TRIM25 Promotes TNF- −α Induced NF-κB Activation through Potentiating the K63-Linked Ubiquitination of TRAF2

This information is current as Yuchun Liu, Kunpeng Liu, Yingqi Huang, Meng Sun, of September 29, 2021. Qingnan Tian, Shoutao Zhang and Yunfei Qin J Immunol 2020; 204:1499-1507; Prepublished online 5 February 2020; doi: 10.4049/jimmunol.1900482

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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. The Journal of Immunology

TRIM25 Promotes TNF-a–Induced NF-kB Activation through Potentiating the K63-Linked Ubiquitination of TRAF2

Yuchun Liu,* Kunpeng Liu,† Yingqi Huang,† Meng Sun,* Qingnan Tian,* Shoutao Zhang,* and Yunfei Qin†

As an important effector in response to various intracellular or extracellular stimuli, the NF-kB family extensively participates in a wide spectrum of biological events, and its dysregulation may result in many pathological conditions, such as microbial infection, tumor progression, and neurodegenerative disorders. Previous investigations showed that multiple types of ubiquitination play critical roles in the modulation of the NF-kB signaling pathway, yet the molecular mechanisms are still poorly understood. In the

current study, we identified TRIM25, an E3 ligase, as a novel positive regulator in mediating NF-kB activation in human Downloaded from embryonic kidney 293T (HEK293T), HeLa cells, THP-1 cells, and PBMCs. The expression of TRIM25 promoted TNF-a–induced NF-kB signaling, whereas the knockdown had the opposite effect. Furthermore, TRIM25 interacted with TRAF2 and enhanced the K63-linked polyubiquitin chains attached to TRAF2. Moreover, TRIM25 bridged the interaction of TRAF2 and TAK1 or IKKb. To our knowledge, our study has identified a previously unrecognized role for TRIM25 in the regulation of NF-kB activation by enhancing the K63-linked ubiquitination of TRAF2. The Journal of Immunology, 2020, 204: 1499–1507. http://www.jimmunol.org/ uclear factor kB is a pivotal transcription factor that may be targeted by multiple polyubiquitin chains, serving as a influences cell proliferation, apoptosis, inflammation, scaffold to recruit TAK1, TAB1, and TAB2. The active TAK1 N and innate immunity. Abnormal regulation of NF-kB further phosphorylates the MAPKs and IKK complex to initiate signaling has been linked to viral infection, tumorigenesis, auto- MAPK and NF-kB cascades. Next, the IKK complex (composed immune diseases, and septic shock (1–4). In unstimulated cells, of IKKa, IKKb, and NEMO) phosphorylates IkB, which results in NF-kB is bound to IkBa (the inhibitory of NF-kB) and the degradation of IkB by proteasome pathway, leading to the retains in the cytoplasm. Several stimuli, including TNF (TNF-a), nuclear translocation of NF-kB (8, 9). Various types of ubiq- IL-1b, and TLRs, can activate NF-kB signaling by regulating uitination are involved in the signal transduction. For example, downstream molecules, although the regulatory mechanisms are TAB2 and TAB3 prefer to bind to K63-linked ubiquitination of by guest on September 29, 2021 still not well illuminated. RIP1, which results in the activation of TAK1 (10). TAK1 can be Ubiquitination is an important modified mode of protein that activated by tripartite motif (TRIM) protein 8 through K63-linked plays critical roles in the regulation of TNF-a–induced NF-kB ubiquitination (11). The K63-linked ubiquitination of NEMO signaling (5–7). A hallmark following TNF-a stimulation is the regulates the assembly of the IKK complex (12, 13). The deg- activation of NF-kB and the transcription of target . The radation of IkB derives from its K48-linked ubiquitination. All binding of TNF-a to TNFR1 leads to the recruitment of TRADD, evidence suggests that ubiquitination plays an important role at and subsequently, TRADD recruits TRAF2, TRAF5, cIAP1, different levels of TNF-a–induced NF-kB signaling. cIAP2, plus RIP1 to form a complex. At this time, these molecules TRIM25, which is an E3- containing a RING finger domain, a B box/coiled-coil domain, and a SPRY domain, is involved in various cellular processes, including cell proliferation *School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China; and innate immunity (14). Previously, TRIM25 was reported to and †Guangdong Provincial Key Laboratory of Liver Disease, Cell- Therapy regulate antiviral innate immunity by delivering K63-linked pol- Translational Medicine Research Center, The Third Affiliated Hospital of Sun Yat- yubiquitin chains to RIG-I (15). A recent report found that Sen University, Guangzhou, Guangdong 510630, China TRIM25 activated RIG-I signaling relying on the isolated 2CARD Received for publication May 14, 2019. Accepted for publication January 9, 2020. of RIG-I, whereas RIPLET could activated full-length RIG-I via This work was supported by the National Natural Science Foundation of China (81970509, 31601135, and 31970419), and Scientific and Technological Innovation dual ubiquitin-dependent and -independent manners, which dem- Leaders in Central Plains (194200510002). Y.Q. is partially supported by the Startup onstrated the different mechanisms of RIG-I signaling activation Research Fund of Sun-Yet Sen University. (16). TRIM25 promoted type I IFN production by promoting the Address correspondence and reprint requests to Dr. Qingnan Tian and Dr. Shoutao degradation of MAVS through the ubiquitin-proteasome system Zhang or Dr. Yunfei Qin, Zhengzhou University, 100 Science Avenue, Zhengzhou, Henan 450001, China (Q.T. and S.Z.) or Guangdong Provincial Key Laboratory of (17). It also has been reported that TRIM25 enhanced K63-linked Liver Disease, Cell-Gene Therapy Translational Medicine Research Center, The ubiquitination of TRAF6 to regulate NF-kB signaling through the Third Affiliated Hospital of Sun Yat-Sen University, 600 Tianhe Road, Guangzhou, MDA5–MAVS antiviral signaling axis (18). Another study dem- Guangdong 510630, China (Y.Q.). E-mail addresses: [email protected] (Q.T.) and b [email protected] (S.Z.) or [email protected] (Y.Q.) onstrated that TRIM25 exerted its function through the TGF- Abbreviations used in this article: AntiSeq, antisense sequencing; HEK293T, human pathway to influence migration and invasion of cancer cells (19). embryonic kidney 293T; IP, immunoprecipitation; KO, knockout; RT-PCR, real-time But the role of TRIM25 is not clear in TNF-a–induced NF-kB PCR; SenseSeq, sense sequencing; siRNA, small interfering RNA; TRIM, tripartite signaling pathway and inflammation. motif; WT, wild type. In this study, we identified TRIM25 as a positive regulator in Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 TNF-a–induced NF-kB signaling through a luciferase assay www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900482 1500 TRIM25 POTENTIATES NF-kB SIGNALING INDUCED BY TNF-a screening system. TRIM25 potentiated the activation of NF-kBby Real-time PCR targeting TRAF2 to promote its K63-linked ubiquitination through Cells were treated with target plasmids or small interfering RNAs (siRNAs) the RING domain and further enhanced the interaction between as indicated, then stimulated by ligands in 24-well plates. The total cellular TRAF2 and TAK1 or IKKb. Knockdown of TRIM25 inhibited RNA was isolated by TRIzol Reagent (Invitrogen) according to the man- NF-kB activation and, subsequently, resulted in the weak inter- ufacturer’s protocol, and the first-strand cDNA was synthesized from total st action between TRAF2 and TAK1. To our knowledge, our results RNA using a PrimeScript 1 strand cDNA Synthesis Kit (Takara Bio). Real-time PCR (RT-PCR) was performed with SYBR Green qPCR Master have identified an unrecognized role for TRIM25 in the positive Mix (Takara Bio). IL-1b, IL-6, and TNF-a induced by NF-kB were ana- regulation of NF-kB signaling by promoting K63-linked ubiq- lyzed by RT-PCR at 94˚C for 5 min, followed by 40 cycles at 94˚C for 20 s, uitination of TRAF2 that will be helpful to keep gaining insight at 55˚C for 20 s, at 72˚C for 20 s, and finally, at 72˚C for 10 min. The into many human diseases, including infection, inflammatory following specific primers were used for RT-PCR: hGAPDH forward primer, 59-ACAACTTTGGTATCGTGGAAGG-39 and hGAPDH reverse diseases, and cancer. primer, 59-GCCATCACGCCACAGTTTC-39; hIL-1b forward primer, 59- ATGATGGCTTATTACAGTGGCAA-39 and hIL-1b reverse primer, 59- Materials and Methods GTCGGAGATTCGTAGCTGGA-39; hIL-6 forward primer, 59-AGAGG- Reagents, Abs, and plasmids CACTGGCAGAAAACAAC-39 and hIL-6 reverse primer, 59-AGG- CAAGTCTCCTCATTGAATCC-39; hTNF-a forward primer, 59- LPS (L4391-1 MG) and IL-1b (SRP3083) were purchased from Sigma- CCAGACCAAGGTCAACCTCC-39 and hTNF-a reverse primer, 59- Aldrich. Recombinant Human TNF-a Protein (10602HNAE25) was CAGACTCGGCAAAGTCGAGA-39; TRIM25 forward primer, 59- purchased from Thermo Fisher Scientific. AGCAGCTACAACAAGAATACACG-39 and TRIM25 reverse primer, HRP–anti-Flag (M2) (A8592) and anti–b-actin (A1978) were purchased 59-GGCTCTGTTCAATCTCCTCCT-39.

from Sigma-Aldrich; HRP–anti-hemagglutinin (12013819001) was purchased Downloaded from from Roche Applied Science; anti-c-Myc-HRP (HT101) was purchased from Knockdown of TRIM25 by RNA interference TransGen Biotech; anti–p-IkBa (2859), anti-IkBa (4814), anti-IKKb (8943), a b LipoRNAiMax (Invitrogen) was used according to the manufacturer’s anti–phospho-IKK / (2697), and anti-TRIM25 (13773), TAK1 (5206) protocols for transfection of siRNAs into HEK293T cells, HeLa cells, were purchased from Cell Signaling Technology; anti-rabbit–HRP IgG THP-1 cells, or PBMCs. The sequences of TRIM25 siRNAs are as (sc-2004), anti-mouse IgG–HRP (sc-2005), and TRAF2 (sc-876, sc-136999) follows: human TRIM25-specific siRNA 1) sense sequencing (SenseSeq): were purchased from Santa Cruz Biotechnology. 59-AGGUCCACCUGAUGUAUAATT-39 and antisense sequencing Empty vector pcDNA3.1 was from Cui Lab at Sun Yat-sen University. 9 9 9 b (AntiSeq): 5 -UUAUACAUCAGGUGGACCUTT-3 ;2)SenseSeq:5- http://www.jimmunol.org/ TRIM25, TRAF2, TRAF5, TAK1, TAB1, TAB2, IKK , and NEMO genes CCCUGAGGCACAAACUAACTT-39 and AntiSeq: 59-GUUAGUUU- were cloned from A549 cDNA and subcloned into the pcDNA3.1 vector. GUGCCUCAGGGTG-39; and 3) SenseSeq: 59-GCAAAUGUUCCC- The plasmids were confirmed by DNA sequencing at Sangon Biotech. AGCACAATT-39 and AntiSeq: 59-UUGUGCUGGGAACAUUUGCTT-39. Cell culture and transfection Statistical analysis Human embryonic kidney 293T (HEK293T) cells, HeLa cells, TRAF6- Data were compared between the different test groups using a Student t test knockout (KO) HEK293T, PBMCs, and THP-1 cells were cultured in and GraphPad Prism 5.0 Software. All the experiments were repeated at DMEM (HyClone) or RPMI 1640 (Life Technologies) with 10% FBS least three times independently, and the differences between groups were (Genestar), and 1% L-glutamine (Life Technologies) at 37˚C in 5% CO2. considered significant when *p , 0.05, **p , 0.01, and ***p , 0.001. HEK293T, HeLa, and THP-1 cells were provided from Dr. X.-F. Qin’s laboratory (Sun Yat-sen University). TRAF6-KO HEK293T cells were by guest on September 29, 2021 kindly provided by Dr. J. Cui (Sun Yat-sen University). Expression plas- Results mids were transfected with Lipofectamine 2000 (Invitrogen), according to TRIM25 is a potent activator of NF-kB signaling the manufacturer’s instructions. To investigate the roles of TRIM family in NF-kBsignaling Luciferase assay induced by TNF-a, we screened the function of a panel of TRIM HEK293T cells were plated in 24-well plates at a density of 6 3 104 cells proteins using luciferase assays. We transfected HEK293T cells per well. After overnight incubation, the cells were cotransfected with with an NF-kB luciferase reporter and an internal control Renilla plasmids encoding NF-kB luciferase reporter (30 ng per well) and the thymidine kinase luciferase, as well as one of the candidate genes internal control Renilla luciferase (10 ng per well) together with 10–25 ng a of empty vector as the control or plasmids encoding HA-TRIM25, Flag- encoding TRIM, and stimulated them with TNF- . Notably, TRAF2, Flag-TRAF5, Flag-TAK1, HA-TAB1, Flag-IKKb, Flag-P65, and TRIM8, TRIM13, and TRIM38, which were previously reported to other plasmids with indicated target genes. After 24 h of transfection, the positively or negatively modulate NF-kB signaling by targeting cells were treated with TNF-a for 6 h, and these cells were harvested in TAK1, NEMO, or TAB2/3, were identified as hits in the screening passive lysis buffer (Promega). The cell lysates were measured using the (Fig. 1A), thus validating this experimental approach (11, 20, 21). Dual-Luciferase Assay Kit according to the manufacturer’s protocol (Promega). All the assays were performed in triplicate and repeated a These data also led to the identification of TRIM25 as a potent minimum of three times. activator of NF-kB signaling. To further determine whether TRIM25 functions in TLR- and/or cytokine-mediated NF-kBacti- Immunoblot and immunoprecipitation analysis vation, we transfected HEK293T or HEK293T/TLR4 cells with an HEK293T cells were plated in 24-well plates at a density of 6 3 104 cells NF-kB luciferase reporter and the internal control Renilla lucifer- per well. Cells were transfected with target plasmids using Lipofectamine ase, as well as an increasing amount of TRIM25 plasmid, and then 2000 according to the manufacturer’s instructions and extracted in 120 ml a b of low-salt lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM stimulated the cells for 6 h with TNF- ,IL-1 , or LPS. As shown k EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100) supplemented in Fig. 1B–D, TRIM25 potently activated NF- B activation induced with 5 mg/ml protease inhibitor (Thermo Fischer Scientific) and Phos- by TNF-a,IL-1b, or LPS in a dose-dependent manner. It is reported phatase Inhibitor Cocktail (Roche), then samples of 20-ml total proteins that TRIM25 can interact TRAF6 and enhance its K63-linked were subjected to SDS-PAGE. ubiquitination, so we speculated that TRIM25 might regulate For immunoprecipitation (IP) experiments, whole cell extracts were b k prepared after transfection or stimulation with ligands as the method IL-1 – or LPS-mediated NF- B signaling through TRAF6 (18). of immunoblot assay, followed by incubation with the anti-Flag agarose Next, we used TNF-a to treat TRAF6 KO cells and found TRIM25 gels (Sigma-Aldrich) overnight at 4˚C. The beads were washed five times also enhances TNF-a–induced NF-kB signaling (Fig. 1E), indi- with low-salt lysis buffer, resuspended with 23 SDS Loading Buffer (FD cating that TRIM25 activated TNF-a–induced NF-kB in a TRAF6- Biotechnology), and boiled for 5 min. The released proteins were sub- jected to SDS-PAGE and transferred onto PVDF membranes with subse- independent manner. quent blocking using 5% skim milk. Membranes were incubated with Next, we used no-tagged or differently tagged TRIM25 to de- specific Abs and detected using chemiluminescence (Millipore). termine the function of TRIM25, and the luciferase assay showed The Journal of Immunology 1501 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. TRIM25 is a potent activator of NF-kB signaling. (A) HEK293T cells were transfected with an empty vector (Ctrl) or plasmids of 15 TRIMs along with NF-kB luciferase reporter and the internal control Renilla luciferase, then mock stimulated or stimulated with TNF-a (5 ng/ml) for 6 h. The cells were analyzed for NF-kB activity by luciferase reporter assay. (B–E) HEK293T or HEK293T/TLR4 or HEK293T/TRAF6-KO cells were transfected with an NF-kB luciferase reporter and the internal control Renilla luciferase, as well as an empty vector or an increasing amount (wedge) of TRIM25 plasmid, and mock stimulated or stimulated the cells for 6 h with IL-1b (B) (10 ng/ml), TNF-a (C and E) (5 ng/ml) or LPS (D) (100 ng/ml). The cells were analyzed for NF-kB activity by luciferase reporter assay. (F) HEK293T cells were transfected with NF-kB luciferase reporter and the internal control Renilla luciferase along with no-tagged or differently tagged TRIM25 expression plasmids, then mock stimulated or stimulated with TNF-a (5 ng/ml) for 6 h. The cells were analyzed for NF-kB activity by a reporter gene assay. (G) HEK293T cells were transfected with an empty vector or HA-TRIM25 and treated by TNF-a (2 ng/ml) for 0, 15, 30, or 60 min. IkBa turnover was detected by Western blotting with indicated Abs. (H) HeLa cells were transfected with an empty vector or HA-TRIM25, then mock stimulated or stimulated with TNF-a (5 ng/ml) for 8 h. The total mRNA was harvested, and IL-1b, IL-6, and TNF-a mRNA abundance were analyzed by quantitative real-time PCR. The empty vector (pcDNA3.1) used to clone TRIM25 was transfected as the “–” for all dates. All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. that they had the same function in activating TNF-a–mediated inhibited the expression of endogenous TRIM25 in protein and NF-kB–luc activation (Fig. 1F). More importantly, we found that mRNA levels (Fig. 2A, 2B). As expected, we found that treat- TRIM25 significantly enhanced the degradation of endogenous ment with TNF-a resulted in much lower p-IKK and p-IkBa in IkBa protein in the presence of TNF-a (Fig. 1G). In addition, we HEK293T cells transfected with TRIM25-specific siRNA than in found that TNF-a treatment resulted in higher expression levels of those transfected with control siRNA (Fig. 2C). In RT-PCR assays, TNF-a, IL-6,orIL-1b mRNA in HeLa cells with TRIM25- we found that TNF-a treatment resulted in lower expression levels overexpression than in those transfected with empty vector of TNF-a, IL-6,orIL-1b mRNA in TRIM25-deficient HeLa cells (Fig. 1H). Taken together, these results suggest that TRIM25 ac- or PBMCs (Fig. 2D, 2E). Hence, these data suggest knockdown of tivates TNF-a–induced NF-kB activation. TRIM25 inhibits NF-kB activity. Knockdown of TRIM25 inhibits NF-kB activation TRIM25 potentiates NF-kB signaling at the level of the We next detected whether endogenous TRIM25 was involved in the TRAF2–TRAF5 complex regulation of TNF-a–mediated NF-kB signaling. We generated To determine the molecular mechanisms by which TRIM25 three pairs of siRNAs specific for TRIM25, all of which efficiently potentiates TNF-a–induced NF-kB signaling, we transfected 1502 TRIM25 POTENTIATES NF-kB SIGNALING INDUCED BY TNF-a Downloaded from http://www.jimmunol.org/

FIGURE 2. Knockdown of TRIM25 inhibits NF-kB activation. (A) The knockdown efficiency of TRIM25-specific siRNAs in protein level. Immunoblot by guest on September 29, 2021 analysis of the knockdown of endogenous TRIM25 in HEK293T cells treated with three pairs of TRIM25-specific siRNAs or control (Ctrl) siRNA (40 nM). b-actin serves as a loading control. (B) The knockdown efficiency of TRIM25-specific siRNAs in mRNA level. The quantitative real-time PCR analysis of endogenous TRIM25 in HEK293T cells treated with TRIM25-specific siRNAs or control siRNA (40 nM). (C) HEK293T cells were transfected with control siRNA or TRIM25-specific siRNA and treated with TNF-a (2 ng/ml) for the indicated time points. TNF-a–induced IKK activation was measured by immunoblotting with indicated Abs. (D and E) HeLa cells (D) or PBMCs (E) were transfected with control siRNA or TRIM25-specific siRNAs for 36 h, then mock stimulated or stimulated with TNF-a (5 ng/ml) for 8 h. The total mRNA was harvested, and IL-1b, IL-6, and TNF-a mRNAs abundance was analyzed by quantitative real-time PCR. All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001, Student t test.

HEK293T cells with TRAF2, TRAF5, TAK1-TAB1, IKKb,or TRIM25 only weakly interacted with TRAF2 in unstimulated p65 subunit together with TRIM25 plus the NF-kB luciferase cells. However, TRIM25 strongly interacted with TRAF2 after reporter. We found that the activation of NF-kB by TRAF2 and TNF-a treatment (Fig. 4C). We also found that endogenous TRAF5 was markedly enhanced by TRIM25 (Fig. 3A). In contrast, TRIM25 interacted with TRAF2 in unstimulated THP-1 cells and TRIM25 did not increase TAK1-TAB1–, IKKb-, or p65-mediated PBMCs, and the treatment of TNF-a could enhance their inter- NF-kB activation (Fig. 3A), suggesting that TRIM25 potentiates action (Fig. 4D, 4E). To identify the domain of TRIM25 respon- the NF-kB pathway upstream of TAK1, most likely targeting the sible for its interaction with TRAF2, we generated three TRIM25 TRAF complex. Consistent with these results, we found that deletion constructs (Fig. 4F), and we found that only ΔRING knockdown of TRIM25 inhibited NF-kB luciferase activity in- domain (KD) interacted with TRAF2 (Fig. 4G). It suggests the duced by TRAF2 and TRAF5 but not TAK1-TAB1, IKKb, or p65 SPRY domain of TRIM25 is critical for binding to TRAF2. In (Fig. 3B). These results suggest that TRIM25 enhances NF-kB addition, we found NF-kB activation, as assessed by a luciferase signaling upstream of TAK1, at the level of the TRAF complex. assay, was increased in the cells transfected with TRIM25 (wild type [WT]) but not its deletion constructs (Fig. 4H). It should TRIM25 interacts with TRAF2 be noted that both the RING and SPRY domain were critical for Next, we sought to determine whether TRIM25 could directly TRIM25-mediated enhancement of TNF-a–induced NF-kB interact with the TRAF complex or other signaling proteins within signaling. the NF-kB signaling pathway. Coimmunoprecipitation experi- ments revealed that TRIM25 specifically interacted with TRAF2 TRIM25 enhances the K63-linked ubiquitination of TRAF2 rather than the TRAF5 or TAK1 complex (Fig. 4A, 4B). To de- Because the RING domain of TRIM25 is critical for its function termine the interaction, we treated HeLa cells with TNF-a and then (Fig. 4H), we examined whether the E3 ligase activity of TRIM25 collected the cell lysates. IP and immunoblot analysis revealed that was essential for its function. An enzymatically inactive mutant, The Journal of Immunology 1503

FIGURE 3. TRIM25 enhances NF-kB signaling at the level of the TRAF2–TRAF5 complex. (A)TRIM25promotedNF-kB activation induced by NF-kB pathway downstream signaling molecules. HEK293T cells transfected with an NF-kB luciferase reporter, together with an empty vector, TRAF2, TRAF5, TAK1-TAB1, IKKb, or P65, along with an empty vector or plasmid of TRIM25. (B) Knockdown of endogenous TRIM25 inhibited TRAF2- or TRAF5-mediated NF-kB activation. Luciferase activity in HEK293T cells transfected with TRIM25-specific siRNA or control siRNA (40 nM) and transfected with an empty vector, TRAF2, TRAF5, TAK1-TAB1, IKKb, or P65 together with an NF-kB luciferase reporter. The empty vector (pcDNA3.1) used to clone TRIM25 was transfected as the “–.” All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. ***p , 0.001, Student t test. Downloaded from

TRIM25-CS (C53S), could not facilitate NF-kB reporter activa- conditions, so it is critical for the healthy host to tightly regulate tion (Fig. 5A), suggesting that TRIM25 may affect the NF-kB TNF-a–induced NF-kB pathway (1, 3). Our study identified pathways through its E3 ligase activity. Next, we tested whether TRIM25 as a positive regulator in TNF-a–induced NF-kB sig-

TRIM25 affected the ubiquitination of TRAF2. We observed that naling pathway, which might shed light on identifying a potential http://www.jimmunol.org/ TRIM25 remarkably enhanced the ubiquitination of TRAF2 rather therapeutic target for inflammation-associated diseases and tumors than TRIM25-CS mutant or TRIM25 DRING domain (Fig. 5B), in the future. and knockdown of TRIM25 limited the ubiquitination of TRAF2 The posttranslation modification of target proteins is important (Fig. 5C). Next, we want to know which kinds of TRAF2 ubiq- in the regulation of the NF-kB pathway. Increasing numbers of uitination can be affected by TRIM25. Our findings revealed proteins that contain many members of TRIM family were that TRIM25 only enhanced K63-ubiquitination of TRAF2 but reported to participate in the regulation of the TNF-a–induced not others (Fig. 5D). This finding was further confirmed in the NF-kB pathway. Just as reported, TRIM8 associates with TAK1, knockdown system (Fig. 5E). Collectively, these results suggest enhances the binding of K63-linked polyubiquitination to TAK1, that TRIM25 enhances the K63-linked ubiquitination of TRAF2 and positively regulates TNF-a– and IL-1b–triggered NF-kB by guest on September 29, 2021 using its E3 ligase activity. activation (11). TRIM13 regulates ubiquitination and turnover of NEMO at the level of IKK complex to suppress the TNF-a– TRIM25 enhances the interaction between TRAF2 and TAK1 induced NF-kB pathway and also potentiates TLR2-mediated or IKKb NF-kB activation via K29-linked polyubiquitination of TRAF2 After TNF-a treatment, TRAF2 could recruit TAK1 and IKK (20, 22). TRIM12c, as a mouse homologue of TRIM5, forms a complex to influence NF-kB signaling, and TRIM25 targeted complex with TRAF6 to enhance the activation of the IFN and TRAF2 through enhancement of its ubiquitination. Therefore, we NF-kB pathways (23). After stimulation by TNF-a, TRIM22, reasoned that TRIM25 may bridge the TRAF2 and TAK1 or the TRIM38, and TRIM39 inhibit NF-kB pathways by targeting IKK complex. As expected, we found that TRIM25 markedly different proteins through different manners of ubiquitination enhanced the interaction between TRAF2 and TAK1 or IKKb but (21, 24, 25). There are still many proteins, such as TRIM45, did not affect the IKK complex (Fig. 6A–C). TRIM25 deficiency TRIM52, RACK1, and so on, that have regulated functions in blocked the interaction between TRAF2 and TAK1 (Fig. 6D). TNF-a–induced NF-kB pathway (26–28). The involvement of so Next, we found that knockdown of TRIM25 reduced the endog- many molecules in the regulation of NF-kB signaling suggests the enous interaction between TRAF2 and TAK1 after TNF-a stim- importance of maintaining its fine regulation. That is to say, the ulation (Fig. 6E). We further found that TRIM25-ΔRING or -CS complicated regulatory network of NF-kB signaling is required to mutant failed to bridge TRAF2 to TAK1 (Fig. 6F) or to facilitate ensure an appropriate immune response. TRAF2-mediated TAK1 activation (Fig. 6G). Collectively, these TRIM25, a member of the TRIM proteins family, is involved in results indicated that the K63-linked ubiquitination of TRAF2 cell proliferation, innate immunity, and cancer. It was reported that mediated by TRIM25 provided the platform to bridge TRAF2 to TRIM25 affected RIG-I–mediated antiviral activity by delivering TAK1 or IKK complex. the K63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I or releasing NEMO and TBK1 complex into the cytosol Discussion through the proteasome degradation of scaffold protein MAVS TNF-a, as one of the pleiotropic cytokines that activate NF-kB (17, 18). However, there was no report about TRIM25 directly signaling, leads to the production of several proinflammatory cy- regulating TNF-a–induced NF-kB signaling. Unlike TRIM25 tokines, such as IL-1b, IL-6, and TNF-a, and further promotes regulates NF-kB signaling by TRAF6 through MDA5-MAVS inflammation and immune responses. Increasing evidence indi- antiviral signaling axis (18), we found TRIM25 facilitated NF-kB cates that dysregulation of NF-kB leads to several inflammation- activity induced by TNF-a, TRAF2, and TRAF5 but not TAK1- associated diseases. Further, proinflammatory cytokines secreted TAB1, IKKb, or p65. Also, it enhanced the degradation of by innate immune cells have been shown to play a pivotal role IkBa and the transcription of proinflammatory cytokines, such in tumor development and progression in chronic inflammation as IL-1b,IL-6,andTNF-a, in response to TNF-a treatment. 1504 TRIM25 POTENTIATES NF-kB SIGNALING INDUCED BY TNF-a Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. TRIM25 interacts with TRAF2. (A and B) TRIM25 interacted with TRAF2. Coimmunoprecipitation and immunoblot analysis of HEK293T cells transfected with various combinations of plasmids, including an empty vector, Flag-TRAF2, Flag-TRAF5 (A), Flag-TAK1, Flag-TAB1, Flag-TAB2 (B), and HA-TRIM25. Flag-tagged proteins were immunoprecipitated with anti-Flag beads and blotted with anti-HA Ab and whole cell lysates (WCL). (C) TRIM25 interacted with TRAF2. HeLa cells were transfected with an empty vector or Flag-TRIM25 and mock treated or treated by TNF-a (5 ng/ml) for 2 h. Flag-tagged TRIM25 was immunoprecipitated with anti-Flag beads and blotted with TRAF2. (D and E) THP-1 cells (D) or PBMCs (E) were treated by TNF-a (5 ng/ml) for 2 h. They were immunoprecipitated with endogenous TRAF2 and blotted with TRIM25 in cell lysates. (F) The domain structure of TRIM25. Numbers indicated the amino acid position in construct. (G) The interaction between HA-TRAF2 and Flag-TRIM25 domains. HEK293T cells transfected with HA-TRAF2 and an empty vector, Flag-TRIM25, or Flag-TRIM25 domains. Whole cell extracts were immunoprecipitated with anti-Flag beads and blotted with anti-HA Ab. (H) The activation of TRIM25 in NF-kB signaling depended on RING and SPRY domains. Luciferase activity of HEK293T cells transfected with plasmids of NF-kB luciferase reporter and an empty vector, Flag-TRIM25, Flag-TRIM25 B Box/CCD-SPRY (DRING), Flag-TRIM25 RING-B Box/CCD (DSPRY), or Flag-TRIM25 B Box/CCD (BBOX), then mock stimulated or stimulated with TNF-a (5 ng/ml) for 6 h. The B Box/CCD was the B box/coiled-coil domain of TRIM25. The empty vector (pcDNA3.1) used to subclone was transfected as the “–” in all dates. All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. **p , 0.01, Student t test. The Journal of Immunology 1505 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. TRIM25 enhances the K63-linked ubiquitination of TRAF2. (A) TRIM25 enhanced NF-kB activation depending on its E3 ubiquitin ligase activity. HEK293T cells were transfected with an NF-kB luciferase reporter, along with empty vector or plasmids for Myc-TRIM25 (WT), Myc-TRIM25 (DRING), or Myc-TRIM25 (C53S), then mock stimulated or stimulated with TNF-a (5 ng/ml) for 6 h. (B) TRIM25 but not its inactive mutants enhanced the ubiquitination of TRAF2. HEK293T cells were transfected with Flag-TRAF2, HA-tagged ubiquitin and empty vector, Myc-TRIM25 (WT), Myc- TRIM25 (DRING), or Myc-TRIM25 (C53S). They have proceeded to IP and immunoblot analysis with anti-Flag beads and were analyzed with anti-HA Ab by immunoblot. (C) Knockdown of TRIM25 reduced the ubiquitination of TRAF2. HEK293T cells were transfected with Flag-TRAF2, HA-tagged ubiquitin and TRIM25-specific siRNA, control siRNA, or Myc-TRIM25 (WT). Flag-tagged proteins were immunoprecipitated with anti-Flag beads and analyzed with anti-HA Ab by immunoblot. (D) TRIM25 enhanced the K63-linked polyubiquitin on TRAF2. HEK293T cells were transfected with plasmids of Flag-TRAF2 and empty vector or Myc-TRIM25, together with HA-tagged ubiquitin, HA-tagged K6, K11, K27, K29, K33, K48, or K63 ubiquitin mutants. Whole cell extracts were immunoprecipitated with anti-Flag beads and blotted with anti-HA Ab. (E) Knockdown of TRIM25 reduced the K63-linked ubiquitin chains of TRAF2. HEK293T cells were transfected with Flag-TRAF2, HA-tagged K63 ubiquitin, along with TRIM25-specific siRNA or control siRNA (40 nM). Flag-tagged proteins were immunoprecipitated with anti-Flag beads and analyzed with anti-HA Ab by immunoblot. The empty vector (pcDNA3.1) used to subclone was transfected as the “–” for all dates. All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. ***p , 0.001, Student t test.

Immunoprecipitation analyses showed that TRIM25 directly TRAF2 and TRIM25 by IP analyses in THP-1 cells and PBMCs, interacted with TRAF2 but not with TRAF5 or TAK1 complex and the interaction was further enhanced with the treatment of and increased the K63-linked ubiquitination of TRAF2. Con- TNF-a. To identify the detailed mechanism that TRIM25 poten- versely, knockdown of TRIM25 reduced the ubiquitination reac- tiates TNF-a–induced NF-kB signaling by targeting TRAF2, we tion to TRAF2. We also confirmed the endogenous interaction of confirmed TRIM25 reinforced the interaction between TRAF2 1506 TRIM25 POTENTIATES NF-kB SIGNALING INDUCED BY TNF-a Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. TRIM25 enhances the interaction between TRAF2 and TAK1 or IKKb.(A–C) HEK293T cells are transfected with different combinations of plasmids for HA-TAK1, HA-IKKb, Flag-TRAF2, Flag-NEMO and Myc-TRIM25 or empty vector. The lysates have proceeded to IP and immunoblot analysis. (D) TRIM25 deficiency blocked the interaction between TRAF2 and TAK1. HEK293T cells were transfected with HA-TAK1 and Flag-TRAF2, along with TRIM25-specific siRNA or control siRNA (40 nM). Flag-tagged proteins were immunoprecipitated with anti-Flag beads and analyzed with anti- HA Ab by immunoblot. (E) THP-1 cells were transfected with control siRNA or TRIM25-specific siRNAs (40 nM), then mock stimulated or stimulated the cells with TNF-a (5 ng/ml) for 2 h. IP of endogenous TRAF2 detected the association with TAK1 in THP-1 cell lysates. (F) TRIM25 inactive mutants reduced the interaction between TRAF2 and TAK1. HEK293T cells were transfected with HA-TAK1 and Flag-TRAF2, along with empty vector, Myc-TRIM25 (WT), Myc-TRIM25 (DRING), or Myc-TRIM25 (C53S) and have proceeded to IP and immunoblot analysis. (G) TRIM25 facilitated TRAF2-TAK1–mediated NF-kB activation. HEK293T cells were transfected with an NF-kB luciferase reporter, together with the vector encoding Flag- TRAF2 and HA-TAK1 plus HA-TAB1, along with empty vector, Myc-TRIM25 (WT), Myc-TRIM25 (DRING), or Myc-TRIM25 (C53S). The empty vector (pcDNA3.1) used to subclone was transfected as the “–” for all dates. All results were representative of three independent experiments. The graphs showed the mean 6 SD of three independent experiments. **p , 0.01, ***p , 0.001, Student t test. and TAK1 or IKKb; it merely had no function to the interaction targeting TAK1 and NEMO (11, 29). From this study, we also between IKKb and NEMO. The enhanced interaction potentiated found that TRIM25 had the interaction with itself by IP, which the activation of TAK1 and IKKb to heighten TNF-a–induced might be helpful to its stabilization, and then strengthened im- NF-kB signaling, whereas TRIM25-ΔRING or TRIM25-CS mutant mune reaction. Previous studies showed that TRAF2 participated could affect the interaction of TRAF2 and TAK1, and knockdown of in the apoptosis pathway, just as TRAF2 is a negative regulator TRIM25 reduced the endogenous interaction between TRAF2 and of TNF-a–induced apoptosis (30), RIPK1 inhibits LPS/TNF– TAK1 after TNF-a stimulation. In this context, it has been reported mediated apoptosis through a TRAF2- and caspase-8–dependent that TRIM8 upregulates TNF-a–andIL-1b–triggered NF-kBsig- pathway (31). Hence TRIM25 may influence the apoptosis naling by targeting TAK1. 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