DOCSLIB.ORG

Specificities of CD40 Signaling: Involvement of TRAF2 in CD40-Induced NF-␬B Activation and Intercellular Adhesion Molecule-1 Up-Regulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Specificities of CD40 Signaling: Involvement of TRAF2 in CD40-Induced NF-␬B Activation and Intercellular Adhesion Molecule-1 Up-Regulation Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1421–1426, February 1999 Cell Biology Specificities of CD40 signaling: Involvement of TRAF2 in CD40-induced NF-kB activation and intercellular adhesion molecule-1 up-regulation HO H. LEE*, PAUL W. DEMPSEY*, THOMAS P. PARKS†‡,XIAOQING ZHU*, DAVID BALTIMORE§, AND GENHONG CHENG*¶ *Department of Microbiology and Molecular Genetics, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California, Los Angeles, CA 90095; †Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877; and §California Institute of Technology, Pasadena, CA 91125 Contributed by David Baltimore, December 22, 1998 ABSTRACT Several tumor necrosis factor receptor- not TRAFs 1, 3, and 4, in transient transfection experiments associated factor (TRAF) family proteins including TRAF2, activates both the NF-kB and stress-activated protein kinase TRAF3, TRAF5, and TRAF6, as well as Jak3, have been (SAPK) signal transduction pathways (7, 8, 11, 13, 16, 17). implicated as potential mediators of CD40 signaling. An To better understand the contributions of the different extensive in vitro binding study indicated that TRAF2 and CD40ct-interacting adapter molecules to specific CD40 sig- TRAF3 bind to the CD40 cytoplasmic tail (CD40ct) with much naling pathways and CD40-mediated biology, we initiated our higher affinity than TRAF5 and TRAF6 and that TRAF2 and studies by examining the relative binding affinities of TRAFs TRAF3 bind to different residues of the CD40ct. Using CD40 2, 3, 5, and 6 and Jak3 with the CD40ct. It was found that the mutants incapable of binding TRAF2, TRAF3, or Jak3, we binding affinities of TRAFs 2 and 3 for the CD40ct are much found that the TRAF2-binding site of the CD40ct is critical for greater than those of TRAFs 5 and 6. Using human CD40 NF-kB and stress-activated protein kinase activation, as well mutant constructs that specifically abolish binding of TRAF2, as the up-regulation of the intercellular adhesion molecule-1 TRAF3, or Jak3 to the CD40ct, we also found that the (ICAM-1) gene, whereas binding of TRAF3 and Jak3 is TRAF2-binding site in the CD40ct is required for CD40- dispensable for all of these functions. Overexpression of a mediated NF-kB and SAPK activation. Finally, we demon- k a dominantly active I B strongly inhibited CD40-induced strate that CD40-mediated up-regulation of ICAM-1, a cell- k NF- B activation, ICAM-1 promoter activity, and cell-surface adhesion molecule important for cell–cell interactions and ICAM-1 up-regulation. These studies suggest a potential inflammation, is mediated through NF-kB signaling by signal transduction pathway from the CD40 receptor to the TRAF2. transcriptional activation of the ICAM-1 gene. MATERIALS AND METHODS CD40 is a 50-kDa glycoprotein, a member of the tumor necrosis factor receptor (TNFR) superfamily, and is expressed Plasmids and Expression Vectors. Full-length wild-type on a variety of cells including B cells. Studies with X-linked human CD40 and its mutant derivatives were generated with hyper-IgM syndrome, a genetic defect in the corresponding PCR end primers and internal primers with appropriate CD40L gene that is expressed predominantly by CD41 T mutations and cloned into the BamHI and SalI sites of the helper cells, and studies with mice lacking either the CD40 or pBABEpuro vector (18, 19). Glutathione S-transferase (GST) CD40L gene, have demonstrated that interaction between fusion proteins all were constructed in the pGex2T prokaryotic CD40 and CD40L during T and B cell contact is essential for gene fusion expression vector (Pharmacia). The construction all events in T cell-dependent antigen responses, including of GST-C17, C10N, C10C, and the full-length GST-mCD40ct germinal center formation, Ig isotype switching, antibody has been described previously (19). Constructs used for the affinity maturation, memory B cell formation, and T cell alanine-scanning mutagenesis were cloned similarly. Expres- activation (1–4). In cell culture systems, stimulation of CD40 sion was confirmed by Coomassie staining. TRAF constructs receptor with either anti-CD40 antibodies or CD40L rescues B were generated by PCR and cloned into the BamHI and NotI cells from apoptosis, promotes B cell proliferation, and induces sites of the PEBB-Flu-tagged mammalian expression vector. Ig isotype switching in the presence of cytokines (2). The TRAF2 and TRAF3 templates were derived from con- The pleiotropic biological roles mediated by CD40 raise an structs used previously (19). pME-TRAF5 template used for important issue regarding how a single receptor generates PCR was a gift from Jun-ichiro Inoue (University of Tokyo). intracellular signals capable of mediating such diverse biolog- The TRAF6 PCR product, generated from a cDNA library, ical phenomena. Studies using yeast two-hybrid screening and was digested with BglII and NotI and cloned into the BamHI in vitro binding assays have led to the identification of TNFR- and NotI sites of the PEBB-Flu vector. The pGL 1.3, pGL associated factor (TRAF) family proteins 2, 3, 5, and 6 as k k 1.3 B-, and the ( B)3-IFN-LUC luciferase reporters have been proteins associated with CD40 (5–9). In addition, Janus kinase described previously (20, 21). The pBABEpuro CD40L con- 3 (Jak3) also has been reported recently to associate with the struct has also been described previously (19). CD40 cytoplasmic tail (CD40ct) (10). The TRAF family consists of six known family members, TRAFs 1–6 (1, 5–9, Abbreviations: TRAF, tumor necrosis factor receptor-associated fac- 11–15), which are capable of interacting with various TNF and tor; Jak, Janus kinase; CD40ct, CD40 cytoplasmic tail; SAPK, stress- non-TNF receptors. Overexpression of TRAFs 2, 5, and 6, but activated protein kinase; JNK, c-Jun N-terminal kinase; ICAM-1, intercellular adhesion molecule 1; IkB-a, inhibitor of NF-kBa; GST, The publication costs of this article were defrayed in part by page charge glutathione S-transferase; CD40L, CD40 ligand (gp39). ‡Present address: Cellegy Pharmaceuticals, Inc., San Carlos, CA payment. This article must therefore be hereby marked ‘‘advertisement’’ in 94070. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. e-mail: genhongc@ PNAS is available online at www.pnas.org. microbio.ucla.edu. 1421 Downloaded by guest on September 30, 2021 1422 Cell Biology: Lee et al. Proc. Natl. Acad. Sci. USA 96 (1999) Cell Culture and Transfection. 293T cells and BOSC pack- aging cells were maintained in DMEM (GIBCOyBRL) sup- plemented with 10% fetal bovine serum (FBS) and were transfected by standard calcium phosphate methods. A549 human lung carcinoma cells, WEHI 231 murine B cell lym- phoma cells, BI-141 murine T cell hybridoma cell lines, and Daudi human Burkitt’s lymphoma B cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillinystreptomycin. Transient transfections with reporter constructs were performed with the Superfect Transfection Reagent (Qiagen, Chatsworth, CA). Harvested samples were assessed for luciferase activity by measurement with Promega luminol substrate. Infections of the BI-141 and WEHI 231 cells FIG. 1. Characterization of the relative binding affinities of TRAFs 2, 3, 5, and 6 for the CD40 cytoplasmic tail and determination of the were performed by the use of BOSC packaging cells as minimal binding regions within the CD40 C17 region. (A) TRAFs 2 described previously (18, 22). For electroporation, Daudi cells y m and 3 bind to the CD40ct with much greater affinity than TRAFs 5 and were electroporated at 250 V 975 F and selected with 1 6. The same amount of glutathione beads bound with the GST-mouse mgyml of active G418 (Mediatech, Washington, DC). CD40ct fusion protein was incubated with extracts from 293T cells In Vitro Binding Assays, Electrophoretic Mobility-Shift transfected with pEBB-Flu TRAF2, pEBB-Flu TRAF3, pEBB-Flu Assays, and in Vitro Kinase SAPK Assays. GST pull-down TRAF5, or pEBB-Flu TRAF6. The beads were washed, and bound assays were performed as described previously (19). For proteins were detected by Western blot analysis with anti-Flu anti- gel-shift and SAPK assays, cells were treated as indicated in bodies (Fig. 1A Upper). Equal amounts of extracts (5% of input used the text. Nuclear extracts were prepared and used in binding for GST pull-down) were analyzed by Western blot analysis with anti-Flu antibodies to ensure similar levels of expression of the reactions with a g-P32-labeled major histocompatibility com- k different Flu-tagged TRAFs (Fig. 1A Lower). (B) The minimum plex II NF- B specific hairpin oligonucleotide (20) using binding regions of TRAF2 and TRAF3 within the C17 region are previously published protocols (23). Endogenous SAPK ac- different. Lysates from 293T cells expressing Flu-TRAF2 or Flu- tivity was assessed by immunoprecipitation with anti-c-Jun TRAF3 were incubated with GST, GST-C17, GST-C10N, or GST- N-terminal kinase 1(JNK1) polyclonal antibodies (Santa Cruz C10C fusion proteins immobilized on glutathione beads. The bound Biotechnology) using previously published protocols (24). proteins then were analyzed by Western blot analysis with anti-Flu Fluorescence-Activated Cell Sorter (FACS) Analysis. To antibodies. assess expression of the human CD40 constructs, BI-141 infectants and parental cells were stained with fluorescein (C17) corresponding to amino acid residues 230–246 of the isothiocyanate (FITC)-conjugated murine anti-human CD40 CD40ct that could bind to TRAF3 as strongly as the entire mAb (clone 5C3; PharMingen) and analyzed by FACScan flow CD40ct (19). This C17 peptide also was capable of binding to cytometer. To assess ICAM-1 induction, WEHI231 or Daudi TRAF2 as strongly as the full-length CD40ct (Fig. 1B). This cells were stimulated for 24 hr with 1 mgyml of G28.5 or with C17 fragment was dissected further into two overlapping soluble CD40L.
Load more

Recommended publications
  • Targeting Tgfβ Signal Transduction for Cancer Therapy
    Signal Transduction and Targeted Therapy www.nature.com/sigtrans REVIEW ARTICLE OPEN Targeting TGFβ signal transduction for cancer therapy Sijia Liu1, Jiang Ren1 and Peter ten Dijke1 Transforming growth factor-β (TGFβ) family members are structurally and functionally related cytokines that have diverse effects on the regulation of cell fate during embryonic development and in the maintenance of adult tissue homeostasis. Dysregulation of TGFβ family signaling can lead to a plethora of developmental disorders and diseases, including cancer, immune dysfunction, and fibrosis. In this review, we focus on TGFβ, a well-characterized family member that has a dichotomous role in cancer progression, acting in early stages as a tumor suppressor and in late stages as a tumor promoter. The functions of TGFβ are not limited to the regulation of proliferation, differentiation, apoptosis, epithelial–mesenchymal transition, and metastasis of cancer cells. Recent reports have related TGFβ to effects on cells that are present in the tumor microenvironment through the stimulation of extracellular matrix deposition, promotion of angiogenesis, and suppression of the anti-tumor immune reaction. The pro-oncogenic roles of TGFβ have attracted considerable attention because their intervention provides a therapeutic approach for cancer patients. However, the critical function of TGFβ in maintaining tissue homeostasis makes targeting TGFβ a challenge. Here, we review the pleiotropic functions of TGFβ in cancer initiation and progression, summarize the recent clinical advancements regarding TGFβ signaling interventions for cancer treatment, and discuss the remaining challenges and opportunities related to targeting this pathway. We provide a perspective on synergistic therapies that combine anti-TGFβ therapy with cytotoxic chemotherapy, targeted therapy, radiotherapy, or immunotherapy.
    [Show full text]
  • A TRAF2 Binding Independent Region of TNFR2 Is Responsible for TRAF2 Depletion and Enhancement of Cytotoxicity Driven by TNFR1
    www.impactjournals.com/oncotarget/ Oncotarget, January, Vol. 5, No. 1 A TRAF2 binding independent region of TNFR2 is responsible for TRAF2 depletion and enhancement of cytotoxicity driven by TNFR1 Lucía Cabal-Hierro1,3, Noelia Artime1, Julián Iglesias1, Miguel A. Prado1,4, Lorea Ugarte-Gil1, Pedro Casado1,5, Belén Fernández-García1, Bryant G. Darnay2 and Pedro S. Lazo1. 1 Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, Oviedo, Spain. 2 University of Texas. MD Anderson Cancer Center. Houston, Texas, USA. 3 Present address: Abramson Cancer Center. Perelman School of Medicine, University of Pennsylvania Philadelphia, PA. USA. 4 Present address: Department of Cell Biology. Harvard Medical School. Boston, MA. USA. 5 Present address: Institute of Cancer, Barts Cancer Institute. Queen Mary University of London. London, UK Correspondence to: Pedro S. Lazo, email:[email protected] Keywords: TNF receptors; Death Receptors; TRAF2; Apoptosis; NF-kappaB Received: October 11, 2013 Accepted: November 27, 2013 Published: November 29, 2013 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT: Tumor Necrosis Factor (TNF) interacts with two receptors known as TNFR1 and TNFR2. TNFR1 activation may result in either cell proliferation or cell death. TNFR2 activates Nuclear Factor-kappaB (NF-kB) and c-Jun N-terminal kinase (JNK) which lead to transcriptional activation of genes related to cell proliferation and survival. This depends on the binding of TNF Receptor Associated Factor 2 (TRAF2) to the receptor.
    [Show full text]
  • Expression of the Tumor Necrosis Factor Receptor-Associated Factors
    Expression of the Tumor Necrosis Factor Receptor- Associated Factors (TRAFs) 1 and 2 is a Characteristic Feature of Hodgkin and Reed-Sternberg Cells Keith F. Izban, M.D., Melek Ergin, M.D, Robert L. Martinez, B.A., HT(ASCP), Serhan Alkan, M.D. Department of Pathology, Loyola University Medical Center, Maywood, Illinois the HD cell lines. Although KMH2 showed weak Tumor necrosis factor receptor–associated factors expression, the remaining HD cell lines also lacked (TRAFs) are a recently established group of proteins TRAF5 protein. These data demonstrate that consti- involved in the intracellular signal transduction of tutive expression of TRAF1 and TRAF2 is a charac- several members of the tumor necrosis factor recep- teristic feature of HRS cells from both patient and tor (TNFR) superfamily. Recently, specific members cell line specimens. Furthermore, with the excep- of the TRAF family have been implicated in promot- tion of TRAF1 expression, HRS cells from the three ing cell survival as well as activation of the tran- HD cell lines showed similar TRAF protein expres- ␬ scription factor NF- B. We investigated the consti- sion patterns. Overall, these findings demonstrate tutive expression of TRAF1 and TRAF2 in Hodgkin the expression of several TRAF proteins in HD. Sig- and Reed–Sternberg (HRS) cells from archived nificantly, the altered regulation of selective TRAF paraffin-embedded tissues obtained from 21 pa- proteins may reflect HRS cell response to stimula- tients diagnosed with classical Hodgkin’s disease tion from the microenvironment and potentially (HD). In a selective portion of cases, examination of contribute both to apoptosis resistance and cell HRS cells for Epstein-Barr virus (EBV)–encoded maintenance of HRS cells.
    [Show full text]
  • TRAF5, a Novel Tumor Necrosis Factor Receptor-Associated Factor Family
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9437-9442, September 1996 Biochemistry TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling (signal transduction/protein-protein interaction/yeast two-hybrid system) TAKAoMI ISHIDA*, TADASHI ToJo*, TSUTOMU AOKI*, NORIHIKO KOBAYASHI*, TSUKASA OHISHI*, TOSHIKI WATANABEt, TADASHI YAMAMOTO*, AND JUN-ICHIRO INOUE*t Departments of *Oncology and tPathology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by David Baltimore, Massachusetts Institute of Technology, Cambridge, MA, May 22, 1996 (received for review March 8, 1996) ABSTRACT Signals emanating from CD40 play crucial called a death domain, suggesting that these receptors could roles in B-cell function. To identify molecules that transduce have either common or similar signaling mechanisms (13). CD40 signalings, we have used the yeast two-hybrid system to Biochemical purification of receptor-associated proteins or the clone cDNAs encoding proteins that bind the cytoplasmic tail recently developed cDNA cloning system that uses yeast of CD40. A cDNA encoding a putative signal transducer genetic selection led to the discovery of two groups of signal protein, designated TRAF5, has been molecularly cloned. transducer molecules. Members of the first group are proteins TRAF5 has a tumor necrosis factor receptor-associated factor with a TRAF domain for TNFR2 and CD40 such as TRAF1, (TRAF) domain in its carboxyl terminus and is most homol- TRAF2 (17), and TRAF3, also known as CD40bp, LAP-1, or ogous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, CRAF1 or CD40 receptor-associated factor (18-20).
    [Show full text]
  • Differential Gene Expression in Oligodendrocyte Progenitor Cells, Oligodendrocytes and Type II Astrocytes
    Tohoku J. Exp. Med., 2011,Differential 223, 161-176 Gene Expression in OPCs, Oligodendrocytes and Type II Astrocytes 161 Differential Gene Expression in Oligodendrocyte Progenitor Cells, Oligodendrocytes and Type II Astrocytes Jian-Guo Hu,1,2,* Yan-Xia Wang,3,* Jian-Sheng Zhou,2 Chang-Jie Chen,4 Feng-Chao Wang,1 Xing-Wu Li1 and He-Zuo Lü1,2 1Department of Clinical Laboratory Science, The First Affiliated Hospital of Bengbu Medical College, Bengbu, P.R. China 2Anhui Key Laboratory of Tissue Transplantation, Bengbu Medical College, Bengbu, P.R. China 3Department of Neurobiology, Shanghai Jiaotong University School of Medicine, Shanghai, P.R. China 4Department of Laboratory Medicine, Bengbu Medical College, Bengbu, P.R. China Oligodendrocyte precursor cells (OPCs) are bipotential progenitor cells that can differentiate into myelin-forming oligodendrocytes or functionally undetermined type II astrocytes. Transplantation of OPCs is an attractive therapy for demyelinating diseases. However, due to their bipotential differentiation potential, the majority of OPCs differentiate into astrocytes at transplanted sites. It is therefore important to understand the molecular mechanisms that regulate the transition from OPCs to oligodendrocytes or astrocytes. In this study, we isolated OPCs from the spinal cords of rat embryos (16 days old) and induced them to differentiate into oligodendrocytes or type II astrocytes in the absence or presence of 10% fetal bovine serum, respectively. RNAs were extracted from each cell population and hybridized to GeneChip with 28,700 rat genes. Using the criterion of fold change > 4 in the expression level, we identified 83 genes that were up-regulated and 89 genes that were down-regulated in oligodendrocytes, and 92 genes that were up-regulated and 86 that were down-regulated in type II astrocytes compared with OPCs.
    [Show full text]
  • Nuclear TRAF3 Is a Negative Regulator of CREB in B Cells
    Nuclear TRAF3 is a negative regulator of CREB in B cells Nurbek Mambetsarieva,b,c,1, Wai W. Linb,1, Laura L. Stunza,d, Brett M. Hansona, Joanne M. Hildebranda,2, and Gail A. Bishopa,b,d,e,f,3 aDepartment of Microbiology, University of Iowa, Iowa City, IA 52242; bImmunology Graduate Program, University of Iowa, Iowa City, IA 52242; cMedical Scientist Training Program, University of Iowa, Iowa City, IA 52242; dHolden Comprehensive Cancer Center, University of Iowa, Iowa City, IA 52242; eInternal Medicine, University of Iowa, Iowa City, IA 52242; and fDepartment of Veterans Affairs Medical Center, Research (151), Iowa City, IA 52246 Edited by Louis M. Staudt, National Cancer Institute, NIH, Bethesda, MD, and approved December 14, 2015 (received for review July 23, 2015) The adaptor protein TNF receptor-associated factor 3 (TRAF3) regu- deficient T cells and macrophages lack the enhanced survival phe- lates signaling through B-lymphocyte receptors, including CD40, notype, although they display constitutive NF-κB2 activation (12, BAFF receptor, and Toll-like receptors, and also plays a critical role 13). TRAF3 degradation is neither necessary nor sufficient for B-cell inhibiting B-cell homoeostatic survival. Consistent withthesefindings, NF-κB2 activation (14). These findings indicate that TRAF3 regu- loss-of-function human TRAF3 mutations are common in B-cell cancers, lates additional important prosurvival pathways in B cells. particularly multiple myeloma and B-cell lymphoma. B cells of B-cell– Nuclear localization of TRAF3 has been reported in several specific TRAF3−/− mice (B-Traf3−/−) display remarkably enhanced sur- nonhematopoietic cell types (15, 16), but the function of TRAF3 vival compared with littermate control (WT) B cells.
    [Show full text]
  • Crystallographic Analysis of CD40 Recognition and Signaling by Human TRAF2
    Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8408–8413, July 1999 Biochemistry Crystallographic analysis of CD40 recognition and signaling by human TRAF2 SARAH M. MCWHIRTER*, STEVEN S. PULLEN†,JAMES M. HOLTON*, JAMES J. CRUTE†,MARILYN R. KEHRY†, AND TOM ALBER*‡ *Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3206, and †Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877-0368 Communicated by Peter S. Kim, Massachusetts Institute of Technology, Cambridge, MA, May 26, 1999 (received for review April 25, 1999) ABSTRACT Tumor necrosis factor receptor superfamily proteins (8, 9). The biological selectivity of signaling also relies members convey signals that promote diverse cellular re- on the oligomerization specificity and receptor affinity of the sponses. Receptor trimerization by extracellular ligands ini- TRAFs. tiates signaling by recruiting members of the tumor necrosis The TNF receptor superfamily member, CD40, mediates factor receptor-associated factor (TRAF) family of adapter diverse responses. In antigen-presenting cells that constitu- proteins to the receptor cytoplasmic domains. We report the tively express CD40, it plays a critical role in T cell-dependent 2.4-Å crystal structure of a 22-kDa, receptor-binding fragment immune responses (10). TRAF1, TRAF2, TRAF3, and of TRAF2 complexed with a functionally defined peptide from TRAF6 binding sites in the 62-aa, CD40 cytoplasmic domain the cytoplasmic domain of the CD40 receptor. TRAF2 forms have been mapped (7, 11). TRAF1, TRAF2, and TRAF3 bind a mushroom-shaped trimer consisting of a coiled coil and a to the same sequence, 250PVQET, and TRAF6 binds to the unique ␤-sandwich domain. Both domains mediate trimer- membrane-proximal sequence, 231QEPQEINF.
    [Show full text]
  • Cell Type-Specific Function of TRAF2 and TRAF3 in Regulating Type I IFN
    Xie et al. Cell Biosci (2019) 9:5 https://doi.org/10.1186/s13578-018-0268-5 Cell & Bioscience RESEARCH Open Access Cell type‑specifc function of TRAF2 and TRAF3 in regulating type I IFN induction Xiaoping Xie1, Jin Jin2, Lele Zhu1, Zuliang Jie1, Yanchuan Li1, Baoyu Zhao3, Xuhong Cheng1, Pingwei Li3 and Shao‑Cong Sun1,4* Abstract Background: TRAF3 is known as a central mediator of type I interferon (IFN) induction by various pattern recognition receptors, but the in vivo function of TRAF3 in host defense against viral infection is poorly defned due to the lack of a viable mouse model. Results: Here we show that mice carrying conditional deletion of TRAF3 in myeloid cells or dendritic cells do not have a signifcant defect in host defense against vesicular stomatitis virus (VSV) infection. However, whole-body inducible deletion of TRAF3 renders mice more sensitive to VSV infection. Consistently, TRAF3 was essential for type I IFN induction in mouse embryonic fbroblasts (MEFs) but not in macrophages. In dendritic cells, TRAF3 was required for type I IFN induction by TLR ligands but not by viruses. We further show that the IFN-regulating function is not unique to TRAF3, since TRAF2 is an essential mediator of type I IFN induction in several cell types, including mac‑ rophages, DCs, and MEFs. Conclusions: These fndings suggest that both TRAF2 and TRAF3 play a crucial role in type I IFN induction, but their functions are cell type- and stimulus-specifc. Keywords: TRAF2, TRAF3, Type I interferon, Antiviral immunity Background IPS-1) serves as an adaptor of RLRs, whereas stimulator Type I interferon (IFN) plays a crucial role in innate of interferon gene (STING) mediates type I IFN induc- immunity against viral infections [1, 2].
    [Show full text]
  • Lipid Rafts Are Important for the Association of RANK and TRAF6
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 35, No. 4, 279-284, August 2003 Lipid rafts are important for the association of RANK and TRAF6 Hyunil Ha1,3, Han Bok Kwak1,2,3, Introduction Soo Woong Lee1,2,3, Hong-Hee Kim2,4, 1,2,3,5 Osteoclasts are multinucleated giant cells responsible and Zang Hee Lee for bone resorption. These cells are differentiated from 1 hematopoietic myeloid precursors of the monocyte/ National Research Laboratory for Bone Metabolism macrophage lineage (Suda et al., 1992). For the dif- 2Research Center for Proteineous Materials 3 ferentiation of osteoclast precursors into mature osteo- School of Dentistry clasts, a cell-to-cell interaction between osteoclast Chosun University, Gwangju 501-759, Korea 4 precursors and osteoblasts/stromal cells are required Department of Cell and Developmental Biology (Udagawa et al., 1990). Recently, many studies have College of Dentistry, Seoul National University provided ample evidences that the TNF family mem- Seoul 110-749, Korea κ 5 ber RANKL (receptor activator of NF- B ligand; also Corresponding author: Tel, 82-62-230-6872; known as ODF, OPGL, and TRANCE) is expressed Fax, 82-62-227-6589; E-mail, [email protected] on the surface of osteoblasts/stromal cells and es- sential for osteoclast differentiation (Anderson et al., Accepted 19 June 2003 1997; Yasuda et al., 1998; Takahashi et al., 1999). When its receptor RANK was stimulated by RANKL, Abbreviations: MAPK, mitogen-activated protein kinase; MCD, several TNF receptor-associated factors (TRAFs), methyl-β-cyclodextrin; RANK, receptor activator of NF-κB; TLR, especially TRAF6, can be directly recruited into RANK Toll-like receptor; TNFR, TNF receptor; TRAF, TNF receptor- cytoplasmic domains and may trigger downstream associated factor signaling molecules for the activation of NF-κB and mitogen activated protein kinases (MAPKs) (Darnay et al., 1998; Wong et al., 1998; Kim et al., 1999).
    [Show full text]
  • Human TRAF-2 Antibody Antigen Affinity-Purified Polyclonal Goat Igg Catalog Number: AF3277
    Human TRAF-2 Antibody Antigen Affinity-purified Polyclonal Goat IgG Catalog Number: AF3277 DESCRIPTION Species Reactivity Human Specificity Detects human TRAF­2 in Western blots. In Western blots, less than 1% cross­reacivity with recombinant human TRAF­1, ­3, ­4, ­5, or ­6 is observed. Source Polyclonal Goat IgG Purification Antigen Affinity­purified Immunogen E. coli­derived recombinant human TRAF­2 Met1­Leu501 Accession # Q12933 Formulation Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. See Certificate of Analysis for details. *Small pack size (­SP) is supplied either lyophilized or as a 0.2 μm filtered solution in PBS. APPLICATIONS Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website. Recommended Sample Concentration Western Blot 0.5 µg/mL See Below Knockout Validated TRAF­2 is specifically detected in HEK293T human embryonic kidney parental cell line but is not detectable in TRAF­2 knockout HEK293T cell line. DATA Western Blot Knockout Validated Detection of Human TRAF­2 by Western Western Blot Shows Human TRAF­2 Blot. Western blot shows lysates of Raji human Specificity by Using Knockout Cell Line. Burkitt's lymphoma cell line and HeLa human Western blot shows lysates of HEK293T cervical epithelial carcinoma cell line. PVDF human embryonic kidney parental cell line and membrane was probed with 0.5 µg/mL Goat Anti­ TRAF­2 knockout HEK293T cell line (KO). Human TRAF­2 Antigen Affinity­purified PVDF membrane was probed with 0.5 µg/mL Polyclonal Antibody (Catalog # AF3277) followed of Goat Anti­Human TRAF­2 Antigen Affinity­ by HRP­conjugated Anti­Goat IgG Secondary purified Polyclonal Antibody (Catalog # Antibody (Catalog # HAF017).
    [Show full text]
  • TRAF6, a Molecular Bridge Spanning Adaptive Immunity, Innate Immunity and Osteoimmunology Hao Wu1* and Joseph R
    Review articles TRAF6, a molecular bridge spanning adaptive immunity, innate immunity and osteoimmunology Hao Wu1* and Joseph R. Arron2 Summary receptor/Toll-like receptor (IL-1R/TLR) superfamily. Gene Tumor necrosis factor (TNF) receptor associated factor targeting experiments have identified several indispen- 6 (TRAF6) is a crucial signaling molecule regulating a sable physiological functions of TRAF6, and structural diverse array of physiological processes, including and biochemical studies have revealed the potential adaptive immunity, innate immunity, bone metabolism mechanisms of its action. By virtue of its many signaling and the development of several tissues including lymph roles, TRAF6 represents an important target in the regu- nodes, mammary glands, skin and the central nervous lation of many disease processes, including immunity, system. It is a member of a group of six closely related inflammation and osteoporosis. BioEssays 25:1096– TRAF proteins, which serve as adapter molecules, coupl- 1105, 2003. ß 2003 Wiley Periodicals, Inc. ing the TNF receptor (TNFR) superfamily to intracellular signaling events. Among the TRAF proteins, TRAF6 is unique in that, in addition to mediating TNFR family Introduction signaling, it is also essential for signaling downstream of The tumor necrosis factor (TNF) receptor associated factors an unrelated family of receptors, the interleukin-1 (IL-1) (TRAFs) were first identified as two intracellular proteins, TRAF1 and TRAF2, associated with TNF-R2,(1) a member of 1Department of Biochemistry, Weill Medical College of Cornell the TNF receptor (TNFR) superfamily. There are currently six University, New York. mammalian TRAFs (TRAF1-6), which have emerged as 2Tri-Institutional MD-PhD Program, Weill Medical College of Cornell important proximal signal transducers for the TNFR super- University, New York.
    [Show full text]
  • Molecular Signatures Differentiate Immune States in Type 1 Diabetes Families
    Page 1 of 65 Diabetes Molecular signatures differentiate immune states in Type 1 diabetes families Yi-Guang Chen1, Susanne M. Cabrera1, Shuang Jia1, Mary L. Kaldunski1, Joanna Kramer1, Sami Cheong2, Rhonda Geoffrey1, Mark F. Roethle1, Jeffrey E. Woodliff3, Carla J. Greenbaum4, Xujing Wang5, and Martin J. Hessner1 1The Max McGee National Research Center for Juvenile Diabetes, Children's Research Institute of Children's Hospital of Wisconsin, and Department of Pediatrics at the Medical College of Wisconsin Milwaukee, WI 53226, USA. 2The Department of Mathematical Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA. 3Flow Cytometry & Cell Separation Facility, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA. 4Diabetes Research Program, Benaroya Research Institute, Seattle, WA, 98101, USA. 5Systems Biology Center, the National Heart, Lung, and Blood Institute, the National Institutes of Health, Bethesda, MD 20824, USA. Corresponding author: Martin J. Hessner, Ph.D., The Department of Pediatrics, The Medical College of Wisconsin, Milwaukee, WI 53226, USA Tel: 011-1-414-955-4496; Fax: 011-1-414-955-6663; E-mail: [email protected]. Running title: Innate Inflammation in T1D Families Word count: 3999 Number of Tables: 1 Number of Figures: 7 1 For Peer Review Only Diabetes Publish Ahead of Print, published online April 23, 2014 Diabetes Page 2 of 65 ABSTRACT Mechanisms associated with Type 1 diabetes (T1D) development remain incompletely defined. Employing a sensitive array-based bioassay where patient plasma is used to induce transcriptional responses in healthy leukocytes, we previously reported disease-specific, partially IL-1 dependent, signatures associated with pre and recent onset (RO) T1D relative to unrelated healthy controls (uHC).
    [Show full text]