Tob2 Inhibits TLR-Induced Inflammatory Responses by Association with TRAF6 and Myd88

Tob2 Inhibits TLR-Induced Inflammatory Responses by Association with TRAF6 and MyD88

This information is current as Guosheng Jiang, Mouchun Gong, Hui Song, Wangnan Sun, of October 2, 2021. Wei Zhao and Lijuan Wang J Immunol published online 1 July 2020 http://www.jimmunol.org/content/early/2020/06/30/jimmun ol.2000057 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 1, 2020, doi:10.4049/jimmunol.2000057 The Journal of Immunology

Tob2 Inhibits TLR-Induced Inﬂammatory Responses by Association with TRAF6 and MyD88

Guosheng Jiang,*,1 Mouchun Gong,†,1 Hui Song,‡,1 Wangnan Sun,* Wei Zhao,‡ and Lijuan Wangx

Optimal activation of TLR pathways is crucial for the initiation of inflammatory responses and eliminating invading micro- organisms. However, excessive of TLR activation may lead to autoimmune and inflammatory diseases. Thus, TLR pathways should be tightly controlled. In this study, we identify Tob2, a Tob/BTG family member, as a suppressor of TLR pathways. Tob2 deficiency enhances TLR-induced NF-kB and MAPK activation and promotes the expression of proinflammatory cytokines in primary peritoneal macrophages of C57BL/6 mice. Furthermore, Tob2-defective C57BL/6 mice may be more susceptible to endotoxemic shock in vivo. Mechanistically, Tob2 interacts with TRAF6 and MyD88 and thus inhibits signaling from the MyD88–TRAF6 complex in primary peritoneal macrophages and HEK293T cells. Therefore, our results uncover a regulatory Downloaded from mechanism of TLR pathways and provide a potential target for the intervention of diseases with excessive TLR activation. The Journal of Immunology, 2020, 205: 000–000.

oll-like receptors are a group of pattern recognition re- (Tob2) is a member of Tob/BTG1 antiproliferative family of ceptors that play crucial roles in inﬂammation and host proteins that plays a crucial role in various physiological defense against invading pathogens (1). Following sensing functions, including bone formation and tumorigenesis (5–11).

T http://www.jimmunol.org/ pathogen-associated molecular patterns, TLRs initiate downstream Like the Tob protein, Tob2 inhibits cell cycle progression signaling by recruiting MyD88 and TIR domain–containing through interaction with CCR4-associated factor 1 (Caf1) (6). adaptor-inducing IFN-b (TRIF) (2). The MyD88-dependent path- Tob2 inhibits PPARg expression by sequestering Smads and way induces the activation of MAPK and NF-kB through C/EBP during adipocyte differentiation (10). Tob2 inhibits the TNFR-associated factor 6 (TRAF6) and TGF-b–activated ki- proliferation of mouse embryonic stem cells and may be in- nase 1 (TAK1), resulting in the induction of proinflammatory volved in the degradation of Id3 in mouse embryonic stem cells cytokines, such as TNF-a and IL-6. The TRIF-dependent path- (12). In addition, Tob2 inhibits RANKL expression and oste- way activates IFN regulatory factor 3 (IRF3) through TRAF3 oclast formation (5). However, the physiological function of and TANK-binding kinase 1 (TBK1), leading to the expression Tob2 in inflammation and TLR signaling remains unclear. by guest on October 2, 2021 of type I IFNs. In addition, TRIF also triggers the activation In this study, we identify Tob2 as a suppressor of TLR pathways. of MAPK and NF-kB via receptor-interacting protein kinase 1 Tob2 interacts with TRAF6 and MyD88 and thereby inhibits (RIP1) and TAK1 (3). signaling from the MyD88–TRAF6 complex. Consequently, Tob2 Although TLR signaling is vital for the immune responses deficiency enhances TLR-induced signaling and the expression of against pathogen infection, uncontrolled TLR activation may lead proinflammatory cytokines. Thus, these results provide a new to detrimental effects, including autoinflammatory disorders. insight into the TLR pathway and identify Tob2 as a specific in- Therefore, TLR signaling must be strictly regulated to avoid hibitor of the MyD88-dependent TLR pathway. Targeting Tob2 excessive inflammatory response (4). Transducer of ErbB2.2 would be beneficial for the development of new strategy to control the diseases with excessive TLR activation.

*Department of Immunology, College of Basic Medical, Binzhou Medical Univer- sity, Yantai 256600, Shandong, China; †Department of General Surgery, Lin’an Materials and Methods ‡ District People’s Hospital, Hangzhou 310013, Zhejiang, China; Department of Mice Immunology, School of Basic Medical Science, Shandong University, Jinan x 250012, Shandong, China; and Pathology Tissue Bank, Qilu Hospital of Shandong Tob2flox/flox mice on a C57BL/6J background (B6; 129-Tob2tm1Nju) were University, Jinan, Shandong 250012, China obtained from Nanjing Biomedical Research Institute of Nanjing Univer- 1G.J., M.G., and H.S. contributed equally to this work. sity. DDX4-Cre (catalog no. 018980) mice were from The Jackson Lab- floxp/+ ORCID: 0000-0001-8440-2274 (W.Z.). oratory. Tob2 mice were crossed with DDX4-Cre transgenic mice to obtain Tob2 deficiency (Tob22/2, Tob2floxp/floxpDDX4-Cre), resulting in a Received for publication January 17, 2020. Accepted for publication June 8, 2020. systemic deletion of Tob2. Tob2flox/flox mice ware used as negative controls This work was supported by grants from the National Natural Science Foundation of (Tob2+/+). C57BL/6 mice were obtained from Beijing Vital River Labo- China (31500699) and the China Postdoctoral Science Foundation (2016M602146). ratory Animal Technology (Beijing, China). All animal experiments were Address correspondence and reprint requests to Dr. Guosheng Jiang or Dr. Lijuan undertaken in accordance with the National Institutes of Health Guide Wang, Department of Immunology, College of Basic Medical, Binzhou Medical for the Care and Use of Laboratory Animals and with the approval of University, Yantai 256600, Shandong, China (G.J.) or Qilu Hospital, Shandong the Scientific Investigation Board of School of Basic Medical Science, University, 107 Wenhuaxi Road, Jinan, Shandong 250012, China (L.W.). E-mail Shandong University, Jinan, Shandong Province, China. addresses: [email protected] (G.J.) or ice_butterfl[email protected] (L.W.) Abbreviations used in this article: IP, immunoprecipitation; LTA, lipoteichoic acid; Cell culture PGN, peptidoglycan; siRNA, small interfering RNA; TAK1, TGF-b–activated kinase 1; TRAF6, TNFR-associated factor 6; TRIF, TIR domain–containing adaptor- To obtain mouse primary peritoneal macrophages, mice were injected i.p. inducing IFN-b; Tob2, transducer of ErbB2.2. with 3% Brewer thioglycolate broth. Three days later, peritoneal exudate cells were harvested and incubated. Two hours later, nonadherent cells Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 were removed, and the adherent monolayer cells were used as peritoneal

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000057 2 Tob2 INHIBITS TLR-INDUCED INFLAMMATORY RESPONSES macrophages (13, 14). HEK293T cells were obtained from the American Tob2; “scrambled” control sequences were 59-UUCUCCGAACGUGU- Type Culture Collection (Manassas, VA). The cells were cultured at 37˚C CACGU-39. under 5% CO2 in DMEM supplemented with 10% FBS (Invitrogen/Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin. RNA quantitation Total RNA was extracted with the RNA Fast 200 RNA Extraction Kit Reagents and Abs according to the manufacturer’s instructions. RNA (500 ng) was reverse LPS (Escherichia coli, 055:B5) was from Sigma-Aldrich (St. Louis, MO); transcribed using Reverse Transcriptase (Takara Bio). The sequences peptidoglycan (PGN), lipoteichoic acid (LTA), and R848 (imidazo- of primers were (forward) 59-CAGATTGGCGAGAAGGGAGTC-39 quinoline compound) were purchased from InvivoGen (San Diego, CA). and (reverse) 59-AAAGAGGCAGTAGTAAAGGTGATGG-39 for Tob2 The final concentrations of agonists were used as follows: 200 ng/ml and (forward) 59-TGTTACCAACTGGGACGACA-39and (reverse) 59- LPS, 10 mg/ml PGN, 1 mg/ml R848, and 5 mg/ml LTA. The Abs specific CTGGGTCATCTTTTCACGGT-39 for b-actin. 396 to anti–p-IRF3 (Ser ) (catalog no.4947), anti–p-IkBa (catalog no.2859), ELISA anti–p-ERK (catalog no.4370), anti-ERK1/2 (catalog no.4695), anti–p-JNK (catalog no.4668), anti-JNK (catalog no.9258), anti–p-p38 (catalog no.4511), The concentrations of mouse TNF-a and mouse IL-6 were measured using anti-p38 (catalog no.8690), and anti-MyD88 (catalog no.4283) were from ELISA kits (Dakewe Biotech, Shenzhen, China) according to the manu- Cell Signaling Technology (Beverly, MA). Anti-Tob2 (catalog no.ab180514) facturer’s instructions. was from Abcam (Cambridge, MA). Anti-HA(catalog no.H3663), anti-Myc (catalog no.M4439), and anti-Flag (catalog no.F1804) were from Sigma- Coimmunoprecitaion and Western blot Aldrich. Anti-TRAF6 (sc-7221), anti–b-actin (sc-81178), and Protein A/G Plus Agarose (sc-2003), used for immunoprecipitation (IP), and For IP, whole-cell extracts were lysed in IP buffer containing 1.0% (v/v) HRP-conjugated secondary Abs were from Santa Cruz Biotechnology Nonidet P-40, 50 mM Tris–HCl (pH 7.4), 50 mM EDTA, 150 mM (Santa Cruz, CA). NaCl, and a protease inhibitor mixture (Merck). After centrifugation for 10 min at 14,000 3 g, supernatants were collected and incubated with Downloaded from Sequences, plasmid constructs, and transfection Protein G Plus Agarose IP reagent together with specific Ab. After 6 h of incubation, beads were washed five times with IP buffer containing 1% N terminus Myc-tagged Tob2 expression plasmid was constructed using (v/v) Nonidet P-40, 50 mM Tris–HCl (pH 7.4), 50 mM EDTA, and PCR-generated fragments. NF-kB reporter plasmid and expression plas- 150 mM NaCl. Immunoprecipitates were eluted by boiling with 1% (w/v) mids for MyD88, TRAF6, TAK1, TAB1, and IKK-b were obtained as SDS sample buffer. For immunoblot analysis, cells were lysed with Pierce previously described (13, 14). For transient silencing, duplexes of small RIPA Buffer (catalog no.89901) supplemented with a protease inhibitor interfering RNA (siRNA) were transfected into cells with INTERFERin mixture, and then protein concentrations in the extracts were measured

reagents (PolyPlus) according to the manufacturer’s instructions. Target with a bicinchoninic acid assay (catalog no.23227; Pierce). Equal amounts http://www.jimmunol.org/ sequences for transient silencing were 59-GCCACCAAAUUUGGUUCC- of extracts were separated by SDS-PAGE and then were transferred onto A-39 (siRNA 1) and 59-GCCACCAAAUUUGGUUCCA-39 (siRNA 2) for nitrocellulose membranes for immunoblot analysis (13, 14). by guest on October 2, 2021

FIGURE 1. Tob2 inhibits TLR-induced cytokine expression. (A and B) Western blot analysis of Tob2 expression in mouse peritoneal macrophages stimulated with LPS (A) or R848 (B) for indicated time periods. (C and D) ELISA analysis of TNF-a (C) and IL-6 (D) in peritoneal macrophages from Tob2+/+ or Tob22/2 mice, followed by stimulation with LPS, R848, LTA, or PGN for 12 h. Data were shown as mean 6 SD (n = 3) of one representative experiment. *p , 0.05, **p , 0.01, ****p , 0.0001. The Journal of Immunology 3

Assay of luciferase activity attenuated Tob2 expression in mouse primary peritoneal macro- Luciferase activity was measured with the Dual-Luciferase Reporter phages (Fig. 1A, 1B). These data indicate that TLR activation Assay System according to the manufacturer’s instructions (Promega), as inhibits Tob2 expression, suggesting its potential roles in TLR described previously (13, 14). Data were normalized for transfection pathways. Next, we investigate the effects of Tob2 on TLR sig- efficiency by division of firefly luciferase activity with that of Renilla naling using Tob2-deficient mice. Tob2 deficiency markedly en- luciferase. hanced LPS, R848, LTA (a TLR2 ligand), and PGN (another LPS-induced cytokine production in mice TLR2 ligand) induced TNF-a and IL-6 expression in mouse pri- 2 2 mary peritoneal macrophages (Fig. 1C, 1D). Taken together, these Tob2+/+ or Tob2 / mice were administered with LPS (10 mg/kg) by i.p. injection for 2 h. Mice were sacrificed, and blood was collected for data indicate that Tob2 negatively regulates TLR-induced proin- measurement of serum cytokines TNF-a and IL-6 by ELISA. flammatory cytokines expression. Statistical analysis Tob2 negatively regulates TLR4-induced NF-kB and MAPK activation All data are presented as mean 6 SD of three or four experiments. Sta- tistical significance between groups was determined by two-tailed Student NF-kB and MAPK activation are required for MyD88-dependent t test with a p value ,0.05 or a Mann–Whitney U test with a p value ,0.1 cytokine production in TLR signaling; thus, we investigated considered statistically significant. whether the activation of NF-kB and MAPK were affected by Tob2. Tob2 deficiency greatly enhanced LPS-induced phos- Results phorylation of IkB-a, ERK, JNK, and p38 in mouse primary Tob2 inhibits TLR-induced proinflammatory peritoneal macrophages (Fig. 2A). To further confirm the reg- cytokine expression ulatory roles of Tob2 on TLR4 signaling, we designed siRNAs Downloaded from To investigate the function of Tob2 in TLR signaling, we initially targeting Tob2 and confirmed the knockdown efficiency in mouse examined its expression following TLR activation. LPS (TLR4 primary peritoneal macrophages (Fig. 2B). Tob2 knockdown ligand) and R848 (TLR7/8 agonist) stimulation both considerably markedly enhanced LPS-induced phosphorylation of IkB-a, ERK, http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Tob2 inhibits TLR-induced NF-kB and MAPK activation. (A) Western blot analysis of the indicated phosphorylated and total signaling proteins in peritoneal macrophages from Tob2+/+ or Tob22/2 mice, followed by stimulation with LPS for the indicated time periods. (B) Western blot and RT-PCR analysis of Tob2 expression in mouse peritoneal macrophages transfected with control (Ctrl) siRNA or Tob2 siRNA 1 and siRNA 2 for 48 h. (C) Western blot analysis of the indicated phosphorylated and total signaling proteins in Ctrl siRNA or Tob2 siRNA 2–transfected mouse peritoneal macrophages, followed by stimulation with LPS for the indicated time periods. (D) Luciferase assay analysis of NF-kB activation in HEK293T cells transfected with NF-kB reporter plasmid and MyD88, together with Tob2 expression plasmid or empty control plasmid. Data were shown as mean 6 SD (n = 4) of one representative experiment. **p , 0.01, ****p , 0.0001. 4 Tob2 INHIBITS TLR-INDUCED INFLAMMATORY RESPONSES p38, and JNK (Fig. 2C). The regulatory role of Tob2 in TLR in LPS-stimulated macrophages (Fig. 3C, 3D). Taken together, signaling was also investigated using a luciferase reporter system. these results show that Tob2 interacts with TRAF6. Tob2 overexpression inhibited MyD88-dependent NF-kB lucifer- MyD88 recruits TRAF6 to form a multiprotein signaling complex ase reporter activity in HEK293T cells (Fig. 2D). Collectively, and activates TAK1, which in turn activates the NF-kB and MAPK these data indicate that Tob2 negatively regulates TLR4-induced pathways (15). Tob2 interacted with both TRAF6 and MyD88 NF-kB and MAPK activation. (Fig. 3C). In addition, the association between Tob2 and MyD88 was observed in both LPS-stimulated macrophages (Fig. 3C, 3D) Tob2 interacts with TRAF6 and MyD88 and inhibits signaling and HEK293T cells (Fig. 3E). Collectively, these results indicate from the MyD88–TRAF6 complex that Tob2 inhibits signaling from the MyD88–TRAF6 complex by To clarify the molecular mechanism by which Tob2 inhibits interacting with TRAF6 and MyD88. MyD88-dependent TLR signaling, we next examined the effects of 2/2 Tob2 on NF-kB activation induced by multiple adaptors involved Tob2 mice may be more susceptible to endotoxemic shock in the TLR pathway. Tob2 overexpression significantly suppressed We next investigated the physiological and pathological rele- TRAF6-induced NF-kB activation, with no effects on TAK1/ vance of the regulatory effects of Tob2 in the context of LPS- TAB1– and IKKb-induced signaling (Fig. 3A). These data suggest induced endotoxemic shock in vivo. Tob2flox/flox (Tob2+/+)and that Tob2 may target TRAF6. Then, we examined the interaction Tob2floxp/floxp DDX4-Cre (Tob22/2) mice were challenged with between Tob2 and TRAF6. Flag-tagged TRAF6 and Myc-tagged LPS by i.p. injection, and systemic inflammation was assessed. Tob2 plasmids were cotransfected into HEK293T cells. Tob2 TNF-a and IL-6 secretion induced by LPS stimulation was much was coprecipitated with TRAF6 (Fig. 3B). Furthermore, the greater in the sera of Tob2-deficient mice than in that of wild- endogenous association between Tob2 and TRAF6 was observed type mice (Fig. 4). These data indicate that Tob2 may be an Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Tob2 interacts with TRAF6 and MyD88 and inhibits signaling from the MyD88–TRAF6 complex. (A) Luciferase assay analysis of NF-kB activation in HEK293T cells transfected with NF-kB reporter plasmid and the indicated adaptors, together with Tob2 expression plasmid or empty control plasmid. Data were shown as mean 6 SD (n = 4) of one representative experiment. (B) Coimmunoprecitation (Co-IP) analysis of lysates from HEK293T cells transfected with Myc–Tob2 and Flag–TRAF6 plasmids. (C and D) Co-IP analysis of lysates from Tob2+/+ and Tob22/2 mouse peritoneal macrophages stimulated with LPS for the indicated time periods. (E) Co-IP analysis of lysates from HEK293T cells transfected with HA–MyD88 and Flag– TRAF6 plasmids, together with Tob2 expression plasmid or empty control plasmid. **p , 0.01. The Journal of Immunology 5

FIGURE 4. Tob2 deﬁciency may enhance TLR4 activation in in vivo ELISA analysis of serum levels of TNF-a and IL-6 of Tob2+/+ or Tob22/2 mice, followed by i.p. injection of LPS (10 mg/kg) for 90 min. Student t test, mean of three samples for TNF-a,and Mann–Whitney U test, mean of three samples, for IL-6. *p , 0.1, **p , 0.05.

important suppressor of TLR-induced proinflammatory responses targets TIR adaptor proteins such as MyD88, Mal, TRIF, and (Fig. 5). TRAM to inhibit TLR downstream signaling (19). In this study, we identified Tob2, a member of the Tob/BTG Discussion family, as a novel suppressor of the TLR pathway. Tob2 inter- TLRs recruit MyD88 and TRIF and then initiate a downstream acts with both MyD88 and TRAF6 in a stimulation-independent signal cascade, resulting in the secretion of proinflammatory cy- manner and thus inhibits the signaling from the MyD88–TRAF6 Downloaded from tokines (including TNF-a, IL-6, etc.) and type I IFNs (16). Ab- complex but without blocking the induced association of these two errant TLR activation causes a variety of inflammatory diseases proteins. Consequently, Tob2 attenuates MyD88-dependent TLR- and autoimmune disorders (4). Thus, fine-tuning of TLR signaling induced proinflammatory cytokine expression. Furthermore, the is vital for the maintenance of immune homeostasis. Control of the physiological relevance of Tob2 in TLR4 regulation was con- formation of functional signaling complexes is an important reg- firmed in an LPS-induced endotoxemic shock model in mice. Of

ulatory mechanism for the regulation of TLR activation (16). For course, there are still some drawbacks in the experiments using http://www.jimmunol.org/ example, BANK1 interacts with TRAF6 and MyD88 to regulate Cre/LoxP mice. Although lack of the negative control (Cre+/LoxP 2 TLR activation (17). Activation of LXR inhibits signaling from Tob2 ) dose did not confound our conclusion, it is a caveat of TLRs to their downstream NF-kB and MAPK effectors through the experimental design. In the future, we will continue to explore disrupting the recruitment of MyD88 and TRAF6 (18). IL-17RD whether Tob2 is involved in the pathogenic conditions of autoimmune diseases through more reasonable grouping and rigorous experimental designs. MyD88 is a key adaptor for TLR signaling, and it is required for activation of NF-kB and MAPK, with subsequent induction of many

proinflammatory cytokines and other proteins important for anti- by guest on October 2, 2021 bacterial immune response. The TLR4–MyD88 pathway also has an important role in endotoxemic shock, and so it is not surprising that negative regulators of MyD88 are present in the cell. Given the pivotal roles of MyD88 in controlling immune responses, our findings will obviously facilitate the development of new strat- egies to control MyD88-mediated inflammatory diseases. In conclusion, we identified Tob2 as a critical negative regulator of TLR-induced inflammatory responses by targeting the MyD88– TRAF6 complex. Therefore, our results outline a novel manner for the control of TLR response and suggest Tob2 as a potential target for the intervention of inflammatory diseases with aberrant TLR activation.

Disclosures The authors have no ﬁnancial conﬂicts of interest.

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