Research ArticleIMMUNOLOGY

Inositol polyphosphate multikinase promotes Toll-like receptor–induced inflammation by stabilizing TRAF6

See allHide authors and affiliations

Science Advances  21 Apr 2017:
Vol. 3, no. 4, e1602296
DOI: 10.1126/sciadv.1602296
  • Fig. 1 Depletion of IPMK in myeloid cells protects against septic responses.

    (A) Survival rate of IpmkWT (n = 12) and IpmkΔMac (n = 9) mice, compiled from two independent experiments after severe CLP-induced sepsis. (B and C) Livers (B) or lungs (C) of IpmkWT and IpmkΔMac mice were harvested 20 hours after CLP or sham operation, sectioned, and stained with hematoxylin and eosin (H&E). Representative images from three mice per group are shown. Scale bars, 100 μm. (D) Survival rate of IpmkWT (n = 8) and IpmkΔMac (n = 8) mice challenged with LPS [30 mg/kg, intraperitoneally (ip)], compiled from two independent experiments. (E to G) IpmkWT (n = 21) and IpmkΔMac (n = 25) mice challenged with LPS (4.5 mg/kg, intraperitoneally), compiled from three independent experiments. LPS-induced changes in body temperature over time (E), reduction in body weight 48 hours after injection of LPS (F), and LPS-induced reduction in food intake over time (G). PBS, phosphate-buffered saline. (H) LPS-induced changes in the weight and size of spleens from IpmkWT (n = 7) and IpmkΔMac (n = 7) mice 48 hours after exposure to LPS (4.5 mg/kg, intraperitoneally). (I) Serum concentrations of IL-6 and TNF were measured 6 hours after LPS (4.5 mg/kg, intraperitoneally) injection. (J and K) Expression of Il-1β, Il-6, and Tnfα mRNA was quantitated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in lung (J) and spleen (K) 6 hours after LPS (4.5 mg/kg, intraperitoneally) injection. Three mice per group were analyzed from two independent experiments (I to K). In all experiments, IpmkWT littermates served as controls for IpmkΔMac mice. Data are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, log-rank test (A and D) or Student’s t test.

  • Fig. 2 IPMK depletion in macrophages blunts TLR-dependent inflammatory responses.

    (A) mRNA expression of the proinflammatory cytokines Il-1β, Il-6, and Tnfα was quantified by RT-qPCR in BMDMs 6 hours after stimulation with LPS (100 ng/ml). (B) Secreted levels of the cytokines IL-1β, IL-6, and TNF in BMDM culture medium were measured by enzyme-linked immunosorbent assay (ELISA) 6 hours after stimulation with LPS (100 ng/ml). (C) Phosphorylation of signaling molecules was analyzed by immunoblotting lysates of BMDMs stimulated for 2 hours with LPS (100 ng/ml). (D) mRNA levels of the proinflammatory cytokines Il-1β, Il-6, and Tnfα were quantified by RT-qPCR in BMDMs 6 hours after stimulation with Pam3CSK4 (100 ng/ml). (E) Secreted levels of the cytokines IL-6 and TNF in BMDM culture medium were measured by ELISA 6 hours after stimulation with Pam3CSK4 (100 ng/ml). (F) Phosphorylation of signaling molecules in BMDMs stimulated for 2 hours with Pam3CSK4 (100 ng/ml) was analyzed by immunoblotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. In all BMDM studies, IpmkWT littermates served as controls for IpmkΔMac mice. Data are representative of at least three independent experiments (A to F) and are presented as means ± SE (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

  • Fig. 3 IPMK deficiency reduces TRAF6 protein levels through K48 ubiquitination–dependent protein degradation, and overexpression of TRAF6 restores TLR signaling.

    (A) TRAF6 protein levels in BMDMs were measured by immunoblot analysis. (B) TRAF6 protein levels were measured in IPMK-depleted RAW 264.7 macrophages by immunoblot analysis. scRNA, scrambled RNA. (C) TRAF6 mRNA levels in BMDMs were measured by RT-qPCR. (D) Immunoblot analysis of TRAF6 protein in BMDMs treated with cycloheximide (CHX) (100 μg/ml) for the indicated times. (E) Immunoblot analysis of TRAF6 protein in BMDMs treated with MG-132 (10 μM) or dimethyl sulfoxide (DMSO) (vehicle control) for 8 hours. (F) HEK293T cells transiently cotransfected with hemagglutinin (HA)–K48 ubiquitin (Ub), FLAG-TRAF6, glutathione S-transferase (GST), or GST-IPMK expression plasmids. Forty-eight hours after transfection, cells were lysed and boiled at 95°C for 15 min and subjected to immunoprecipitation (IP) with an anti-FLAG antibody followed by an immunoblot analysis with anti-FLAG, anti-GST, or anti-HA antibodies. (G) Levels of endogenous TRAF6 K48 ubiquitination in IpmkWT and IpmkΔMac BMDMs. The cells were lysed and boiled at 95°C for 15 min and subjected to immunoprecipitation with an anti-TRAF6 antibody followed by an immunoblot analysis with anti-K48 ubiquitin–specific antibodies. (H) Levels of endogenous TRAF6 K48 ubiquitination in IPMK-depleted RAW 264.7 macrophages. The cells were lysed and boiled at 95°C for 15 min and subjected to immunoprecipitation with an anti-TRAF6 antibody followed by an immunoblot analysis with anti-K48 ubiquitin–specific antibodies. (I) IPMK-depleted RAW 264.7 cells overexpressing FLAG-TRAF6 or vector control (FLAG only) were stimulated with LPS (100 ng/ml) for 2 hours, and phosphorylation levels of signaling molecules were assessed by immunoblotting. (J) IPMK-depleted RAW 264.7 cells overexpressing FLAG-TRAF6 or vector control (FLAG only) were stimulated with LPS (100 ng/ml) for 6 hours. mRNA levels of the proinflammatory cytokines Il-1β, Il-6, and iNos were measured by RT-qPCR. In all BMDM studies, IpmkWT littermates served as controls for IpmkΔMac mice. All blots are representative of at least three independent experiments; densitometric quantitation results were normalized to controls. Data are means ± SE (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test; N.S., not significant.

  • Fig. 4 IPMK regulates TRAF6 stability and TLR signaling independent of its catalytic activity.

    (A) Schematic depiction of human IPMK domains. IPMK fragments used for binding studies are indicated below, with numbers corresponding to the amino acids in the full-length protein. Key domains for inositol binding (green), kinase activity (SSLL in blue and IDF in yellow), and the nuclear localization signal sequence (purple). ATP, adenosine 5′-triphosphate. (B) IpmkΔMac BMDMs were either mock-transduced or transduced with FLAG–WT-IPMK or FLAG–KA-IPMK. TRAF6 protein levels were analyzed by immunoblotting and quantified densitometrically using tubulin as a normalization control. (C) IpmkΔMac BMDMs were either mock-transduced or transduced with FLAG–WT-IPMK or FLAG–KA-IPMK. Cells were stimulated with LPS (100 ng/ml) for 6 hours. mRNA levels of the proinflammatory cytokines Il-6 and Tnfα were measured by RT-qPCR. (D) IpmkΔMac BMDMs were either mock-transduced or transduced with FLAG–WT-IPMK or FLAG–KA-IPMK. Cells were stimulated with LPS (100 ng/ml) for 2 hours, and phosphorylation of signaling molecules was detected by immunoblotting. (E) HEK293T cells were transfected with FLAG-TRAF6 and GST, GST–WT-IPMK, or GST–KA-IPMK, with or without HA–K48 ubiquitin. Forty-eight hours after transfection, cells were lysed and boiled at 95°C for 15 min and subjected to immunoprecipitation with an anti-FLAG antibody followed by an immunoblot analysis with anti-FLAG, anti-GST, or anti-HA antibodies. (F) Levels of endogenous TRAF6 K48 ubiquitination were measured in either mock-transduced or FLAG–WT-IPMK– or FLAG–KA-IPMK–transduced IpmkΔMac BMDMs. The cells were lysed and boiled at 95°C for 15 min and subjected to immunoprecipitation with an anti-TRAF6 antibody followed by an immunoblot analysis with anti-K48 ubiquitin–specific antibodies. All blots are representative of at least three independent experiments. Results are means ± SE (n = 3). *P < 0.05; **P < 0.01, one-way analysis of variance (ANOVA) followed by Tukey’s post-test.

  • Fig. 5 IPMK, a TRAF6-binding protein, regulates TRAF6 protein stability.

    (A) HEK293T cells were cotransfected with GST-IPMK and FLAG-TRAF6 or vector control (FLAG only), followed by immunoprecipitation and immunoblot analysis. IgG, immunoglobulin G. (B) TRAF6 was immunoprecipitated from RAW 264.7 macrophages, and coimmunoprecipitated IPMK was detected by immunoblot analysis. (C) TRAF6 was immunoprecipitated from IpmkΔMac and littermate IpmkWT BMDMs, and coimmunoprecipitated IPMK was detected by immunoblot analysis. (D) Recombinant IPMK and in vitro translated FLAG-TRAF6 were coincubated and then immunoprecipitated and analyzed by immunoblotting. (E) Mapping the IPMK domain responsible for binding TRAF6. GST, GST-IPMK, or GST-IPMK fragments were pulled down from HEK293T cells cotransfected with FLAG-TRAF6. TRAF6 proteins in IPMK pull-down experiments were detected by immunoblotting. (F) HEK293T cells cotransfected with plasmids encoding FLAG-TRAF6, Myc-IPMK, and either GST or GST–DN-IPMK were subjected to immunoprecipitation and immunoblot analysis. (G to J) Wild-type BMDMs were mock-transduced or transduced with FLAG–DN-IPMK. TRAF6 protein levels were determined by immunoblot analysis and quantified densitometrically using GAPDH as a normalization control (G). Levels of K48-linked ubiquitinated TRAF6 were measured by immunoprecipitation and immunoblot analysis and quantified densitometrically using GAPDH as a normalization control (H). Cells were stimulated with LPS (100 ng/ml) for 6 hours, after which Il-1β, Il-6, and Tnfα mRNA levels were measured by RT-qPCR (I). Cells were stimulated with LPS (100 ng/ml) for 2 hours, after which phosphorylation of signaling molecules was analyzed by immunoblotting (J). All blots are representative of at least three independent experiments. Results are means ± SE (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test.

  • Fig. 6 IPMK-TRAF6 binding is negatively regulated by IRAK1.

    (A) HEK293T-TLR4 cells were transfected with GST-IPMK and FLAG-TRAF6 or vector control (FLAG only). Cells were stimulated with LPS (100 ng/ml) for 15 min, followed by immunoprecipitation and immunoblot analysis. Densitometric quantitation of GST-IPMK bound to FLAG-TRAF6 was normalized to immunoprecipitated FLAG-TRAF6. (B and C) Wild-type BMDMs (B) and RAW 264.7 macrophages (C) were incubated with or without LPS (100 ng/ml) for 15 min, followed by immunoprecipitation and immunoblot analysis. (D) TRAF6 contains a RING domain, zinc finger repeats, and a C-terminal TRAF (TRAF-C) domain. (E) HEK293T cells were transfected with GST-IPMK or FLAG-TRAF6 fragments (RING domain–deleted 132–530 and N-terminal 1–289), followed by immunoprecipitation and immunoblot analysis. (F) HEK293T cells were transfected with GST-IPMK, FLAG-TRAF6, HA-IRAK1, or vector control (HA only). HA-IRAK1 and GST-IPMK in each FLAG-TRAF6 immunoprecipitate were detected by immunoblotting. (G) HEK293T-TLR4 cells were transfected with GST-IPMK and FLAG-TRAF6 or FLAG-TRAF6[1–289] and incubated with or without LPS (100 ng/ml) for 15 min. GST-IPMK in each FLAG immunoprecipitate was detected by immunoblotting. Data are representative of at least three independent experiments. (H) Model depicting the regulation of TLR signaling by IPMK. In unstimulated macrophages, IPMK binds TRAF6 and protects it from K48 ubiquitination and subsequent degradation. Upon TLR activation, TRAF6 dissociates from IPMK and engages IRAK1, thereby transducing TLR signaling cascades. IPMK-deficient conditions in macrophages lead to increased K48 ubiquitination and degradation of TRAF6. Data in (A) are means ± SE (n = 3). ***P < 0.001, Student’s t test. KO, knockout.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/4/e1602296/DC1

    fig. S1. Generation of myeloid lineage-specific conditional IPMK-null mice.

    fig. S2. Validation of myeloid lineage-specific conditional IPMK-null mice.

    fig. S3. IPMK depletion in macrophages blunts TLR-dependent inflammatory responses.

    fig. S4. IPMK depletion in RAW 264.7 macrophages down-regulates TLR-dependent inflammatory responses.

    fig. S5. IPMK depletion in macrophages does not alter TLR3-dependent inflammatory responses.

    fig. S6. Upstream TLR signaling regulators are not altered in IPMK-depleted macrophages.

    fig. S7. Overexpression of IPMK reduces TRAF6 ubiquitination.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Generation of myeloid lineage-specific conditional IPMK-null mice.
    • fig. S2. Validation of myeloid lineage-specific conditional IPMK-null mice.
    • fig. S3. IPMK depletion in macrophages blunts TLR-dependent inflammatory responses.
    • fig. S4. IPMK depletion in RAW 264.7 macrophages down-regulates TLR-dependent inflammatory responses.
    • fig. S5. IPMK depletion in macrophages does not alter TLR3-dependent inflammatory responses.
    • fig. S6. Upstream TLR signaling regulators are not altered in IPMK-depleted macrophages.
    • fig. S7. Overexpression of IPMK reduces TRAF6 ubiquitination.

    Download PDF

    Files in this Data Supplement:

Navigate This Article