Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway

Nature Immunology volume 4, pages 1144–1150 (2003)Cite this article

Abstract

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll–interleukin 1 receptor (TIR) domain–containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain–containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-β production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cloning and characterization of human TRAM and targeted disruption of the gene encoding mouse TRAM.
Figure 2: IL-1-induced responses in TRAM-deficient cells.
Figure 3: Impaired TLR4-mediated cytokine production and B cell activation in TRAM-deficient mice.
Figure 4: Defects in TLR4-mediated MyD88-independent responses in TRAM-deficient mice.
Figure 5: TRAM-deficient cells lack TLR4-mediated MyD88-independent signal transduction.
Figure 6: Participation of TIR domain–containing adaptor molecules in TLR3 and TLR4 signaling pathways.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376 (2003).

    Article  CAS  Google Scholar 

  2. Janeway, C.A. Jr. & Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002).

    Article  CAS  Google Scholar 

  3. Takeuchi, O. et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169, 10–14 (2002).

    Article  CAS  Google Scholar 

  4. Alexopoulou, L. et al. Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat. Med. 8, 878–884 (2002).

    Article  CAS  Google Scholar 

  5. Takeuchi, O. et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11, 443–51 (1999).

    Article  CAS  Google Scholar 

  6. Alexopoulou, L., Holt, A.C., Medzhitov, R. & Flavell, R.A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

    Article  CAS  Google Scholar 

  7. Hoshino, K. et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749–3752 (1999).

    CAS  PubMed  Google Scholar 

  8. Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

    Article  CAS  Google Scholar 

  9. Hayashi, F. et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103 (2001).

    Article  CAS  Google Scholar 

  10. Takeuchi, O. et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13, 933–940 (2001).

    Article  CAS  Google Scholar 

  11. Hemmi, H. et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196–200 (2002).

    Article  CAS  Google Scholar 

  12. Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

    Article  CAS  Google Scholar 

  13. Wesche, H., Henzel, W.J., Shillinglaw, W., Li, S. & Cao, Z. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7, 837–847 (1997).

    Article  CAS  Google Scholar 

  14. Janssens, S. & Beyaert, R. Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members. Mol. Cell. 11, 293–302 (2003).

    Article  CAS  Google Scholar 

  15. Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998).

    Article  CAS  Google Scholar 

  16. Kawai, T., Adachi, O., Ogawa, T., Takeda, K. & Akira, S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11, 115–122 (1999).

    Article  CAS  Google Scholar 

  17. Kawai, T. et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J. Immunol. 167, 5887–5894 (2001).

    Article  CAS  Google Scholar 

  18. Kaisho, T., Takeuchi, O., Kawai, T., Hoshino, K. & Akira, S. Endotoxin-induced maturation of MyD88-deficient dendritic cells. J. Immunol. 166, 5688–5694 (2001).

    Article  CAS  Google Scholar 

  19. Horng, T., Barton, G.M. & Medzhitov, R. TIRAP: an adapter molecule in the Toll signaling pathway. Nat. Immunol. 2, 835–841 (2001).

    Article  CAS  Google Scholar 

  20. Fitzgerald, K.A. et al. Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction. Nature 413, 78–83 (2001).

    Article  CAS  Google Scholar 

  21. Yamamoto, M. et al. Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420, 324–329 (2002).

    Article  CAS  Google Scholar 

  22. Horng, T., Barton, G.M., Flavell, R.A. & Medzhitov, R. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420, 329–333 (2002).

    Article  CAS  Google Scholar 

  23. Yamamoto, M. et al. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J. Immunol. 169, 6668–6672 (2002).

    Article  CAS  Google Scholar 

  24. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T. & Seya, T. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-β induction. Nat. Immunol. 4, 161–167 (2003).

    Article  CAS  Google Scholar 

  25. Yamamoto, M. et al. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301, 640–643 (2003).

    Article  CAS  Google Scholar 

  26. Hoebe, K. et al. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424, 743–748 (2003).

    Article  CAS  Google Scholar 

  27. Sharma, S. et al. Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151 (2003).

    Article  CAS  Google Scholar 

  28. Fitzgerald, K.A. et al. IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496 (2003).

    Article  CAS  Google Scholar 

  29. Mink, M., Fogelgren, B., Olszewski, K., Maroy, P. & Csiszar, K. A novel human gene (SARM) at chromosome 17q11 encodes a protein with a SAM motif and structural similarity to Armadillo/β-catenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans. Genomics 74, 234–244 (2001).

    Article  CAS  Google Scholar 

  30. O'Neill, L.A., Fitzgerald, K.A. & Bowie, A.G. The Toll-IL-1 receptor adaptor family grows to five members. Trends. Immunol. 24, 286–290 (2003).

    Article  Google Scholar 

  31. Bin, L.H., Xu, L.G. & Shu, H.B. TIRP, a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling. J. Biol. Chem. 278, 24526–24532 (2003).

    Article  CAS  Google Scholar 

  32. Kopydlowski, K.M. et al. Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo. J. Immunol. 163, 1537–1544 (1999).

    CAS  PubMed  Google Scholar 

  33. Ohmori, Y. & Hamilton, T.A. Requirement for STAT-1 in LPS-induced gene expression in macrophages. J. Leukoc. Biol. 69, 598–604 (2001).

    CAS  PubMed  Google Scholar 

  34. Toshchakov, V. et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT-1α/β-dependent gene expression in macrophages. Nat. Immunol. 3, 392–398 (2002).

    Article  CAS  Google Scholar 

  35. Doyle, S. et al. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17, 251–263 (2002).

    Article  CAS  Google Scholar 

  36. Sakaguchi, S. et al. Essential role of IRF-3 in lipopolysaccharide-induced interferon-beta gene expression and endotoxin shock. Biochem. Biophys. Res. Commun. 306, 860–866 (2003).

    Article  CAS  Google Scholar 

  37. Hacker, H. et al. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192, 595–600 (2000).

    Article  CAS  Google Scholar 

  38. Schnare, M., Holt, A.C., Takeda, K., Akira, S. & Medzhitov, R. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr. Biol. 10, 1139–1142 (2000).

    Article  CAS  Google Scholar 

  39. Vasilakos, J.P. et al. Adjuvant activities of immune response modifier R-848: comparison with CpG ODN. Cell Immunol. 204, 64–74 (2000).

    Article  CAS  Google Scholar 

  40. Grosshans, J., Schnorrer, F. & Nusslein-Volhard, C. Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal. Mech. Dev. 81, 127–38 (1999).

    Article  CAS  Google Scholar 

  41. Jiang, Z. et al. Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex. J. Biol. Chem. 278, 10952–10956 (2003).

    Article  CAS  Google Scholar 

  42. Yu, K.Y. et al. Cutting edge: mouse pellino-2 modulates IL-1 and lipopolysaccharide signaling. J. Immunol. 169, 4075–4078 (2002).

    Article  CAS  Google Scholar 

  43. Jensen, L.E. & Whitehead, A.S. Pellino2 activates the mitogen activated protein kinase pathway. FEBS Lett. 545, 199–202 (2003).

    Article  CAS  Google Scholar 

  44. Jensen, L.E. & Whitehead, A.S. Pellino3, a novel member of the Pellino protein family, promotes activation of c-Jun and Elk-1 and may act as a scaffolding protein. J. Immunol. 171, 1500–1506 (2003).

    Article  CAS  Google Scholar 

  45. Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction. Immunity 13, 539–548 (2000).

    Article  CAS  Google Scholar 

  46. Sato, S. et al. A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways. Int. Immunol. 14, 783–791 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Golenbock and H. Tomizawa for providing the NF-κB reporter plasmid and R-848, respectively. We also thank T. Kawai, H. Sanjo and H. Kuwata for discussions; M. Hashimoto for secretarial assistance; N. Okita and N. Iwami for technical assistance; and P. Lee for critical reading of the manuscript. Supported by grants from Special Coordination Funds; the Ministry of Education, Culture, Sports, Science and Technology; Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists; The Uehara Memorial Foundation; The Naito Foundation; and The Junior Research Associate from RIKEN.

Author information

Authors and Affiliations

  1. Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, 565-0871, Osaka, Japan

    Masahiro Yamamoto, Hiroaki Hemmi, Satoshi Uematsu, Tsuneyasu Kaisho, Osamu Takeuchi, Kiyoshi Takeda & Shizuo Akira

  2. ERATO, Japan Science and Technology Corporation, 3-1 Yamada-oka, Suita, 565-0871, Osaka, Japan

    Shintaro Sato, Kiyoshi Takeda & Shizuo Akira

  3. RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Kanagawa, Japan

    Katsuaki Hoshino & Tsuneyasu Kaisho

Authors
  1. Masahiro Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shintaro Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiroaki Hemmi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satoshi Uematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Katsuaki Hoshino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tsuneyasu Kaisho
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Osamu Takeuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kiyoshi Takeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shizuo Akira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shizuo Akira.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1.

Anti-mTRAM specifically recognized mouse TRAM and human TRAM 293 cells were transfected with empty (5 μg), mTRAM (1 μg), hTRAM (1 μg), and MyD88 (5 μg) vectors. The total amount of DNA (5 μg) was kept constant by adding the empty vector. Cell lysates were immunoprecipitated (IP) with anti-Myc (9B11, Cell signaling), and immunoblotted (IB) with anti-mTRAM or anti-Myc (9E10, Santa Cruz). (PDF 128 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yamamoto, M., Sato, S., Hemmi, H. et al. TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway. Nat Immunol 4, 1144–1150 (2003). https://doi.org/10.1038/ni986

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni986

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing