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

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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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)

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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). https://doi.org/10.1038/ni1383

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