11
LIND/ABIN-3 Is a Novel Lipopolysaccharide-inducible Inhibitor of NF-B Activation * S Received for publication, August 7, 2006 Published, JBC Papers in Press, November 6, 2006, DOI 10.1074/jbc.M607481200 Andy Wullaert ‡1,2 , Lynn Verstrepen ‡3 , Sofie Van Huffel ‡4 , Minou Adib-Conquy § , Sigrid Cornelis , Marja Kreike , Mira Haegman , Karim El Bakkouri , Matthew Sanders , Kelly Verhelst ‡1 , Isabelle Carpentier ‡5 , Jean-Marc Cavaillon § , Karen Heyninck ‡6 , and Rudi Beyaert ‡7 From the Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium and the § Cytokines & Inflammation Unit, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-B (NF-B). Although LPS-induced activation of NF-B is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-B signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-B activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-B activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-B-de- pendent gene expression in response to LPS/TLR4 at a level down- stream of TRAF6 and upstream of IKK. NF-B inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-B activity in the liver, thereby par- tially protecting mice against LPS/D-( )-galactosamine-induced mor- tality. Taken together, these results implicate ABIN-3 as a novel nega- tive feedback regulator of LPS-induced NF-B activation. The innate immune response to microbial pathogens begins when pathogen-associated molecular patterns meet their cognate Toll-like receptors (TLRs) 8 on effector cells of the immune system, such as monocytes and macrophages (1). Lipopolysaccharide (LPS), an integral cell wall component of Gram-negative bacteria and one of the most potent stimulators in innate immunity, is recognized by the TLR4-MD2 receptor complex (2). In the past years, much progress has been made in understanding the intra- cellular signaling cascades that are initiated when LPS stimulates TLR4 (reviewed in Refs. 3 and 4). Ligation of the TLR4-MD2 com- plex by LPS initially results in the recruitment of myeloid differen- tiation factor (MyD)88 and MyD88-adaptor like (Mal), also called TIRAP, to the receptor cytoplasmic domain. MyD88 then facili- tates recruitment of the serine/threonine kinases IL-1R-associated kinase (IRAK)-1 and -4, thus enabling IRAK4 to phosphorylate IRAK1. The latter subsequently dissociates from the receptor complex and associates with tumor necrosis factor (TNF) receptor-associated factor (TRAF)6, constituting a cytoplasmic signaling complex. Upon ubiquitination, TRAF6 activates transforming growth factor--activated kinase 1, which in turn activates the inhibitor of B kinase (IKK) complex that consists of the regulatory subunit IKK (also known as NEMO) and the kinases IKK and IKK. The latter eventually phosphorylates the inhibitory IB proteins, resulting in their ubiquitination and degradation. This allows the transcription factor NF-B to translocate to the nucleus and initiate transcription of inflam- matory cytokines, such as TNF, which contribute to mounting an inflammatory response. Apart from this MyD88-dependent signaling pathway, TLR4 also initiates a MyD88-independent signaling pathway that is mediated by the adaptor proteins Toll/IL-1 receptor domain domain-containing adaptor-induc- ing interferon- (TRIF; also known as TICAM-1) and TRIF- related adaptor molecule (also known as TICAM-2). Although * This work was supported in part by grants from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO), the Interuniversitaire Attractiepolen, the Emman- uel Vanderschueren stichting, and the Geconcerteerde Onderzoeksacties. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement ” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1 Performed this work as predoctoral fellows, with the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-technologisch Onderzoek in de Industrie (IWT). 2 Current address: Dept. of Mouse Genetics and Inflammation, Institute for Genetics, University of Cologne, Zu ¨ lpicher Str. 47, D-50674 Cologne, Germany. 3 Predoctoral fellow with the FWO. 4 Current address: Membrane Biology Lab, Institute of Molecular and Cell Biol- ogy, 61 Biopolis Drive, Proteos, Singapore 138673. 5 Postdoctoral fellow with the IWT. 6 Postdoctoral fellow with the FWO. 7 To whom correspondence should be addressed. Tel.: 32-9-331-3770; Fax: 32-9-331-3609; E-mail: [email protected]. 8 The abbreviations used are: TLR, Toll-like receptor; ABIN, A20-binding inhib- itor of NF-B activation; AHD, ABIN homology domain; GalN, D-()-galac- tosamine; GFP, green fluorescent protein; IB, inhibitory protein of B; IKK, IB kinase; IL, interleukin; IL-1R, interleukin-1 receptor; IRAK, IL-1R-associ- ated kinase; LPS, lipopolysaccharide; Luc, luciferase; Mal, MyD88-adaptor- like; MyD88, myeloid differentiation factor 88; NF-B, nuclear factor-B; PBMC, peripheral blood mononuclear cells; RIP, receptor interacting pro- tein; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; TRIF, Toll/IL-1 receptor domain-containing adaptor inducing interferon-; LIND, Listeria INDuced; CMV, cytomealovirus; E1, ubiquitin-activating enzyme; E3, ubiquitin-protein isopeptide ligase; pfu, plaque-forming unit(s); TPA, 12-O-tetradecanoylphorbol-13-acetate; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 1, pp. 81–90, January 5, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. JANUARY 5, 2007 • VOLUME 282 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 81 by guest on February 3, 2019 http://www.jbc.org/ Downloaded from

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LIND/ABIN-3 Is a Novel Lipopolysaccharide-inducibleInhibitor of NF-�B Activation*□S

Received for publication, August 7, 2006 Published, JBC Papers in Press, November 6, 2006, DOI 10.1074/jbc.M607481200

Andy Wullaert‡1,2, Lynn Verstrepen‡3, Sofie Van Huffel‡4, Minou Adib-Conquy§, Sigrid Cornelis‡, Marja Kreike‡,Mira Haegman‡, Karim El Bakkouri‡, Matthew Sanders‡, Kelly Verhelst‡1, Isabelle Carpentier‡5,Jean-Marc Cavaillon§, Karen Heyninck‡6, and Rudi Beyaert‡7

From the ‡Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, FlandersInteruniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgiumand the §Cytokines & Inflammation Unit, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France

Recognition of lipopolysaccharide (LPS) by Toll-like receptor(TLR)4 initiates an intracellular signaling pathway leading to theactivation of nuclear factor-�B (NF-�B). Although LPS-inducedactivation of NF-�B is critical to the induction of an efficientimmuneresponse, excessiveorprolongedsignaling fromTLR4canbe harmful to the host. Therefore, the NF-�B signal transductionpathway demands tight regulation. In the present study, wedescribe the human protein Listeria INDuced (LIND) as a novelA20-binding inhibitorofNF-�Bactivation(ABIN)that is relatedtoABIN-1 and -2 and, therefore, is further referred to as ABIN-3.Similar to the other ABINs, ABIN-3 binds to A20 and inhibitsNF-�B activation induced by tumor necrosis factor, interleukin-1,and 12-O-tetradecanoylphorbol-13-acetate. However, unlike theother ABINs, constitutive expression of ABIN-3 could not bedetected in different human cells. Treatment of humanmonocyticcells with LPS strongly induced ABIN-3 mRNA and proteinexpression, suggestingarole forABIN-3 intheLPS/TLR4pathway.Indeed, ABIN-3 overexpression was found to inhibit NF-�B-de-pendent gene expression in response toLPS/TLR4at a level down-stream of TRAF6 and upstream of IKK�. NF-�B inhibition wasmediated by theABIN-homology domain 2 andwas independent ofA20binding.Moreover, in vivoadenoviral gene transferofABIN-3 inmice reduced LPS-induced NF-�B activity in the liver, thereby par-tiallyprotectingmiceagainstLPS/D-(�)-galactosamine-inducedmor-tality.Takentogether, theseresults implicateABIN-3asanovelnega-tive feedback regulator of LPS-inducedNF-�Bactivation.

The innate immune response to microbial pathogens beginswhen pathogen-associatedmolecular patternsmeet their cognateToll-like receptors (TLRs)8oneffector cells of the immunesystem,such as monocytes and macrophages (1). Lipopolysaccharide(LPS), an integral cell wall component of Gram-negative bacteriaand one of the most potent stimulators in innate immunity, isrecognized by the TLR4-MD2 receptor complex (2). In the pastyears, much progress has been made in understanding the intra-cellular signaling cascades that are initiated when LPS stimulatesTLR4 (reviewed inRefs. 3 and4). Ligationof theTLR4-MD2com-plex by LPS initially results in the recruitment ofmyeloid differen-tiation factor (MyD)88 andMyD88-adaptor like (Mal), also calledTIRAP, to the receptor cytoplasmic domain. MyD88 then facili-tates recruitmentof the serine/threoninekinases IL-1R-associatedkinase (IRAK)-1 and -4, thus enabling IRAK4 to phosphorylateIRAK1. The latter subsequently dissociates from the receptorcomplex and associates with tumor necrosis factor (TNF)receptor-associated factor (TRAF)6, constituting a cytoplasmicsignaling complex. Upon ubiquitination, TRAF6 activatestransforming growth factor-�-activated kinase 1, which in turnactivates the inhibitor of �B kinase (IKK) complex that consistsof the regulatory subunit IKK� (also known as NEMO) and thekinases IKK� and IKK�. The latter eventually phosphorylatesthe inhibitory I�B proteins, resulting in their ubiquitinationand degradation. This allows the transcription factor NF-�B totranslocate to the nucleus and initiate transcription of inflam-matory cytokines, such as TNF, which contribute to mountingan inflammatory response. Apart from this MyD88-dependentsignaling pathway, TLR4 also initiates a MyD88-independentsignaling pathway that is mediated by the adaptor proteinsToll/IL-1 receptor domain domain-containing adaptor-induc-ing interferon-� (TRIF; also known as TICAM-1) and TRIF-related adaptor molecule (also known as TICAM-2). Although

* This work was supported in part by grants from the Fonds voor WetenschappelijkOnderzoek-Vlaanderen (FWO), the Interuniversitaire Attractiepolen, the Emman-uel Vanderschueren stichting, and the Geconcerteerde Onderzoeksacties. Thecosts of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 Performed this work as predoctoral fellows, with the Vlaams Instituut voorde Bevordering van het Wetenschappelijk-technologisch Onderzoek in deIndustrie (IWT).

2 Current address: Dept. of Mouse Genetics and Inflammation, Institute forGenetics, University of Cologne, Zulpicher Str. 47, D-50674 Cologne, Germany.

3 Predoctoral fellow with the FWO.4 Current address: Membrane Biology Lab, Institute of Molecular and Cell Biol-

ogy, 61 Biopolis Drive, Proteos, Singapore 138673.5 Postdoctoral fellow with the IWT.6 Postdoctoral fellow with the FWO.7 To whom correspondence should be addressed. Tel.: 32-9-331-3770; Fax:

32-9-331-3609; E-mail: [email protected].

8 The abbreviations used are: TLR, Toll-like receptor; ABIN, A20-binding inhib-itor of NF-�B activation; AHD, ABIN homology domain; GalN, D-(�)-galac-tosamine; GFP, green fluorescent protein; I�B, inhibitory protein of �B; IKK,I�B kinase; IL, interleukin; IL-1R, interleukin-1 receptor; IRAK, IL-1R-associ-ated kinase; LPS, lipopolysaccharide; Luc, luciferase; Mal, MyD88-adaptor-like; MyD88, myeloid differentiation factor 88; NF-�B, nuclear factor-�B;PBMC, peripheral blood mononuclear cells; RIP, receptor interacting pro-tein; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; TRIF,Toll/IL-1 receptor domain-containing adaptor inducing interferon-�; LIND,Listeria INDuced; CMV, cytomealovirus; E1, ubiquitin-activating enzyme;E3, ubiquitin-protein isopeptide ligase; pfu, plaque-forming unit(s); TPA,12-O-tetradecanoylphorbol-13-acetate; RT, reverse transcription; GAPDH,glyceraldehyde-3-phosphate dehydrogenase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 1, pp. 81–90, January 5, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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the TRIF/TRIF-related adaptor molecule pathway may con-tribute to delayed NF-�B activation, it is mainly responsible forinterferon regulatory factor 3 transcription factor activation viaIKK� and TANK-binding kinase 1.

Although the LPS-induced inflammatory response is indis-pensable for controlling the growth of pathogenic microorga-nisms (5), excessive cytokine production can be harmful to thehost and may even contribute to a life-threatening conditiontermed septic shock (6). In addition, TLR4-initiated signalingpathways have recently been implicated in the pathogenesis ofvarious autoimmune and chronic inflammatory diseases. Forinstance, activation of TLR4 has been shown to contribute toexperimental models of autoimmune encephalomyelitis,asthma, and atherosclerosis (7–9). This universal and inher-ently dangerous role of TLR4 in inflammation emphasizes theneed for tight regulation of TLR4-initiated signaling pathways.As such, it is not surprising that the host acquired several

proteins that can hold LPS-induced NF-�B activation in check(reviewed in Ref. 10). One of these negative feedback regulatorsis the zinc finger protein A20. This protein was originally iden-tified as an inhibitor of TNF-induced NF-�B activation,because mice with a functional deletion of the A20 gene dieprematurely due to unrestrained TNF-induced inflammation(11). However, the observation that mice doubly deficient inbothA20 andTNF (orTNF receptor-1) developed spontaneousinflammation, similar tomice deficient in A20 alone, suggestedthat A20 also has a negative role in LPS-induced NF-�B activa-tion. Indeed, A20-deficient macrophages show a prolongedNF-�B response to LPS, and reconstitution experiments withthese cells in mice showed that A20 is required for preventingendotoxic shock, pointing to A20 as an important down-regu-lator of pro-inflammatory signals initiated by LPS (12).In the present study, we identify Listeria INDuced (LIND), a

protein that is induced in humanmononuclear phagocytes by Lis-teria infection (13), as a novel LPS-inducible A20-binding inhibi-tor of NF-�B activation. As LINDwas found to share sequence aswell as functional homology with two previously identified A20-binding inhibitors of NF-�B, ABIN-1, and ABIN-2 (14, 15), wenamed it ABIN-3. Interestingly, ABIN-3 expressionwas inducibleby LPS in the monocytic cell line THP-1 as well as in primaryhumanmonocytes.Moreover, becauseABIN-3 could inhibit LPS-induced activation of NF-�B in vitro as well as in vivo, our resultsidentify ABIN-3 as a novel player in the negative feedback regula-tion of NF-�B activation in response to LPS.

MATERIALS AND METHODS

Cell Lines and Reagents—Human embryonic kidney cells(HEK293T) were a kind gift from Dr. M. Hall (University of Bir-mingham, Birmingham,UK) andwere grown inDulbecco’smod-ifiedEagle’smediumsupplementedwith10% fetal bovine serum,2mM L-glutamine, 0.4 mM sodium pyruvate, and antibiotics. Thehuman THP-1 myelomonocytic cell line was obtained from theAmerican Type Culture Collection and was grown in RPMI 1640supplemented with 10% fetal bovine serum, 0.4mM sodium pyru-vate, 2mM L-glutamine, 4�M �-mercaptoethanol, and antibiotics.The murine RAW264.7 macrophage cell line was obtained fromthe ATCC (Manassas, VA) and was cultured in Dulbecco’s modi-fied Eagle’s medium supplemented with 10% fetal calf serum.

Recombinant human TNF and recombinant murine IL-1� wereproduced inEscherichia coli in our laboratory andwere purified toat least 99% homogeneity. TNF had a specific biological activity of2.3� 107 IU/mg of purified protein, as determinedwith the inter-national standard code 87/650 (National Institute for BiologicalStandards and Control, Potters Bar, UK). IL-1� had a specificactivity of 3.65 � 108 IU/mg of purified protein, as determinedwith the international standardcode93/668.LPS fromSalmonellaabortus equiwas obtained from Sigma.Cloning of ABIN-3—TBLASTN searches with the region of

homology between ABIN-1 and ABIN-2 were conducted usingthe NCBI online service, in the non-redundant and expressedsequence tag data base. A “full insert sequence” of a clone ofhuman coronary artery smooth muscle cells (accession numberAK024815) was identified as a potential homologue. 5� Rapidamplification of cDNA ends (RACE) was performed on HeLamRNA, using the SMARTRACE kit (Clontech Laboratories, Paloalto, CA) according to the instructions from the manufacturer.Primers for first round and nested PCR were 5�-cgttccttttcc-ttctcctcccgctgca-3� and 5�-ctctgcctctgatgcggatccttctccc-3�,respectively. Full-length cDNA was amplified using forward (5�-ggagatctgcggccgctatggcacattttgtacaagg-3�) andreverse (5�-gagatc-tctacggatggactttctttactgagg-3�) primers. The open reading frameof ABIN-3was cloned in-frame with an N-terminal E tag into themammalian expression vector pCAGGS. The cloned fragmentwas sequenced on both strands with a cycle sequencer (AppliedBiosystems, Foster City, CA). Deletion mutants were generatedby PCR and cloned in pCAGGS using the following primers:5�-tgcgaacaaggaaaagatcaagtgttcattttccgagg-3� and 5�-gaaaatgaac-acttgatcttttccttgttcgcaagag-3� for ABIN-3�AHD1 and 5�-gaacag-aaatggaagttcttaatcaagagaaagaggagc-3� and 5�-tctttctcttgattaagaa-cttccatttctgttctcatc-3� for ABIN-3 �AHD2.Plasmids and Adenoviruses—Plasmids coding for GFP and

FLAG-tagged A20 have been described previously (16, 17). Theplasmid encoding TLR4 was a kind gift from Dr. M. Muzio(Dept. of Immunology and Cell Biology, Mario Negri Institute,Milano, Italy) (18), and plasmids encodingMyD88, IRAK1, andTRAF6 were kind gifts from Dr. J. Tschopp (Institute of Bio-chemistry, University of Lausanne, Switzerland) (19, 20). Theplasmid pNFconluc (21), encoding the luciferase (Luc) genedriven by a minimal NF-�B-responsive promoter, was a giftfromDr. A. Israel (Institut Pasteur, Paris, France). The plasmidpUT651, encoding �-galactosidase, was supplied by Eurogen-tec (Seraing, Belgium). For the production of a recombinantABIN-3 adenovirus, the ABIN-3 cDNA, N-terminally fused toanE tag,was cloned into the pACpLpA.CMVshuttle vector andcotransfected with the rescue plasmid pJM17 (which encodesthe adenovirus dl309 genome, lacking E1 and E3 functions) intoHEK293 cells using calcium phosphate coprecipitation (22).Recombinant plaques were isolated and expression of ABIN-3from the ubiquitously active cytomegalovirus (CMV) promoterwas confirmed by Western blotting. Control viruses withouttransgene (AdRR5) or expressing the �-galactosidase gene(AdLacZ) were generated with the same pJM17 adenoviralbackbone vector. A virus expressing an NF-�B luciferasereporter gene (AdNF�BLuc) (23) was obtained from Dr. B.McGray (University of Iowa College of Medicine, Iowa City,IA). High titer virus stocks were prepared in HEK293 cells and

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purified by single CsCl banding. Titers were determined byplaque assay in HEK293 cells and calculated as plaque formingunits (pfu) per milliliter of virus stock.Isolation and Culture of PrimaryMonocytes—Peripheral blood

mononuclear cells (PBMCs)were prepared from fresh blood sam-ples of healthy donors drawn on citrate/phosphate/dextrose(Etablissement Francais du Sang, Paris, France). Bloodwas diluted1:2 in RPMI 1640 Glutamax medium (BioWhittaker, Verviers,Belgium) and centrifuged over Ficoll (MSL, Eurobio, Les Ulis,France) for 20min at 15 °C and 600� g. Humanmonocytes wereselected from PBMCs by adherence. PBMCs were plated at 6 �106 cells/ml and allowed to adhere for 1 h at 37 °C in a 5%CO2 airincubator in a humidified atmosphere. Non-adherent cells wereremoved; adherent cells were washed with RPMI and cultured inRPMI supplemented with antibiotics (100 IU/ml penicillin and100g/ml streptomycin) and0.2%normalhumanserum(BioWhit-taker). Monocytes were stimulated with 100 ng/ml LPS from S.abortus equi (Alexis, San Diego, CA).Transfection, Coimmunoprecipitation, and Western Blotting—

2 � 106 HEK293T cells were seeded in 10-cm Petri dishes andtransfected with a total of 5 �g of DNA per plate using the DNAcalcium phosphate coprecipitation method, as described (24).After 24 h, the cells were lysed in 500 �l of lysis buffer (50 mMHepes, pH 7.6, 250mMNaCl, 5mM EDTA, 0.1%Nonidet P-40),supplemented with protease and phosphatase inhibitors.Immunoprecipitation was performed with a monoclonal anti-FLAG M2 antibody (Sigma), and immunocomplexes werebound to protein A-trisacryl beads (Pierce). Beads were washedtwice with lysis buffer, twice with the same buffer containing 1MNaCl, and again twice with lysis buffer. Binding proteins wereeluted with Laemmli buffer and analyzed by 12.5% SDS-PAGEandWestern blotting. Detection of co-precipitating and trans-fected proteins was achieved with a monoclonal anti-E tag(Amersham Biosciences) or anti-FLAG tag (Sigma) antibody,each of which was coupled to horseradish peroxidase. Immu-noreactivity was revealed with a Renaissance-enhanced chemi-luminescence system (PerkinElmer Life Sciences).Reporter Gene Assays for NF-�B—2� 105 HEK293T cells were

grown in6-well plates and transiently transfectedbyDNAcalciumphosphate coprecipitation with a total of 1 �g of DNA. The DNAmixture comprised 100 ng of pUT651, 100 ng of pNFconluc, and800 ng of specific expression plasmids. After 24 h, the cells wereseeded in 24-well plates. Another 24 h later, cells were leftuntreated or were stimulated with TNF (1000 IU/ml), IL-1� (40ng/ml), or TPA (200 ng/ml) for 6 h. For RAW264.7 cells, 5 � 105cells were transfected using Lipofectamine 2000 and Opti-MEM(Invitrogen) with 250 ng of an NF-�B-dependent luciferasereporter plasmid and 100 ng of a plasmid encoding �-galactosid-ase, togetherwith250ngof specific expressionplasmids.After 6h,the cells were stimulated with 100 ng/ml LPS for 3 h. In all of theabove cases, cells were lysed after stimulation in 200 �l of lysisbuffer (25 mM Tris-phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM1,2-cyclohexaminediaminetetraacetic acid, 10% glycerol, and 1%TritonX-100). After addition of substrate buffer to a final concen-tration of 470�M luciferin, 270�McoenzymeA, and530�MATP,luciferase (Luc) activity was measured in a Topcount microplatescintillationreader (Packard InstrumentCo.,Meriden,CT).�-Ga-lactosidase activitywas assayedusing chlorophenol red�-D-galac-

topyranoside substrate (Roche Molecular Biochemicals, Mann-heim, Germany) or the Galactostar reporter gene assay system(Applied Biosystems). Luc values were normalized for �-galacto-sidase values to correct for differences in transfection efficiency(plotted as Luc/�-galactosidase).To determine NF-�B activation induced by overexpression of

specific signaling proteins, 4� 104 HEK293T cells were seeded in24-wellplatesandtransiently transfectedwith20ngofpUT651,20ng of pNFconluc, and a total of 160 ng of specific expression plas-mids. After 24 h, the cells were lysed and analyzed as describedabove. For in vivoNF-�Banalysis by luciferase reporter gene assay,mice were infected with a total of 5� 109 pfu adenoviruses, com-prising 25% AdNF�BLuc, 25% AdLacZ, and 50% AdRR5 orAdABIN-3. Three days after infection, mice were injected intrap-eritoneally with 200 ng of LPS or vehicle. After 4 h, mice werekilled, liverhomogenatesweremade,andNF-�Bpromoteractivitywas determined by measuring luciferase (Luc) activity in tissueextracts as described above. Luc values were normalized for�-ga-lactosidase values to correct for differences in infection efficiency(plotted as Luc/�-galactosidase).IL-8 Determination—IL-8 levels in cell supernatants were

determined via specific enzyme-linked immunosorbent assay(BDPharmingen) according to themanufacturer’s instructions.ABIN-3 mRNA and Protein Expression Analysis—For multi-

ple tissue RT-PCR, a human “Rapid Scan cDNA panel” waspurchased from Origene Technologies (Rockville, MD). PCRwas performed with gene-specific primers (5�-actggacgccgcgg-aaagat-3� and 5�-tggcggaagctggtcaagag-3�) that amplify a691-bp fragment of the ABIN-3 open reading frame.ForRT-PCRonTHP-1monocytes, 5� 105 cellswere seeded in

10-cmPetri dishes and allowed to grow for 48 h. At the end of thisperiod thecellswereeither leftuntreatedor stimulatedwithLPS (1�g/ml)orTNF(1000 IU/ml).TotalRNAofTHP-1cellswas isolatedby the guanidium isothiocyanate-phenol-chloroform method (25),and first strandcDNAwassynthesizedusing theSuperScriptTMfirst-strand synthesis system forRT-PCR (Invitrogen).For RT-PCR on PBMC, total RNA was prepared using the

RNeasy Mini Kit (Qiagen) and reverse-transcribed with Super-script II RNase H (Invitrogen) according to the manufacturer’sprotocol. cDNA samples were amplified by PCR with gene-specific primers (5�-ggagatctgcggccgctatggcacattttgtacaagg-3�and 5�-ggagatctctacggatggactttctttactgagg-3�) that amplify thecomplete open reading frame ofABIN-3. As a control for cDNAintegrity, either RT-PCR for a �-actin fragment was performedusing 5�-gaactttgggggatgctcgc-3� and 5�-tggtgggcatgggtcagaag-3� primers, or RT-PCR for GAPDHwas performed using 5�-tgaa-ggtcggagtcaacggatttggt-3� and 5�-catgtgggccatgaggtccac-cac-3� primers. For ABIN-3 protein expression analysis in THP-1monocytes, 7 � 106 cells were seeded in 10-cm Petri dishes andwereeither leftuntreatedor stimulatedwithLPS(1�g/ml)orTNF(1000 IU/ml) for various time periods. Subsequently, cell lysateswere prepared and immunoblotted with a rabbit polyclonalABIN-3 antibody raised against an ABIN-3-specific peptide(NH2-CDVQHKANGLSSVKKVHP-COOH) coupled to keyholelimpet hemocyanin.For real-time quantitative PCR, total RNA of PBMC was pre-

pared using the RNeasy Mini Kit (Qiagen). Purified RNA wasreverse-transcribed with Superscript II RNase H (Invitrogen)

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according to themanufacturer’s protocol. The expression levels ofABIN-3 and GAPDH were determined by real-time quantitativePCR, using a LightCycler FastStart DNAMasterPLUS SYBRGreenI kit (Roche Applied Science). Forward and reverse primers forhuman ABIN-3 were, respectively, 5�-caaaggaaaaggaacattac-3�and 5�-tgctgtagctcctctttctc-3�. Primers for GAPDH were the RT2

PCR primer set from SuperArray (Frederick, MD). The cDNAcopynumberof eachgenewasdeterminedusinga six-point stand-ard curve. Standard curves were run with each set of samples, thecorrelation coefficients (r2) for the standard curves being �0.98.All results were normalized with respect to the expression ofGAPDH.Toconfirmthe specificityof thePCRproducts, themelt-ing profile of each sample was determined using the LightCycler,and by heating the samples from 60 °C to 95 °C at a linear rate of0.10 °C/s while measuring the fluorescence emitted. Analysis ofthe melting curve demonstrated that each pair of primers ampli-fied a single product. In all cases, the PCR products were checkedfor sizebyagarosegel separationandethidiumbromidestaining toconfirm that a single product of the predicted size was amplified.ForABIN-3, each run consisted of an initial denaturation timeof 5minat95 °Cand40cyclesat95 °C for8s, 56 °C for8s, and72 °Cfor15 s. For GAPDH, the run consisted of 40 cycles at 95 °C for 15 s,58 °C for 15 s, and 72 °C for 25 s.Animal Treatment Protocols— Female C57BL/6 mice (8–12

weeks old) were purchased from Charles River (Sulzfield, Ger-many). All animals were maintained under standard specificpathogen-free conditions and received humane care in con-cordance with the National Institutes of Health guidelines andwith the legal requirements in Belgium. All animal experimentswere performed in accordance with protocols approved by theInstitutional Animal Care and Research Advisory Committee.For adenovirus infection, mice were intravenously injectedwith a total of 5 � 109 pfu of virus diluted in pyrogen-freephosphate-buffered saline. In preliminary experiments, wefound that adenoviral transgene expression in the liver is max-imal 3 days after infection. Therefore, mice were challengedwith LPS/GalN 3 days after infection. In the LPS/GalN-inducedmodel of acute lethal hepatitis, mice were injected intraperito-neal with 200 ng of LPS in combination with 20 mg of GalN(Sigma), corresponding to the LD100 determined in preliminarystudies.Statistics—All data represent at least three independent

experiments and are expressed as mean values � S.D. Sur-vival curve was compared using a log rank �2 test, and thelevel of probability was noted (*, p � 0.05; **, p � 0.01; and*** p � 0.0001).

RESULTS

Identification of LINDas anABIN—Theprotein sequences ofthe A20-binding inhibitors of NF-�B ABIN-1 and ABIN-2show significant homology over a region of�70 amino acids. Itis in this region that the previously described ABIN homologydomain 1 (AHD1) andAHD2are located (26) (Fig. 1).Using thishomologous region in BLAST searches, we identified LIND, aprotein that is induced in human mononuclear phagocytesinfected with Listeria (13), as a potential ABIN protein. Com-parison of the full-length protein sequence of LIND with thesequences of ABIN-1 and ABIN-2 revealed that LIND was

much more homologous to ABIN-1 than to ABIN-2. Besidesthe AHD1 and AHD2 regions of homology, LIND and ABIN-1also share a third region of strong homology, indicated asAHD3, which is not present in ABIN-2 (Fig. 1). Because of thisstrong sequence homology with ABIN-1 and ABIN-2, we willhenceforth refer to LIND as ABIN-3 (TNIP3).To test ifABIN-3, besides sequencehomology, also shows func-

tional homology with ABIN-1 and ABIN-2, we investigatedwhether ABIN-3 could interact with the zinc finger protein A20.Therefore, expression plasmids for E-tagged ABIN-3 and FLAG-tagged A20 were cotransfected in HEK293T cells, followed byimmunoprecipitation with an anti-FLAG tag antibody. Immuno-blotting with anti-E tag revealed that ABIN-3 indeed coimmuno-precipitated with A20, indicating that both proteins can associatewith each other inmammalian cells (Fig. 2A). In addition to inter-actingwithA20,ABIN-1andABIN-2are also characterizedby theability to inhibit the activation of NF-�B in response to TNF,IL-1�, and TPA (14, 15). To test if ABIN-3 shares this NF-�Binhibiting activity, we coexpressed ABIN-3 with an NF-�B-dependent luciferase reporter gene in HEK293T cells. The effectsof the NF-�B inhibitor A20 and the irrelevant protein GFP wereused as positive and negative controls, respectively. ABIN-3 wasindeed able to inhibit NF-�B-dependent luciferase expressioninduced by TNF, IL-1�, or TPA (Fig. 2B). Taken together, thisstrong sequence and functional homology with ABIN-1 andABIN-2 identifies LIND as a novel A20-binding inhibitor ofNF-�B activation, named ABIN-3.ABIN-3 Is an LPS-inducible Protein—Tissue distribution of

ABIN-3mRNAwas investigated by PCR amplification of a cDNApanel containing first strand cDNA samples from 24 differenthuman tissues. No ABIN-3 mRNA could be detected in heart,salivary gland, adrenal gland, pancreas, ovary, or fetal brain. Highlevels ofABIN-3mRNAweredetected inmostof theother tissues,except for kidney and bonemarrow, both of which showed only alow expression level of ABIN-3mRNA (Fig. 3A).We subsequently analyzed ABIN-3 mRNA expression by

semi-quantitative RT-PCR on mRNA isolated from varioushuman cell lines such as THP-1, HEK293, and HepG2. Consti-tutive expression of ABIN-3 mRNA could not be detected inany of the cell lines (data not shown). However, a clear induc-tion of ABIN-3 mRNA could be observed in THP-1 monocytesafter stimulation for 3 h with LPS (Fig. 3B). In contrast, stimu-lation of THP-1 cells with TNF only led to a slight induction ofABIN-3 mRNA. To investigate if the observed induction ofABIN-3 mRNA in THP-1 cells was also reflected at the proteinlevel, polyclonal antibodies against ABIN-3 were generated inrabbits, andused to analyze the expression ofABIN-3 protein inTHP-1 cells treated with LPS or TNF. Consistent with the RT-PCR data, ABIN-3 protein could not be detected in unstimu-lated cells. However, after 6-h LPS treatment ABIN-3 proteinwas clearly visible (Fig. 3C). In contrast, stimulation of THP-1cells with TNF did not lead to detectable expression levels ofABIN-3 protein (data not shown). We also evaluated LPS-in-ducible expression of ABIN-3 in primary human monocytes,which were selected by adherence from peripheral bloodmononuclear cells of healthy donors. These monocytes werestimulated either with vehicle or with LPS for 2 or 20 h. TotalRNA was isolated, and expression of ABIN-3 mRNA was ana-

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lyzed by semi-quantitative RT-PCR (Fig. 3D) as well as by real-time quantitative PCR (Fig. 3E). In both cases, expression ofABIN-3mRNAwas induced already slightly after 2-h treatmentwith LPS, and was more pronounced after 20 h. All together,these data demonstrate that LPS is a potent inducer of ABIN-3expression in monocytic cells.ABIN-3 Inhibits LPS/TLR4-induced NF-�B Activation—The

above results show that expression of ABIN-3 is inducible by

LPS and thus suggest a role forABIN-3 in the LPS/TLR4-inducedpathway to NF-�B. To investigatewhether ABIN-3 prevents LPS/TLR4-induced NF-�B activity, wefirst tested the effect of ABIN-3overexpression on NF-�B-depend-ent luciferase reporter gene expres-sion in response to transient TLR4overexpression in HEK293T cells,which as such is already sufficient toactivate NF-�B. The upper panel ofFig. 4A illustrates that ABIN-3expression significantly reducedTLR4-induced NF-�B-dependentluciferase gene expression. To eval-uate whether ABIN-3 also inhibitsthe expression of an endogenousNF-�B target gene, we also analyzedthe TLR4-induced production ofIL-8 in the same experiment. IL-8levels in the HEK293T cell superna-tant were increased 6-fold uponTLR4 expression (Fig. 4A, lowerpanel). Consistent with the NF-�Binhibitory effect of ABIN-3 in theluciferase reporter assay and the factthat IL-8 expression is known to beat least partially NF-�B-dependent(18, 27), coexpression of ABIN-3significantly reduced the expressionof IL-8 in response to TLR4. Toinvestigate the effect of ABIN-3 in amore physiologically relevant cellline, we next investigated whetherABIN-3 also inhibits LPS/TLR4-in-duced expression of an NF-�B-de-pendent luciferase reporter gene inthe RAW264.7 macrophage cellline. As can be seen in Fig. 4B,ABIN-3 expression indeed signifi-cantly reduced LPS-induced lucifer-ase expression in RAW264.7 mac-rophages, further establishing thefunction of ABIN-3 as an inhibitorof LPS/TLR4-induced NF-�B-de-pendent gene expression.For the previously described

NF-�B inhibitors ABIN-1 andABIN-2, it was shown that their

AHD2 region is essential for NF- �B inhibition (26).9 In addi-tion, ABIN-1 and ABIN-2 have been shown to bind A20through the more upstream AHD1. To elucidate whetherAHD1 and AHD2 of ABIN-3 have a similar function,

9 A. Wullaert, L. Verstrepen, S. Van Huffel, M. Adib-Conquy, S. Cornelis, M.Kreike, M. Haegman, K. El Bakkouri, M. Sanders, K. Verhelst, I. Carpentier,J.-M. Cavaillon, K. Heyninck, and R. Beyaert, unpublished results.

FIGURE 1. Sequence homology of LIND/ABIN-3 with ABIN-1 and ABIN-2. Alignment of the amino acidsequences of human ABIN-1, ABIN-2, and ABIN-3. Identical amino acids are shaded in black, homologous aminoacids in gray. The regions of strong homology AHD1, AHD2, and AHD3 are indicated with &, *, and $,respectively.

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we made deletion mutants of ABIN-3 that either lacked AHD1(ABIN-3 �AHD1) or AHD2 (ABIN-3 �AHD2) and evaluatedthe binding of these mutants to A20 as well as their NF-�B-inhibiting potential. Transient overexpression of FLAG-taggedA20 together with E-tagged ABIN-3 WT, ABIN-3 �AHD1, orABIN-3 �AHD2, followed by immunoprecipitation of A20with an anti-FLAGantibody, clearly demonstrated thatABIN-3�AHD2 and ABIN-3WT bind equally well to A20. In contrast,ABIN-3 �AHD1 did not coprecipitate with A20, showing thatthe binding of ABIN-3 to A20 requires AHD1 (Fig. 5A). Similarresults were obtained when binding was studied via yeast two-hybrid experiments (data not shown). The NF-�B-inhibitingeffect of theABIN-3 deletionmutantswas analyzed bymeans ofan NF-�B luciferase reporter gene test in HEK293T cells.Whereas both ABIN-3 WT and ABIN-3 �AHD1 significantlyreduced TLR4-induced expression of an NF-�B-dependentluciferase reporter gene, ABIN-3 �AHD2 had no effect any-more (Fig. 5B). This indicates that AHD2 is essential for the

NF-�B inhibiting function of ABIN-3, whereas AHD1 is not.Because AHD1 is essential for ABIN-3/A20 binding, theseresults also indicate that ABIN-3 does not need to bind A20 toprevent NF-�B activation.We next investigated the level in the NF-�B signaling path-

way at which ABIN-3 interferes with LPS/TLR4-inducedNF-�B activation. Therefore, we analyzed the effect of ABIN-3coexpression on NF-�B activation induced by overexpressionof the TLR4 signaling proteins MyD88, IRAK1, and TRAF6, aswell as by IKK�, which is acting more downstream in the path-way and mediates NF-�B activation by all stimuli that activatethe “classic” NF-�B pathway. As shown in Fig. 6, ABIN-3 inhib-ited NF-�B activation induced by MyD88, IRAK1, and TRAF6but not that induced by IKK�. This suggests that ABIN-3 inter-feres with LPS/TLR4-induced NF-�B activation at a leveldownstream of TRAF6 but upstream of IKK�.ABIN-3 Inhibits LPS-induced NF-�B Activation in the Liver

and Protects Mice against LPS/GalN-induced Mortality—Tovalidate the NF-�B inhibitory function of ABIN-3 in vivo, wealso tested the effect of ABIN-3 on LPS-induced NF-�B activa-tion inmouse liver. For this purpose,micewere infectedwith anadenovirus expressing anNF-�B-dependent luciferase reportergene, together with an adenovirus expressing either an ABIN-3transgene (AdABIN-3), or no transgene (AdRR5) as a control.Three days after AdABIN-3 infection, ABIN-3 transgeneexpression was clearly detectable in total liver cell extracts byWestern blotting (Fig. 7A,upper part). LPS injection of AdRR5-infected mice resulted in a 13-fold increase in NF-�B-depend-ent luciferase activity in the liver (Fig. 7A, lower part). However,consistent with the NF-�B inhibitory effect of ABIN-3 in vitro,LPS-induced NF-�B activity was substantially lower in the liverof AdABIN-3-infected mice.Because these data indicate that ABIN-3 can inhibit LPS-

inducedNF-�B activity in the liver, we investigated the effect ofadenoviral gene transfer ofABIN-3 in themurinemodel of LPS/GalN-induced acute liver failure. Therefore, C57BL/6 micewere injected intravenously with 5 � 109 pfu of AdRR5 orAdABIN-3. Three days later, mice were challenged intraperito-neally with a lethal dose of LPS/GalN. In the control group, allmice died within 10 h after LPS/GalN injection. In contrast,AdABIN-3-infected mice were significantly (p 0.0038) pro-tected against LPS/GalN-induced mortality, as one-third ofthem survived the LPS/GalN challenge (Fig. 7B). These obser-vations clearly demonstrate that ABIN-3-mediated NF-�Binhibition in the liver is associated with a protective effectagainst LPS/GalN-induced liver failure.

DISCUSSION

Although essential to combat bacterial infections, LPS-inducedactivation of NF-�B acts as a double-edged sword. Inappropriateor prolonged activation of NF-�B can lead to an exaggeratedimmune response, which might be harmful to the host. There-fore, to prevent excessive immune responses to LPS, the hostmay acquire mechanisms that dampen the response to LPS oreven confer unresponsiveness to successive triggers with LPS, aphenomenonnamedLPS tolerance (28).Down-regulating LPS-induced responses can at least partially be accomplished by theLPS-induced production of NF-�B inhibitory proteins, which

FIGURE 2. Functional homology of LIND/ABIN-3 with ABIN-1 and ABIN-2.Coimmunoprecipitation of ABIN-3 with A20. A, 2 � 106 HEK293T cells weretransiently transfected with 3 �g of E-tagged ABIN-3 and 1.5 �g of FLAG-tagged A20 expression vectors, as indicated. Immunoprecipitation (IP) of A20was performed with anti-FLAG tag antibody, and coprecipitating ABIN-3 wasdetected by immunoblotting (WB) with anti-E tag antibody (upper panel).Aliquots of total lysates (TL) were analyzed for expression of ABIN-3 (middlepanel) and A20 (lower panel) by immunoblotting with anti-E tag and anti-FLAG tag, respectively. B, effect of ABIN-3 on NF-�B-dependent gene expres-sion. 2 � 105 HEK293T cells were transiently transfected with 300 ng ofexpression plasmid for GFP, A20, or ABIN-3, each time with 100 ng of pUT651and 100 ng of pNFconluc. Cells were left untreated or stimulated with 200ng/ml TPA, 40 ng/ml IL-1�, or 1000 IU/ml TNF for 6 h. All cells were lysed 24 hafter transfection. Cell lysates were assayed for Luc and �-galactosidase activ-ity. Luc values were normalized for �-galactosidase values to adjust for differ-ences in transfection efficiency (plotted as Luc/Gal). Each bar represents themean � S.D. of three samples.

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then provide a negative feedback loop (reviewed in Refs. 10 and29). For example, LPS-inducible alternative splicing of MyD88can shut down LPS-induced NF-�B activation by preventingthe recruitment of IRAK4 to MyD88 (30, 31). Several otherLPS-inducible proteins were shown to inhibit NF-�B activationby targeting different steps in the TLR4 signaling pathway andinclude, among others, A20 (12), SOCS1 (32, 33), IRAK-M (34),and ST2 (35). Here we identify human ABIN-3 as a novel pro-tein that fulfils two essential criteria to be implicated in thenegative feedback regulation of LPS-induced NF-�B activation.First, ABIN-3 expression was induced by LPS in monocytic cells.Second,wecould showthat expressionofABIN-3 inhibitsNF-�B-dependentgeneexpression in response toLPS,both in vitroaswellas in vivo. HumanABIN-3 shows partial sequence homologywithABIN-1 and ABIN-2 and shares with these proteins the ability tobind A20 and to inhibit TNF-, IL-1-, and LPS-induced NF-�Bactivation upon overexpression in HEK293T cells (14, 15).9These overlapping activities suggest that the function ofABIN-1, -2, and -3 might be at least partially redundant. Thefact that ABIN-2-deficient mice are normal and do not showany defect in NF-�B activation in response to different stimulimight also reflect such redundancy (36). However, we cannotexclude cell type- or stimulus-specific effects of distinct ABINs

on NF-�B signaling. The morerestricted expression of ABIN-3 inspecific tissues, as well as its induc-ibility by LPS, suggests that ABIN-3might indeed have a unique func-tion. In this respect, it is worthmen-tioning that coimmunoprecipita-tion experiments have shown thatABIN-3 does not compete with theother ABINs for binding to A20(data not shown). Moreover, ourongoing yeast two-hybrid experi-ments demonstrate different pro-tein-protein interactions for eachABIN family member.It is still unclear how ABINs

interfere with NF-�B signaling. Ourfinding that ABIN-3 still preventsTRAF6-induced NF-�B activation,but no longer IKK�-induced NF-�Bactivation, indicates that ABIN-3interferes with LPS/TLR4 signalingat the level of or downstream ofTRAF6 but upstream of IKK�. Asimilar effect on proximal signalingwas previously shown for ABIN-1and -2, which inhibit TNF-inducedNF-�B activation downstream ofTRAF2 and upstream of IKK�. Thebinding of ABIN-3 to A20 suggeststhat its NF-�B inhibitory effectmight be mediated by A20. A20 wasrecently proposed to inhibit NF-�Bactivation by de-ubiquitinating sev-eral proteins, including TRAF6,

RIP, and IKK� (12, 37–39). In fact, while this report was pre-pared, ABIN-1 was described to physically link A20 to IKK� bydirectly binding IKK�, thus facilitating A20-mediated de-ubiq-uitination of IKK� (39). However, our data demonstrate thatthe NF-�B inhibitory potential of ABIN-3 does not correlatewith ABIN-3/A20 binding, as an AHD1-deletion mutant ofABIN-3, which can no longer bind A20, is still fully capable ofinhibiting NF-�B activation. Although we cannot exclude thatABINs somehow regulate or modulate the function of A20,these findings make it unlikely that the NF-�B inhibitory effectof ABIN-3 is exclusively mediated by A20.Another model that could explain the NF-�B inhibitory

effect of ABIN-3 implicates the possibility that ABIN-3 pre-vents the formation of specific protein-protein interactions inthe cell. Deletion analysis of ABIN-3 showed that, like inABIN-1 and -2 (26),9 the AHD2 region is necessary for itsNF-�B inhibiting function. In this context it is worth mention-ing that AHD2 shows strong sequence homology with a regionin IKK� that was recently shown to mediate the binding ofIKK� to polyubiquitin chains (26, 40, 41). This allows IKK� tobind polyubiquitinated receptor interacting protein (RIP) 1 inthe TNF signaling pathway, which is essential for TNF-inducedNF-�B activation. Although IKK�/RIP1 binding most likely

FIGURE 3. Expression profiling of ABIN-3. A, PCR was performed on cDNA from different human tissues to amplifya 691-bp fragment of the ABIN-3 open reading frame (upper panel). PCR for �-actin served as a control (lower panel).B, expression of ABIN-3 mRNA in THP-1 monocytes, either untreated or stimulated for 3 h with 1000 IU/ml TNF or 1�g/ml LPS. First strand cDNA was prepared, and PCR was performed to amplify the complete open reading frame ofABIN-3 (upper panel). PCR for �-actin served as a control (lower panel). C, expression of ABIN-3 protein in THP-1 cellsafter treatment with LPS for various time periods as indicated. Total cell lysates were prepared and Western blottingwas performed with anti-ABIN-3 antibodies. As loading controls, two nonspecific bands (*) are shown. D, expressionof ABIN-3 mRNA in human primary monocytes stimulated for 2 h or 20 h with 100 ng/ml LPS. One representativeRT-PCR experiment out of three performed on different donors is shown. E, mRNA expression of ABIN-3 in humanprimary monocytes that were stimulated for 2 or 20 h with LPS was analyzed by real-time quantitative PCR. Thefigure represents the mean � S.D. of three independent experiments performed with different donors. Expressionof ABIN-3 was normalized to that of GAPDH.

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also involves other surrounding amino acids that provide fur-ther specificity, similar ubiquitin-dependent protein-proteininteractions might be mediated by ABINs via their AHD2. Inthis way, ABIN-3 might also compete with IKK� or other sig-naling proteins to form crucial protein-protein interactions inresponse to TLR4 triggering. Because RIP1 is not involved inthe LPS/TLR4-induced MyD88-dependent signaling pathwaythat is inhibited by ABIN-3 (42), ubiquitin-dependent protein-

FIGURE 4. ABIN-3 inhibits LPS/TLR4-induced NF-�B-dependent geneexpression. A, effect of ABIN-3 on NF-�B-dependent gene expression inresponse to TLR4 overexpression in HEK293T cells. 4 � 104 cells were tran-siently transfected with 20 ng of pUT651 and 20 ng of pNFconluc. Whereindicated, cells were also transfected with 20 ng of a TLR4 expression plasmidand/or 40 ng of an ABIN-3 expression plasmid. Total amounts of transfectedDNA were kept constant by adding empty vector. 24 h after transfection, cellsupernatants were collected and cell lysates were prepared. Cell lysates wereassayed for Luc and �-galactosidase activity (upper panel). IL-8 concentrationsin the supernatant were measured by enzyme-linked immunosorbent assay(lower panel). Luc values and IL-8 levels were normalized for �-galactosidasevalues to adjust for differences in transfection efficiency. Each bar representsthe mean � S.D. of three samples. B, effect of ABIN-3 on LPS-induced NF-�Bactivation in RAW264.7 macrophages. 5 � 105 cells were transiently trans-fected with 100 ng of pUT651 and 250 ng of pNFconluc, together with 250 ngof either empty vector (/) or an ABIN-3 expression plasmid. 6 h after transfec-tion, cells were stimulated with 100 ng/ml LPS for 3 h. Cell lysates wereassayed for Luc and �-galactosidase activity. Luc values were normalized for�-galactosidase values to adjust for differences in transfection efficiency(plotted as Luc/Gal). Each bar represents the mean � S.D. of three samples.

FIGURE 5. Role of AHD1 and AHD2 for ABIN-3/A20 binding and ABIN-3-mediated inhibition of TLR4-induced NF-�B activation. A, coimmu-noprecipitation of ABIN-3 deletion mutants with A20. 1.2 � 106 HEK293Tcells were transiently transfected with 1 �g of FLAG-tagged A20 togetherwith 1 �g of E-tagged ABIN-3 WT, ABIN-3 �AHD1, or ABIN-3 �AHD2, asindicated. Immunoprecipitation (IP) of A20 was performed with anti-FLAGtag antibody, and coprecipitating ABIN-3 WT or deletion mutants weredetected by immunoblotting (WB) with anti-E tag antibody (upper panel).Aliquots of total lysates (TL) were analyzed for expression of ABIN-3 WTand deletion mutants (middle panel) and A20 (lower panel) by immuno-blotting with anti-E tag and anti-FLAG tag, respectively. B, effect of ABIN-3deletion mutants on TLR4-induced NF-�B activation. 4 � 104 HEK293Tcells were transiently transfected with 20 ng of pUT651, 20 ng of pNFcon-luc, and 20 ng of empty vector (/), or an expression plasmid for TLR4. Ineach case, cells were also transfected with 40 ng of an expression plasmidfor ABIN-3 WT, ABIN-3 �AHD1, or ABIN-3 �AHD2. All cells were lysed 24 hafter transfection. Cell lysates were assayed for Luc and �-galactosidaseactivity. Luc values were normalized for �-galactosidase values to adjustfor differences in transfection efficiency (plotted as Luc/Gal). Each bar rep-resents the mean � S.D. of three samples.

FIGURE 6. ABIN-3 inhibits NF-�B activation downstream of TRAF6 andupstream of IKK�. 4 � 104 HEK293T cells were transiently transfectedwith 20 ng of pUT651, 20 ng of pNFconluc, and 40 ng of an expressionplasmid for a specific TLR4-signaling protein, as indicated. In each case,cells were cotransfected with 60 ng of an expression plasmid for ABIN-3 orGFP (negative control). All cells were lysed 24 h after transfection, Luc and�-galactosidase activities were determined and plotted as Luc/Gal. Eacherror bar represents the mean � S.D. of three samples.

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protein interactions different from RIP1/IKK� must be impli-cated as potential targets for ABIN-3.We were unable to further establish the role of ABIN-3 in LPS

signaling by RNA interference-mediated knockdown of ABIN-3,becausewecouldobtainnosufficient reductionofABIN-3expres-sion (data not shown). Elucidation of the role of ABIN-3 by gener-ating ABIN-3 knock-out mice by homologous recombinationmight be an alternative approach. However, whereas ABIN-1 andABIN-2 are expressed inmurine aswell as inhumancells,wewereunable to identify a functional murine orthologue of the humanABIN-3 gene. Data base searches revealed a gene termed “weaklysimilar toABIN-3,” but closer examinationof its sequence showed

that it encodes a smaller protein that does not contain the com-pleteAHD2.Moreover, as overexpression of thismurineABIN-3-like protein did not inhibit NF-�B activation (data not shown), itdoes not qualify as a true ABIN. Although a functional murineABIN-3 gene might not exist, it is worth mentioning that expres-sionof humanABIN-3 is able topreventLPS-inducedNF-�Bacti-vation inmurine cells as reflectedbyour experimentswithmurineRAW264.7macrophagesaswell asour invivomouseexperiments.

Multiple stimuli can activate NF-�B by partially overlappingsignaling pathways. Therefore, ABIN-3 might also affect theactivation of NF-�B by other stimuli than the ones tested in thisstudy (TNF, IL-1, TPA, and LPS). In this respect, it is worthmentioning that ABIN-3 has previously been described asLIND (Listeria INDuced), a protein that is induced inmononu-clear phagocytes infected with Listeria (13). Because Listeria isnot a Gram-negative bacterium and thus has no LPS, a TLRagonist other than LPS must be responsible for inducingABIN-3 expression, raising the possibility that ABIN-3 acts as anegative regulator of inflammatory responses initiated by awide range of TLRs. In addition, it cannot be excluded thatABIN-3 also regulates pathways different from NF-�B, such asthe activation of interferon regulatory factor 3 and AP-1. Incontrast to its ability to inhibit TPA-induced NF-�B activation(Fig. 2B), ABIN-3 did not prevent TPA-induced AP-1 activa-tion in the same cells (data not shown), demonstrating thatABIN-3 does not act in a nonspecific way. Further studies willbe needed to reveal the complex interplay betweenABIN-3 andother signaling proteins in the regulation of different signalingpathways.In addition to the NF-�B inhibitory potential of ABIN-3 in cul-

tured cells, we were able to show that adenoviral gene transfer ofABIN-3 inhibits LPS-induced expression of an NF-�B-dependentluciferase reporter gene in the liver, and partially protects miceagainst LPS/GalN-induced mortality. In this model of acute liverfailure, LPS induces the production and release of several cyto-kines, including TNF, IL-1, and IL-6, whose production is knowntobeNF-�B-dependent.These cytokines subsequently contributeto the pathogenesis of hepatic liver failure (43). As studies withNF-�Bdecoyoligonucleotideshavepreviously been shown topre-vent LPS-induced fatal liver failure (44), the NF-�B inhibitoryeffect of ABIN-3 is most likely responsible for the observed pro-tection against LPS/GalN-induced mortality. This suggestion isreinforced by our observation that human ABIN-3 also inhibitsNF-�B activation inmurinemacrophages, which are the predom-inant cytokine-producing cells after LPS challenge (45). On theother hand, other NF-�B-independent effects of ABIN-3 mightalso account for the protection of mice against LPS/GalN, as theclosely homologous ABIN-1 protein was recently shown to pos-sess an anti-apoptotic effect in hepatocytes, enabling it to protectmice against TNF/GalN-induced mortality (46). However, wewere unable to show a similar anti-apoptotic effect for ABIN-3(data not shown).In conclusion, we identified ABIN-3 as a novel player in the

negative feedback regulation of LPS-induced NF-�B activation.Because NF-�B has an important role in the development andprogress of septic shock and different autoimmune and chronicinflammatory diseases, strategies that increase the expression ortheactivityofABIN-3mighthaveanimportanttherapeuticpotential.

FIGURE 7. ABIN-3 inhibits NF-�B activation in the liver and protects miceagainst LPS/GalN-induced mortality. A, effect of ABIN-3 on LPS-inducedexpression of an NF-�B-driven luciferase reporter gene in mouse liver. Micewere infected with AdNF-�BLuc and AdLacZ, together with either controlAdRR5 (n 4) or AdABIN-3 (n 5) adenovirus. Three days later, mice wereinjected intraperitoneally with 200 ng of LPS or vehicle. Four hours after chal-lenge, mice were killed and liver homogenates were prepared. Adenoviralexpression of the ABIN-3 transgene in the liver was analyzed by Western blot-ting using an anti-E tag antibody (upper panel; each lane represents a differentmouse). NF-�B activity was analyzed by determining Luc and �-galactosidaseactivities (lower panel). Luc values were normalized for �-galactosidase valuesto adjust for differences in infection efficiency (plotted as Luc/Gal). B, ABIN-3partially protects mice against LPS/GalN-induced mortality. Mice wereinjected intravenously with 5 � 109 pfu of AdABIN-3 (f) or AdRR5 (�) as acontrol and challenged 3 days later with 200 ng of LPS plus 20 mg of GalN.Survival is presented as a combined Kaplan-Meyer plot of three independentexperiments (n 15 in total). Mortality was counted over a period of 30 h,after which there were no further deaths. **, p � 0.01.

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Acknowledgments—We thank Drs. B. De Geest, B. McGray, M.Klinkenberg, J. Tschopp, and A. Israel for providing adenoviruses andplasmids. A. Meeuws is thanked for technical assistance.

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Negative Regulation of TLR4-induced NF-�B Activation

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Verhelst, Isabelle Carpentier, Jean-Marc Cavaillon, Karen Heyninck and Rudi BeyaertCornelis, Marja Kreike, Mira Haegman, Karim El Bakkouri, Matthew Sanders, Kelly

Andy Wullaert, Lynn Verstrepen, Sofie Van Huffel, Minou Adib-Conquy, SigridActivation

BκLIND/ABIN-3 Is a Novel Lipopolysaccharide-inducible Inhibitor of NF-

doi: 10.1074/jbc.M607481200 originally published online November 6, 20062007, 282:81-90.J. Biol. Chem. 

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