cIAP1-dependent TRAF2 degradation regulates the differentiation of

PHAGOCYTES, GRANULOCYTES, AND MYELOPOIESIS

cIAP1-dependent TRAF2 degradation regulates the differentiation of monocytesinto macrophages and their response to CD40 ligandAlban Dupoux,1,2 Jessy Cartier,1,2 Severine Cathelin,1,2 Rodolphe Filomenko,1,2 Eric Solary,1,2 and Laurence Dubrez-Daloz1,2

1Inserm Unite Mixte de Recherche 866, Dijon; and 2Faculty of Medicine, Institut Federatif de Recherche, University of Burgundy, Dijon, France

Peripheral blood monocytes are plastic cellsthat migrate to tissues and differentiate intovarious cell types, including macrophages,dendritic cells, and osteoclasts. We havedescribed the migration of cellular inhibitorof apoptosis protein 1 (cIAP1), a member ofthe IAP family of proteins, from the nucleusto the Golgi apparatus in monocytes under-goingdifferentiation intomacrophages.Herewe show that, once in the cytoplasm, cIAP1is involved in the degradation of the adaptorprotein tumor necrosis factor receptor–

associated factor 2 (TRAF2) by the proteoso-mal machinery. Inhibition of cIAP1 preventsthe decrease in TRAF2 expression that char-acterizesmacrophageformation.Wedemon-strate that TRAF2 is initially required formacrophage differentiation as its silencingprevents I�-B� degradation, nuclearfactor-�B (NF-�B) p65 nuclear transloca-tion, and the differentiation process. Then,we show that cIAP1-mediated degradationof TRAF2 allows the differentiation processto progress. This degradation is required for

the macrophages to be fully functional asTRAF2 overexpression in differentiated cellsdecreases the c-Jun N-terminal kinase–mediated synthesis and the secretion ofproinflammatory cytokines, such as interleu-kin-8 and monocyte chemoattractant pro-tein 1 (MCP-1) in response to CD40 ligand.We conclude that TRAF2 expression andsubsequent degradation are required for thedifferentiation of monocytes into fully func-tional macrophages. (Blood. 2009;113:175-185)

Introduction

Tumor necrosis factor receptor (TNFR)–associated factors (TRAFs)form an evolutionarily conserved family of intracellular adaptors thatbind directly or indirectly to members of the TNFR and the interleukin-1(IL-1)/Toll-like receptor (TLR) families.1,2 They participate in thetransduction of signals from these receptors to downstream events thatregulate cell proliferation, differentiation, and death.

The member of this family known as TRAF2 directly bindsCD27, CD30, CD40, CD137, TNFR2, and receptor activator ofnuclear factor-�B (RANK). TRAF2 can also bind TNFR1indirectly, through interaction with TNFR-associated deathdomain protein.3

On receptor engagement, TRAF2 is recruited in a receptor-associated multiprotein complex4-6 where it contributes to stimulatespecific downstream signaling pathways. Depending on cell type,differentiation stage, and stimulated receptors, these signalingpathways can involve c-jun N-terminal kinase (JNK), nuclearfactor �B (NF-�B), and p38 mitogen-activated protein kinase(p38MAPK).4,5,7-9 TRAF2 is also a key regulator of TNFR1-mediated apoptosis.10-13 TRAF2 activity is regulated by its interac-tion with protein partners, such as TRAF1,14-16 subcellular localiza-tion,7,8,15 ubiquitylation, and degradation by the proteasomepathway.8,12,13,17-19

A yeast 2-hybrid screen of proteins able to bind TRAF2identified a direct interaction with cIAP1 (cellular inhibitor ofapoptosis protein 1, also named BIRC2, HIAP2), a member of theIAP family of proteins.20,21 Thanks to the presence of a C-terminalzinc finger domain (RING domain) that displays an E3-ubiquitinligase activity, cIAP1 was demonstrated to promote TRAF2ubiquitylation and to target the protein for proteasome-mediateddegradation.12,13,22-24

We have previously shown that cIAP1 was required formacrophage differentiation.25 We have also shown that cIAP1migrated from the nucleus to the cytoplasm to concentrate at thesurface of the Golgi apparatus in monocytes undergoing differentia-tion into macrophages.26 However, the role of cIAP1 and thefunctional significance of its differentiation-associated redistribu-tion remained unknown. Here we show that TRAF2 is initiallyrequired for the differentiation of monocytes into macrophages.Then, cIAP1 triggers its proteosomal degradation, which appears tobe required for the normal outcome of the differentiation process.cIAP1 also maintains a low level of TRAF2 in differentiatedmacrophages, which favors the secretion of proinflammatorycytokines on exposure to CD40 ligand (CD40L).

Methods

Antibodies

The antihuman cIAP1 and antihuman HSC70 mouse monoclonal antibodieswere obtained from BD Biosciences (Le Pont de Claix, France) and SantaCruz Biotechnology (Santa Cruz, CA), respectively. The following rabbitpolyclonal antibodies were used: antihuman cIAP1, antihuman X-linkedinhibitor of apoptosis protein (XIAP; R&D Systems, Lille, France),antihuman TRAF2 (StressGen, Victoria, BC), antihuman poly(ADP-ribose)polymerase (Santa Cruz Biotechnology), antihuman JNK/stress-activatedprotein kinase (SAPK), antihuman phospho-JNK/SAPK, antihuman I�B�(Cell Signaling Technology, Ozyme, Saint-Quentin-en-Yvelines, France).For immunofluorescence experiments, antihuman NF-�B p65 (Santa CruzBiotechnology) and fluorescein isothiocyanate (FITC)–conjugated antihu-man GM-130 (Transduction Laboratories, Lexington, KY; BD Biosciences,San Jose, CA) were used. For flow cytometry experiments, we used FITC or

Submitted February 5, 2008; accepted August 1, 2008. Prepublished online asBlood First Edition paper, September 30, 2008; DOI 10.1182/blood-2008-02-137919.

The online version of this article contains a data supplement.

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allophycocyanin (APC)–conjugated anti-CD11b or anti-CD71 antibodies(BD Biosciences PharMingen). Secondary antibodies used included goathorseradish peroxidase (HRP)–conjugated antimouse or antirabbit antibod-ies (Jackson ImmunoResearch Laboratories, West Grove, PA) for Westernblot analysis and goat 488 or 568–Alexa Fluor antirabbit or antimouseantibodies (Molecular Probes, Eugene, OR) for immunofluorescence studies.

Chemicals

Recombinant human (rh) macrophage colony-stimulating factor (M-CSF)was obtained from PeproTech (Neuilly-sur-seine, France) and rhCD40Lfrom Abcys (Paris, France). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA)was from Sigma-Aldrich (St Quentin Fallavier, France), Smac-N7 peptide,lactacystin, and kinase inhibitor SP600125 from Calbiochem (Fontenaysous Bois, France), and Z-leu-leu-leu-H-(aldehyde) (MG132) from PeptideInstitute (Osaka, Japan). Leptomycin B (LMB) was kindly provided by DrM. Yoshida (University of Tokyo, Tokyo, Japan).

Cell culture and differentiation

U937 and THP-1 were obtained from the ATCC (Manassas, VA) anddifferentiated by 20 nM TPA exposure as described.27 Human peripheralblood monocytes were obtained from healthy donors with informed consentand purified using a CD14� monocyte isolation kit with a light-scatteringcolumn according to the manufacturer’s instructions (Miltenyi Biotec,Paris, France) and then incubated (0.5 � 106/mL) for up to 6 days in RPMI1640 medium (Lonza, Verviers, Belgium), 10% fetal calf serum (Lonza), inthe presence of 100 ng/mL M-CSF to trigger their differentiation intomacrophages. The differentiated phenotype was identified by a flowcytometric analysis of the cell surface expression of CD11b (U937 andTHP-1 cells) or CD71 (human macrophages) as described.28

Cell extracts, immunoprecipitation, cell fractionation, andimmunoblot analysis

Whole-cell lysates were prepared by incubating the cells in lysis buffer(50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Triton X-100) in the presenceof protease inhibitors and then sonicated. Extracts for phosphoproteinanalysis were prepared by lysing the cells in lysis buffer (50 mM Tris-HCl,pH 7.4, 50 mM NaCl, 15 mM Na4P2O7, 5 mM NaF, 150 mM Na3VO4, 1%Triton X-100) in the presence of protease inhibitors and then centrifuged toremove cellular debris. Cell fractionation and immunoprecipitation experi-ments were performed as described.26,28 All extracts were stored at �80°Cuntil analysis. Protein concentration was measured using the Bio-Rad DCProtein Assay Kit (Ivry sur Seine, France). Forty micrograms of proteinswas incubated in loading buffer (125 mM Tris-HCl, pH 6.8, 10%�-mercaptoethanol, 4.6% sodium dodecyl sulfate, 20% glycerol, 0.003%bromophenol blue), separated by sodium dodecyl sulfate–polyacrylamidegel electrophoresis (SDS-PAGE), and electroblotted to nitrocellulosemembrane (Whatman, Schleicher & Schuell, Versailles, France). Afterblocking nonspecific binding sites for 2 hours by 2% of bovine serumalbumin in Tris-buffered saline (TBS)–Tween (0.05 M Tris base, 0.9%NaCl, pH 7.6, 0.05% Tween 20), the membrane was incubated overnight at�4°C with the primary antibody, washed, incubated with horseradishperoxidase-conjugated secondary antibodies for 45 minutes at roomtemperature, and then washed again 3 times in TBS-Tween. The immuno-blot was revealed using an enhanced chemiluminescence detection kit (GEHealthcare, Little Chalfont, United Kingdom) by autoradiography.

Plasmid constructs, siRNAs, and cell transfection

Six-day–differentiated human blood monocytes and 48-hour–differentiatedTHP-1 cells were transfected with pBABE-TRAF2 wt (kindly provided byOlivier Micheau, Inserm UMR 866, Dijon, France) or related control vector(3 �g/106 cells) using Lipofectamine LTX reagent (Invitrogen) or withsiRNA (2 �g/106 cells) using Oligofectamine reagent (Invitrogen). Undiffer-entiated THP-1 and U937 cells and human blood monocytes werenucleoporated with small interfering RNA (siRNA) using Amaxa nucleofec-tor kit (Amaxa Biosystems, Gaithersburg, MD) following the manufactur-er’s instructions. The siRNA sequences were cIAP1 siRNA sequence

1 (forward: 5�-GGCCAAGAGUUUGUUGAUtt-3�; reverse: 5�-AUCAA-CAAACUCUUGGCCtt-3�; Eurogentec, Seraing, Belgium); cIAP1 se-quence 2 (121288) designed and purchased from Ambion (Courtaboeuf,France), TRAF2 siRNA sequence 1 (Hs_TRAF2_4) and sequence2 (Hs_TRAF2_5) both designed and provided by QIAGEN (Courtaboeuf,France).

Retroviral supernatant production and transduction protocol

A mutated form of TRAF2 within the RING and the fourth zinc fingerdomain (TRAF2*) (kindly provided by Ze’ev Ronai, Ruttenberg CancerCenter, New York, NY), cIAP1 wt, and cIAP1-RING (deleted for theRING domain) cDNAs were inserted into a bicistronic retroviral pMX-IGvector (kindly provided by Toshio Kitamura, Advanced Clinical ResearchCenter, Institute of Medical Science, Tokyo, Japan) that harbors the internalribosomal entry site and simultaneously expresses green fluorescent protein(GFP) with the protein of interest. HEK 293T cells (ATCC) were grown inDulbecco modified Eagle medium (DMEM)–10% fetal calf serum to 70%confluence in a 6-well culture plates and transfected using Lipofectamine2000 (Invitrogen) following the manufacturer’s protocol with plasmidDNA of interest in the presence of MLV gag-pol and ecotropic envelopeencoding plasmids. Viral particles containing supernatant were collectedevery day for 3 days. THP-1 and U937 cells were incubated in viralsupernatant in the presence of 4 �g/mL polybrene (Sigma-Aldrich). Cellswere subjected to 3 runs of virus infection at a 1-day interval. The efficiencyof infection is checked by analysis of GFP-positive cells by flow cytometry.We observed a 1.4- to 1.6-fold increase in the mean of fluorescenceintensity of whole cell population in pMX-IG compared with control-transduced cells.

Phagocytic assay

To evaluate the phagocytic activity of monocytic cells, U937 and THP-1cells were differentiated in a 24-well dish during 48 to 72 hours by 20 nMTPA treatment and incubated with FITC-conjugated Escherichia coliBioParticles (Invitrogen) in fresh medium at 37°C. After incubation, cellswere immediately put on ice to stop phagocytosis and washed 3 times inice-cold Dulbecco phosphate-buffered saline (DPBS; Lonza). The fluores-cence of FITC-conjugated E coli BioParticles, which were absorbed ontothe cell surface, was faded by incubated cells for 1 minute in 0.25 mg/mLTrypan blue solution. Cells were then washed and slightly scraped in DPBS.The FITC-conjugated E coli BioParticles engulfed were evaluated by a flowcytometry analysis (LSRII, BD Biosciences). A negative control wasperformed by incubating cells with E coli BioParticles at 4°C.

Cytokine antibody blot array and ELISA

Monocyte-derived macrophages were first transfected with DNA constructsas previously described. Twenty hours after, cells were washed andincubated in fresh medium containing 500 ng/mL CD40L. The cell culturesupernatant was collected 24 hours after CD40L stimulation and stored at�80°C until analysis. The presence of cytokines in macrophage supernatantwas assessed using human cytokine antibody arrays panel A (R&DSystems) following the manufacturer’s instructions. The autoradiographyfilms were digitalized and analyzed by densitometry software ImageJ(http://rsb.info.nih.gov/ij/index.html). Monocyte chemoattractant protein1 (MCP-1) and IL-8 were quantified using an enzyme-linked immunosor-bent assay (ELISA) kit from eBioscience and Biosource (BiosourceEurope, Nivelles, Belgium), respectively, following the manufacturer’sinstructions.

RNA purification and quantitative real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen) according to themanufacturer’s instructions. RNA was reverse transcribed by Moloneymurine leukemia virus (MMLV) reverse transcriptase with oligo(dT)primers (Promega, Madison, WI). Specific cDNAs were amplified on a7500 FAST thermocycler (Applied Biosystems, Foster City, CA) using theSyBr Green detection protocol as outlined by the manufacturer (AppliedBiosystems). Specific forward and reverse primers were: cyclophilin,

176 DUPOUX et al BLOOD, 1 JANUARY 2009 � VOLUME 113, NUMBER 1




5�-GTCGACGGCGAGCCC-3� and 5�-TCTTTGGGACCTTGTCTG-CAA-3� used as a standardizing control; TNF-�, 5�-GGAGAAGGGTGAC-CGACTCA-3� and 5�-TGCCCAGACTCGGCAAAG-3�; MCP-1, 5�-TCTCTGCCGCCCTTCTGT-3� and 5�-GCATCTGGCTGAGCGAGC-3�;IL-8, 5�-CTGGCCGTGGCTCTCTTG-3� and 5�-CTTGGCAAAACTG-CACCTTCA-3�; TRAF2 5�-TGGCTGGCCGCATACC-3� and 5�-TGTAGC-CGTACCTGCTGGTGTA-3�; and cIAP1 5�-CCTGGAGATAGGGTAGC-CTGC-3� and 5�-TGACATAGCATCATCCTTTGGTTC-3� (Eurogentec).

Immunofluorescence studies

Cells were fixed in paraformaldehyde (PFA; 2%) for 10 minutes at roomtemperature, washed twice, saturated in DPBS containing 0.1% saponin and5% nonfat milk, and incubated overnight at room temperature in thepresence of primary antibody diluted in DPBS containing 0.1% saponin and0.5% bovine serum albumin (BSA). After washing, cells were incubated for30 minutes with secondary antibody and washed again with DPBS. Nucleiwere stained by Hoechst 33342 (Sigma-Aldrich). To rule out nonspecificsignal, cells were incubated in the presence of an irrelevant mouse or rabbitimmunoglobulin and a specific secondary antibody. Analysis was per-formed using either a fluorescence (Nikon Eclipse 80i; Nikon, Champigny,France) or a confocal (Leica TCS SP2; Leica, Bron, France) microscope(objective 50�; original magnification �500). The images were capturedby a 3 CCD (charge-coupled device) color video camera (Sony, Paris,France), digitally saved using Archimed-Pro software (Microvision Instru-ments, Evry, France), and further processed using Photoshop software(Adobe Systems France, Paris, France).

Electrophoretic mobility shift assay

Nuclear fractions were obtained by incubating the cells in lysis buffer(10 mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid [HEPES],pH 7.8, 10 mM KCl, 0.1 mM ethylenediaminetetraacetic acid [EDTA],0.1 mM ethyleneglycoltetraacetic acid [EGTA], 1 mM dithiothreitol[DTT], 0.6% Nonidet P40) in the presence of protease inhibitors. Celllysate was centrifuged at 1200g for 10 minutes, and the pellet waswashed once in lysis buffer and then resuspended in a buffer containing20 mM HEPES, pH 7.8, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, inthe presence of complete protease inhibitor mixture for 30 minutes onice. Nuclear extracts were cleared by centrifugation at 20 000g for30 minutes; then 5 �g was incubated with 100 000 cpm 32P–end-labeled NF-�B (5�-AGTTGAGGGGCTTTCCCAGGC-3�) consensusoligonucleotide (Promega) in a reaction buffer containing 5 �L HNB(0.5 M sucrose, 15 mM Tris, pH 7.5, 60 mM KCl, 0.25 mM EDTA, pH 8,0.125 mM EGTA, pH 5, 0.15 mM spermin, 0.5 mM spermidine, 1 mMDTT), 2 �L MgSp (10 mM MgCl2, 80 mM spermidine), 1.5 �L NaPi(10 mM NaPi, 1 mM EDTA), 10 mM DTT, and 0.2 �g poly(dI-dC).After 30 minutes, the DNA-protein complexes were separated from freeoligonucleotides by electrophoresis in a 4% polyacrylamide gel anddetected by a PhosphorImager.

Results

Monocyte differentiation into macrophages is associated with adecrease in TRAF2 expression

We performed an immunoblot analysis of TRAF2 expression inhuman peripheral blood monocytes undergoing differentiation intomacrophages on exposure to M-CSF. We observed an increase inTRAF2 expression at day 1, then a time-dependent decrease in theprotein level (Figure 1A, 2 independent samples). A similarobservation was made in THP-1 and U937 cell lines, 2 monoblasticcell lines whose differentiation into macrophages was induced byexposure to the phorbol ester TPA. In these cell lines, TRAF2expression increased during the first hours of the differentiationprocess and then progressively decreased (Figure 1A). In THP-1cells (Figure 1B), addition of the proteasome inhibitor MG132 to

the culture medium 16 hours after the beginning of TPA exposureprevented the subsequent decrease in TRAF2 expression as ob-served 7 hours later, that is, 23 hours after initiation of thedifferentiation. A similar inhibition of TRAF2 protein level de-crease was obtained by inhibiting the proteasome with MG132 inTPA-treated U937 cells (data not shown). A quantitative real-timePCR analysis demonstrated that exposure of peripheral bloodmonocytes to M-CSF induced a rapid and strong increase inTRAF2 mRNA level. Then, this mRNA remained highly expressedcompared with nondifferentiated cells while the protein wasprogressively degraded (Figure 1C).

Figure 1. TRAF2 expression decreases along differentiation into macrophages.(A) Immunoblot analysis of TRAF2 expression in peripheral blood monocytesexposed to 100 ng/mL M-CSF and in THP-1 and U937 cells exposed to 20 nM TPA forthe indicated times (sample A and sample B represent 2 independent experiments).HSC70 indicates loading control. (B) Immunoblot analysis of TRAF2 expression inTHP-1 cells exposed for the indicated times to 20 nM TPA. In indicated samples(�MG), 30 �M MG132 was added 7 hours before preparing extracts and immunoblotanalysis, ie, 16 hours after initiation of the differentiation by TPA. HSC70 indicatesloading control. (C) Real-time PCR analysis of mRNA expression of TRAF2 inperipheral blood monocytes exposed to 100 ng/mL M-CSF (mean � SD of indepen-dent triplicates). AU indicates arbitrary units.

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cIAP1 is involved in the differentiation-associated TRAF2degradation

The RING domain containing protein cIAP1 was demonstrated tobe an E3 enzyme in the ubiquitylation process that leads to TRAF2degradation by the proteasome machinery.12,13,23 Coimmunoprecipi-tation experiments demonstrated that cIAP1 interacted with TRAF2in undifferentiated cells. This interaction was no more detected interminally differentiated cells (Figure 2A), in accordance with thedecreased expression of TRAF2 protein (Figure 1A). Addition ofthe proteasome inhibitor MG132 to the culture medium 7 hoursbefore the coimmunoprecipitation experiment permitted the TRAF2/

cIAP1 interaction to be partially recovered (Figure 2A). Todetermine whether cIAP1 was involved in the degradation ofTRAF2 that accompanies the macrophagic differentiation, we firstexposed the cells to the IAP inhibitor Smac-N7, a cell-permeablepeptide made of the 7 N-terminal amino acids of the endogenousIAP inhibitor, Smac. This inhibitory peptide was shown to preventcIAP1-TRAF2 interaction.29 Its addition to the culture mediumpartially prevented the differentiation-associated degradation ofTRAF2 (Figure 2B). We also used a retrovirus vector to introducein THP-1 cells an E3-defective mutant of cIAP1 in which the RINGdomain has been deleted (cIAP1-RING). The expression of thiscIAP1 dominant-negative mutant12 delayed the degradation ofTRAF2 in THP-1 cells undergoing differentiation into macro-phages (Figure 2C). In contrast, retrovirus-mediated overexpres-sion of wild-type cIAP1 decreased the basal level of TRAF2expression in undifferentiated cells (Figure 2C). Lastly, the use of 2cIAP1-targeting siRNAs that specifically decreased cIAP1 expres-sion (Figure 2D left panel) prevented the differentiation-associatedTRAF2 depletion (Figure 2D right panel). Interestingly, whenintroduced into macrophages obtained by ex vivo differentiation ofperipheral blood monocytes by M-CSF, the 2 cIAP1 siRNAsinduced a drastic increase in TRAF2 level (Figure 2E), suggestingthat cIAP1 was also required for maintaining a low level of TRAF2protein in differentiated macrophages.

Differentiation-associated TRAF2 degradation occurs in theGolgi apparatus–associated compartment

We previously showed that, in cells undergoing differentiation,cIAP1 migrated from the nucleus to the cytoplasm through anuclear export signal (NES)–chromosome maintenance region 1(CRM1)-dependent mechanism, to concentrate at the Golgi appara-tus surface,26 which was confirmed in Figure 3A,E. The expressionof cIAP1 in the cytoplasm is inversely correlated with TRAF2expression (Figure 3A). Leptomycin B blocked the nuclear exportof cIAP1 (Figure 3B top panel) and prevented the decrease inTRAF2 protein expression (Figure 3B) associated with differentia-tion. In undifferentiated cells, TRAF2 is expressed both in thenuclear and in the cytoplasm-enriched fractions (Figure 3C). Alongmacrophage formation, TRAF2 protein expression first increases inthe cytoplasm (Figure 3C), which is in accordance with resultsobtained in whole cell extracts (Figure 1A), then decreases whencIAP1 accumulates in this cellular compartment. Conversely,nuclear TRAF2 expression remains unchanged (Figure 3C). Fluo-rescence microscopy analyses confirmed that, in undifferentiatedcells, TRAF2 was expressed in both the cytoplasm and the nucleus(Figure 3D,E). During the first hours of TPA-induced differentia-tion, TRAF2 accumulated at the cytoplasm periphery and formedclusters (Figure 3D). Then, TRAF2 labeling gets progressively lessbright, coinciding with TRAF2 depletion in the cytoplasm. After24 hours of TPA treatment, TRAF2 was mainly detected in thenuclear compartment (Figure 3D,E). Inhibition of the proteasomemachinery by addition of MG132 for 7 hours or lactacystin for4 hours before immunofluorescence staining revealed that theadaptor protein colocalized with cIAP1 and the Golgi apparatusmarker GM130 in a cytoplasm perinuclear structure (Figures 3E,S1 for controls, available on the Blood website; see the Supplemen-tal Materials link at the top of the online article). These resultssuggest that TRAF2 is degraded in the Golgi apparatus–associatedcompartment by a cIAP1 and proteasome-dependent process.

Figure 2. cIAP1 is responsible for differentiation-associated TRAF2 depletion.(A) Coimmunoprecipitation of cIAP1 and TRAF2 in U937 cells exposed for theindicated times to 20 nM TPA. (Right panel) MG132 was added during the last7 hours of exposure to TPA (IP with an anti-cIAP1 antibody; immunoblot withanti-TRAF2 and anti-cIAP1 antibodies; Ig indicates irrelevant immunoglobulin).(B) U937 cells were left untreated or treated with 20 nM TPA for 48 hours in theabsence or presence of 50 �M Smac-N7 peptide before immunoblot analysis ofTRAF2. (C) U937 cells were transduced with murine stem cell virus–based retroviralvector, either empty (Control) or encoding cIAP1 or RING (a cIAP1 constructdeleted of the C-terminal E3-ubiquitin ligase domain), and cIAP1 expression waschecked by immunoblotting (top panel) before inducing cell differentiation byexposure to 20 nM TPA for the indicated times and analyzing TRAF2 expression byimmunoblot (bottom panel). (D) THP-1 cells were transfected with either negative-control (Co) or 2 cIAP1 targeting siRNA sequences (1 and 2), 48 hours beforeexposing the cells to 20 nM TPA. (Left panel) Immunoblot analysis of cIAP1, 48 hoursafter siRNA treatment. (Right panel) Immunoblot analysis of TRAF2 at indicatedtimes after TPA exposure (20 nM). (E) Macrophages were obtained by exposure ofhuman blood monocytes for 6 days to M-CSF (100 ng/mL) and then transfected witheither negative-control (Co) or cIAP1 targeting siRNAs (1 and 2). cIAP1 (left panel)and TRAF2 (right panel) expression was studied by immunoblotting, 48 hoursafter siRNA transfection in independent samples (A-C). (B-E) HSC70 used asloading control.





TRAF2 is a positive regulator of macrophage differentiation

The initial increase in TRAF2 mRNA and protein expression(Figure 1) and its clustering at the cell periphery (Figure 3D)suggested that TRAF2 might play a role in the first phases of thedifferentiation process. This TRAF2 expression increase wasefficiently prevented by the use of 2 independent and specificsiRNAs in human blood monocytes and THP-1 cells (Figure 4Atop panel). TRAF2-targeting siRNAs prevented their differentia-tion induced by M-CSF (CD71 expression) and TPA (CD11b ex-pression) exposure, respectively (Figure 4A bottom panel). We28

and others30 previously showed that macrophage differentiationrequired a moderate and transient activation of NF-�B transcrip-tion factor. Here we show that macrophage differentiation isassociated with a nuclear translocation of p65 NF-�B subunit(Figure 4B) and a decrease in I�-B� expression (Figure 4C).

The 2 TRAF2-targeting siRNAs prevent both I�-B� decrease(Figure 4D) and p65 nuclear redistribution (Figure 4E).

TRAF2 degradation is required for terminal differentiation ofmacrophages

cIAP1 knockdown by 2 specific siRNAs blocks TPA-induceddifferentiation of THP-1 cells (not shown) and M-CSF–induceddifferentiation of human peripheral blood monocytes (Figure 5A).Monocytic cells were transduced with a murine stem cell virus-based retroviral vector encoding simultaneously TRAF2 and GFPthanks to the presence of an internal ribosome entry site, and theninduced to differentiate by TPA exposure. A flow cytometricanalysis of GFP-positive cells indicated that approximately 70% ofthe cells had been efficiently transduced (Figure S2). To reduce thedifferentiation-associated degradation of exogenous TRAF2, we

Figure 3. TRAF2 is degraded in the cytoplasm.(A) Immunoblot analysis of cIAP1 in nucleus andcytoplasm-enriched fractions and TRAF2 in whole-cellextracts from THP-1 cells undergoing differentiation onTPA exposure (20 nM for the indicated times). HSC70indicates loading control. (B) THP-1 cells were induced todifferentiate into macrophages by exposure to 20 nMTPA for 24 hours, in the absence or presence of 100 nMleptomycin B (LMB) before analyzing cIAP1 localizationby fluorescence microscopy (top panel, c-IAP1 in green).Nuclei were stained using Hoechst blue. (Bottom panel)Immunoblot analysis of TRAF2 expression. HSC70 indi-cates loading control. (C) Immunoblot analysis of TRAF2in nucleus and cytoplasm-enriched fractions from TPA-treated THP-1 cells (20 nM for the indicated times).Poly(ADP-ribose) polymerase was used as a nucleusfraction marker and X-linked inhibitor of apoptosis proteinas a cytoplasm fraction marker. HSC70 indicates loadingcontrol. (D) Fluorescence microscopic analysis of TRAF2expression (green) in THP-1 cells exposed for the indi-cated times to 20 nM TPA (original magnification �400).(E) Confocal fluorescence microscopic analysis of TRAF2(red) and cIAP1 (green) or TRAF2 (red) and GM130(green) expression in undifferentiated (Control) and TPA-differentiated (24 hours) THP-1 cells, in the absence orpresence of 30 �M MG132 or 10 �M lactacystin addedfor the 7 or 4 last hours of treatment, respectively. Nucleiwere stained using Hoechst blue (original magnification�500).





used a mutated form of the protein (TRAF2*) that was shown toresist more efficiently than wild-type protein to proteasome-mediated degradation.7 Retrovirus-mediated expression of TRAF2*induced an approximately 1.5-fold increase in TRAF2 proteinexpression compared with cells transduced with the empty vector(Figure 5B). Although degradation of endogenous TRAF2 oc-curred along the differentiation, TRAF2 expression always re-mained higher in TRAF2*-transduced cells (Figure 5B), and thisoverexpression was sufficient to prevent macrophage differentia-tion, as evidenced by studying CD11b expression at the cell surface(Figure 5C). TRAF2 overexpression also induced an approxi-mately 20% decrease in the ability of cells to engulf bacteria, adifferentiation-associated property (Figure 5D). An electrophoreticmobility shift assay (EMSA) also suggested that overexpression ofTRAF2* or the cIAP1-RING construct partially prevented thedown-regulation of NF-�B activity that is required for the differen-tiation into macrophages to progress (Figure 5E).

TRAF2 modulates the response of mature macrophages toCD40L

As low level of TRAF2 expression was maintained through acIAP1-dependent mechanism in macrophages (Figure 2E), wewondered whether this regulation was involved in macrophage-specific functional properties. Macrophages obtained by a 6-dayexposure of peripheral blood monocytes to M-CSF were transientlytransfected with an empty vector or a wild-type TRAF2 encodingvector. In these conditions, TRAF2-protein level in transfectedmacrophages was almost similar to that observed in primarymonocytes (Figure 6A). These cells were stimulated for 24 hourswith 500 ng/mL CD40L, and cell supernatants were analyzed bythe use of a cytokine antibody array (Figure 6B). Of the 36cytokines analyzed, 15 were detected in CD40L-stimulated macro-phage supernatants. The level of 11 of them, including C5a, IL-1�,IL-1ra, IL-8, IL-16, IP-10, I-TAC, MCP-1, MIF, Serpin E1, and

Figure 4. TRAF2 is required for macrophage differen-tiation. (A,B,D,E) THP-1 cells or human monocyteswere transfected with either negative-control (Co) orTRAF2 targeting siRNAs (sequences 1 and 2), 48 hoursbefore exposing the cells to differentiating agents (20 nMTPA for THP-1 and 100 ng/mL M-CSF for humanmonocytes). (A) (Top panel) Immunoblot analysis ofTRAF2, 48 hours after siRNA treatment. (Bottom panel)Flow cytometry analysis of CD11b or CD71 membraneexpression, 48 hours after addition of differentiationinducers. Gray histogram represents negative controlsiRNA; white histograms, TRAF2-targeting siRNA se-quence 1 (black line) and 2 (gray line). One representa-tive of 3 experiments is shown. (B) Fluorescence micros-copy analysis of NF-�B p65 subunit (green) in THP-1cells exposed for the indicated times to 20 nM TPA(original magnification �400). Nuclei were stained usingHoechst (blue). (C) Immunoblot analysis of I�-B� inTHP-1 cells exposed for the indicated times to 20 nMTPA. (D) Immunoblot analysis of I�-B� in THP-1 cellsexposed for the indicated times to 20 nM TPA,48 hours after transfection with indicated siRNAs.(E) Percentage of cells with nuclear p65 quantified byimmunofluorescence in THP-1 cells exposed for theindicated times to 20 nM TPA, 48 hours after treatmentwith scramble (black) or TRAF2-specific (gray repre-sents sequence 1; white, sequence 2) siRNAs. Data aremean plus or minus SD of 3 independent experiments.





TNF-�, was lower in the supernatant of TRAF2-overexpressingmacrophages than in the supernatant of macrophages transfectedwith the empty vector (Figure 6B). An ELISA quantificationconfirmed that TRAF2 restoration inhibited MCP-1 (�30%) andIL-8 (�35%) secretion (Figure 6C), and this effect was maintainedover time (Figure 6D). siRNA-mediated decrease in cIAP1 expres-sion also decreased the secretion of IL-8 and MCP-1 (Figure 6E).

A quantitative real-time–PCR analysis showed that TRAF2overexpression (Figure 7A) as well as siRNA-mediated decrease incIAP1 expression (Figure 7C) decreased the expression of IL-8 andMCP-1 mRNA, which was confirmed with time (Figure 7B). JNKwas shown to be one of the key pathways that control the cytokinesecretion in response to CD40L in macrophages.31 Inhibition ofJNK by SP600123 abolished CD40L-induced increase in MCP-1,and IL-8 mRNA (Figure 7D). In this setting, TRAF2 silencing didnot interfere with CD40-induced JNK activation (not shown), butTRAF2 overexpression delayed and rapidly down-regulated JNKactivation (Figure 7E). Altogether, these data suggest that TRAF2,although being required for optimal differentiation at the beginningof the process, must be subsequently down-regulated for an optimalcytokine response of macrophages to CD40L and c-IAP1 isinvolved in this down-regulation.

Discussion

Once in the tissues, peripheral blood monocytes evolve into a variety ofterminally differentiated cells, depending on signals received from themicroenvironment. The molecular mechanisms that sustain the differen-tiation processes and confer to differentiated cells their specific proper-ties are only partially depicted. Here we show that the adaptor moleculeTRAF2 is both a positive (initial phase) and a negative (secondaryphase) regulator of the differentiation of monocytes into macrophages.The protein must be degraded by a proteasome-dependent mechanismthat involves cIAP1 for terminal differentiation of monocytes intomacrophages and for mature macrophages to optimally respond tostimulation with CD40L.

TRAF2 is an important regulator of cell signaling response toTNFR-mediated extracellular stimuli, leading to cell proliferation,cell activation, and cytokine secretion.2 Ex vivo studies haverevealed that TRAF2 could also have a role in the regulation ofdifferentiation in 2 specific cell types. First, TRAF2 was proposedto modulate B-cell maturation by mediating CD40-mediated immu-noglobulin class switching.32,33 Second, TRAF2 plays an importantrole in RANK and TNFRs signals that trigger osteoclast differentia-tion.34 Here we show that TRAF2 is also a regulator of macrophagedifferentiation and activity. TRAF2 acts as an adaptor molecule thatconnects several plasma membrane receptors to downstreamsignaling pathways.2 TRAF2 was shown to be recruited to mem-brane complexes and translocated to raft microdomains to partici-pate to signaling events before being degraded by the proteasomesystem.6,8,14,19 Here we show that, in monocytes induced todifferentiate into macrophages, TRAF2 mRNA rapidly increasesand the protein forms clusters at the periphery of the cell. Thisredistribution of TRAF2 protein coincides with I�-B� degradationand the nuclear translocation of NF-�B p65 subunit, 2 events thatare prevented by TRAF2 silencing. These results suggest thatTRAF2 takes parts in the canonical NF-�B activating pathwayduring initial phase of monocyte differentiation into macrophages.

Later on in the differentiation process, TRAF2 is degraded bythe proteasome system, which was demonstrated to be an importantregulatory event in various signaling pathways.8,11,13,17,19 Overex-pression of a TRAF2 mutant known to resist to proteasome-mediated degradation prevent the progression of macrophagedifferentiation and interfere with the down-regulation of NF-�Bactivity. Altogether, our results suggest that initial accumulation ofTRAF2 and its subsequent degradation by a cIAP1-dependentprocess play a role in the time-dependent modulation of NF-�Bactivity along the differentiation process.28,30

Figure 5. TRAF2 degradation is required for macrophage differentiation.(A) (Top panel) Immunoblot analysis of c-IAP1 expression in human monocytestransfected 48 hours before with control (Co) or cIAP1-specific (1 and 2) siRNAs.(Bottom panel) Flow cytometry analysis of CD71 expression at the surface of humanmonocytes transfected with control (gray area) or cIAP1 targeting siRNA sequences1 (black line) and 2 (dark gray line) and exposed 48 hours later to 100 ng/mL M-CSFfor 48 hours. (B-E) THP-1 cells were transduced with an empty retroviral vector (Co)or a vector encoding a proteasome-resistant TRAF2 mutant (TRAF2*) or a vectorencoding a cIAP1RING mutant (RING), then induced to differentiate into macro-phages by exposure to 20 nM TPA for the indicated times. (B) Immunoblot analysis ofTRAF2 expression in transduced cells exposed for the indicated times to 20 nM TPA.HSC70 indicates loading control. (Bottom panel) TRAF2/HSC70 ratio of signalintensities quantified by densitometry (E indicates control vector; and F, TRAF2*).(C) Transduced cells were left untreated (top panel) or exposed to 20 nM TPA for 48hours (bottom panel) before flow cytometry analysis of CD11b membrane expres-sion. Gray area represents control vector; black lines, TRAF2*-transfected cells. (D)Engulfment activity of labeled bacteria E coli was measured in transduced cellsexposed for 48 hours to 20 nM TPA. Results were normalized to control cells(mean � SD of 3 independent experiments). *Statistically significant differences(P .05, Student test). (E) NF-kB DNA-binding activity was assessed by EMSA inTHP-1-transduced cells exposed for the indicated times (hours) to 20 nM TPA.A representative experiment is shown.





TRAF2 is targeted for proteolytic degradation by ubiquityla-tion. The molecule is composed of an N-terminal RING domain35

that characterizes E3 ubiquitin ligase activity36 and was proposedto promote its own ubiquitylation.19 Siah2 protein is also aRING-containing protein that can mediate TRAF2 ubiquitylationto target the protein for degradation under stressful conditions.17

cIAP1 is another RING-containing ubiquitin ligase that can triggerTRAF2 ubiquitylation and proteasome-dependent degradation, eg,on engagement of TNFR2.8,12,13 We have previously shown thatcIAP1 is required for optimal differentiation of monocytes intomacrophages.25 We show here that cIAP1 is one of the E3 ligasesinvolved in the proteasome-dependent degradation of TRAF2along this differentiation process. Because the effect of cIAP1inhibition on differentiation-associated TRAF2 down-regulationwas only partial, we cannot rule out a function for TRAF2 itself,Siah2, or other E3 in this event.

We also observed that the degradation of TRAF2 in monocytesundergoing macrophage differentiation was associated with thetranslocation of the protein to a perinuclear structure. By inhibitingthe proteasome, we demonstrated that TRAF2 colocalizes withcIAP1 and proteins of the Golgi apparatus-associated compart-ment.26 TRAF2 was shown also to colocalize with endoplasmicreticulum markers, including the cognate E2 enzyme Ubc6.Interestingly, Ubc6 behaves as the E2 enzyme for cIAP1 E3activity, leading to TRAF2 degradation.8 Altogether, these observa-tions indicate that cIAP1 interaction with TRAF2 that leads to itsproteasome-mediated degradation along macrophage formationmight occur in the Golgi-associated compartment.

TRAF2 depletion sensitizes specific cells to TNF-�-induced apopto-sis.6,11,14,18,19 The premature death of TRAF2-deficient mice was relatedto an increased sensitivity of thymocytes and hematopoietic progenitorsto TNF-induced cell death.10 Accordingly, cIAP1-mediated TRAF2degradation, for example, in response to TNFR2 stimulation, sensitizesspecific cells to TNFR1-mediated death.12,13 Interestingly, cIAP1-mediated TRAF2 degradation observed in monocytes undergoingmacrophage differentiation did not sensitize these cells to TNF-inducedapoptosis (data not shown) but was required for an optimal response ofdifferentiated macrophages to CD40L. CD40 is expressed on antigen-presenting cells that include macrophages, whereas the expression of itsligand is almost restricted to activated CD4� T cells. T cell–dependentstimulation of CD40 in macrophages induces their activation, enhancestheir survival,37 up-regulates the expression of costimulatory mol-ecules,31,38,39 and regulates their antimicrobial activity.40 It was previ-ously shown that trimeric CD40 cytoplasmic domain could formmultiprotein complexes that contained TRAF2 and cIAP1, together withother TRAFs.41,42 Association of cIAP1 with the CD40 cytoplasmicdomain complex was dependent on the presence of an intact TRAF1/2/3-binding site, but the role of cIAP1 in this complex remainedundetermined.41,42 We demonstrate that cIAP1-mediated TRAF2 deple-tion associated with macrophage differentiation facilitates the cytokinesecretion in response to CD40 stimulation. Accordingly, an elevatedlevel of TNF-� has been observed in sera from TRAF2-deficient mice10

that is responsible for the premature death of the animals.43

The TRAF-dependent signaling pathway activated by CD40Ldepends on the cell type and the differentiation stage.44 Inmacrophages, CD40 proinflammatory signals involve the adaptor

Figure 6. Influence of TRAF2 on CD40-mediated cytokine secretion in macro-phages. Macrophages were obtained by exposure of human blood monocytes for6 days to M-CSF (100 ng/mL) and then transfected with an empty vector (Co) or aTRAF2-expressing vector (TRAF2). Twenty-four hours after transfection, macro-phages were treated with 500 ng/mL CD40L. (A) Immunoblot analysis of TRAF2expression, 24 hours after transfection. Peripheral blood monocytes were used as acontrol. (B) (Top panel) Cytokines were detected in the culture supernatant of control(Co) and TRAF2-transfected cells treated with CD40L for 24 hours using an antibodyarray. (Bottom panel) Spots were quantified by densitometry analysis and reported tointernal control (C�). The ratio between cytokine quantities detected in control andTRAF2-transfected macrophage supernatants was quantified. (C-E) ELISA quantifi-cation of IL-8 and MCP-1 secretion in (C) control (Co) and TRAF2-

transfected cells (TRAF2) treated with CD40L for 24 hours (each line indicates anindependent sample) or (D) control (�) and TRAF2-transfected cells (f) treated withCD40L for the indicated times (hours). Results are the mean plus or minus SD of3 measurements in one sample. (E) Control (Co) and cIAP1 siRNA-transfected cells(2 sequences 1 and 2) treated with CD40L for 24 hours (mean � SD of 3 independentsamples normalized to the control).





TRAF6 and the ERK and JNK-dependent pathways.31,39,45 Macro-phages that harbor a CD40 mutant unable to bind TRAF2 stillproduced cytokine in response to CD40L showing that TRAF2 isdispensable for its function.31,46 The present results show thatTRAF2 actually negatively regulates this pathway by preventingJNK activation and downstream production of MCP-1 and IL-8 inresponse to CD40L. This negative effect of TRAF2 on macrophageresponse to CD40L could possibly be mediated by heterodimeriza-

tion with another TRAF, such as TRAF6, as it has been observed inepithelial cells.9,16,47

Although TRAF2 is a positive regulator of the differentiation ofmonocytes into macrophages, its down-regulation by cIAP1 inmature macrophages may be part of a coordinated response thatprepares these cells to their various functions, ie, appropriateresponse to CD40 ligand. When associated with the Golgi appara-tus, cIAP1 could regulate the expression of other signaling

Figure 7. Influence of TRAF2 on CD40-mediatedcytokine mRNA expression in macrophages. Macro-phages were obtained by exposure of human bloodmonocytes for 6 days to M-CSF (100 ng/mL) and thentransfected with an empty vector (Co) or a TRAF2-expressing vector (TRAF2) or cIAP1 siRNA sequence 1and 2 and treated with CD40L 24 hours later. (A-D)Real-time PCR analyses of mRNA expression of MCP-1and IL-8 (A) in Co and TRAF2-transfected macrophagestreated for 24 hours with 500 ng/mL CD40L (each linerepresent an independent sample). (B) Control (opensymbols) and TRAF2-transfected (closed symbols) mac-rophages treated for the indicated times with 500 ng/mLCD40L. (C) Control and c-IAP1 targeting siRNAs macro-phages treated for 24 hours with 500 ng/mL CD40L(mean � SD of 3 independent experiments normalizedto controls). (D) Macrophages treated by 500 ng/mLCD40L for 24 hours in the absence (Co) or presence of10 �M SP600123 (SP) (mean � SD of 3 independentexperiments normalized to controls). (E) Immunoblotanalysis of JNK and phosphorylated JNK (p-JNK) incontrol (Co) and TRAF2-overexpressing macrophagesexposed to CD40L for the indicated times. One represen-tative of 3 independent experiments is shown.





regulators that include NEMO/IKK,48 RIP1,49 ASK1,50 andNIK,51 which were demonstrated to be cIAP1 ubiquitin targets.Further studies will determine whether these kinases are cIAP1targets in this differentiation setting and how their modulationscontribute to the macrophage functions.

Acknowledgments

We thank Arlette Hammann and Lydie Desoches for technicalassistance, Nathalie Droin for efficient help in real-time PCRanalysis, Minoru Yoshida for providing LMB, Toshio Kitamura forpMXs-IG expressing retroviral vector, Ze’ev Ronai for TRAF2mutant cDNA, and Olivier Micheau for wt TRAF2 expressingvector.

This work was supported by grants from the Ligue NationaleContre le Cancer (Equipe Labellisee), the Comite de Cote d’Or ofthe Ligue Nationale Contre le Cancer, the Association Centpoursan-

glavie, the Agence Nationale de la Recherche, and the NationalInstitute of Cancer.

Authorship

Contribution: A.D. performed experiments and analyzed data;J.C. performed experiments; S.C. performed immunoprecipita-tion experiments; R.F. performed EMSA experiments; E.S.supervised the research group and corrected the paper; andL.D.D. supervised the experiments and research team, analyzeddata, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Laurence Dubrez-Daloz, Institut National dela Sante et de la Recherche Medicale, Unite Mixte de Recherche866, Faculte de Medecine, 7 boulevard Jeanne d’Arc, 21079 Dijoncedex, France; e-mail: [email protected].

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online September 30, 2008 originally publisheddoi:10.1182/blood-2008-02-137919

2009 113: 175-185

Dubrez-DalozAlban Dupoux, Jessy Cartier, Séverine Cathelin, Rodolphe Filomenko, Eric Solary and Laurence monocytes into macrophages and their response to CD40 ligandcIAP1-dependent TRAF2 degradation regulates the differentiation of

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