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. 2011 Jan 25;108(4):1427-32.
doi: 10.1073/pnas.1014156108. Epub 2011 Jan 10.

Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation

Ken-ichi Fujita  1 , Daisuke Maeda, Qi Xiao, Srinivasa M Srinivasula
Affiliations

Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation

Ken-ichi Fujita et al. Proc Natl Acad Sci U S A. .

Abstract

Toll-like receptors (TLRs) play a crucial role in several innate immune responses by regulating autophagy, but little is known about how TLR signaling controls autophagy. Here we demonstrate that p62/SQSTM1 is required for TLR4-mediated autophagy, which we show as selective autophagy of aggresome-like induced structures (ALIS). Treatment with LPS or Escherichia coli induced LC3(+) dot-like structures, and their assembly, but not lysosomal degradation, occurred independently of classic autophagic machinery. Microscopic and ultrastructural analyses showed that p62 is a component of the induced LC3(+) dots and these TLR4-induced p62(+) structures resemble ALIS. The levels of p62 mRNA and protein were increased in TLR4-activated cells and knockdown of p62 suppressed the ALIS formation and LC3-II conversion. The accumulation of p62 and ALIS required activation of Nrf2 by reactive oxygen species-p38 axis-dependent TLR4/MyD88 signaling, suggesting a link between innate immune and oxidative-stress responses. These findings indicate that TLR4-driven induction of p62 plays an essential role in the formation and the autophagic degradation of ALIS, which might be critical for regulating host defense.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Formation of TLR4-mediated LC3+ dots occurs independently of classic autophagic machinery. (A and B) Immunoblots of extracts from RAW cells treated with LPS (0.1 or 1 μg/mL) or K12 (0.5 or 5 × 107) in the presence or absence of PepA/E64d for 24 h (A), or cells transfected with ATG5 siRNAs (NS, #1, or #2) and treated with LPS (B) probed for the indicated proteins. (C) Confocal images of cellular localization of endogenous LC3 in cells treated with LPS. (Scale bars, 5 μm.) (D–J) Percentage of cells with GFP-LC3+ dots. RAW cells expressing GFP-LC3WT (D–J) or -LC3G120A (J) were treated with LPS at the indicated times (D), along with a series of threefold dilutions of PMB (E), in the presence or absence of PepA/E64d (F), in the absence (Cont) or presence of the indicated compounds for 16 h (G), at the indicated times after cells transfected with ATG5 siRNAs (H), and for 24 h (J). The fluorescent images (F, Left and J, Left) and immunoblots of extracts from cells transfected with ATG5 siRNAs (I) are also included. (Scale bars, 20 μm.) The results shown are means ± SD; *P < 0.005; ns, not significant.
Fig. 2.
Fig. 2.
LPS-mediated transcriptional up-regulation of p62 controls ALIS formation and autophagy. (A) Confocal images of cellular localization of p62 in GFP-LC3 expressing RAW cells (Left) and BMDMs (Right) stimulated with LPS (1 μg/mL) for 16 h. (Scale bars, 5 μm.) (B) Fluorescent images (Upper) and percentage of cells with GFP-LC3+ dots (Lower) of RAW cells expressing GFP-LC3WT or -LC3K51A treated with LPS. (Scale bars, 20 μm.) (C) Immunoelectron micrographs of LPS-stimulated RAW cells. Cells treated with LPS were immunolabeled for p62 (Upper). (Scale bars, 2 μm.) (Lower) Magnified image of selected region. (Scale bar, 0.5 μm.) (D) Confocal images of localization of ubiquitin and p62 in RAW cells stimulated with LPS. (Scale bars, 5 μm.) (E–G) Immunoblots of detergent-soluble (Sol) and -insoluble (Ins) fractions of 0.5, 1, or 2% Triton X-100 (TX)-containing buffers from RAW cells treated with LPS (E), of the detergent (2%)-soluble fractions from RAW cells (Left) and BMDMs (Right) treated with LPS for the indicated times (F), and of the detergent-soluble and -insoluble fractions from RAW cells treated with LPS in the presence or absence of PepA/E64d (G), probed for the indicated proteins. Ubs, ubiquitinated proteins. (H) The levels of p62 mRNA in RAW cells (Upper) and BMDMs (Lower) stimulated with LPS for 16 and 2 h, respectively. The results shown are means ± SD; *P < 0.005.
Fig. 3.
Fig. 3.
Requirement of p62 for LPS-induced selective autophagy of ALIS. (A–E) Cells transfected with p62 siRNAs (NS, #1, or #2) were treated with LPS (1 μg/mL) for 16 h. (A) Percentage of cells with GFP-LC3+ dots (Upper) and immunoblots of the extracts for the indicated proteins (Lower) in RAW cells (Left) and BMDMs (Right) expressing GFP-LC3. The results shown are means ± SD; * Indicates statistical significance compared with NS (P < 0.005). nd, Not detectable. (B–D) Confocal images of cellular localization of the indicated molecules in BMDMs (B) and RAW cells (C and D). (Scale bars, 5 μm.) (E) Immunoblots of the detergent-soluble and -insoluble fractions from RAW cells treated with or without PepA/E64d probed for the indicated proteins. Ubs, ubiquitinated proteins.
Fig. 4.
Fig. 4.
MyD88-dependent activation of p38 and Nrf2 is required for p62 induction. (A–C) Cells transfected with siRNAs (NS, #1, or #2) for the indicated genes were stimulated with LPS (1 μg/mL, 16 h). The levels of mRNA (A) and protein (B) of p62 in RAW cells, and percentage of cells with GFP-LC3+ dots (C) in GFP-LC3 expressing RAW are shown. (D and E) RAW cells incubated without (Cont) or with the indicated inhibitors were stimulated with LPS. The levels of p62 mRNA (D) and immunoblots of the detergent-soluble and -insoluble fractions probed for the indicated proteins (E) are shown. Ubs, ubiquitinated proteins. (F–H) Cells transfected with Nrf2 siRNAs (NS, #1, or #2) were stimulated with LPS. The levels of mRNAs of p62 and NQO1 (F), immunoblots of the cellular extracts probed for the indicated proteins (G), and percentage of cells with GFP-LC3+ dots (H) are shown. (I) RAW cells incubated without (Cont) or with the indicated inhibitors were stimulated with LPS. The levels of NQO1 mRNA are shown. The results shown are means ± SD; *statistical significance compared with NS or Cont (P < 0.005).
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References

    1. Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature. 2004;430:257–263. - PubMed
    1. Kawai T, Akira S. TLR signaling. Cell Death Differ. 2006;13:816–825. - PubMed
    1. Thimmulappa RK, et al. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest. 2006;116:984–995. - PMC - PubMed
    1. Virgin HW, Levine B. Autophagy genes in immunity. Nat Immunol. 2009;10:461–470. - PMC - PubMed
    1. Saitoh T, Akira S. Regulation of innate immune responses by autophagy-related proteins. J Cell Biol. 2010;189:925–935. - PMC - PubMed

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