TumorNecrosisFactor(TNF)Receptor-associatedFactor7Is ... · and GST-c-JUN 1–79 fusion protein for...

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Tumor Necrosis Factor (TNF) Receptor-associated Factor 7 Is Required for TNF-induced Jun NH 2 -terminal Kinase Activation and Promotes Cell Death by Regulating Polyubiquitination and Lysosomal Degradation of c-FLIP Protein * Received for publication, September 2, 2011, and in revised form, December 27, 2011 Published, JBC Papers in Press, January 3, 2012, DOI 10.1074/jbc.M111.300137 Ivan Scudiero ‡§1 , Tiziana Zotti ‡§1 , Angela Ferravante ‡§ , Mariangela Vessichelli ‡§ , Carla Reale ‡§ , Maria C. Masone ‡§ , Antonio Leonardi , Pasquale Vito ‡§2 , and Romania Stilo ‡§ From the Dipartimento di Scienze per la Biologia, la Geologia e l’Ambiente, Università degli Studi del Sannio, Via Port’Arsa 11, Benevento 82100, the § Biogem Consortium, Via Camporeale, Ariano Irpino 83031, and the Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli “Federico II,” Via Pansini 5, Napoli 80131, Italy Background: TRAF proteins function as signal transducers for receptors involved in innate and adaptive immune responses. Results: TRAF7 is crucial for activation of JNK and regulates the expression level of the anti-apoptotic protein c-FLIP. Conclusion: A lysosomal pathway is involved in the turnover of c-FLIP. Significance: The results shown are important for anticancer strategies based on down-regulation of c-FLIP. The pro-inflammatory cytokine tumor necrosis factor (TNF) signals both cell survival and death. The biological outcome of TNF treatment is determined by the balance between survival factors and Jun NH 2 -terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated fac- tors (TRAFs) family of proteins, is essential for activation of JNK following TNF stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti- apoptotic protein c-FLIP L and demonstrate that degradation of c-FLIP L also occurs through a lysosomal pathway. RNA interfer- ence-mediated depletion of TRAF7 correlates with increased c-FLIP L expression level, which, in turn, results in resistance to TNF cytotoxicity. Collectively, our results indicate an impor- tant role for TRAF7 in the activation of JNK following TNF stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death. Death receptors are cell surface receptors that belong to the tumor necrosis factor (TNF) 3 receptor superfamily, the most well known members of which are tumor necrosis factor recep- tor 1 (TNFR1), the CD95/Fas receptor, and TNF-related apo- ptosis-inducing ligand (TRAIL) receptors DR4/TRAIL-R1 and DR5/TRAIL-R2 (1). Upon ligand binding, activated death receptors oligomerize, thereby inducing the formation of the death-inducing signaling complex (DISC) on their intracellular parts (2). The DISC consists of oligomerized death receptor, the adaptor molecule FADD, two isoforms of procaspase-8, pro- caspase-10, and c-FLIP proteins (2, 3). Upon binding to the DISC, procaspase-8 undergoes oligomerization, which results in processing of the zymogen (3–5). In responsive cells, acti- vated caspase-8 is then able to activate effector caspases, thereby initiating apoptosis (3–5). Cellular FLICE-inhibitory protein (c-FLIP), also known as Casper, iFLICE, FLAME-1, CASJ, CLARP, MRIT, or usurpin, is known as a crucial inhibitor of death receptor-mediated apo- ptosis by interfering with caspase-8 activation at the DISC sig- naling (6). c-FLIP exists as three splice variants, which give rise to a long form of c-FLIP (c-FLIP L ) polypeptide of 55 kDa (7, 8), the short form (c-FLIP S ) polypeptide of 26 kDa (7, 8), and a third 23-kDa form, called c-FLIP R (9). As a characteristic fea- ture, c-FLIP proteins contain tandem death effector domains, and all three isoforms of c-FLIP can be recruited to the DISC through an interaction of their tandem death effector domains with the adaptor protein FADD. Due to its structural similarity to caspase-8 and caspase-10, c-FLIP can therefore remain bound to FADD and inhibit complete caspase-8 processing and activation (8). Consistent with its role of crucial negative regulator of the apoptotic pathway, c-FLIP has been found overexpressed in different types of cancer. Increased levels of c-FLIP have been observed in a large number of tumor cell lines of various type, including carcinoma, gastric adenocarcinoma, pancreatic car- cinoma, melanoma, ovarian carcinoma, and prostate carci- noma (10, 11). In primary tumors, an elevated level of c-FLIP has been observed in B-cell chronic lymphocytic leukemia (12, 13), bladder urothelial carcinoma (14), lung adenocarcinoma (15), gallbladder carcinoma (16), and hepatocellular carcinoma (17). Generally, a high level of c-FLIP expression correlates with * This work was supported by Telethon Grant GGP08125B. 1 Both authors share the first authorship. 2 To whom correspondence should be addressed. E-mail: P.V.vito@unisannio. it. 3 The abbreviations used are: TNF, tumor necrosis factor; TNFR1, TNF receptor 1; TRAIL, TNF-related apoptosis-inducing ligand; DISC, death-inducing sig- naling complex; c-FLIP, cellular FLICE; TRAF, TNF receptor-associated fac- tor; EF, embryonic fibroblast; shTRAF7, shRNA targeting human TRAF7. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 8, pp. 6053–6061, February 17, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. FEBRUARY 17, 2012 • VOLUME 287 • NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 6053 by guest on January 11, 2020 http://www.jbc.org/ Downloaded from

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Tumor Necrosis Factor (TNF) Receptor-associated Factor 7 IsRequired for TNF�-induced Jun NH2-terminal KinaseActivation and Promotes Cell Death by RegulatingPolyubiquitination and Lysosomal Degradation of c-FLIPProtein*

Received for publication, September 2, 2011, and in revised form, December 27, 2011 Published, JBC Papers in Press, January 3, 2012, DOI 10.1074/jbc.M111.300137

Ivan Scudiero‡§1, Tiziana Zotti‡§1, Angela Ferravante‡§, Mariangela Vessichelli‡§, Carla Reale‡§, Maria C. Masone‡§,Antonio Leonardi¶, Pasquale Vito‡§2, and Romania Stilo‡§

From the ‡Dipartimento di Scienze per la Biologia, la Geologia e l’Ambiente, Università degli Studi del Sannio, Via Port’Arsa 11,Benevento 82100, the §Biogem Consortium, Via Camporeale, Ariano Irpino 83031, and the ¶Dipartimento di Biologia e PatologiaCellulare e Molecolare, Università degli Studi di Napoli “Federico II,” Via Pansini 5, Napoli 80131, Italy

Background: TRAF proteins function as signal transducers for receptors involved in innate and adaptive immuneresponses.Results: TRAF7 is crucial for activation of JNK and regulates the expression level of the anti-apoptotic protein c-FLIP.Conclusion: A lysosomal pathway is involved in the turnover of c-FLIP.Significance: The results shown are important for anticancer strategies based on down-regulation of c-FLIP.

The pro-inflammatory cytokine tumor necrosis factor (TNF)� signals both cell survival and death. The biological outcome ofTNF� treatment is determined by the balance between survivalfactors and Jun NH2-terminal kinase (JNK) signaling, whichpromotes cell death. Here, we show that TRAF7, the mostrecently identified member of the TNF receptor-associated fac-tors (TRAFs) family of proteins, is essential for activation of JNKfollowing TNF� stimulation. We also show that TRAF6 andTRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIPL and demonstrate that degradation ofc-FLIPL also occurs through a lysosomal pathway. RNA interfer-ence-mediated depletion of TRAF7 correlates with increasedc-FLIPL expression level, which, in turn, results in resistance toTNF� cytotoxicity. Collectively, our results indicate an impor-tant role for TRAF7 in the activation of JNK following TNF�stimulation and clearly point to an involvement of this proteinin regulating the turnover of c-FLIP and, consequently, celldeath.

Death receptors are cell surface receptors that belong to thetumor necrosis factor (TNF)3 receptor superfamily, the mostwell knownmembers of which are tumor necrosis factor recep-tor 1 (TNFR1), the CD95/Fas receptor, and TNF-related apo-ptosis-inducing ligand (TRAIL) receptors DR4/TRAIL-R1 andDR5/TRAIL-R2 (1). Upon ligand binding, activated deathreceptors oligomerize, thereby inducing the formation of the

death-inducing signaling complex (DISC) on their intracellularparts (2). TheDISC consists of oligomerized death receptor, theadaptor molecule FADD, two isoforms of procaspase-8, pro-caspase-10, and c-FLIP proteins (2, 3). Upon binding to theDISC, procaspase-8 undergoes oligomerization, which resultsin processing of the zymogen (3–5). In responsive cells, acti-vated caspase-8 is then able to activate effector caspases,thereby initiating apoptosis (3–5).Cellular FLICE-inhibitory protein (c-FLIP), also known as

Casper, iFLICE, FLAME-1, CASJ, CLARP,MRIT, or usurpin, isknown as a crucial inhibitor of death receptor-mediated apo-ptosis by interfering with caspase-8 activation at the DISC sig-naling (6). c-FLIP exists as three splice variants, which give riseto a long form of c-FLIP (c-FLIPL) polypeptide of 55 kDa (7, 8),the short form (c-FLIPS) polypeptide of 26 kDa (7, 8), anda third 23-kDa form, called c-FLIPR (9). As a characteristic fea-ture, c-FLIP proteins contain tandem death effector domains,and all three isoforms of c-FLIP can be recruited to the DISCthrough an interaction of their tandem death effector domainswith the adaptor protein FADD. Due to its structural similarityto caspase-8 and caspase-10, c-FLIP can therefore remainbound to FADD and inhibit complete caspase-8 processing andactivation (8).Consistent with its role of crucial negative regulator of the

apoptotic pathway, c-FLIP has been found overexpressed indifferent types of cancer. Increased levels of c-FLIP have beenobserved in a large number of tumor cell lines of various type,including carcinoma, gastric adenocarcinoma, pancreatic car-cinoma, melanoma, ovarian carcinoma, and prostate carci-noma (10, 11). In primary tumors, an elevated level of c-FLIPhas been observed in B-cell chronic lymphocytic leukemia (12,13), bladder urothelial carcinoma (14), lung adenocarcinoma(15), gallbladder carcinoma (16), and hepatocellular carcinoma(17). Generally, a high level of c-FLIP expression correlateswith

* This work was supported by Telethon Grant GGP08125B.1 Both authors share the first authorship.2 To whom correspondence should be addressed. E-mail: P.V.vito@unisannio.

it.3 The abbreviations used are: TNF, tumor necrosis factor; TNFR1, TNF receptor

1; TRAIL, TNF-related apoptosis-inducing ligand; DISC, death-inducing sig-naling complex; c-FLIP, cellular FLICE; TRAF, TNF receptor-associated fac-tor; EF, embryonic fibroblast; shTRAF7, shRNA targeting human TRAF7.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 8, pp. 6053–6061, February 17, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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a more aggressive form of the tumor and with a poor prognosis(18). For this, c-FLIP represents a promising target for cancertherapy, and a large amount of work is currently underway tovalidate c-FLIP as a therapeutic target to restore and/orincrease an apoptotic response in cancer cells (10, 11).Transcriptionally, c-FLIP expression is regulated by several

transcription factors, including NF-�B (19, 20) and p53 (21).However, in addition to transcriptional regulation, c-FLIPexpression level is also greatly regulated by post-transcriptionalmechanisms. In fact, c-FLIP expression is regulated by heatstress (22), by the JNK-mediated phosphorylation and activa-tion of the E3 ubiquitin ligase Itch (23), and by the ubiquitin/proteasomal pathway (24). In addition, JNK-independent deg-radative mechanisms of c-FLIP also have been described(25–27).TRAF7 is the most recently identified member of the TVF

receptor-associated factor (TRAF) proteins, a family of cyto-plasmic regulatorymolecules that functions as signal transduc-ers for receptors involved in both innate and adaptive humoralimmune responses (28–30). Functionally, TRAF7 potentiatesMEKK3-mediated signaling and regulates activation of NF-�Btranscription factor by promoting K29-linked polyubiquitina-tion of NEMO and p65 (28–31).Here, we show that TRAF7 is essential for activation of JNK

following TNF� stimulation. We also show that TRAF7 pro-motes cell death bymodulating the expression level of the anti-apoptotic protein c-FLIPL.

EXPERIMENTAL PROCEDURES

Cell Culture, Plasmids, and Antibodies—HEK293 and HeLacells were cultured inDulbecco’smodified Eagle’smedium sup-plemented with 10% FCS. Jurkat cells were cultured in RPMImedium supplemented with 10% FCS. HEK293 cells weretransfected by calcium phosphate precipitation. TRAF2�/�

embryonic fibroblasts (EFs) were provided by Drs. T. W. MakandW.C. Yeh (32), JNKDK0 EFswere provided byDr. R. Davis(33), and p65�/� EFs were provided by Dr. G. Franzoso (34).Cells were cultured in Dulbecco’s modified Eagle’s medium(Invitrogen) supplementedwith 10% FCS, 100 units/ml penicil-lin, and 100 �g/ml streptomycin.

Lentiviral vectors expressing shTRAF7 RNAs were obtainedfrom Sigma and used according to the manufacturer’s instruc-tions. Plasmids encoding mutant ubiquitins were a kind gift ofDr. C. Sasakawa, University of Tokyo.Sources of antisera and monoclonal antibodies were the fol-

lowing: anti-c-FLIP and anti-Fas from Alexis; anti-FLAG andanti-�-actin from Sigma; anti-phosho-Jun, anti-phosho-p38, anti-p38, and anti-JNK1/3 (anti-JNK) from Cell Signaling;and anti-ubiquitin (P4D1), anti-phosho-JNK, and anti-HAfrom Santa Cruz Biotechnology. TNF�, MG132, leupeptin, andcycloheximide were from Sigma.Immunoblot Analysis andCoprecipitation—Cell lysates were

made in lysis buffer (150 mM NaCl, 20 mM Hepes, pH 7.4, 1%Triton X-100, 10% glycerol, and a mixture of protease inhibi-tors). Proteinswere separated by SDS-PAGE, transferred onto anitrocellulosemembrane, and incubatedwith primary antibod-ies followed by horseradish peroxidase-conjugated secondaryantibodies (Amersham Biosciences). Blots were developed

using the ECL system (Amersham Biosciences). For coimmu-noprecipitation experiments, cells were lysed in lysis buffer,and immunocomplexes were bound to protein A/G (RocheApplied Science), resolved by SDS-PAGE, and analyzed byimmunoblot assay.Luciferase Assay—To assess AP1 activation, HEK293 cells

were transfected with the indicated plasmidic DNAs togetherwith pAP-1-luc (Clontech) in 6-well plates. 24 h after transfec-tion, luciferase activity was determined with the LuciferaseAssay System (Promega). Plasmids expressing RSV-�-galacto-sidase or TK-Renillawere used in transfectionmixtures to nor-malize for efficiency of transfection.InVitroKinaseAssay—In vitro kinase assaywas performed as

reported by Herr et al. (35). Briefly, following stimulation, celllysates were first immunoprecipitated with rabbit polyclonalanti-JNK1/3 (Santa Cruz Biotechnology) conjugated with pro-tein A-Sepharose beads. Then, samples were spun down andwashed to remove nonspecifically bound proteins. The kinasereaction was carried out in the presence of nonradioactive ATPand GST-c-JUN 1–79 fusion protein for 25 min at 30 °C. Thereaction was terminated by adding Laemmli buffer, and sam-ples were resolved by 12% SDS-PAGE. After blotting ontonitrocellulose membrane, phosphorylated c-Jun protein wasdetected using a rabbit polyclonal antibody specifically raisedagainst phosphorylated Ser-73. Signals were developed by ECL.ATPLite Assay—A total of 8� 103 cells per well was cultured

in a flat-bottom 96-well plate in quadruplicates in 10% FBS/DMEM medium. Cell viability was determined using anATPLiteTM (PerkinElmer Life Sciences) kit according to themanufacturer’s instructions.Statistical Analysis—Results are expressed as mean � S.D.

from a number of samples as indicated in the correspondingfigure legends. Student’s t test was used to determine statisticalsignificance of two-group comparisons. Multiple comparisonsstatistical significancewas verified by analysis of variancewith aone-tailed F-test, the posthoc Tukey honestly significant differ-ence test, and Bonferroni and Dunnett corrections.

RESULTS

We and others have previously reported that ectopic expres-sion of TRAF7 induces activation of AP1 transcription factor(28–31). To verify whether TRAF7 plays a physiological role inTNFR1 signaling, we determined the effect of RNA interfer-ence-mediated depletion of TRAF7 on TNF�-induced AP1activation, as assessed by an AP1-luciferase reporter assay. Forthis, HEK293 cells, transfected with a vector encoding for ashort hairpin RNA (shRNA) designed to target human TRAF7(30) or a scramble shRNA, were exposed to TNF� for 6 h, andluciferase activity was determined. As shown in Fig. 1A, deple-tion of TRAF7 reduces AP1 transcriptional activity induced byTNF� exposure. This effect was specific for the TNFR1 signal-ing, because AP1 activity induced by a constitutively activemutant of MEKK7 was not altered by TRAF7 depletion in thesame cells (Fig. 1A). Next, we determined whether induction ofAP1 activity due to TRAF7 expression occurs through activa-tion of JNK. To this end, HEK293 cells were transfected with anexpression vector empty or encoding for TRAF7, and the acti-vation state of JNK was monitored by in vitro kinase assay. As

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shown in Fig. 1B, TRAF7 expression induces activation of JNK,independently of TNF� stimulation. Activation of JNK follow-ing TRAF7 expression was confirmed by immunoblot assaysprobed with anti phospho-JNK antibodies (Fig. 1C).Finally, we determined whether TRAF7 function is required

for JNK activation following TNF� stimulation. Thus, HEK293cells depleted of TRAF7 were exposed to TNF�. Cell lysateswere then immunoprecipitated with anti-JNK antibody, andthe immunoprecipitates were subjected to in vitro kinaseassays. The result of the experiment, shown in Fig. 1D, indicatesthat depletion of TRAF7 reduces activation of JNK followingTNF� stimulation. Overall, from these experiments we con-cluded that TRAF7 expression is sufficient for JNK activation,and its function is required for complete activation of JNK fol-lowing TNF� stimulation.

It is known that TNF�-mediated JNK activation acceleratesturnover of c-FLIPL via induction of the E3 ubiquitin ligase Itch,which specifically polyubiquitinates c-FLIPL, thereby inducingits proteasomal degradation (23). In addition, we have recentlyshown that TRAF7 mediates polyubiquitination events thatplay a key role in regulating cellular functions (31). Taking intoaccount that TRAF7 expression induces cell death (29, 31), weexamined whether TRAF7 influences the ubiquitination stateof c-FLIP. For this, we transfected HEK293 cells with TRAF7and assessed the ubiquitination state of endogenous c-FLIPL by

immunoblot experiments. As shown in Fig. 2A, TRAF7 expres-sion promotes polyubiquitination of endogenous c-FLIPL andcorrelates with a reduction in the expression level of c-FLIPL(Fig. 2B). Because ubiquitin possesses seven lysines (Lys-6, Lys-11, Lys-27, Lys-29, Lys-33, Lys-48 and Lys-63), and the fate ofubiquitinated proteins depends on the K-type of ubiquitin link-age, we used a series of ubiquitin mutants possessing singlelysine residues to investigate the nature of TRAF7-mediatedpolyubiquitination of c-FLIPL. In the presence of TRAF7,c-FLIPL was polyubiquitinated by Lys-29-, Lys-33-, Lys-63-,and, to a lesser extent, by Lys-48-linked ubiquitin mutants (Fig.2C). Given this result, we examined whether also TRAF6,another member of the TRAF family that promotes polyubiq-uitination (36), was capable of inducing polyubiquitination ofc-FLIPL. In fact, as shown in Fig. 2D, TRAF6 induced polyubiq-uitination of c-FLIPL, although with a pattern different thanthat used byTRAF7. Indeed, TRAF6 triggered Lys-27-, Lys-29-,and, to a lesser extent, Lys-48-linked polyubiquitination ofc-FLIPL. Interestingly, TRAF6, but not TRAF7, also promoted aweak polyubiquitination of JNK (Fig. 2E).It is known that, although Lys-48 polyubiquitination targets

proteins for proteasomal degradation, unconventional Lys-29polyubiquitination promotes lysosomal protein degradation(37). Thus, we tried to determine whether, in addition to pro-teasomal degradation, also lysosomal mechanisms regulate the

FIGURE 1. TRAF7 is required for JNK activation following TNF� stimulation. A, HEK293 cells were transfected with vectors encoding for an shRNA targetinghuman TRAF7 (shTRAF7) (30) or a control sequence (scrambled), along with an AP1-luciferase reporter plasmid. 16 h later, cells were treated with TNF� (30ng/ml) for 6 h, and luciferase activity was determined. Data shown represent relative normalized luciferase activity and are representative of six independentexperiments done in triplicate. B, HEK293 cells were transfected with an expression vector empty or encoding TRAF7. 24 h later, cells were stimulated withTNF�, and the activation state of JNK was monitored by in vitro kinase assay. Cells exposed to anisomycin served as a positive control for JNK activation.C, HEK293 cells were transfected as indicated and stimulated with TNF�. The activation state of JNK and p38 was monitored by immunoblot assay. D, HEK293cells were infected with lentiviral vectors encoding for shTRAF7 or a scramble control sequence. Cells were stimulated with TNF�, and the activation state ofJNK was monitored by in vitro kinase assay.

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FIGURE 2. TRAF7 promotes polyubiquitination of c-FLIPL. A, HEK293 cells were transfected with an expression vector encoding for HA-ubiquitin along witha vector empty or expressing a FLAG-tagged version of TRAF7. 24 h later, cell lysates were immunoprecipitated with anti c-FLIP antibody, separated bySDS-PAGE, and transferred onto membranes subsequently probed with anti-HA. B, HEK293 cells transfected with an expression vector encoding for TRAF7were monitored for c-FLIP expression by immunoblot assay. c-FLIPL expression levels were quantitated by ImageJ (lower panel). C and D, HEK293 cells werecotransfected with HA-tagged ubiquitin mutants and FLAG-tagged TRAF7 (C) or TRAF6 (D). The numbers indicate the only lysine residue remaining in theubiquitin molecule. Immunoprecipitates with anti-c-FLIP antibody were resolved by SDS-PAGE and blotted onto a membrane subsequently probed withanti-HA. E, HEK293 cells were transfected as indicated, and the ubiquitination state of JNK was monitored by immunoblot assay probed with an anti-ubiquitinantibody (P4D1).

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expression level of c-FLIPL. To do this, we used a very sensitivecellular experimental system, the EFs from TRAF2�/� mice. Infact, these cells are particularly sensitive to the cytotoxic activ-ity of TNF�, because they express very low levels of c-FLIPL dueto a rapid degradation of this protein (38). Thus, we monitoredthe expression levels of c-FLIPL in TRAF2�/� EFs untreated ortreated with lysosomal (leupeptin � NH4Cl) or proteasomal(MG132) inhibitors. As shown in Fig. 3A, TRAF2�/� EFsexpress almost undetectable levels of c-FLIPL. Treatment withMG132 restores c-FLIPL expression, confirming the existenceof a proteasomal-mediated degradation of the protein (24, 36).Interestingly, c-FLIPL expression was also restored in cellsexposed to lysosomal inhibitors. Significantly, also treatmentwith cycloheximide, which inhibits the lysosomal proteasescathepsins, restores c-FLIPL expression in TRAF2�/� EFs. Col-lectively, these results show, for the first time, that degradationof c-FLIPL also occurs through a lysosomal pathway, which,together with the proteasomal pathway, regulates the expres-sion level of this protein. The recovery of c-FLIPL due to pro-

teasomal and lysosomal inhibitors is functionally relevant,because TRAF2�/� EFs treated with these inhibitors are con-siderably resistant to TNF� cytotoxicity (Fig. 3B) and show alower level of caspase-8 activation following TNF� stimulation(Fig. 3C). To verify that the protective effect exerted by lyso-somal inhibitors was not limited to the TRAF2�/� murine EFs,we depletedTRAF2 from the human cell lineHEK293 via infec-tion with a retroviral vector encoding a shRNA targetinghuman TRAF2. As shown in Fig. 3D, also in this experimentalsystem lysosomal inhibitors confer resistance toTNF� cytotox-icity. Lysosomal inhibitors treatment induces accumulation ofc-FLIPL also in the mouse embryonic fibroblast cell line NIH-3T3 (Fig. 3E).Next, we examined whether TRAF7 was involved in the deg-

radation of c-FLIP in TRAF2�/� EFs. To do this, we abrogatedTRAF7 expression in TRAF2�/� EFs via infection with a retro-viral vector encoding two different shRNA designed to targetmurine TRAF7. As shown in Fig. 4A, depletion of TRAF7 inTRAF2�/� EFs correlated with an increase in the level of

FIGURE 3. Lysosome inhibitors restore c-FLIPL expression in TRAF2�/� EF. A, TRAF2�/� EFs were treated for 12 h with the indicated inhibitors (2.5 �M

MG132, 1.25 nM leupeptin, 2.5 �M NH4Cl), and normalized lysates were examined for c-FLIPL expression by immunoblot analysis. Lysates from NIH-3T3 cellsserved as positive control c-FLIPL expression. B, TRAF2�/� EFs were pretreated for 1 h with the indicated inhibitors, and TNF� was added at the indicatedconcentration. 16 h later, cell viability was determined with the ATPLiteTM assay. Each data point represents the mean � S.D. cell survival expressed as apercentage of untreated cells in six replicates. C, TRAF2�/� EFs pretreated for 1 h with the indicated inhibitors were stimulated with TNF� for the indicated timeperiods, and caspase-8 activation was monitored by immunoblot assay. D, HEK293 cells were infected with retroviral vectors encoding for a shRNA targetinghuman TRAF2 or a control scramble sequence. Cells were pretreated for 1 h with lysosomes inhibitors, and then TNF� was added at the indicated concentra-tion. 16 h later, cell viability was determined with the ATPLiteTM assay. Each data point represents the mean � S.D. cell survival expressed as a percentage ofuntreated cells in four replicates. E, NIH-3T3 cells were left untreated or treated with lysosomes inhibitors for indicated time periods. c-FLIPL expression levelswere monitored by Western blot analysis and quantitated by ImageJ. Each data point shown in the graph represents relative normalized optical densitypercentage.

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c-FLIPL expression, which, in turn, correlated with resistanceto the cytotoxic activity of TNF� (Fig. 4B). The same result (Fig.4C) was obtained using p65�/� EFs, which, similarly to theTRAF2�/� EFs, are sensitive to the cytotoxic activity of TNF�(39).We also determined whether the resistance to TNF� cyto-

toxicity observed following abrogation of TRAF7 is due to thepositive effect that TRAF7 plays on JNK activation. For this, weused EFs derived from mice deficient for both JNK1 and JNK2(JNK�/�double knock out), which donot displayTNF�-induc-ible c-FLIPL proteasomal degradation (23). Normally, thesecells are more resistant than normal EFs to the cytotoxic actionof TNF�, yet cell death can be observed following exposure toTNF� in the presence of cycloheximide. As shown in Fig. 5,TRAF7 depletion resulted in increased survival of JNK�/�

DKO EFs following treatment with TNF�, indicating thatresistance to apoptosis due to TRAF7 deficiency is independentof JNK function.

Finally, we examined whether the observation obtainedusing cell lines derived from knockout mice was extensible toother cell types. For this, we used two well known cellular sys-tems, namely Jurkat cells and HeLa cells. As shown in Fig. 6A,Jurkat cells depleted ofTRAF7were significantlymore resistantto the cytotoxic activity of both anti-Fas andTNF�. In addition,TRAF7-depleted Jurkat cells showed a remarkable accumu-lation of c-FLIPL following TNF� stimulation (Fig. 6B). Simi-larly, TRAF7-depleted HeLa cells showed an increased expres-sion level of both c-FLIPL and c-FLIPS following TNF�stimulation (Fig. 6C).

DISCUSSION

There are several aspects that make particularly interestingthe finding we report herein. The first one is the demonstrationthat TRAF7 is required for activation of JNK following TNF�stimulation. It is well established that, following TNF� stimu-lation, the balance between NF-�B and JNK activities deter-mines the outcome of TNFR1 signaling, such that NF-�Bpromotes cell survival, whereas JNK activation enhancesTNF�-induced death (40). Therefore, the discovery here thatTRAF7 has an essential role in activation of JNK followingTNF� stimulation is, per se, valuable information that adds afurther element in themap of knowledgewe have about TNFR1signaling.At least in part, JNK activity controls TNF�-induced death

through the proteasomal processing of c-FLIPL via activation ofthe ubiquitin ligase Itch (23). This brings us to the secondimportant finding contained in this report: i.e., the evidence fora lysosomal pathway that controls c-FLIPL turnover. This dis-covery is significant and potentially of great consequence,because many in vitro studies have demonstrated the impor-tance of the role of c-FLIPL in resistance to apoptosis inducedby death receptors and to conventional chemotherapy (10, 11).Elevated expression of c-FLIP is often identified in malignantcancers, and it strongly correlates with a poor prognosis (10,

FIGURE 4. TRAF7 depletion restores c-FLIPL expression in TRAF2�/� EF. A, TRAF2�/� EFs were infected with retroviral vectors encoding for two differentshRNAs targeting murine TRAF7 (shTRAF7#5 and shTRAF7#2) or a control sequence (scramble). After selection, cells were left untreated or stimulated with TNF�(10 ng/ml) for 6 h, and then normalized lysates were examined for c-FLIPL expression by immunoblot analysis. B, TRAF2�/� EFs retrovirally infected as indicatedwere treated with TNF� at the indicated concentrations for 16 h, and then cell viability was determined with the ATPLiteTM assay. Lysates from NIH-3T3 cellsserved as positive control for c-FLIPL expression. Each data point represents the mean � S.D. cell survival expressed as a percentage of untreated cells in sixreplicates. Statistical analysis was by the one-tailed unpaired Student’s test. C, p65�/� EFs retrovirally infected as indicated were treated as in B.

FIGURE 5. TRAF7 depletion protects cells independently of JNK function.JNK�/� DKO EFs retrovirally infected as indicated were treated with TNF�plus cycloheximide (1 �g/ml) at the indicated concentration for 16 h, andthen cell viability was determined with the ATPLiteTM assay. Each data pointrepresents the mean � S.D. cell survival expressed as a percentage ofuntreated cells in six replicates. Statistical analysis was by the one-tailedunpaired Student’s test.

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11). Additionally, preclinical data clearly indicate that selectiveinhibitors of c-FLIP, in combination with conventional chem-otherapy, could represent an effective antitumor therapy (41–43). Therefore, the finding here, that a lysosomal degradationpathway regulates cellular turnover of c-FLIPL, offers addi-tional tools for anticancer strategies based on down-regulationof c-FLIP.The last, but not less important aspect emphasized in this

report, is that TRAF7 is involved in regulating the expressionlevel of c-FLIPL to different degrees. First, TRAF7 could regu-late degradation of c-FLIPL via activation of JNK and, thus,through the pathway JNK/Itch/proteasome described by

Chang et al. (23). The evidence that TRAF7 promotes Lys-48-linked polyubiquitination of c-FLIP (Fig. 2B) is consistent withthis possibility. However, it should definitely be noted thatTRAF7 promotes Lys-29-linked polyubiquitination of c-FLIPL,and this type of polyubiquitination has been associated withlysosomal degradation of proteins (37). Also striking is the evi-dence that lysosomal degradation of proteins by Lys-29-linkedpolyubiquitination occurs through the E3 ubiquitin ligase Itch(37).In addition, because TRAF6 promotes polyubiquitination of

c-FLIPL, it would be interesting to see if it regulates the expres-sion level of c-FLIPL as well. However, this analysis is compli-

FIGURE 6. TRAF7 depletion in Jurkat and HeLa cells. A, Jurkat cells were infected with retroviral vectors encoding either for an shRNA targeting human TRAF7or for a scrambled sequence. Cells were treated with either anti-Fas (upper panel) or TNF� (lower panel) at the indicated concentrations plus cycloheximide (1�g/ml). 16 h later, cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Each data point represents the mean � S.D. cellsurvival expressed as percentage of untreated cells in four replicates. Statistical analysis was by the one-tailed unpaired Student’s test. B, Jurkat cells infectedand treated with TNF� as in A were monitored for c-FLIPL expression levels by Western blot and quantitated by ImageJ. Data shown represent relativenormalized optical density percentage and are representative of five independent experiments. C, HeLa cells infected and treated with TNF� as indicated weremonitored for c-FLIPL and c-FLIPS expression levels.

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cated by the fact that TRAF6 is a potent activator of NF-�B,which, in turn, positively regulates transcription of c-FLIP (19,20). Also intriguing is the experiment conducted on theJNK�/� DKO EFs, which would suggest that the positive effectexerted by TRAF7 depletion on c-FLIPL turnover is indepen-dent of JNK function.Clearly, ourwork opensmany interesting questions concern-

ing TRAF7 function that require further investigation. In thiscontext, the generation of animal models genetically modifiedin the locus encoding for TRAF7 will certainly be of enormousvalue to finally define the physiological role of this protein.

Acknowledgments—We thank Drs. Antonella Fierro and LiberatoPanza for technical assistance, Drs. Angela Zampelli and FrancescoMorra for critical reading of the manuscript, and Drs. C. Sasakawaand H. Ashida at the University of Tokyo for kindly providing theplasmids encoding mutant ubiquitins used in this study.

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Maria C. Masone, Antonio Leonardi, Pasquale Vito and Romania StiloIvan Scudiero, Tiziana Zotti, Angela Ferravante, Mariangela Vessichelli, Carla Reale,

Regulating Polyubiquitination and Lysosomal Degradation of c-FLIP Protein-terminal Kinase Activation and Promotes Cell Death by2-induced Jun NH

αTumor Necrosis Factor (TNF) Receptor-associated Factor 7 Is Required for TNF

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