Smad6 negatively regulates interleukin 1-receptor–Toll-like receptor signaling through direct interaction with the adaptor Pellino-1


Transforming growth factor-β1 (TGF-β1) is a potent cytokine with pleiotropic effects, including anti-inflammatory activity. Here we show that the signaling protein Smad6 bound to Pellino-1, an adaptor protein of mammalian interleukin 1 receptor (IL-1R)–associated kinase 1 (IRAK1), and thereby promoted TGF-β-mediated anti-inflammatory effects. Smad6–Pellino-1 interaction abrogated signaling mediated by a complex of IRAK1, Pellino-1 and adaptor protein TRAF6 that formed after stimulation by IL-1β treatment. Blockade of IRAK1–Pellino-1–TRAF6 signaling prevented degradation of the inhibitor IκBα and subsequent nuclear translocation of transcription factor NF-κB and thus expression of proinflammatory genes. 'Knockdown' of endogenous Smad6 expression by RNA interference reduced anti-inflammatory activity mediated by TGF-β1 or the TGF-β family member BMP-4. Thus Smad6 is a critical mediator of the TGF-β–BMP pathway that mediates anti-inflammatory activity and negatively regulates IL-1R–Toll-like receptor signals.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Smad6 binds to Pellino-1.
Figure 2: Smad6 blocks IRAK1-mediated signaling complex formation induced by IL-1β treatment.
Figure 3: Smad6 inhibits NF-κB-mediated reporter gene induction by IL-1β or LPS.
Figure 4: Smad6 inhibits IκBα degradation and nuclear translocation of NF-κB.
Figure 5: Expression of Smad6 reduces the expression of proinflammatory genes induced by IL-1β treatment.
Figure 6: Inhibition of endogenous Smad6 reduces the anti-inflammatory effects of TGF-β1 or BMP-4.
Figure 7: Smad6 induction by TGF-β1 is required for the inhibition of proinflammatory gene expression.


  1. 1

    Roberts, A.B. & Sporn, M.B. Physiological actions and clinical applications of transforming growth factor-β (TGF-β). Growth Factors 8, 1–9 (1993).

    CAS  Article  Google Scholar 

  2. 2

    Whitman, M. Smads and early developmental signaling by the TGF-β superfamily. Genes Dev. 12, 2445–2462 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Blobe, G.C., Schiemann, W.P. & Lodish, H.F. Role of transforming growth factor-β in human disease. N. Engl. J. Med. 342, 1350–1358 (2000).

    CAS  Article  Google Scholar 

  4. 4

    de Caestecker, M.P., Piek, E. & Roberts, A.B. Role of transforming growth factor-β signaling in cancer. J. Natl. Cancer Inst. 92, 1388–1402 (2000).

    CAS  Article  Google Scholar 

  5. 5

    Hayashi, H. et al. The MAD-related protein Smad7 associates with the TGF-β receptor and functions as an antagonist of TGF-β signaling. Cell 89, 1165–1173 (1997).

    CAS  Article  Google Scholar 

  6. 6

    Imamura, T. et al. Smad6 inhibits signalling by the TGF-β superfamily. Nature 389, 622–626 (1997).

    CAS  Article  Google Scholar 

  7. 7

    Nakao, A. et al. Identification of Smad7, a TGF-β-inducible antagonist of TGF-β signalling. Nature 389, 631–635 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Monteleone, G. et al. Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease. J. Clin. Invest. 108, 601–609 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Dong, C. et al. Deficient Smad7 expression: a putative molecular defect in scleroderma. Proc. Natl. Acad. Sci. USA 99, 3908–3913 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Asano, Y., Ihn, H., Yamane, K., Kubo, M. & Tamaki, K. Impaired Smad7-Smurf–mediated negative regulation of TGF-β signaling in scleroderma fibroblasts. J. Clin. Invest. 113, 253–264 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Kavsak, P. et al. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF-β receptor for degradation. Mol. Cell 6, 1365–1375 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Ebisawa, T. et al. Smurf1 interacts with transforming growth factor-β type I receptor through Smad7 and induces receptor degradation. J. Biol. Chem. 276, 12477–12480 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Murakami, G., Watabe, T., Takaoka, K., Miyazono, K. & Imamura, T. Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads. Mol. Biol. Cell 14, 2809–2817 (2003).

    CAS  Article  Google Scholar 

  14. 14

    Ulloa, L., Doody, J. & Massague, J. Inhibition of transforming growth factor-β/SMAD signalling by the interferon-γ/STAT pathway. Nature 397, 710–713 (1999).

    CAS  Article  Google Scholar 

  15. 15

    Bitzer, M. et al. A mechanism of suppression of TGF-β/SMAD signaling by NF-κB/RelA. Genes Dev. 14, 187–197 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Topper, J.N. et al. Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium. Proc. Natl. Acad. Sci. USA 94, 9314–9319 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Bai, S., Shi, X., Yang, X. & Cao, X. Smad6 as a transcriptional corepressor. J. Biol. Chem. 275, 8267–8270 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Bai, S. & Cao, X. A nuclear antagonistic mechanism of inhibitory Smads in transforming growth factor-β signaling. J. Biol. Chem. 277, 4176–4182 (2002).

    CAS  Article  Google Scholar 

  19. 19

    Lin, X. et al. Smad6 recruits transcription corepressor CtBP to repress bone morphogenetic protein-induced transcription. Mol. Cell. Biol. 23, 9081–9093 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Grosshans, J., Schnorrer, F. & Nusslein-Volhard, C. Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal. Mech. Dev. 81, 127–138 (1999).

    CAS  Article  Google Scholar 

  21. 21

    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).

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

    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).

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

    Akira, S., Takeda, K. & Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Kimbrell, D.A. & Beutler, B. The evolution and genetics of innate immunity. Nat. Rev. Genet. 2, 256–267 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Dinarello, C.A. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int. Rev. Immunol. 16, 457–499 (1998).

    CAS  Article  Google Scholar 

  28. 28

    O'Neill, L. The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defense. Biochem. Soc. Trans. 28, 557–563 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Martin, M.U. & Wesche, H. Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family. Biochim. Biophys. Acta 1592, 265–280 (2002).

    CAS  Article  Google Scholar 

  30. 30

    Afrakhte, M. et al. Induction of inhibitory Smad6 and Smad7 mRNA by TGF-β family members. Biochem. Biophys. Res. Commun. 249, 505–511 (1998).

    CAS  Article  Google Scholar 

  31. 31

    Maric, I. et al. Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats. J. Cell. Physiol. 196, 258–264 (2003).

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Dinarello, C.A. Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147 (1996).

    CAS  PubMed  Google Scholar 

  34. 34

    Medzhitov, R. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145 (2001).

    CAS  Article  Google Scholar 

  35. 35

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

    CAS  Article  Google Scholar 

  36. 36

    O'Neill, L.A. & Dinarello, C.A. The IL-1 receptor/Toll-like receptor superfamily: crucial receptors for inflammation and host defense. Immunol. Today 21, 206–209 (2000).

    CAS  Article  Google Scholar 

  37. 37

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

    CAS  Article  Google Scholar 

  38. 38

    Lorenz, E., Mira, J.P., Frees, K.L. & Schwartz, D.A. Relevance of mutations in the TLR4 receptor in patients with Gram-negative septic shock. Arch. Intern. Med. 162, 1028–1032 (2002).

    CAS  Article  Google Scholar 

  39. 39

    Aliprantis, A.O. et al. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science 285, 736–739 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Hou, L., Sasaki, H. & Stashenko, P. Toll-like receptor 4-deficient mice have reduced bone destruction following mixed anaerobic infection. Infect. Immun. 68, 4681–4687 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Cook, D.N., Pisetsky, D.S. & Schwartz, D.A. Toll-like receptors in the pathogenesis of human disease. Nat. Immunol. 5, 975–979 (2004).

    CAS  Article  Google Scholar 

  42. 42

    McCartney-Francis, N., Jin, W. & Wahl, S.M. Aberrant Toll receptor expression and endotoxin hypersensitivity in mice lacking a functional TGF-β1 signaling pathway. J. Immunol. 172, 3814–3821 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Neumann, D., Kollewe, C., Martin, M.U. & Boraschi, D. The membrane form of the type II IL-1 receptor accounts for inhibitory function. J. Immunol. 165, 3350–3357 (2000).

    CAS  Article  Google Scholar 

  44. 44

    Brint, E.K. et al. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol. 5, 373–379 (2004).

    CAS  Article  Google Scholar 

  45. 45

    Wald, D. et al. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4, 920–927 (2003).

    CAS  Article  Google Scholar 

  46. 46

    Kobayashi, K. et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191–202 (2002).

    CAS  Article  Google Scholar 

  47. 47

    Burns, K. et al. Inhibition of interleukin 1 receptor/Toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4. J. Exp. Med. 197, 263–268 (2003).

    Article  Google Scholar 

  48. 48

    Kinjyo, I. et al. SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Immunity 17, 583–591 (2002).

    CAS  Article  Google Scholar 

  49. 49

    Naiki, Y. et al. Transforming growth factor-β differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling. J. Biol. Chem. 280, 5491–5495 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Kawai, T. et al. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11, 115–122 (1999).

    CAS  Article  Google Scholar 

  51. 51

    Ju, E.M. et al. Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-β1 depends on the enhanced TGF-β signaling and requires the decreased phosphorylation of PKB/Akt. Biochem. Biophys. Res. Commun. 328, 1170–1181 (2005).

    CAS  Article  Google Scholar 

  52. 52

    Kim, B.C. et al. Jab1/CSN5, a component of the COP9 signalosome, regulates transforming growth factor-β signaling by binding to Smad7 and promoting its degradation. Mol. Cell. Biol. 24, 2251–2262 (2004).

    CAS  Article  Google Scholar 

  53. 53

    Fujii, M. et al. Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Mol. Biol. Cell 10, 3801–3813 (1999).

    CAS  Article  Google Scholar 

  54. 54

    Kim, B.C., Mamura, M., Choi, K.S., Calabretta, B. & Kim, S.J. Transforming growth factor-β1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells. Mol. Cell. Biol. 22, 1369–1378 (2002).

    CAS  Article  Google Scholar 

  55. 55

    Park, S.H. et al. Mechanism of induction of transforming growth factor-β type II receptor gene expression by v-Src in murine myeloid cells. Cell Growth Differ. 12, 9–18 (2001).

    CAS  PubMed  Google Scholar 

  56. 56

    Cardosa, M.J., Gordon, S., Hirsch, S., Springer, T.A. & Porterfield, J. Interaction of West Nile Virus with primary murine macrophage: Role of cell activation and receptors for antibody and complement. J. Virol. 57, 952–959 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank G. Merlino and A. Hobbie for critical reading of the manuscript. Supported by Korea Research Foundation Grant funded by the Korean Government (KRF-2003-015-C00528 to S.H.P.; laboratory equipment, R08-2003-000-10077-0 to S.H.P.), the intramural research fund of the National Cancer Center of Korea (C.-H.L. and I.-H.K) and the Intramural Research Program of the National Cancer Institute.

Author information




K.-C.C., Y.S.L., S.L., H.K.C., C.-H.L., E.-K.L. and S.H. did the experimental work and analyzed data; I.-H.K. participated in the study design; S.H.P. and S.-J.K. designed and conceptualized the research, supervised the experimental work, analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Seong-Jin Kim or Seok Hee Park.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Smad6 inhibits the expression of the NF-κB-promoter reporter induced by LPS in RAW264.7 cells. (PDF 687 kb)

Supplementary Fig. 2

Schematic representation of Smad6 'knockdown' plasmid (pU6-siSmad6) by siRNA. (PDF 757 kb)

Supplementary Fig. 3

Inhibition of endogenous Pellino-1 by siRNA abrogates NF-κB activation induced by IL-1β treatment. (PDF 711 kb)

Supplementary Fig. 4

Stable expression of Peli1 siRNA decreases NF-κB activation upon treatment of IL-1β. (PDF 186 kb)

Supplementary Fig. 5

LPS induced IRAK1-mediated signaling complexes are disrupted by pre-treatment with TGF-β1. (PDF 201 kb)

Supplementary Fig. 6

IL-1β-induced endogenous TRAF6-mediated signaling complexes are disrupted by pre-treatment with TGF-β1. (PDF 199 kb)

Supplementary Fig. 7

Models for the negative regulation of IL-1R–TLR signaling by Smad6. (PDF 201 kb)

Supplementary Table 1

Primer sequences. (PDF 33 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Choi, K., Lee, Y., Lim, S. et al. Smad6 negatively regulates interleukin 1-receptor–Toll-like receptor signaling through direct interaction with the adaptor Pellino-1. Nat Immunol 7, 1057–1065 (2006).

Download citation

Further reading


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