TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-κB Activation and IL-10 Production This information is current as of September 26, 2021. Liang-Guo Xu and Hong-Bing Shu J Immunol 2002; 169:6883-6889; ; doi: 10.4049/jimmunol.169.12.6883 http://www.jimmunol.org/content/169/12/6883 Downloaded from References This article cites 59 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/169/12/6883.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TNFR-Associated Factor-3 Is Associated With BAFF-R and Negatively Regulates BAFF-R-Mediated NF-␬B Activation and IL-10 Production Liang-Guo Xu* and Hong-Bing Shu2*† TALL-1 is a member of the TNF family that is critically involved in B cell survival, maturation, and progression of lupus-like autoimmune diseases. TALL-1 has three receptors, including BCMA, TACI, and BAFF-R, which are mostly expressed by B lymphocytes. Gene knockout studies have indicated that BAFF-R is the major stimulatory receptor for TALL-1 signaling and is required for normal B cell development. The intracellular signaling mechanisms of BAFF-R are not known. In this report, we attempted to identify BAFF-R-associated downstream proteins by yeast two-hybrid screening. This effort identified TNFR-asso- ciated factor (TRAF)3 as a protein specifically interacting with BAFF-R in yeast two-hybrid assays. Coimmunoprecipitation Downloaded from experiments indicated that BAFF-R interacts with TRAF3 in B lymphoma cells and this interaction is stimulated by TALL-1 treatment. Domain mapping experiments indicated that both a 6-aa membrane proximal region and the C-terminal 35 aa of BAFF-R are required for its interaction with TRAF3. Moreover, overexpression of TRAF3 inhibits BAFF-R-mediated NF-␬B activation and IL-10 production. Taken together, our findings suggest that TRAF3 is a negative regulator of BAFF-R-mediated NF-␬B activation and IL-10 production. The Journal of Immunology, 2002, 169: 6883–6889. http://www.jimmunol.org/ ALL-1, also called BAFF, Blys, THANK, and zTNF4, is uria, and glomerulonephritis, phenotypes that mimic those of sys- a member of the TNF family of ligands identified by us temic lupus erythemia (3, 5, 14, 29, 30). Conversely, it has been T and others (1–5). TALL-1 is specifically and constitu- shown that recombinant soluble TACI-Ig fusion proteins can sig- tively expressed by monocytes, macrophages, and dendritic cells nificantly inhibit progression of lupus-like autoimmune syndrome (1–3, 6), and is induced by IFN-␥ and IL-10 (3, 6). Like most and collagen-induced arthritis in animal models (27, 31). Recently, members of the TNF family, the extracellular domain of TALL-1 gene knockout studies further confirmed that TALL-1 is required can be cleaved to form a soluble cytokine (2). Crystal structure for normal B cell development (26). Surprisingly, gene inactiva- studies suggest that soluble TALL-1 (sTALL-1)3 contains an tion studies also indicated that BAFF-R, but not TACI and BCMA, unique “flap” region that is important for its virus-like assembly, is required for TALL-1-triggered B cell development (26, 32–34). by guest on September 26, 2021 receptor binding, and biological activities (7–9). Taken together, these studies suggest that the TALL-1/BAFF-R TALL-1 signals through three receptors, including BCMA, signaling plays critical roles in regulation of B cell function and TACI, and BAFF-R, which are members of the TNFR family (5, autoimmune diseases. 10–24). All three receptors are mainly expressed by B lympho- The signal transduction pathways triggered by BCMA, TACI, cytes, while TACI is also induced in a subset of T cells following and BAFF-R are poorly characterized. Like many other members their activation (17–24). Early studies suggest that sTALL-1 can of the TNFR family, BCMA and TACI can bind to TNFR-asso- potently stimulate B lymphocyte proliferation in vitro, either alone ciated factor (TRAF) proteins and activate the transcription factor or in synergy with anti-IgM (2, 3, 10–13). It is now believed that NF-␬B and the serine/threonine protein kinase JNK (10–13, 16, sTALL-1 promotes either differentiation of B cells or survival of 35). In addition, it has also been shown that TACI can activate the mature B cells (25–28). Administration of recombinant sTALL-1 transcription factor NF-AT (36). or overexpression of sTALL-1 in mice leads to increased numbers The intracellular signaling pathways and downstream effectors of mature B lymphocytes, splenomegaly, anti-DNA Abs, protein- triggered by BAFF-R are not known. In this study, we identified TRAF3 as a BAFF-R-associated protein. Our findings suggest that TRAF3 negatively regulates BAFF-R-mediated NF-␬B activation *Department of Immunology, National Jewish Medical and Research Center, Uni- versity of Colorado Health Sciences Center, Denver, CO 80206; and †Department of and IL-10 production. Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, People’s Republic of China Received for publication July 12, 2002. Accepted for publication October 11, 2002. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Reagents and cell culture charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Mammalian-derived recombinant Flag-tagged human sTALL-1 (10) and 1 This work was supported by grants from the Ellison Medical Foundation, National Escherichia coli-derived recombinant His6-tagged human sTALL-1 (7) Institutes of Health (1RO1 AI49992-01), Arthritis Foundation, National Natural Sci- were previously described. The human embryonic kidney 293 (Dr. Z. Cao, ence Foundation of China (39925016), Chinese “863” Program (2001AA221281), Tularik, South San Francisco, CA), the B lymphoma Bjab (Dr. J. Hagman, and Special Funds for Major State Basic Research of China (G19990539). National Jewish Medical and Research Center, Denver, CO) and RPMI 2 Address correspondence and reprint requests to Dr. Hong-Bing Shu, Department of 8226 (American Type Culture Collection (ATCC), Manassas, VA) cells, Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, the mAbs against Flag (Sigma-Aldrich, St. Louis, MO) and hemagglutinin K516c, Denver, CO 80206. E-mail address: [email protected] (HA) (Covance, Berkeley, CA) epitopes, and the rabbit polyclonal anti- 3 Abbreviations used in this paper: sTALL-1, soluble TALL-1; T2, transitional type II; TRAF3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) were obtained LT␤R, lymphotoxin-␤ receptor; TRAF, TNFR-associated factor; HA, hemagglutinin. from the indicated sources. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 6884 TRAF3 IS INVOLVED IN BAFF-R SIGNALING Bjab and RPMI 8226 cells were cultured in RPMI 1640 medium con- minescent kit (Tropix, Medford, MA). Luciferase activities were normal- taining 10% FBS. 293 cells were maintained in high glucose DMEM con- ized on the basis of ␤-galactosidase expression levels. taining 10% FCS. ELISA Yeast two-hybrid screening Human IL-10 ELISA were performed using the human IL-10 ELISA To construct a BAFF-R bait vector, a cDNA fragment encoding for aa Ready-Set-Go kit (eBioscience, San Diego, CA) by following procedures 97–184 of BAFF-R was inserted in-frame into the Gal4 DNA-binding do- recommended by the manufacturer. main vector pGBT (Clontech Laboratories, Palo Alto, CA). The human B cell cDNA library (ATCC) was screened as described (37, 38). Results Identification of TRAF3 as a specific BAFF-R-interacting Coimmunoprecipitation and Western blot protein 293 cells (5 ϫ 106) were transfected with the indicated plasmids. Twenty- four hours after transfection, cells were lysed in 1 ml lysis buffer (20 mM The intracellular signaling pathways mediated by BAFF-R are un- Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 ␮g/ml aprotinin, known. Similar to other members of the TNFR family, BAFF-R 10 ␮g/ml leupeptin, 1 mM PMSF). For each immunoprecipitation, 0.4 ml does not have intrinsic enzymatic activity and is believed to trans- aliquot of lysates was incubated with 0.5 ␮g of the indicated mAb or duce signals through physical interaction with downstream signal- ␮ control mouse IgG, and 25 l of a 1:1 slurry of GammaBind G Plus Sepha- ing proteins. To identify BAFF-R-associated signaling proteins, rose (Amersham Pharmacia Biotech, Piscataway, NJ) for at least 1 h. The Sepharose beads were washed three times with 1 ml lysis buffer containing we used the yeast two-hybrid system to screen a human B cell 500 mM NaCl. The precipitates were fractionated on SDS-PAGE and sub- cDNA library with the intracellular domain of BAFF-R as bait. We sequent Western blot analyses were performed as described (37, 38). screened a total of 5 million independent clones and obtained 78 ␤ Downloaded from Vectors -galactosidase-positive clones.