87-501), or mutant FLAG-TRAF6 (289-522) and treated with LIGHT for 15 min. Cell extracts were applied to SDS-PAGE for Western blotting with anti-HA, anti-FLAG, and antiactin antibodies. (B) The dominant negative form of TRAF2 (87-501) inhibits LIGHT-induced NF-{kappa}B activation. Cells were transfected with NF-{kappa}B-LUC, ß-galactosidase, and pcDNA or mutant FLAG-TRAF2 (87-501). After 24 h, cells were treated with or without LIGHT (50 ng/ml) for 8 h. Luciferase activities were then measured using a luciferase assay system, and values were normalized based on ß-galactosidase activities. The data represent triplicate experiments. (C) The dominant negative form of TRAF2 (87-501) induces JNK activation by LIGHT treatment. HA-JNK1 and pcDNA, mutant FLAG-TRAF2 (87-501), or mutant FLAG-TRAF6 (289-522) were transfected and treated with LIGHT (50 ng/ml) for 15 min. Cell extracts were quantified by protein assay and were then either immunoprecipitated with anti-HA antibody to perform a kinase assay or resolved on SDS-PAGE for Western blotting with anti-FLAG and anti-HA antibodies.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LIGHT-induced NF-{kappa}B and JNK activation is mediated by LTßR. Cytokines of the TNF family can activate multiple signaling pathways, including NF-{kappa}B and JNK pathways in most types of cells. Previous studies reported that LIGHT is a potent inducer of NF-{kappa}B activation (38), but it remained unclear whether LIGHT can activate JNK. To study the molecular mechanism of LIGHT signaling, we examined the activation of NF-{kappa}B and JNK following recombinant LIGHT engagement in HeLa cells. As shown in Fig. 1A, LIGHT induced the degradation of I{kappa}B{alpha} within 15 min after treatment and the protein level of I{kappa}B{alpha} was recovered by 60 min. We also measured the NF-{kappa}B activity by EMSA and found that LIGHT-induced NF-{kappa}B activation was quick and prolonged (Fig. 1B). As controls of nuclear protein content, the activity of the transcription factor Oct1 and the Sp1 protein level in the same nuclear extracts were examined by EMSA and Western blotting, respectively. Consistent with previous reports, these results indicated that LIGHT activates NF-{kappa}B in HeLa cells. To test whether LIGHT activates JNK, we performed an in vitro kinase assay following LIGHT treatment, using glutathione S-transferase-c-Jun as the substrate (35). As shown in Fig. 1C, LIGHT was cable of activating JNK, and this activation was transient and peaked at 15 min after treatment. Taken together, these data suggest that LIGHT, like other members of the TNF family, including TNF and TRAIL, can induce activation of both NF-{kappa}B and JNK pathways.



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  		FIG. 1. Activation of NF-{kappa}B and JNK following LIGHT engagement in HeLa cells. (A) Degradation of I{kappa}B{alpha} induced by LIGHT in HeLa cells. HeLa cells were treated with LIGHT (50 ng/ml) and incubated for various times as indicated. Cell extracts were applied to SDS-PAGE for Western blotting using anti-I{kappa}B{alpha} and antiactin antibodies. (B) Kinetics of NF-{kappa}B activation by LIGHT. Cells were treated with LIGHT for various times. Nuclear extracts were prepared, and 5 µg of the nuclear exact from each sample was used to analyze NF-{kappa}B activity by EMSA with an NF-{kappa}B probe. As controls, Oct1 oligonucleotides and anti-Sp1 antibody were used in EMSA and Western blotting, respectively. (C) LIGHT-induced JNK activation. Cells were treated with LIGHT for various times as indicated, and cell extracts were quantified by protein assay and were then either immunoprecipitated with anti-JNK1 antibody to perform a kinase assay or resolved on SDS-PAGE for Western blotting with anti-JNK1 antibody.

 
Although LIGHT signaling could be mediated by two receptors, LTßR and HVEM, in this study we focused on the LIGHT-LTßR signaling axis. To do so, we examined LIGHT-induced activation of NF-{kappa}B and JNK in WT and LTßR null (LTßR–/–) MEF cells (17). As shown in Fig. 2A, top panel, LIGHT treatment resulted in potent NF-{kappa}B activation in WT MEF cells, as measured by EMSA. In contrast, little activity of NF-{kappa}B was detected in LTßR–/– MEF following LIGHT treatment. As a control, LIGHT-induced NF-{kappa}B activation was also measured in TNFR1–/– cells, and no decrease of NF-{kappa}B activity was observed in these cells, suggesting that the observed decrease of NF-{kappa}B activity in LTßR–/– cells is due to the absence of LTßR. When TNF was used to treat the same types of MEF cells, it activated NF-{kappa}B equally well in both WT and LTßR–/– cells but failed to do so in TNFR1–/– cells (Fig. 2A, bottom panel). These results indicate that LTßR plays a major role in LIGHT-induced NF-{kappa}B activation in MEF cells. Similarly, we also measured LIGHT-induced JNK activation in LTßR–/– cells by in vitro kinase assay. As shown in Fig. 2B, LIGHT treatment did not lead to the activation of JNK, while JNK activation was observed in WT and TNFR1–/– MEF cells. Therefore, these data imply that LTßR is the dominant receptor for LIGHT-induced NF-{kappa}B and JNK activation in MEF cells.



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  		FIG. 2. LTßR is the dominant receptor for LIGHT-induced NF-{kappa}B and JNK activation in MEF cells. (A) LIGHT-induced NF-{kappa}B activation is dependent on LTßR. Mouse WT, LTßR–/–, and TNFR1–/– fibroblasts were stimulated with LIGHT (50 ng/ml) or TNF-{alpha} (30 ng/ml) for various times as indicated. NF-{kappa}B activity was measured by EMSA as described in the legend for Fig. 1. (B) Activation of JNK was impaired in LTßR–/– fibroblasts. Mouse WT, LTßR–/–, and TNFR1–/– fibroblasts were either left untreated or treated with LIGHT for 15 min for the JNK kinase assay. Cell extracts were normalized according to protein assay results and then either immunoprecipitated with anti-JNK1 antibody to perform a kinase assay or resolved on SDS-PAGE for Western blotting with anti-JNK1 antibody.

 
TRAF2 has an essential, nonredundant role in LIGHT-induced activation of NF-{kappa}B and JNK. Previous studies suggested that several proteins, including TRAF2 and TRAF3, can interact with LTßR to form an LTßR signaling complex and mediate LTßR signaling (30, 52). To test whether these proteins are recruited to LTßR in response to LIGHT treatment, we performed coimmunoprecipitation experiments with an anti-LTßR antibody. As LIGHT triggers a stronger activation of NF-{kappa}B and JNK in HeLa cells than in MEF cells, we carried out these experiments in HeLa cells. As shown in Fig. 3 (panels 1 and 2), both TRAF2 and TRAF3 coprecipitated with LTßR, and the presence of these two proteins in the LTßR complex could be detected as early as 5 min after LIGHT treatment. Interestingly, the recruitment of TRAF2 to the LTßR complex was quite sustained and was still noticeable at 8 h after LIGHT treatment. However, receptor interacting protein (RIP), one of the key effector molecules of TNF signaling, was not present in the LTßR complex (Fig. 3, panel 3). As previous studies indicated that IKK is essential for LTßR-mediated NF-{kappa}B activation (40), we then tested whether the two catalytic subunits of IKK, IKK{alpha} and IKKß, are recruited to the receptor complex. As shown in Fig. 3, panels 4 and 5, while the recruitment of IKK{alpha} to LTßR was robust and prolonged following LIGHT treatment, the appearance of IKKß in the receptor complex was weak and transient. This observation is consistent with previous reports that IKK{alpha} plays a specific and significant role in LTßR signaling compared to IKKß (54).

Although it had been suggested that TRAF2 may be involved in LTßR signaling, the exact role of TRAF2 in LIGHT-LTßR signaling remained largely unknown. To investigate whether TRAF2 is required for LIGHT-induced NF-{kappa}B and JNK activation, we first tested the effect of the dominant negative mutant TRAF2 (87-501), a mutant lacking the ring finger (35), on LIGHT-LTßR signaling. To examine the effect on NF-{kappa}B activation, both I{kappa}B{alpha} degradation and the induction of NF-{kappa}B reporter activity were measured. As shown in Fig. 4A and B, the expression of TRAF2 (87-501) blocked LIGHT-induced I{kappa}B{alpha} degradation and NF-{kappa}B reporter activity. Similarly, the expression of TRAF2 (87-501) also inhibited LIGHT-induced JNK activation as measured in an in vitro kinase assay (Fig. 4C). To show that the effect of the TRAF2 mutant on LIGHT signaling is specific, the dominant negative mutant of TRAF6, TRAF6 (289-522), was used as a control and the expression of TRAF6 (289-522) had no detectable effect on LIGHT signaling (Fig. 4A and C). These results indicated that TRAF2 may be indeed a key effector molecule in LIGHT-induced NF-{kappa}B and JNK activation.

To further confirm this conclusion, we examined LIGHT-induced NF-{kappa}B and JNK activation in TRAF2–/– MEF cells. Since RIP and TRAF5 have been reported as key effector molecules of TNF signaling, we also tested whether these proteins are required for LIGHT signaling by using RIP–/– and TRAF5–/– MEF cells. The absence of the expression of TRAF2, RIP, and TRAF5 in respective null cells is documented in Fig. 5A. For NF-{kappa}B activation, as shown in Fig. 5B, LIGHT was able to activate NF-{kappa}B to a similar extent in WT, RIP–/–, and TRAF5–/– MEF cells, but failed to do so in TRAF2–/– cells. Sp1 protein levels in these four types of cells were used as a protein content control. Oct1 activity was used as a control for the EMSA. When LIGHT-induced JNK activation was examined in all these four types of cells, the failure of JNK activation in response to LIGHT treatment was only observed in TRAF2–/– cells (Fig. 5C and data not shown). To rule out the possibility that the inability of LIGHT signaling in TRAF2–/– cells is due to some defect, other than the absence of TRAF2 protein, we tested whether the ectopic expression of TRAF2 in TRAF2–/– cells could reconstitute LIGHT signaling. Using two previously established stable cell lines of TRAF2–/– cells, TRAF2–/–/TRAF2 and TRAF2–/–/TRAF2 (87-501) (10), the latter one expressing the truncated TRAF2 (87-501) (Fig. 6A), we found that LIGHT-induced NF-{kappa}B and JNK activation was restored by WT TRAF2 (Fig. 6B and C). Interestingly, the expression of the truncated TRAF2, TRAF2 (87-501), in TRAF2–/– cells failed to restore the LIGHT-induced NF-{kappa}B activation (Fig. 6B). Taken together, these results suggested that TRAF2, but not RIP or TRAF5, is essential for LIGHT-induced NF-{kappa}B and JNK activation, and the RING finger domain of TRAF2 is essential for this function. More importantly, unlike in TNF signaling, TRAF2 has a nonredundant role in LIGHT-LTßR signaling. These findings further support the notion that TRAF3 is essential for LTßR-mediated apoptosis but is dispensable for LTßR-mediated NF-{kappa}B activation (15, 30, 52).



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  		FIG. 5. TRAF2 has a critical role in LIGHT-induced activation of NF-{kappa}B and JNK. (A) Expression levels of TRAF2, RIP, TRAF5, and actin in WT, TRAF2–/–, RIP–/–, and TRAF5–/– fibroblasts. The same amount of cell extracts from each cell line was applied to SDS-PAGE for Western blotting assays with anti-TRAF2, anti-RIP, anti-TRAF5, and antiactin antibodies. (B) LIGHT-induced NF-{kappa}B activation. Mouse WT, TRAF2–/–, RIP–/–, and TRAF5–/– fibroblasts were treated with 50 ng of LIGHT/ml for various times as indicated. NF-{kappa}B activity was measured by EMSA as described in the legend for Fig. 1. (C) Activation of JNK was impaired in TRAF2–/– fibroblasts. Mouse WT and TRAF2–/– fibroblasts were treated with LIGHT for the indicated time periods, and cell extracts were quantified by protein assay and then either immunoprecipitated with anti-JNK1 antibody to perform a kinase assay or resolved on SDS-PAGE for Western blotting with anti-JNK1 antibody.

 


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  		FIG. 6. Restoration of LIGHT-induced NF-{kappa}B and JNK activation in TRAF2–/–/TRAF2 cells. (A) Protein expression levels in WT, TRFA2–/–, TRAF2–/–/TRAF2, and TRAF2–/–/TRAF2 (87-501) cells. The same amount of cell extracts from WT, TRAF2–/–, and two stable cell lines of TRAF2–/–, TRAF2–/–/TRAF2 and TRAF2–/–/TRAF2 (87-501), was applied to SDS-PAGE for Western blotting with anti-TRAF2 antibody. (B) Reconstitution of LIGHT-induced NF-{kappa}B activation in TRAF2–/–/TRAF2 cells. Mouse WT, TRAF2–/–, TRAF2–/–/TRAF2, and TRAF2–/–/TRAF2 (87-501) cells were treated with LIGHT (50 ng/ml) for various times as indicated. NF-{kappa}B activity was measured by EMSA. (C) Restoration of LIGHT-induced JNK activation in TRAF2–/–/TRAF2 cells. Mouse WT, TRAF2–/–, and TRAF2–/–/TRAF2 cells were treated with LIGHT for 15 min and collected for an in vitro kinase assay. The activation of JNK was measured by using a phosphorimager.

 
TRAF2 is also required for LIGHT- and LTßR-mediated NF-{kappa}B2 activation. It was reported recently that the engagement of LTßR leads to the activation of NF-{kappa}B2 through processing of p100 to p52, a distinct feature of the alternative NF-{kappa}B pathway (54). Compared to NF-{kappa}B1 activation, the kinetics of this pathway is relatively slow. While IKKß plays a major role in NF-{kappa}B1 activation, IKK{alpha} and the MAP3K, NF-{kappa}B-inducing kinase, have all been suggested to play a critical role in NF-{kappa}B2 activation (42, 54). Since our data indicated that TRAF2 was essential for LIGHT-LTßR-mediated NF-{kappa}B and JNK activation, it was important to define its role in p100 processing in response to LTßR ligation. As shown in Fig. 7A, the engagement of LTßR by an agonistic anti-LTßR antibody (14) resulted in the processing p100 to p52, as evidenced by Western blotting with an anti-p52/p100 antibody. To test the involvement of TRAF2 in this process, we examined p100 processing in TRAF2–/– cells at 8 h after anti-LTßR treatment. As in the previous experiments, RIP–/– and TRAF5–/– cells were also used. As shown in Fig. 7B, LTßR-mediated NF-{kappa}B2 activation was normal in RIP–/– and TRAF5–/– cells, in accordance with the results shown Fig. 5B. However, there was no detectable processing of p100 to p52 in TRAF2–/– cells following anti-LTßR antibody treatment. We also tested LIGHT-induced processing of p100 in these cells and obtained similar results (Fig. 7C and data not shown). Since the RING finger domain of TRAF2 is critical for LIGHT-induced NF-{kappa}B activation (Fig. 6B), we then tested whether the RING domain of TRAF2 is also essential for LIGHT-induced NF-{kappa}B2 activation. To do so, we examined the processing of p100 in the two reconstituted cell lines of TRAF2–/– cells, TRAF2–/–/TRAF2 and TRAF2–/–/TRAF2 (87-501). As shown in Fig. 7C, while the ectopic expression of full-length TRAF2 restored the processing of p100 in response to LIGHT treatment, the truncated TRAF2 (87-501) failed to do so. Therefore, we conclude that TRAF2 is also a key effector in LIGHT-LTßR-mediated NF-{kappa}B2 activation and that the RING domain of TRAF2 is indispensable.



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  		FIG. 7. TRAF2 is required for LIGHT-LTßR-mediated NF-{kappa}B2 activation. (A) LTßR engagement induces processing of p100 to generate p52 in WT MEF. Mouse WT fibroblasts were treated with an agonistic LTßR antibody (1 µg/ml) for various times as indicated. Cell extracts were applied to SDS-PAGE for Western blotting with anti-p100/p52 and antiactin antibodies. (B) TRAF2 but not RIP or TRAF5 is required to trigger p100 processing. Mouse WT, RIP–/–, TRAF2–/–, and TRAF5–/– fibroblasts were either left untreated or treated with an agonistic LTßR antibody (1 µg/ml) for 8 h. (C) The RING finger domain of TRAF2 is essential for LIGHT-induced NF-{kappa}B2 activation. Mouse WT, TRAF2–/–, TRAF2–/–/TRAF2, and TRAF2–/–/TRAF2 (87-501) cells were treated with LIGHT (50 ng/ml) for 8 h and collected for Western blotting.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As with several other members of the TNF superfamily, LIGHT is implicated in costimulation and control of T-cell homeostasis, the function most likely mediated by the HVEM receptor (41, 56). Additionally, LIGHT, along with LT{alpha}/LTß, appears to play a role in organogenesis of some lymphoid tissues (43), and this partly redundant function may be mediated by the LTßR, which is expressed on stromal cells of lymphoid tissues (51). The molecular mechanism of LIGHT-LTßR signaling is not fully understood and, in particular, the role of TRAF2 protein in the downstream functions had remained unknown. In this report, we demonstrate for the first time that TRAF2 has an essential, nonredundant role in LIGHT-LTßR-mediated activation of both the transcription factor NF-{kappa}B and the MAP kinase JNK. It has been shown that the engagement of LTßR by recombinant LIGHT or anti-LTßR antibodies can induce apoptosis and NF-{kappa}B activation (4, 52), but the activation of JNK by LIGHT through LTßR has not yet been reported. Since the cytoplasmic domain of LTßR does not contain consensus sequences characteristic of a protein kinase, it is believed that LTßR signaling is mediated by the formation of an LTßR complex (52). Recently, it was suggested that LTßR can interact with several effector proteins, including TRAF2 and TRAF3, and that TRAF3 plays a key role in LTßR-induced apoptosis (15, 44). However, the effector molecule that is responsible for LTßR-mediated NF-{kappa}B activation has not been identified. In the present study, we have demonstrated that LIGHT activates both NF-{kappa}B and JNK in HeLa and MEF cells, and this signaling is mainly mediated by LTßR in MEF cells. In response to LIGHT, the LTßR signaling complex is quickly formed by recruiting other proteins to LTßR. Consistent with earlier reports (30, 52), we found that both TRAF2 and TRAF3 are present in the LTßR complex. Since TRAF3 is essential for LTßR-mediated apoptosis but not for NF-{kappa}B activation and TRAF2 is known to mediate NF-{kappa}B and JNK activation in several other signaling pathways, it was important to determine the role of TRAF2 in LIGHT-LTßR signaling. Our studies with the dominant negative mutant TRAF2 (87-501) and employing TRAF2–/– cells suggest that TRAF2 is essential for LIGHT-LTßR-mediated NF-{kappa}B and JNK activation. Using TRAF2–/– MEF cells, we also found that TRAF2 is essential for NF-{kappa}B2 activation, as the processing of p100 to p52 is severely impaired in these cells. Therefore, our data identify TRAF2, an important effector molecule of TNF signaling, as playing a critical role in LIGHT-LTßR signaling.

TRAF2 was initially identified as a component of the TNFR2 complex and later as a key effector molecule of TNFR1 signaling (45). Although TRAF2 is dispensable for TNF-induced apoptosis, it plays a critical role in TNF-induced activation of NF-{kappa}B and JNK (55). Studies with TRAF2 and TRAF5 null mice suggested that TRAF2 and TRAF5 are functionally redundant in TNF signaling (50). In this study, however, we found that TRAF2, but not TRAF5, is specifically required for LIGHT-LTßR-mediated NF-{kappa}B and JNK activation. Unlike in TNF signaling, TRAF2 function has nonredundancy in LIGHT-LTßR signaling. Our early studies indicated that the role of TRAF2 in TNF signaling is to recruit downstream molecules, such as IKK, to the TNFR1 complex (10). It now appears that TRAF2 may fulfill a similar role in LIGHT and LTßR signaling, since IKK and TRAF2 are both present in the same LTßR complex (Fig. 3) and the truncated TRAF2, TRAF2 (87-501), is unable to restore LIGHT signaling in TRAF2–/– cells (Fig. 6). Interestingly, while TRAF2 and both IKK{alpha} and IKKß are recruited to LTßR at the early time points, only TRAF2 and IKK{alpha} are present in the LTßR complex at 8 h after LIGHT treatment (Fig. 3), which is the time point when the processing of p100 occurs. In addition, our data suggest that the RING figure domain of TRAF2 is required for both the classic and alternative NF-{kappa}B pathways induced by LIGHT (Fig. 6 and 7). These observations imply that TRAF2 may recruit different downstream targets to initiate distinct pathways: IKK{alpha} and IKKß in the case of NF-{kappa}B1 activation and IKK{alpha} for p100 processing.

Recent studies also suggested that TRAF2 is involved in TNF- or reactive oxygen species-induced necrotic cell death. However, since LIGHT did not induce cell death in MEF cells, the role of TRAF2 in LIGHT-induced cell death was not addressed in this study. However, since TRAF3 is known to be essential for LTßR-mediated cell death, it is possible that TRAF2 may play a less important role in this process. Furthermore, a recent study reported that TRAF3 is degraded during the activation of NF-{kappa}B2 by BAFF and CD40 and that the TRAF3 degradation is important for p100 processing (32). To examine whether such a regulation of TRAF3 stability exists in LIGHT-induced NF-{kappa}B2 activation, we examined the TRAF3 protein level in response to LIGHT treatment. While p100 processing was detected at 8 h after LIGHT treatment (Fig. 7C), we failed to observe any obvious change of the TRAF3 protein level (Fig. 3). However, we found that after it is recruited to the LTßR signaling complex, TRAF3 is dissociated from the complex at the time when the NF-{kappa}B2 pathway is activated (Fig. 3). Therefore, it seems that TRAF3 is regulated differently in LIGHT-induced NF-{kappa}B2 activation compared to BAFF and CD40 signaling.

Previous studies demonstrated that TNF signaling needs another critical effector molecule, RIP (11, 28). RIP is a death domain kinase, and it is required for TNF-induced activation of both NF-{kappa}B and MAP kinases (11). Recently, it has been reported that RIP is also essential for TNF-induced necrotic cell death (34). However, in this study we found no role for RIP in LIGHT-LTßR-mediated activation of NF-{kappa}B and JNK. We also examined whether RIP2, a homologue of RIP, is involved in LIGHT-LTßR signaling. Our preliminary results from coimmunoprecipitation experiments indicated that RIP2 is not recruited to the LTßR complex in response to LIGHT treatment (data not shown). Therefore, it appears that LIGHT-LTßR signaling may not require a RIP-like molecule. Nevertheless, as several signaling pathways that use TRAF proteins as their effector molecules require a protein kinase effector, for instance, RIP for TNF signaling, IRAK for IL-1 signaling, and RIP2 for TLR signaling (29), it is likely that a functionally similar molecule may also be required for LIGHT and LTßR signaling. Identification of such a molecule should be addressed in future studies.

Regarding the non-NF-{kappa}B gene activation pathways, this study documents that LIGHT is a potent activator of the MAP kinase JNK pathway. As a key modulator of the transcription factor AP-1, the activation of JNK may play a critical role in LIGHT-mediated cellular responses, in particular those involved in the development and maintenance of the lymphoid tissues. While our present work has made progress in understanding mechanisms of LIGHT-LTßR signaling, many important issues remain elusive. For instance, it will be important to determine how NF-{kappa}B and JNK pathways are specifically regulated by TRAF2 in response to LIGHT and what is the respective role of these two pathways in LIGHT-mediated cellular responses. Addressing these issues will help us to better understand the physiological functions of LIGHT, specifically those mediated by the LTßR.


   ACKNOWLEDGMENTS
 
We thank W. C. Yeh and T. W. Mak for the TRAF2–/– fibroblasts, M. Kelliher for RIP–/– fibroblasts, H. Nakano for TRAF5–/– fibroblasts, and J. Browning for the agonistic anti-LTßR antibody. We thank M. Drutskaya for help with MEF preparations. We are also grateful to Swati Choksi for her critical reading of the manuscript.

S.A.N. is funded in part with U.S. Federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-12400.

The contents of this publication do not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.


   FOOTNOTES
 
* Corresponding author. Mailing address: Cell and Cancer Biology Branch, NCI, NIH, Bldg. 10, Rm. 6N105, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-3390. Fax: (301) 402-1997. E-mail: zgliu{at}helix.nih.gov. Back


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 Discussion
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Molecular and Cellular Biology, March 2005, p. 2130-2137, Vol. 25, No. 6
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