Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ

Abstract

Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IκBζ (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IκB family member Bcl-3 (refs 1–6). Transcription of IκBζ is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IκBζ is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IκBζ-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-α. Endogenous IκBζ specifically associates with the p50 subunit of NF-κB, and is recruited to the NF-κB binding site of the IL-6 promoter on stimulation. Moreover, NF-κB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IκBζ-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IκBζ-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IκBζ occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IκBζ.

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Figure 1: Specific induction of IκBζ on stimulation by TLR/IL-1R ligands.
Figure 2: Immune responses in IκBζ-/- cells and kinetics of IκBζ induction.
Figure 3: In vitro analysis of IκBζ on the Il6 promoter.
Figure 4: The TLR/IL-1R responses in NF-κB1/p50-deficient cells and microarray analysis of IκBζ-/- cells a, NF-κB1+/+ (filled symbols) and NF-κB1-/- (open symbols) peritoneal macrophages were cultured with 10 ng ml-1 LPS in the presence of 30 ng ml-1 IFN-γ for 24 h.
Figure 5: In vivo cytokine production in IκBζ-/- mice.

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Acknowledgements

We thank T. Kitamura, A. Aderem, D. Golenbock and H. Tomizawa for providing Plat-E packaging cell lines, flagellin, the ELAM1 reporter plasmid, and R-848, respectively. We also thank T. Kawai and K. Ishii for discussions; M. Hashimoto for secretarial assistance; and N. Okita and N. Iwami for technical assistance. This work was 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.

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Correspondence to Shizuo Akira.

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Supplementary information

Supplementary Discussion 1

Targeted Disruption of murine IκB-ζ gene. (DOC 20 kb)

Supplementary Discussion 2

Relation between the κB sequences of the gene tested and IκB-ζ requirement and regulation of IκB-ζ expression. (DOC 20 kb)

Supplementary Figure 1

Targeted Disruption of murine IκB-ζ gene. (JPG 82 kb)

Supplementary Figure 2

Histological analysis on IκB-ζ-/- mice. (JPG 164 kb)

Supplementary Figure 3

IL-1β-induced activation of signaling cascades IκB-ζ-/- MEFs. (JPG 50 kb)

Supplementary Figure 4a

Gene expression analysis of LPS-inducible genes with statistically selected genes. (JPG 154 kb)

Supplementary Figure 4b-d

Gene expression analysis of LPS-inducible genes with statistically selected genes. (JPG 89 kb)

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(DOC 27 kb)

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Yamamoto, M., Yamazaki, S., Uematsu, S. et al. Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ. Nature 430, 218–222 (2004). https://doi.org/10.1038/nature02738

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