Autophagy Suppresses Interleukin-1β (IL-1β) Signaling by Activation of p62 Degradation via Lysosomal and Proteasomal Pathways*

Background: ATG16L1 is associated with the increased susceptibility to Crohn disease. Results: ATG16L1 suppresses the IL-1β signaling via regulation of p62 stability and mediates ubiquitination of p62. Conclusion: ATG16L1 suppresses IL-1β signaling by down-regulating p62 levels via both autolysosomal and proteasomal pathways. Significance: p62 can be a target of intervention for Crohn patients. ATG16L1 is an essential component of the autophagasome. The T300A allele of ATG16L1 is associated with the increased susceptibility to Crohn disease. In this study, we identified a novel function of ATG16L1, which suppresses signaling of the pro-inflammatory cytokine IL-1β. Deletion of ATG16L1 in mouse embryonic fibroblasts significantly amplifies IL-1β signal transduction cascades. This amplification is due to elevated p62 levels in ATG16L1-deficient cells. We found that ATG16L1 regulates p62 levels via both autolysosomal and proteasomal pathways. For proteasomal degradation, we found that Cullin-3 (Cul-3) is a E3 ubiquitin ligase of p62 and that ATG16L1 is essential for neddylation of Cul-3, a step required for Cul-3 activation. Taken together our data indicate that loss-of-function of ATG16L1 results in a hyper-responsiveness to the IL-1β signaling because of the increased p62 level.

ATG16L1 is an essential component of the autophagasome. The T300A allele of ATG16L1 is associated with the increased susceptibility to Crohn disease. In this study, we identified a novel function of ATG16L1, which suppresses signaling of the pro-inflammatory cytokine IL-1␤. Deletion of ATG16L1 in mouse embryonic fibroblasts significantly amplifies IL-1␤ signal transduction cascades. This amplification is due to elevated p62 levels in ATG16L1-deficient cells. We found that ATG16L1 regulates p62 levels via both autolysosomal and proteasomal pathways. For proteasomal degradation, we found that Cullin-3 (Cul-3) is a E3 ubiquitin ligase of p62 and that ATG16L1 is essential for neddylation of Cul-3, a step required for Cul-3 activation. Taken together our data indicate that loss-of-function of ATG16L1 results in a hyper-responsiveness to the IL-1␤ signaling because of the increased p62 level.
Autophagy is a process that removes damaged proteins and organelles in response to cellular stresses such as starvation. Autophagy also plays a role in cell defense by removing intracellular pathogens (1,2). The ATG16L1 protein is essential for autophagy and the allele of this gene, T300A, is implicated in the susceptibility to Crohn disease (CD) 3 (3), a form of inflammatory bowel disease (IBD). ATG16L1 regulates the granule exocytosis pathway in paneth cells (4), and suppresses induction of IL-1␤ in response to LPS in macrophages (5). These and other yet unknown functions of ATG16L1 may explain its role in intestinal inflammation.
Ubiquitinated protein aggregates are found in various human diseases, including neurodegenerative, liver, and muscle disorders (6). These protein aggregates contain typically p62 (Sequestosome-1, SQSTM1). p62 acts as a selective autophagy receptor for the ubiquitinated protein aggregates (6). In addition to its role in autophagy, p62 acts as an important scaffold in the IL-1␤ signaling pathway by promoting oligomerization of ubiquitinated TRAF6 (7) and MyD88 (8), and as an adaptor protein in Nrf2induced expression of anti-oxidative response genes (9).
Here we report a novel function of ATG16L1 in the IL-1␤ signaling cascade. We discovered that ATG16L1 suppresses IL-1␤ signaling by promoting degradation of p62 via Cul-3mediated proteasomal as well as via autolysosomal degradation.
Transfection of MEF-Transfection of MEF with siRNA or plasmid DNA was done by electroporation with Nucleofector (Lonza, Basel, Switzerland) using MEF2 solution and protocol T-020.
Stable Transfection of p62 shRNA-Control and p62 shRNA bearing lentivirus particles were purchased from Santa Cruz Biotechnology. Two days post-infection, cells were selected with puromycin (1 g/ml) for several passages before usage.   ACT TCC ATC CAG TT  AAG TAG GGA AGG CCG TGG TT  KC  CTG GGA TTC ACC TCA AGA AC  GAA GCC AGC GTT CAC CAG AC  p62  TGA AAC ATG GAC ACT TTG GCT  ACA TTG GGA TCT TCT GGT GGA  IB␣  TGG CCT TCC TCA ACT TCC AGA ACA  TCA GGA TCA CAG CCA GCT TTC AGA Plasmids-Flag-ATG16L1 is described elsewhere (1), and human p62 cDNA was PCR cloned at Bgl2/NotI sites into a modified pCMV (Clontech) containing eGFP.
V5-tagged Cul-3 plasmid was obtained from Michael Freeman (Vanderbilt University) (16). Human Myc-Nedd8 expression vector was purchased from OriGene (Rockville, FIGURE 2. Autophagy mediates both proteasomal and lysosomal degradation of p62. A, p62 protein level is elevated in ATG16L1-deficient and ATG5 KO MEF. The indicated cell lines were stimulated with IL-1␤ (1 ng/ml), and the levels of the indicated proteins were measured by IB. B, p62 is degraded by both proteasome and autolysosome. WT MEF were treated with MG132 (10 M), bafiliomycin A1 (BA1, 100 nM), 3-methyladenine (3MA, 1 mM) and the indicated proteins were measured by IB (exp.: exposure). All above data are representative of at least five independent experiments. C, autophagy regulates degradation of p62 by the ubiquitin-proteasome pathway. The cell lines were treated as indicated and p62 and actin levels were measured by IB. To remove the noise from the p62-interacting ubiquitinated proteins, similar amounts of p62 were immunoprecipitated (rather than the same amount of total extracts), subjected to SDS-PAGE, and probed with anti-ubiquitin antibody. D, p62 is ubiquitinated. WT MEF were transfected with Flag-ubiquitin, and either p62 or Flag-ubiquitin was immunoprecipitated and probed for Flag or p62, respectively. All data are representative of at least two independent experiments. E, GFP-p62 is ubiquitinated in a ATG16L1-dependent manner. WT or ATG16L1-deficient MEF were transfected with GFP-p62 and treated with MG132 (10 M) for 1 h. The lysates were harvested under the denaturing condition (the buffer containing 20 mM HEPES, pH 7.5, 0.15 M NaCl, 1 mM DTT, 1 mM MEM (N-ethylmaleimide), 1% Triton X-100, 1% SDS, protease mixture (Sigma)), and immediately boiled for 10 min. The samples were diluted 3ϫ with the same buffer without SDS (final SDS concentration: 0.33%), p62 was immunoprecipitated with a monoclonal anti-GFP antibody and subjected to IB with anti-ubiquitin and anti-p62 antibodies successively.

MD). The Flag-ubiquitin construct was obtained from M. Karin (UCSD).
Quantitative PCR (qPCR)-Quantitative PCR was performed as described with SYBR Green and GAPDH as control (15) ( Table 1).
Densitometry-Densitometry of all blots was performed with Image J software.
Statistical Significance-Statistical significances were calculated with one-way ANOVA or Student's t test. p values less than 0.05 were considered significant.
Immunofluorescence-Immunofluorescence was performed as described (15). Briefly, MEF grown in chamber slides were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS/Triton X-100 (1%, PTX buffer) twice, incubated with the indicated primary antibody for 1 h in PTX, washed twice with PTX, incubated with a secondary antibody conjugated with Alexa Fluor 488 or 546 (Invitrogen) for 1 h, washed twice in PTX, and mounted for confocal imaging (Olympus FV1000).

RESULTS
Autophagy Suppresses IL-1␤ Signal Transduction-To investigate the role of autophagy in IL-1␤ signaling, we used WT, ATG16L1-deficient (described in related figures as ATG16L1 ⌬/⌬ or ⌬/⌬) MEF (mouse embryonic fibroblasts) (5) and ATG5 KO cells (14). While WT cells produced little IL-6, both ATG16L1-deficient and ATG5-KO cells produced it at a high level in response to IL-1␤ stimulation (Fig. 1A). Consistently, the levels of IL-1␤ activated downstream signal trans-ducers were significantly higher in ATG16L1-deficient cells compared with those in WT cells (3-fold in NF-B activation and 30 -50-fold in MAPKs) (Fig. 1B). In subsequent studies, we used activation of ERK as a surrogate for all MAPKs. Similarly, activation of IL-1␤-induced NF-B and ERK in ATG5 KO cells was higher, 2-and 6-fold, respectively, compared with WT cells (Fig. 1C). Additionally, the levels of IL-1␤-induced IB␣, IL-6, and KC (keratinocyte-derived chemokine) mRNA were significantly higher in ATG16L1-deficient cells than in WT cells (Fig.  1D). However, the impact of ATG16L1 on the signaling of LPS or TNF-␣ was minimal (supplemental Fig. S1). Collectively, these data demonstrate that IL-1␤ signaling is amplified in autophagy-deficient MEF, resulting in transcriptional and translational activation of downstream targets. Because the impact of ATG16L1 or ATG5 deficiency on IL-1␤ signaling is similar, most of the subsequent studies were performed in ATG16L1-deficient MEF.
ATG16L1 suppresses IL-1␤ signal transduction via downregulation of p62. The fact that both NF-B and MAPK pathways are affected by ATG16L1 deletion indicated that its regulation of IL-1␤ signaling by ATG16L1 must be at or above the divergence of both pathways, such as at the level of MyD88-IRAK-TRAF6-TAK1 (17). We therefore searched for molecules that are affected by ATG16L1 and influence the MyD88-IRAK-TRAF6-TAK1 activation. p62 accumulates in cells with a defective autophagasome formation (18), and acts as a signaling hub through its ability to recruit and oligomerize signaling molecules (19). For instance, it promotes IL-1␤ signaling by its association with TRAF6, promoting the oligomerization of ubiquitinated TRAF6 (7,20).
The p62 level in ATG16L1-deficient and ATG5 KO MEF was 20 and 6 fold higher than that observed in WT MEF, respectively ( Fig. 2A). However, the p62 transcript levels were not affected by ATG16L1 deficiency (supplemental Fig. S2A). Although it is known that p62 is primarily degraded by the autolysosome, we found that p62 is also degraded by the proteasome: Both proteasome inhibition (5-fold) and autolysosome inhibition (2-fold) induced p62 accumulation (Fig. 2B). Furthermore, ectopically expressed GFP-p62 was also accumulated upon proteasomal inhibition (supplemental Fig.  S2B), indicating that the accumulation of p62 by proteasomal inhibition was not due to the transcriptional activation of p62. Both proteasomal and lysosomal p62 degradation was dependent on autophagy (ATG5 and ATG16L1) (Fig. 2C), but these two degradation pathways were distinct because only the proteasomal pathway involved ubiquitination of p62 (Fig. 2C). We further demonstrated the ubiquitination of p62 with overexpression of Flag-tagged ubiquitin by immunoprecipitation and immunoblotting (Fig. 2D). In addition, GFP-p62 underwent ubiquitination in a ATG16L1dependent manner under the denaturing conditions (Fig.  2D). These data strongly indicate that p62 itself undergoes ubiquitination in an autophagy-dependent manner.
We next explored the role of p62 in IL-1␤ signaling in vivo. We measured serum cytokine levels induced by IL-1␤ in WT or p62 KO mice and found that TNF␣ or IL-6 production in p62 KO mice was significantly lower than that in WT mice (Fig. 3C), confirming the amplifying role p62 in IL-1␤ signaling.
To determine the neddylation of Cul-3, we overexpressed V5-Cul-3 and myc-Nedd8, and analyzed the neddylation by immunoblotting. We detected in WT cell lysate a distinct band above Cul-3 (i.e. Nedd8-Cul-3), which was absent in ATG16L1deficient MEF lysate (Fig. 5H). Using immunoprecipitation, we confirmed that endogenous Cul-3 is neddylated only in WT but not in ATG16L1-deficient cells. Both IL-1␤-induced and constitutive neddylation of Cul-3 was completely dependent on ATG16L1 (Fig. 5I). Collectively, these data demonstrate that ATG16L1 is required for Cul-3 neddylation and thus for Cul-3-dependent ubiquitination and degradation of p62.

DISCUSSION
Our data provide the molecular mechanism by which ATG16L1 suppresses a potent pro-inflammatory signal. As summarized in Fig. 6, ATG16L1, most likely in complex with ATG5-ATG12 (22), suppresses IL-1␤ signaling by down-regulating p62 levels. p62 was proposed to regulate the assembly and delivery of polyubiquitinated, misfolded, or aggregated proteins, or dysfunctional organelles for their clearance through autophagy (23). In addition, it promotes aggregation of ubiquitinated proteins such as TRAF6 for the activation of IL-1␤ signaling (7). The expression level of p62 is important for these biological functions and it is thought to be regulated solely by autophagy (18). Our data demonstrate that p62 also undergoes proteasomal degradation through Cul-3-mediated ubiquitination and that both ATG16L1 and ATG5 play a key role in both processes. A recent report showed that a significant portion of p62 co-elutes with proteasome when either proteasome or lysosome function is inhibited, and that ATG16L1 associates with proteasome under steady state or upon proteasomal inhibition (24). ATG16L1 deletion in mouse macrophages causes over-production of IL-1␤ protein upon LPS stimulation (5), which may explain the heightened susceptibility of mice carrying ATG16L1-deficient macrophages in an animal model of ulcerative colitis (5). On the other hand, the reduced expression of ATG16L1 in intestinal epithelial cells impedes the release of antimicrobial peptides from Paneth cells, potentially resulting in altered microbial communities adjacent to epithelial crypts (4). A recent study proposed that viral infection in a host carry-ing the susceptible ATG16L1 allele can serve as an initial trigger for a CD-like intestinal inflammation of the colon (25).
Our data provide novel insight to underlying molecular mechanisms and demonstrate how ATG16L1 suppresses directly a pro-inflammatory signal. Our model predicts that IL-1␤ overproduced by macrophages with dysfunctional ATG16L1 (5) also provokes a hyper-inflammatory response in autophagy-deficient cells due to the enhanced TRAF6/p62 oligomerization, which amplifies the downstream signal transduction (Fig. 6). It also suggests that the threshold of IL-1␤ protein levels to induce inflammatory responses would be much lower FIGURE 5. ATG16L1 mediates ubiquitination of p62 by Cul-3. A, p62 co-localizes with Cul-3. GFP-p62 and V5-Cul-3 were expressed in WT MEF and IF was performed with anti-V5 antibody. (Scale bar represents 20 m.) B, Cul-3 interacts with p62. WT MEF were transfected with either GFP-p62 alone or with GFP-62 plus V5-Cul-3. V5-Cul-3 was immunoprecipitated with anti-V5 antibody, and the precipitates were immunoblotted for p62 and V5-Cul-3 successively. C, endogenous ATG16L1, p62, and Cul-3 form an immunoprecipitable complex. WT or ATG16L1-deficient MEF were treated as indicated, p62 was immunoprecipitated and the levels of ATG16L1 and Cul-3 were measured successively by IB. D, Cul-3 silencing increases the p62 level and amplifies IL-1␤ signaling. WT MEF were transfected with either control or Cul-3 siRNA. The levels of p62, pERK, ERK, Cul-3, Nrf-2, and SMA were determined by IB and NF-B by EMSA. E, Cul-3 overexpression in WT MEF decreases the p62 level and suppresses IL-1␤ signaling. WT MEF were transfected with either GFP or V5-Cul-3. The levels of p62, pERK, ERK, Cul 3, and SMA were measured by IB and NF-B was measured by EMSA. F, Cul-3 overexpression in ATG16L1-deficient MEF does not affect the p62 level or IL-1␤ signaling. ATG16L1-deficient MEF were transfected with either GFP or V5-Cul-3. The levels of p62, pERK, Cul-3, and SMA were determined by IB, and NF-B by EMSA. G, expression of Cul-3 induces ubiquitination of p62. WT MEF were transfected with V5-Cul-3 and treated with MG132 as indicated. p62 was immunoprecipitated and immunoblotted for ubiquitin. All data are representative of more than two independent experiments. H, ATG16L1 is required for Cul-3 neddylation. WT or ATG16L1-deficient MEF were transfected with V5-Cul-3 plus Myc-Nedd8, and the levels of the indicated proteins were measured by IB. I, ATG16L1 is required for Cul-3 neddylation. WT or ATG16L1-deficient MEF were stimulated with IL-1␤ as indicated, Cul-3 was immunoprecipitated, and probed for Nedd8 or Cul-3 by IB. FIGURE 6. ATG16L1 is a negative regulator of IL-1␤ signaling. Constitutive degradation of p62 by the autolysosome and the proteasome in the presence of WT ATG16L1 restrains IL-1␤ signaling cascades and the subsequent inflammatory response. In the absence of ATG16L1, p62 levels are increased. This increase in p62 levels promotes oligomerization and activation of TRAF6 (7,19), resulting in overactivation of NF-〉 and MAPKs upon IL-1␤ stimulation that leads to a hyper-inflammatory response.
in the hosts carrying a CD-susceptible ATG16L1 allele (T300A) than that in those with a CD-resistant allele. Finally, our results suggest that CD patients who carry the mutant form of ATG16L may benefit from the inhibition of IL-1␤ signaling by neutralizing IL-1␤ levels or by blocking IL-1R.