A20 is a cytoplasmic protein required for the termination of tumor necrosis factor (TNF)–induced signals. We show here that mice doubly deficient in either A20 and TNF or A20 and TNF receptor 1 developed spontaneous inflammation, indicating that A20 is also critical for the regulation of TNF-independent signals in vivo. A20 was required for the termination of Toll-like receptor–induced activity of the transcription factor NF-κB and proinflammatory gene expression in macrophages, and this function protected mice from endotoxic shock. A20 accomplished this biochemically by directly removing ubiquitin moieties from the signaling molecule TRAF6. The critical function of this deubiquitinating enzyme in the restriction of TLR signals emphasizes the importance of the regulation of ubiquitin conjugation in innate immune cells.
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*Note: In the version of this article originally published online, the last sentence of the legend to Figure 7 was incorrect and should be deleted. This error has been corrected for the HTML and print versions of this article.
Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335–376 (2003).
Janeway, C.A., Jr. & Medzhitov, R. Innate immune recognition. Annu. Rev. Immunol. 20, 197–216 (2002).
Akira, S., Takeda, K. & Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680 (2001).
Barton, G.M. & Medzhitov, R. Toll-like receptor signaling pathways. Science 300, 1524–1525 (2003).
Akira, S. Toll-like receptor signaling. J. Biol. Chem. 278, 38105–38108 (2003).
Kobayashi, K. et al. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell 110, 191–202 (2002).
Kinjyo, I. et al. SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Immunity 17, 583–591 (2002).
Nakagawa, R. et al. SOCS-1 participates in negative regulation of LPS responses. Immunity 17, 677–687 (2002).
Zhang, G. & Ghosh, S. Negative regulation of Toll-like receptor-mediated signaling by Tollip. J. Biol. Chem. 277, 7059–7065 (2002).
Wald, D. et al. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4, 920–927 (2003).
Brint, E. 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).
Chuang, T.-H. & Ulevitch, R. Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors. Nat. Immunol. 5, 495–502 (2004).
Opipari, A.W., Jr., Boguski, M.S. & Dixit, V.M. The A20 cDNA induced by tumor necrosis factor α encodes a novel type of zinc finger protein. J. Biol. Chem. 265, 14705–14708 (1990).
Krikos, A., Laherty, C.D. & Dixit, V.M. Transcriptional activation of the tumor necrosis factor α-inducible zinc finger protein, A20, is mediated by κB elements. J. Biol. Chem. 267, 17971–17976 (1992).
Lee, E.G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000).
Heyninck, K. & Beyaert, R. The cytokine-inducible zinc finger protein A20 inhibits IL-1-induced NF-κB activation at the level of TRAF6. FEBS Lett. 442, 147–150 (1999).
Cooper, J.T. et al. A20 blocks endothelial cell activation through a NF-κB-dependent mechanism. J. Biol. Chem. 271, 18068–18073 (1996).
Nau, G.J. et al. Human macrophage activation programs induced by bacterial pathogens. Proc. Natl. Acad. Sci. USA 99, 1503–1508 (2002).
Nomura, F. et al. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface Toll-like receptor 4 expression. J. Immunol. 164, 3476–3479 (2000).
Makarova, K.S., Aravind, L. & Koonin, E.V. A novel superfamily of predicted cysteine proteases from eukaryotes, viruses and Chlamydia pneumoniae. Trends Biochem. Sci. 25, 50–52 (2000).
Evans, P.C. et al. A novel type of deubiquitinating enzyme. J. Biol. Chem. 278, 23180–23186 (2003).
Borodovsky, A. et al. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem. Biol. 9, 1149–1159 (2002).
Balakirev, M.Y., Tcherniuk, S.O., Jaquinod, M. & Chroboczek, J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517–522 (2003).
Weissman, A.M. Themes and variations on ubiquitylation. Nat. Rev. Mol. Cell. Biol. 2, 169–178 (2001).
Lomaga, M.A. et al. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13, 1015–1024 (1999).
Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).
Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).
Baud, V. et al. Signaling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is sufficient for JNK and IKK activation and target gene induction via an amino-terminal effector domain. Genes Dev. 13, 1297–1308 (1999).
Ohashi, K., Burkart, V., Flohe, S. & Kolb, H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the Toll-like receptor-4 complex. J. Immunol. 164, 558–561 (2000).
Okamura, Y. et al. The extra domain A of fibronectin activates Toll-like receptor 4. J. Biol. Chem. 276, 10229–10233 (2001).
Evans, P.C. et al. Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity. Biochem. J. 378, 727–734 (2004).
Beyaert, R, Heyninck, K & VanHuffel, S. A20 and A20-binding proteins as cellular inhibitors of nuclear factor-κB-dependent gene expression and apoptosis. Biochem. Pharmacol. 60, 1143–1151 (2000).
Song, H.Y., Rothe, M. & Goeddel, D.V. The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-κB activation. Proc. Natl. Acad. Sci. USA 93, 6721–6725 (1996).
Brummelkamp, T.R., Nijman, S.M., Dirac, A.M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).
Kovalenko, A. et al. The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination. Nature 424, 801–805 (2003).
Trompouki, E. et al. CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members. Nature 424, 793–796 (2003).
Bignell, G.R. et al. Identification of the familial cylindromatosis tumour-suppressor gene. Nat. Genet. 25, 160–165 (2000).
Wertz, I.E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004).
Burkett, P. et al. IL-15Rα expression on CD8+ T cells is dispensable for T cell memory. Proc. Natl. Acad. Sci. USA 100, 4724–4729 (2003).
Chutkow, W.A. et al. Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels. J. Clin. Inv. 110, 203W. A. 208 (2002).
Hofmann, R.M. & Pickart, C.M. In vitro assembly and recognition of Lys-63 polyubiquitin chains. J. Biol. Chem. 276, 27936–27943 (2001).
We thank I. Wertz, V. Dixit, and B. Malynn for reagents, discussions and critical reading of the manuscript. Supported by the National Institutes of Health (RO1AI53224 and R01DK52751 to A.M.), Digestive Disease Research Center (DK42086), Burroughs Welcome (E.M.), Crohn's and Colitis Foundation of America (D.B.) and Medical Scientist Training Program (GM07281; E.T., R.-C.A. and M.W.).
The authors declare no competing financial interests.
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