Limits...
TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.

Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, Akira S - Nat. Immunol. (2009)

Bottom Line: Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling.TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages.Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defense, World Premier International Immunology Frontier Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

ABSTRACT
The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

Show MeSH

Related in: MedlinePlus

TANK controls TRAF6 ubiquitination in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10μM R-848 for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-IRAK1. The kinase activities in the immunoprecipitates were measured using an in vitro kinase assay. (b) Macrophages from wild-type and Tank−/− mice were stimulated with 10 μM R-848 for the indicated periods. Whole cell lysates were subjected to immunoblot analysis with anti-IRAK1. Immunoblots ofβ-tubulin are shown as a loading control. (c) Cell lysates of macrophages treated with R-848 for the indicated periods were immunoprecipitated with anti-TRAF6, followed by immunoblot analysis with anti-Ub. Immunoblots of TRAF6 are shown as a loading control. Data of two independent experiments are shown. (d) HEK293 cells were cotransfected with Flag-TRAF6 and Myc-TANK. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblot analysis with anti-Ub. Immunoblots of β-tubulin are shown as a loading control. The data shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2910115&req=5

Figure 5: TANK controls TRAF6 ubiquitination in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10μM R-848 for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-IRAK1. The kinase activities in the immunoprecipitates were measured using an in vitro kinase assay. (b) Macrophages from wild-type and Tank−/− mice were stimulated with 10 μM R-848 for the indicated periods. Whole cell lysates were subjected to immunoblot analysis with anti-IRAK1. Immunoblots ofβ-tubulin are shown as a loading control. (c) Cell lysates of macrophages treated with R-848 for the indicated periods were immunoprecipitated with anti-TRAF6, followed by immunoblot analysis with anti-Ub. Immunoblots of TRAF6 are shown as a loading control. Data of two independent experiments are shown. (d) HEK293 cells were cotransfected with Flag-TRAF6 and Myc-TANK. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblot analysis with anti-Ub. Immunoblots of β-tubulin are shown as a loading control. The data shown are representative of three independent experiments.

Mentions: The above-described results indicate that TANK negatively regulates TLR-induced activation of NF-κB and AP-1. Activation of IRAK-1 in response to R-848 was not enhanced in Tank−/− macrophages (Fig. 5a). Furthermore, IRAK-1 was degraded after R-848 stimulation with similar kinetics in wild-type and Tank−/− macrophages (Fig. 5b), indicating that TANK regulates signaling downstream of IRAKs. TANK has been reported to interact with the TRAF family members TRAF1, 2, 3, 5 and 6. Among these, TRAF6 is needed for TLR signaling. Since TRAF6 is ubiquitinated in response to TLR stimulation, we examined whether TANK modifies the ubiquitination of TRAF6. As shown in Fig. 5c, induction of TRAF6 ubiquitination in response to R-848 stimulation was enhanced in Tank−/− macrophages compared with wild-type cells. Reciprocally, overexpression of TANK in HEK293 cells inhibited the ubiquitination of TRAF6 (Fig. 5d). Taken together, these results indicate that TANK inhibits TLR-induced NF-κB and AP-1 activation by suppressing TRAF6 ubiquitination.


TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.

Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, Akira S - Nat. Immunol. (2009)

TANK controls TRAF6 ubiquitination in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10μM R-848 for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-IRAK1. The kinase activities in the immunoprecipitates were measured using an in vitro kinase assay. (b) Macrophages from wild-type and Tank−/− mice were stimulated with 10 μM R-848 for the indicated periods. Whole cell lysates were subjected to immunoblot analysis with anti-IRAK1. Immunoblots ofβ-tubulin are shown as a loading control. (c) Cell lysates of macrophages treated with R-848 for the indicated periods were immunoprecipitated with anti-TRAF6, followed by immunoblot analysis with anti-Ub. Immunoblots of TRAF6 are shown as a loading control. Data of two independent experiments are shown. (d) HEK293 cells were cotransfected with Flag-TRAF6 and Myc-TANK. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblot analysis with anti-Ub. Immunoblots of β-tubulin are shown as a loading control. The data shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2910115&req=5

Figure 5: TANK controls TRAF6 ubiquitination in response to TLR7 stimulation in macrophages(a) Peritoneal macrophages from wild-type (WT) and Tank−/− mice were stimulated with 10μM R-848 for the indicated periods. Cell lysates were prepared and immunoprecipitated with anti-IRAK1. The kinase activities in the immunoprecipitates were measured using an in vitro kinase assay. (b) Macrophages from wild-type and Tank−/− mice were stimulated with 10 μM R-848 for the indicated periods. Whole cell lysates were subjected to immunoblot analysis with anti-IRAK1. Immunoblots ofβ-tubulin are shown as a loading control. (c) Cell lysates of macrophages treated with R-848 for the indicated periods were immunoprecipitated with anti-TRAF6, followed by immunoblot analysis with anti-Ub. Immunoblots of TRAF6 are shown as a loading control. Data of two independent experiments are shown. (d) HEK293 cells were cotransfected with Flag-TRAF6 and Myc-TANK. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblot analysis with anti-Ub. Immunoblots of β-tubulin are shown as a loading control. The data shown are representative of three independent experiments.
Mentions: The above-described results indicate that TANK negatively regulates TLR-induced activation of NF-κB and AP-1. Activation of IRAK-1 in response to R-848 was not enhanced in Tank−/− macrophages (Fig. 5a). Furthermore, IRAK-1 was degraded after R-848 stimulation with similar kinetics in wild-type and Tank−/− macrophages (Fig. 5b), indicating that TANK regulates signaling downstream of IRAKs. TANK has been reported to interact with the TRAF family members TRAF1, 2, 3, 5 and 6. Among these, TRAF6 is needed for TLR signaling. Since TRAF6 is ubiquitinated in response to TLR stimulation, we examined whether TANK modifies the ubiquitination of TRAF6. As shown in Fig. 5c, induction of TRAF6 ubiquitination in response to R-848 stimulation was enhanced in Tank−/− macrophages compared with wild-type cells. Reciprocally, overexpression of TANK in HEK293 cells inhibited the ubiquitination of TRAF6 (Fig. 5d). Taken together, these results indicate that TANK inhibits TLR-induced NF-κB and AP-1 activation by suppressing TRAF6 ubiquitination.

Bottom Line: Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling.TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages.Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Host Defense, World Premier International Immunology Frontier Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

ABSTRACT
The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

Show MeSH
Related in: MedlinePlus