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K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5

Nature Immunology volume 15, pages 231–238 (2014)Cite this article

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Abstract

Although interleukin 1 (IL-1) induces expression of the transcription factor IRF1 (interferon-regulatory factor 1), the roles of IRF1 in immune and inflammatory responses and mechanisms of its activation remain elusive. Here we found that IRF1 was essential for IL-1-induced expression of the chemokines CXCL10 and CCL5, which recruit mononuclear cells into sites of sterile inflammation. Newly synthesized IRF1 acquired Lys63 (K63)-linked polyubiquitination mediated by the apoptosis inhibitor cIAP2 that was enhanced by the bioactive lipid S1P. In response to IL-1, cIAP2 and the sphingosine kinase SphK1 (the enzyme that generates S1P) formed a complex with IRF1, which led to its activation. Thus, IL-1 triggered a hitherto unknown signaling cascade that controlled the induction of IRF1-dependent genes that encode molecules important for sterile inflammation.

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Figure 1: IRF1 is critical for IL-1-induced expression of CXCL10 and CCL5.
Figure 2: IRF1 is required for the recruitment of mononuclear cells into sites of sterile inflammation.
Figure 3: IL-1-induced K63-linked polyubiquitination of IRF1 is mediated by cIAP2 in the presence of S1P.
Figure 4: IL-1 induces the formation of a TRAF6-cIAP2-SphK1 complex that also contains IRF1.
Figure 5: SphK1 activity is critical for activation of the expression of CXCL10 and CCL5 by IL-1.
Figure 6: IL-1-mediated upregulation of the expression of CXCL10 and CCL5 requires cIAP2.
Figure 7: S1P directly binds to cIAP2 and promotes ubiquitination of IRF1.

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Acknowledgements

We thank B. Darnay (MD Anderson) for the plasmid encoding wild-type TRAF2; X.-Y. Wang (Virginia Commonwealth University) for the plasmid encoding histidine-tagged TRAF6; Z. Chen (University of Texas Southwestern Medical Center) for the plasmids encoding hemagglutinin-tagged ubiquitins; C. Duckett (University of Michigan) for the plasmid encoding hemagglutinin-tagged cIAP2 and for Birc3−/− MEFs; X. Wang (University of Texas Southwestern Medical Center) for the mimetic SMAC; R. Proia (US National Institutes of Health) for Sphk1−/− mice; A. Larner (Virginia Commonwealth University) for Stat1−/− mice; K. Fitzgerald (University of Massachusetts) for the INF-β reporter gene; P. Knapp (Virginia Commonwealth University) for mouse astrocytes; and J. Almenara for tissue processing, sectioning and staining. Supported by the US National Institutes of Health (1R01AI093718 to T.K.; 5R37GM043880 and 1U19AI077435 to S.S.; R01CA160688 to K.T.; 5P30NS047463 to the Virginia Commonwealth University Microscopy Facility; and P30CA16059 to the Massey Cancer Center for support of the Lipidomics Developing Shared Resource and the Flow Cytometry Cores) and the National Natural Science Foundation of China (91029704 to C.L.).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Kuzhuvelil B Harikumar, Jessie W Yester, Michael J Surace, Clement Oyeniran, Megan M Price, Wei-Ching Huang, Nitai C Hait, Jeremy C Allegood, Akimitsu Yamada, Reetika Bhardwaj, Kazuaki Takabe, Sheldon Milstien, Sarah Spiegel & Tomasz Kordula

  2. Department of Surgery and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA

    Akimitsu Yamada & Kazuaki Takabe

  3. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

    Xiangqian Kong & Cheng Luo

  4. Department of Medicine, Department of Molecular Microbiology and Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

    Helen M Lazear & Michael S Diamond

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Contributions

K.B.H. planned and did experiments, with assistance from J.W.Y., M.J.S., C.O., M.M.P., W.-C.H., N.C.H., J.C.A., A.Y., H.M.L., R.B., K.T. and M.S.D.; X.K. and C.L. did molecular docking; S.M., S.S. and T.K. conceived of the study and contributed to planning of the experiments; and T.K. wrote the initial draft of the manuscript, which was subsequently edited by all other authors.

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Correspondence to Tomasz Kordula.

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Integrated supplementary information

Supplementary Figure 1 IL-1 induces expression of cytokines in astrocytes.

(a) Primary human astrocytes were stimulated with IL-1 for 8h, and expression of IL-6, IL-8, and CCL4, mRNA was analyzed by TaqMan qPCR. Data were normalized to the expression of GAPDH and presented relative to the expression in untreated cells. (b) Astrocytes were stimulated with IL-1 for 2h, and expression of CXCL10 and CCL5 mRNA was analyzed as above. Error bars represent s.d. *** P<0.001 (a,b, Student's t-test).

Supplementary Figure 2 IRF-1 is indispensable for the recruitment of mononuclear cells into sites of sterile inflammation.

Irf1−/− mice (n=5) or wild-type littermates (n=7) were injected s.c. with 50 μl turpentine. Tissues at the site of injection were collected after 24h. Infiltrating T cells (a) and macrophages (b) were visualized by immunofluorescence using anti-F4/80 and anti-CD90.2 antibodies, respectively. Nuclei were stained with Hoechst. (c) Quantification of images (b and c). Error bars represent s.d. * P<0.05. (d) Cells were isolated from the spleen, blood, and the bone marrow of untreated animals and analyzed by flow cytometry. Error bars represent s.e.m. *** P<0.001 (one-way ANOVA).

Supplementary Figure 3 Schematic representation of the interaction of S1P with cIAP2.

(a) The interaction of S1P with cIAP2 calculated by LIGPLOT. Thatched semi-circles indicate van der Waals contacts between hydrophobic protein residues and S1P. Hydrogen bonds are shown as green dashed lines. Note that Lys596 and Thr594 residues of cIAP2 may stabilize the phosphate group of S1P.

Supplementary Figure 4 Working model of cIAP2-mediated activation of IRF1.

Upon stimulation with IL-1, the IL-1R recruits MyD88 adapter, IRAK4, IRAK1, MEKK3, and TRAF6. Phosphorylation of IRAK1, and a series of TRAF6-dependent K63 polyubiquitinations allow for the recruitment of the TAK/TAB1/TAK2 and IKKα/IKKβ/IKKγ complexes, and consequent activation of MAP kinases and NF-κB, respectively (“fast” TRAF6-RING-dependent response). Subsequently, NF-κB translocates to the nucleus and induces the expression of IRF1, cIAP2 and cytokines, such as IL-8 and IL-6. The newly-synthesized IRF1 is then K63 polyubiquitinated by TRAF6-associated cIAP2. This K63-linked polyubiquitination is regulated by intracellular S1P that is generated by IL-1-activated SphK1 (“delayed” cIAP2/SIP-regulated response). In turn, IRF1 translocates to the nucleus and activates expression of IRF1-dependent genes, such as CCL5 and CXCL10.

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Harikumar, K., Yester, J., Surace, M. et al. K63-linked polyubiquitination of transcription factor IRF1 is essential for IL-1-induced production of chemokines CXCL10 and CCL5. Nat Immunol 15, 231–238 (2014). https://doi.org/10.1038/ni.2810

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