Free Access
J Innate Immun 2011;3:437–446

Regulation of Lipopolysaccharide-Induced Translation of Tumor Necrosis Factor-Alpha by the Toll-Like Receptor 4 Adaptor Protein TRAM

Wang L.a · Trebicka E.a · Fu Y.a · Waggoner L.d · Akira S.e · Fitzgerald K.A.d · Kagan J.C.b, c · Cherayil B.J.a, b
aMucosal Immunology Laboratory, Division of Pediatric Gastroenterology, Massachusetts General Hospital, Charlestown, Mass., bDepartment of Pediatrics, Harvard Medical School, and cDivision of Gastroenterology, Children’s Hospital, Boston, Mass., dDivision of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Mass., USA; eLaboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan
email Corresponding Author

 goto top of outline Key Words

  • Macrophage
  • Lipopolysaccharide
  • Toll-like receptor
  • Inflammation
  • Signal transduction

 goto top of outline Abstract

Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-α requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair – Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) – to TNF-α expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-α protein expression even at times when TNF-α mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-α 3′ untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-α translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-α 3′ untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-α protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-α protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-α translation.

Copyright © 2011 S. Karger AG, Basel

 goto top of outline References
  1. Lembo A, Kalis C, Kirschning CJ, Mitolo V, Jirillo E, Wagner H, Galanos C, Freudenberg MA: Differential contribution of Toll-like receptors 4 and 2 to the cytokine response to Salmonella enterica serovar Typhimurium and Staphylococcus aureus in mice. Infect Immun 2003;71:6058–6062.
  2. Li Q, Cherayil BJ: Role of Toll-like receptor 4 in macrophage activation and tolerance during Salmonella enterica serovar Typhimurium infection. Infect Immun 2003;71:4873–4882.
  3. Royle M, Totemeyer S, Aldridge LC, Maskell DJ, Bryant CE: Stimulation of Toll-like receptor 4 by lipopolysaccharide during cellular invasion by live Salmonella typhimurium is a critical but not exclusive event leading to macrophage responses. J Immunol 2003;170:5445–5454.
  4. Weiss DS, Raupach B, Takeda K, Akira S, Zychlinsky A: Toll-like receptors are temporally involved in host defense. J Immunol 2004;172:4463–4469.
  5. Kawai T, Akira S: The role of pattern recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010;11:373–384.
  6. Sheedy FJ, O’Neill LA: The troll in Toll: Mal and TRAM as bridges for TLR2 and TLR signaling. J Leukoc Biol 2007;82:196–203.
  7. Oshiumi H, Sasai M, Shida K, Fujita T, Matsumoto M, Seya T: TIR-containing adaptor molecule (TICAM)-2, a bridging adaptor recruiting to Toll-like receptor 4 TICAM-1 that induces interferon β. J Biol Chem 2003;278:49751–49762.
  8. McGettrick AF, Brint EK, Palsson-McDermott EM, Rowe DC, Golenbock DT, Gay NJ, Fitzgerald KA, O’Neill LA: TRIF-related adaptor molecule is phosphorylated by PKC(ε) during Toll-like receptor 4 signaling. Proc Natl Acad Sci USA 2006;103:9196–9201.
  9. Rowe DC, McGettrick AF, Latz E, Monks BG, Gay NJ, Yamamoto M, Akira S, O’Neill LA, Fitzgerald KA, Golenbock DT: The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction. Proc Natl Acad Sci USA 2006;103:6299–6304.
  10. Kagan JC, Su T, Horng T, Chow A, Akira S, Medzhitov R: TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon β. Nat Immunol 2008;9:361–368.
  11. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S: Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 2003;301:640–643.
  12. Yamamoto M, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T, Takeuchi O, Takeda K, Akira S: TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol 2003;4:1144–1150.
  13. Anderson P: Post-transcriptional control of cytokine production. Nat Immunol 2008;9:353–359.
  14. Falvo JV, Tsytsykova AV, Goldfeld AE: Transcriptional control of the TNF gene. Curr Dir Autoimmun 2010;11:27–60.
  15. Takeuchi O, Akira S: Pattern recognition receptors and inflammation. Cell 2010;140:805–820.
  16. Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, Takeda K, Akira S: Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol 2002;169:6668–6672.
  17. Wang L, Johnson EE, Shi HN, Walker WA, Wessling-Resnick M, Cherayil BJ: Attenuated inflammatory responses in hemachromatosis reveal a role for iron in the regulation of macrophage cytokine translation. J Immunol 2008;181:2723–2731.
  18. Wang L, Harrington L, Trebicka E, Shi HN, Kagan JC, Hong CC, Lin HY, Babitt JL, Cherayil BJ: Selective modulation of TLR4-activated inflammatory responses by altered iron homeostasis. J Clin Invest 2009;119:3322–3328.
  19. Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC: The C282Y mutation causing hereditary hemochromatosis does not produce a null allele. Blood 1999;194:9–11.
  20. Pietrangelo A: Hereditary hemochromatosis: pathogenesis, diagnosis and treatment. Gastroenterology 2010;139:393–408.
  21. Zhou XY, Tomatsu S, Flemin RE, Parkkila S, Waheed A, Jiang J, Fei Y, Brunt EM, Ruddy DA, Prass CE, Schatzman RC, O’Neill R, Britton RS, Bacon BR, Sly WS: HFE gene knock-out produces a mouse model of hereditary hemochromatosis. Proc Natl Acad Sci USA 1998;95:2492–2497.
  22. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, Kim SO, Goode J, Mann N, Mudd S, Crozat K, Sovath S, Han J, Beutler B: Identification of Lps2 as a key transducer of MyD88-independent TLR signalling. Nature 2003;424:743–748.
  23. Vasudevan S, Steitz JA: AU-rich element-mediated upregulation of translation by FXR1 and Argonaute 2. Cell 2007;128:1105–1118.
  24. Carpenter S, O’Neill LA: Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signalling proteins. Biochem J 2009;422:1–10.
  25. Ronkina N, Menon MB, Schwermann J, Tiedje C, Hitti E, Kotlyarov A, Gaestel M: MAPKAP kinases MK2 and MK3 in inflammation: complex regulation of TNF biosynthesis via expression and phosphorylation of tristetraprolin. Biochem Pharmacol 2010;80:1915–1920.
  26. Han J, Brown T, Beutler B: Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level. J Exp Med 1990;171:465–475.
  27. Hel Z, Di Marco S, Radzioch D: Characterization of the RNA binding proteins forming complexes with a novel putative regulatory region in the 3′ UTR of TNFα mRNA. Nucl Acids Res 1998;26:2803–2812.
  28. Covert MW, Leung TH, Gaston JE, Baltimore D: Achieving stability of lipopolysaccharide-induced NF-ĸB activation. Science 2005;309:1854–1857.
  29. Gais P, Tiedje C, Altmayr F, Gaestel M, Weighardt H, Holzmann B: TRIF signaling stimulates translation of TNFα mRNA via prolonged activation of MK2. J Immunol 2010;184:5842–5848.
  30. Neininger A, Kontoyiannis D, Kotlyarov A, Winzen R, Ecker R, Volk HD, Holtmann H, Kollias G, Gaestel M: MK2 targets AU-rich elements and regulates TNF and IL-6 independently at different post-transcriptional levels. J Biol Chem 2002;277:3065–3068.
  31. Jiang Z, Georgel P, Du X, Shamel L, Sovath S, Mudd S, Huber M, Kalis C, Keck S, Galanos C, Freudenberg M, Beutler B: CD14 is required for MyD88-independent LPS signaling. Nat Immunol 2005;6:565–570.
  32. Tseng PH, Matsuzawa A, Zhang W, Mino T, Vignali DAA, Karin M: Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines. Nat Immunol 2010;11:70–75.

 goto top of outline Author Contacts

Dr. Bobby J. Cherayil
Pediatric Gastroenterology Unit, Massachusetts General Hospital
Building 114, 16th Street
Charlestown, MA 02129 (USA)
Tel. +1 617 726 4170, E-Mail

 goto top of outline Article Information

Received: November 19, 2010
Accepted after revision: February 3, 2011
Published online: April 14, 2011
Number of Print Pages : 10
Number of Figures : 8, Number of Tables : 1, Number of References : 32

 goto top of outline Publication Details

Journal of Innate Immunity

Vol. 3, No. 5, Year 2011 (Cover Date: August 2011)

Journal Editor: Herwald H. (Lund), Egesten A. (Lund)
ISSN: 1662-811X (Print), eISSN: 1662-8128 (Online)

For additional information:

Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.