MLN4924, a First-In-Class NEDD8-Activating Enzyme Inhibitor, Attenuates IFN-Β Production

MLN4924, a First-in-Class NEDD8-Activating Enzyme Inhibitor, Attenuates IFN-β Production

This information is current as Hui Song, Wanwan Huai, Zhongxia Yu, Wenwen Wang, of September 28, 2021. Jing Zhao, Lining Zhang and Wei Zhao J Immunol published online 19 February 2016 http://www.jimmunol.org/content/early/2016/02/19/jimmun ol.1501752 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 19, 2016, doi:10.4049/jimmunol.1501752 The Journal of Immunology

MLN4924, a First-in-Class NEDD8-Activating Enzyme Inhibitor, Attenuates IFN-b Production

Hui Song, Wanwan Huai, Zhongxia Yu, Wenwen Wang, Jing Zhao, Lining Zhang, and Wei Zhao

Neddylation is a posttranslational protein modiﬁcation that conjugates ubiquitin-like protein neural precursor cell–expressed developmentally downregulated protein 8 (NEDD8) to target proteins and regulates diverse cellular processes. MLN4924, a novel NEDD8 activating enzyme inhibitor, which has emerged as a promising anticancer drug, has a multifaceted function by inhibiting the process of neddylation. However, the potential roles of MLN4924 and neddylation in IFN-b production remain unknown. In this study, we show that MLN4924 inhibits TLR3/4- and retinoic acid–inducible gene-I–induced IFN-b expression in different cells, whereas NEDD8 knockdown had no effects on IFN-b expression. The ability of the MLN4924 to inhibit IFN-b production

was confirmed in vivo, as mice treated with MLN4924 exhibited decreased levels of IFN-b upon LPS or polyinosinic-polycytidylic Downloaded from acid stimulation. Furthermore, we show that MLN4924 inhibits IFN regulatory factor 3 (IRF3) transcriptional activation and prevents IRF3 binding to IFN-b promoter. Our findings suggest that MLN4924 inhibits TLR3/4- and retinoic acid–inducible gene- I–induced IFN-b expression by preventing IRF3 binding to the IFN-b promoter, with a neddylation-independent manner. Therefore, our results provide new insight into the mechanism of MLN4924 and may have significant implications for the treatment of MLN4924. The Journal of Immunology, 2016, 196: 000–000. http://www.jimmunol.org/

attern recognition receptors, including TLRs and retinoic Although type I IFNs are important for the elimination of invading acid–inducible gene-I (RIG-I)–like helicases (RIG-I–like microorganisms, overproduction of type I IFNs results in adverse P receptors), signal viral infection and activate immune cells pathogenic effects characteristic of many autoimmune disorders, to produce type I IFNs (IFN-a/b), which are involved in the such as systemic lupus erythematosus (7, 8). Thus, understanding elimination of viral infection (1–3). TLRs and RIG-I–like recep- the mechanisms how type I IFN production is limited is critical to tors recruit different adaptor proteins, including TLR/IL-1R do- protecting against such harmful effects. main–containing adaptor protein inducing IFN-b (TRIF, also Neural precursor cell–expressed developmentally downregu- called TICAM-1) and mitochondrial antiviral signaling protein lated protein 8 (NEDD8) is a conserved ubiquitin-like protein (9). by guest on September 28, 2021 (MAVS, also called IPS-1, Cardif, or VISA) to initiate IFN-b Posttranslational modification by the attachment of NEDD8 is signaling. Recruitment of TRIF and MAVS promotes the activa- known as neddylation (9). Neddylation is an ATP-dependent en- tion of TANK-binding kinase 1 (TBK1) (1–4). Activated TBK1 zymatic process in which NEDD8 is activated by an E1 enzyme then phosphorylates IFN regulatory factor 3 (IRF3), triggers its known as NEDD8 activating enzyme (NAE) and is subsequently dimerization and nuclear translocation, where they form active transferred to the E2 enzyme, Ubc12. NEDD8 E3 ligases catalyze transcriptional complexes that bind to IFN stimulation response the transfer of NEDD8 from the E2 enzyme onto the target protein elements and activate type I IFN genes expression (1–5). Secreted (9). MLN4924 is a first-in-class small-molecule NAE inhibitor, type I IFN binds to IFN-a/b receptor and triggers the production which is reported to inhibit the process of neddylation and has of numerous antiviral genes through the JAK/STAT pathway (6). entered clinical trials as a cancer drug (10–12). MLN4924 has been demonstrated to suppress the growth of multiple human tu- Department of Immunology, Shandong University School of Medicine, Jinan, Shan- mors by targeting neddylation (12). MLN4924 has a multifaceted dong 250012, China mechanism of action by antagonizing NEDD8-mediated protein Received for publication August 3, 2015. Accepted for publication January 21, 2016. degradation, such as induction of DNA re-replication and DNA This work was supported by National Natural Science Foundation of China Grants damage, elevation of oxidative stress, inhibition of NF-kB activity, 31370017 and 31570867, Shandong Provincial Nature Science Foundation for apoptotic cell death, and cellular senescence (12). Besides its at- Distinguished Young Scholars Grant JQ201420, the Key Research and Development Program of Shandong Province (Grant 2015GSF118159), and by National 973 tractive function in cancer therapy, MLN4924 possesses regula- Program of China Grant 2011CB503906. tory functions in a number of cell signaling pathways. For Address correspondence and reprint requests to Prof. Wei Zhao, Department of example, MLN4924 increases phosphorylated IkBa in B cells and Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, myeloid leukemia cells and reduces the expression of several NF- Shandong 250012, China. E-mail address: [email protected] kB target genes in B cells (13, 14). In macrophages and dendritic Abbreviations used in this article: ChIP, chromatin immunoprecipitation; h, human; cells, MLN4924 could prevent IkBa degradation, increase HEK, human embryonic kidney; IFIT, IFN-induced protein with tetratricopeptide repeats; IRF3, IFN regulatory factor 3; ISG, IFN-stimulated gene; m, murine; MAVS, phosphorylated IkBa, and then repress TLR4-induced proin- mitochondrial antiviral signaling protein; NAE, NEDD8 activating enzyme; NEDD8, flammatory cytokine (TNF-a and IL-6) expression (15, 16). neural precursor cell–expressed developmentally downregulated protein 8; NEDP1, NEDD8-specific protease 1; poly(I:C), polyinosinic-polycytidylic acid; RIG-I, reti- MLN4924 blocks lentiviral infection in myeloid cells by dis- noic acid–inducible gene-I; SeV, Sendai virus; siRNA, small interfering RNA; TBK1, rupting neddylation-dependent Vpx-mediated SAMHD1 degra- TANK-binding kinase 1; TRIF, Toll/IL-1R domain–containing adaptor protein induc- dation, indicating the potential efficacy of inhibiting neddylation ing IFN-b; VSV, vesicular stomatitis virus. as an antiretroviral strategy (17). MLN4924 inhibits the NEDD8 Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 cascade, blocks the action of Vif, and thus has potent anti-HIV

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501752 2 MLN4924 INHIBITS IFN-b PRODUCTION activity (18). However, the potential roles of MLN4924 and murine IFN-stimulated gene (ISG)15, 59-TGCTGAGATGGACTGTGAGGAA- neddylation in pattern recognition receptor–mediated IFN-b pro- 39 and 59-TCTTGGCGATAGGCTACGACTG-39 for murine IFN-induced protein duction and antiviral innate immune responses remain unknown. with tetratricopeptide repeats (IFIT)1, 59-CCTAAACAGTTACTCCACCTTCG-39 and 59-TTGCTGACCTCCTCCATTCT-39 for mIFIT2, 59-GACTTGTCTGC- In this study, we demonstrate that MLN4924 attenuates TLR3/4- TACTTGGAATGC-39 and 59-TTGGTTGAGGAAGAGAGGGCT-39 for and RIG-I–mediated IFN-b production by preventing IRF3 binding mIFN-a,59-TGTTACCAACTGGGACGACA’3 and 59-CTGGGTCATCTTTT- to the IFN-b promoter, with a neddylation-independent manner. CACGGT-39 for mb-actin, 59-CAACAAGTGTCTCCTCCAAAT-39 and 59- TCTCCTCAGGGATGTCAAAG-39 for hIFN-b,59-ACGGCGTACTTCCAGA- TGG-39 and 59-CTCGGTTCAAGATCCAGGT-39 for VSV, and 59-GGAA- Materials and Methods ATCGTGCGTGACATTAA-39, and 59-AGGAAGGAAGGCTGGAAGAG- 39 for hb-actin. Data are normalized to b-actin expression in each sample. Mice and cell culture Female C57BL/6 mice (5–6 wk of age) were obtained from Joint Ventures Western blot Sipper BK Experimental Animal Company (Shanghai, China). All animal Cells were lysed with M-PER protein extraction reagent (Pierce, Rockford, experiments were undertaken in accordance with the National Institutes of IL) supplemented with a protease inhibitor mixture, and then protein con- Health Guide for the Care and Use of Laboratory Animals, with the ap- centrations in the extracts were measured with a bicinchoninic acid assay proval of the Scientiﬁc Investigation Board of Medical School of Shan- (Pierce). Nuclear proteins were extracted by NE-PER protein extraction dong University (Jinan, Shandong Province, China). To obtain mouse reagent (Pierce) according to the manufacturer’s instructions. Equal amounts primary peritoneal macrophages, C57BL/6J mice were injected i.p. with of extracts were separated by SDS-PAGE and then were transferred onto 3% Brewer’s thioglycollate broth. Three days later, peritoneal exudate nitrocellulose membranes for immunoblot analysis (19–21). cells were harvested and incubated. Two hours later, nonadherent cells were removed and the adherent monolayer cells were used as peritoneal

Luciferase assay Downloaded from macrophages (19–21). THP-1, HeLa, and human embryonic kidney (HEK) 293T cells were obtained from American Type Culture Collection (Man- Luciferase activities were measured with a Dual-Luciferase reporter assay assas, VA). The cells were cultured at 37˚C under 5% CO2 in DMEM system (Promega) according to the manufacturer’s instructions (19–21). supplemented with 10% FCS (Invitrogen/Life Technologies), 100 U/ml Data are normalized for transfection efficiency by subtracting firefly lu- penicillin, and 100 mg/ml streptomycin. ciferase activity with that of Renilla luciferase.

Reagents Chromatin immunoprecipitation assay LPS (Escherichia coli, 055:B5) and polyinosinic-polycytidylic acid [poly Chromatin from macrophages was fixed and immunoprecipitated using the http://www.jimmunol.org/ (I:C)] were from Sigma-Aldrich (St. Louis, MO). The concentrations of chromatin immunoprecipitation (ChIP) assay kit as recommended by the agonists were used as follows: LPS at 100 ng/ml and poly(I:C) at 10 mg/ml. manufacturer (Upstate Biotechnology) (20). The purified chromatin was MLN4924 was from Selleck Chemicals. IFN-b was from Sino Biological m m 396 immunoprecipitated using 2 g anti-IRF3 or 2 g irrelevant Ab (anti- (Beijing, China). Anti-IRF3 (4302), anti–p-IRF3 (Ser ) (4947), anti–p-JNK actin) Abs. The input fraction corresponded to 0.1 and 0.05% of the (4668), anti–p-p38 (4511), anti–p-ERK (4370), anti-p38 (8690), anti-ERK1/ chromatin solution before immunoprecipitation. After DNA purification, 2 (4695), anti–p-IkBa (2859), and anti–p-STAT1 (7649) were from Cell the presence of the selected DNA sequence was assessed by PCR. The Signaling Technology (Beverly, MA). Anti-NEDD8 (ab81264) was from primers were 59- CCAGGAGCTTGAATAAAATGAA-39 and 59- TGCA- Abcam (Cambridge, MA). Anti-Smurf1 (55175-1-AP) was from Proteintech. GTGAGAATGATCTTCCTT -39 for IFN-b promoter (2200 to 241) and Anti–lamine A/B (BS1446) was from Bioworld Technology. Anti–b-actin 59-GCTACTCTGCCTGGCTTTTCA-39 and 59- TACAGTTTCACCAAT- (sc-81178) and HRP-conjugated secondary Abs were from Santa Cruz TGCTGGAG-39 for IFN-b promoter (2391 to 2261). The PCR program by guest on September 28, 2021 Biotechnology (Santa Cruz, CA). Sendai virus (SeV) was purchased from was 94˚C for 3 min, followed by 94˚C for 30 s, 55˚C for 30 s, and 72˚C for China Center for Type Culture Collection (Wuhan University, Wuhan, 30s for a total of 40 cycles, and then 72˚C for 10 min. PCR products were China) and the multiplicity of infection used was 1. NF-kB reporter plasmid resolved in 10% acrylamide gels. The average size of the sonicated DNA was purchased from Stratagene. IFN-b and IRF3 reporter plasmids and fragments subjected to immunoprecipitation was 500 bp as determined by vesicular stomatitis virus (VSV)–GFP were gifts from Dr. Xuetao Cao ethidium bromide gel electrophoresis. (Second Military Medical University, Shanghai, China). FLAG–NEDD8- specific protease 1 (NEDP1) was a gift from Edward Yeh (Addgene plas- Statistical analysis mid no. 18066) (22). Expression plasmids for RIG-I, MDA5, MAVS, TRIF, TBK1, and IRF3 5D were described before (19, 21). All experiments were independently performed three times. Data are pre- sented as means 6 SD of three or four experiments. Analysis was performed Transfection using a Student t test or ANOVA. The p values ,0.05 were considered to be statistically significant. For transient transfection of plasmids into HeLa and HEK293T cells, Lipofectamine 2000 reagents (Invitrogen) were used. For transient silencing, duplexes of small interfering RNA (siRNA) were transfected into Results cells with the INTERFERin reagent (Polyplus-transfection, New York, NY) b according to the standard protocol. Target sequences for transient silencing MLN4924 negatively regulates IFN- production were 59-AGAGGAGGAGGUGGUCUUA-39 (siRNA 1), 59-GUGGCAAG- To investigate the potential roles of MLN4924 on IFN-b production, CAGAUGAAUGA-39 (siRNA 2), and 59-GCUUCCCUCUCUUAUGACU- 39 (siRNA 3) for human (h)NEDD8, 59-GGAGGAAGGUUUGGACUAU-39 mouse peritoneal macrophages were pretreated with MLN4924, for mSmurf1, and “scrambled” control sequences, all of which were pur- followed by stimulation with LPS, poly(I:C), or infection with SeV. chased from Ribobio (Guangzhou, China). MLN4924 treatment significantly attenuated LPS-, poly(I:C)-, and SeV-induced IFN-b production at both protein and mRNA levels ELISA (Fig. 1A, 1B). Furthermore, MLN4924 inhibited LPS- and SeV- The concentration of IFN-b was measured with ELISA kits (BioLegend, induced IFN-b production in a dose-dependent manner (Fig. 1C, San Diego, CA). The concentrations of TNF-a and IL-6 were measured 1D), and a concentration as low as 100 nM could significantly with ELISA kits (Dakewe Biotech Company, Shenzhen, China). inhibit IFN-b expression (Fig. 1D). MLN4924 also inhibited LPS-, RNA quantitation poly(I:C)-, and SeV-induced IFN-b production in human THP-1 Total RNA was extracted with RNAfast2000 RNA extraction kit according cells (Fig. 1E). Furthermore, MLN4924 negatively regulated IFN-b to the manufacturer’s instructions (Invitrogen). A LightCycler (ABI Prism promoter activation in both HeLa and HEK293T cells (Fig. 1F, 1G). 7000) and a SYBR RT-PCR kit (Takara Bio) were used for quantitative Collectively, these results demonstrate that MLN4924 attenuates real-time RT-PCR analysis. Specific primers used for RT-PCR assays were IFN-b production in different cells. 59-ATGAGTGGTGGTTGCAGGC-39 and 59-TGACCTTTCAAATGCAG- TAGATTCA-39 for murine (m)IFN-b,59-CACCACTCCCTGCTGCTTTG-39 To determine whether MLN4924 specifically attenuates the and 59-ACACTTGGCGGTTCCTTCG-39 for mRANTES, 59-AGAAGCAG- production of IFN-b, we investigated its regulatory roles in the ATTGCCCAGAAG-39 and 59-TGCGTCAGAAAGACCTCATAGA-39 for production of proinflammatory cytokines. As shown in Fig. 1H, The Journal of Immunology 3

MLN4924 greatly inhibited LPS-induced TNF-a and IL-6 ex- also attenuated LPS- and poly(I:C)-induced MAPK activation pression in mouse peritoneal macrophages, which was consistent (Fig. 3A). Next, we investigated whether MLN4924 could regulate with previous reports (15). IRF3 nuclear translocation. As shown in Fig. 3C, no considerable differences of IRF3 level in the nucleus were observed between MLN4924 inhibits IRF3 transcriptional activation and its DMSO- and MLN4924-treated macrophages. binding to IFN-b promoter Previously, we found that MLN4924 inhibited IRF3 luciferase To determine the mechanism and molecular targets of MLN4924 reporter activation (Fig. 2B–D). The IRF3 reporter used in this in TLR3/4- and RIG-I–induced IFN-b production, the effects of study includes two plasmids (24). One is a fusion expression MLN4924 on IFN-b promoter activation mediated by TRIF, RIG- plasmid that IRF3 fused to GAL4-DNA binding domain. Another I, MDA5, MAVS, TBK1, and IRF3 5D were examined in lucif- is a luciferase reporter with a promoter fragment that allows erase assays. MLN4924 treatment signiﬁcantly inhibited TRIF-, GAL4 binding. Therefore, activation of IRF3 will allow IRF3-GAL4 RIG-I–, MDA5-, MAVS-, TBK1-, and IRF3 5D–induced IFN-b binding and drive the expression of the luciferase. We then per- promoter activation (Fig. 2A). Previous investigations showed that formed ChIP assay to investigate whether MLN4924 could reg- wild-type IRF-3 induces marginal levels of type I IFN (23). In ulate IRF3 binding to the IFN-b promoter.IRF3boundtotheIFN-b contrast, the IRF-3 5D mutant, in which residues at positions 396, promoter region (nt 2200 to 241) from peritoneal primary mac- 398, 402, 404, and 405 were replaced by the phosphomimetic rophages activated with LPS (Fig. 3D), whereas unstimulated aspartate amino acid, induces strong activation of the IFN-b controls did not demonstrate this DNA binding. Using MLN4924 promoter (23). IRF3 5D–induced IFN-b promoter activation treatment, IRF3 DNA binding to the IFN-b promoter was greatly was inhibited by MLN4924 treatment (Fig. 2A). Therefore, we decreased in LPS-stimulated primary macrophages (Fig. 3D). As Downloaded from presume that MLN4924 targets the IRF3 or IRF3 downstream negative controls, IRF3 could not bind to the IFN-b promoter pathway. We then observed the effect of MLN4924 on IRF3 region (nt 2391 to 2260) from peritoneal primary macrophages activation using IRF3 lucifersase assay. TRIF-, RIG-I–, MDA5-, activated with LPS, indicating speciﬁc binding of IRF3 to the MAVS-, and TBK1-induced IRF3 activation was substantially promoter region from nt 2200 to 241. Collectively, these data attenuated by MLN4924 treatment (Fig. 2B). Additionally, MLN4924 suggest that MLN4924 inhibits IFN-b transcription by preventing

treatment also inhibited poly(I:C)- and SeV-induced IRF3 and NF- IRF3 binding to the IFN-b promoter. http://www.jimmunol.org/ kB activation (Fig. 2C, 2D). Therefore, we conclude that MLN4924 b inhibits IRF3 transcriptional activation. To further confirm the in- MLN4924 inhibits IFN- production in a neddylation- hibitory roles of MLN4924 on IRF3 activation, RANTES (another independent manner IRF3-dependent cytokine) expression was determined in MLN4924- It has been reported that MLN4924 could use a neddylation- pretreated macrophages. As shown in Fig. 2E, MLN4924 negatively dependent mechanism to regulate several signal pathways (12). regulated LPS-, poly(I:C)-, and SeV-induced RANTES expression in To determine whether neddylation is involved in MLN4924- mouse peritoneal macrophages. mediated inhibition of TLR- and RIG-I–induced IFN-b produc- Phosphorylation and subsequent nuclear translocation of IRF3 tion, NEDD8 knockdown experiments were performed. The ex- are crucial for its transcriptional activation. We then investigated pression of NEDD8 was greatly decreased with transfection of by guest on September 28, 2021 whether MLN4924 could regulate IRF3 phosphorylation and nu- NEDD8-specific siRNA in HeLa and HEK293T cells (Fig. 4A). clear translocation. As shown in Fig. 3A and 3B, MLN4924 treat- NEDD8 siRNA 2 and 3, which have a higher efficiency to knock- ment had no effect on IRF3 phosphorylation induced by LPS or down NEDD8 expression, were used in the following experiments. poly(I:C). As parallel controls, MLN4924 enhanced LPS- and poly(I:C)- NEDD8 knockdown had no effects on poly(I:C)-, SeV-, and TRIF- induced IkBa phosphorylation (Fig. 3A). Furthermore, MLN4924 induced IFN-b promoter activation (Fig. 4B, 4C). Similarly,

FIGURE 1. MLN4924 negatively regulates IFN-b production. (A and B) ELISA (A) or RT-PCR (B) analyses of IFN-b production in peritoneal macrophages pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS, poly(I:C), or infected with SeV for indicated time periods. (C and D) ELISA (C) or RT-PCR (D) analyses of IFN-b production in peritoneal macrophages pretreated with increasing concentrations of MLN4924 for 2 h and then stimulated with LPS or infected with SeV for indicated time periods. (E) RT-PCR analyses of IFN-b production in THP-1 cells pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS, poly(I:C), or infected with SeV for indicated time periods. (F and G) HeLa (F) or HEK293T (G) cells were transfected with IFN-b reporter plasmid, and then the cells were treated with DMSO or MLN4924 for 2 h and stimulated as indicated. The luciferase activity was measured. (H) ELISA analyses of TNF-a and IL-6 production in peritoneal macrophages pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS for 6 h. Data are shown as mean 6 SD (n = 6) of one representative experiment. **p , 0.01. 4 MLN4924 INHIBITS IFN-b PRODUCTION

FIGURE 2. MLN4924 inhibits IRF3 activation. (A and B) HEK293T cells were transfected with adaptor plasmids as indicated, along with IFN-b (A)or IRF3 (B) reporter plasmid. Eight hours later, cells were treated with DMSO or MLN4924 for 16 h and luciferase activity was measured. (C and D) HeLa (C) Downloaded from or HEK293T (D) cells were transfected with IRF3 or NF-kB reporter plasmid, and then the cells were treated with DMSO or MLN4924 for 2 h and then stimulated as indicated. The luciferase activity was measured. (E) RT-PCR analyses of RANTES production in peritoneal macrophages pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS, poly(I:C), or infected with SeV for indicated time periods. Data are shown as mean 6 SD (n = 6) of one representative experiment. **p , 0.01.

NEDD8 knockdown had no effects on LPS-induced IFN-b ex- as ISGs, including IFN-induced 15-kDa protein (ISG15), ISG56 http://www.jimmunol.org/ pression in THP-1 cells (Fig. 4D). (also called IFIT1), and ISG54 (also called IFIT2), play critical NEDP1 (also known as DEN1 or SENP8), a cysteine protease roles in the immune responses against viral infection (27). The specific for NEDD8, could remove NEDD8 from its substrates and fact that MLN4924 attenuates type I IFN production prompted us then inhibit neddylation process (25, 26). By coexpressing NEDP1 to investigate the function of MLN4924 in the regulation of type I with IRF3 5D and IFN-b luciferase plasmids, we observed that IFN–activated gene expression and antiviral immunity. However, NEDP1 overexpression and NEDD8 knockdown had no effects on MLN4924 had no influence on the expression of ISG15, IFIT1, IRF3 5D–induced IFN-b promoter activation (Fig. 4E). Although and IFIT2 induced by LPS and poly(I:C) (Fig. 5A). Additionally, NEDD8 knockdown had no effects on SeV-induced IFN-b promoter MLN4924 also had no significant effects on the replication of activation (Fig. 4B, 4F), MLN4924 inhibited SeV-induced IFN-b VSV (Fig. 5B, 5C), a type of ssRNA virus recognized by RIG-I. by guest on September 28, 2021 promoter activation in NEDD8 knockdown HEK293T cells (Fig. 4B, Secreted type I IFN binds to IFN-a/b receptor and triggers the 4F). Taken together, these data indicate that neddylation is not in- production of antiviral genes through the JAK/STAT pathway (6). volved in the regulation of IFN-b expression and MLN4924 inhibits To elucidate the potential mechanisms by which MLN4924 has no IFN-b production in a neddylation-independent manner. effects on the expression of ISGs, we examined the phosphorylation of STAT1 at Tyr701, which is crucial for the IFN-mediated MLN4924 has no effects on VSV replication signaling (6). As shown in Fig. 5D, MLN4924 treatment sub- MLN4924 treatment significantly attenuated both IFN-a and IFN-b stantially enhanced IFN-b–induced phosphorylation of STAT1 at expression (Figs. 1, 5A). Type I IFNs and the activated genes, such Tyr701. It has been reported that E3 ligase Smurf1 could inhibit

FIGURE 3. MLN4924 inhibits IRF3 transcriptional activation and its binding to IFN-b promoter. (A) Mouse peritoneal macrophages were pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS or poly(I:C) for the indicated time period. Phosphorylated and total signaling proteins were examined by Western blot analysis. (B) Western blot analysis of phosphorylated IRF3 and total IRF3 in peritoneal macrophages pretreated with increasing concentrations of MLN4924 for 2 h and then stimulated with LPS. (C) Mouse peritoneal macrophages were pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS. Nuclear fractions were extracted and IRF3 was examined by Western blot analysis. (D) Mouse peritoneal macrophages were pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS for 1 h. ChIP assay was used to assess the binding of IRF3 to the IRF3 binding sites within the 2200 to 241 region of the murine IFN-b promoter. Total extract was used as a loading control. PCR products from the amplication of an IRF3 site–free region within 2391 to 2261 of the murine IFN-b promoter were used as speciﬁcity controls. The Journal of Immunology 5

FIGURE 4. MLN4924 inhibits IFN-b production in a neddylation-independent manner. (A) Western blot analysis of NEDD8 expression in HeLa or Downloaded from HEK293T cells transfected with scramble siRNA, NEDD8 siRNA 1, NEDD8 siRNA 2, or NEDD8 siRNA 3 for 48 h. (B)HeLaorHEK293Tcellswere transfected with scramble siRNA, NEDD8 siRNA 2, or NEDD8 siRNA 3 for 24 h and then transfected with IFN-b reporter plasmid for the next 24 h. The cells were treated as indicated and the luciferase activity was measured. (C) HEK293T cells were transfected with scramble siRNA, NEDD8 siRNA 2, or NEDD8 siRNA 3 for 24 h and then transfected with IFN-b reporter plasmid, along with TRIF plasmid, for the next 24 h. The luciferase activity was measured. (D)RT- PCR analyses of IFN-b,TNF-a, and IL-6 production in THP-1 cells transfected with scramble siRNA, NEDD8 siRNA 2, or NEDD8 siRNA 3 and then stimulated with LPS for 2 h. (E) HEK293T cells were transfected with scramble siRNA, NEDD8 siRNA 2, or NEDD8 siRNA 3 for 24 h and then transfected with IFN-b reporter plasmid, along with IRF3 5D and IRF3 5D plus NEDP1, for the next 24 h. (F) HEK293T cells were transfected with scramble siRNA, http://www.jimmunol.org/ NEDD8 siRNA 2, or NEDD8 siRNA 3 for 24 h and then transfected with IFN-b reporter plasmid for the next 24 h. The cells were treated as indicated and the luciferase activity was measured. Data are shown as means 6 SD (n = 6) of one representative experiment. **p , 0.01, :p . 0.05.

STAT1 phosphorylation (28), and neddylation is crucial for (17). Multiple chemical inhibitors could exert their functions with the activation of Smurf1 (29). We then investigated whether atypical mechanisms. Previously, we have reported that lithium, a the promoting effects of MLN4924 on STAT1 phosphorylation classical glycogen synthase kinase-3b inhibitor and long-term mood were Smurf1-dependent. siRNA targeting Smurf1 was used to stabilizer in the treatment of psychiatric diseases, could attenuate inhibit endogenous Smurf1 expression in peritoneal macro- TLR3/4- and RIG-I–mediated IFN-b production and antiviral re- by guest on September 28, 2021 phages (Fig. 5E). MLN4924-mediated enhancement of STAT1 sponse via inhibition of TBK1 kinase activity, with a glycogen phosphorylation was greatly inhibited in Smurf1 knockdown synthase kinase-3b–independent manner (33). LY294002, a PI3K macrophages (Fig. 5F). Furthermore, MLN4924 also inhibited inhibitor, could inhibit TLR3/4-mediated IFN-b production via LPS-induced ISG15 expression in Smurf1 knockdown macro- inhibition of IRF3 activation with a PI3K-independent mechanism phages (Fig. 5G). Taken together, these data indicated that (34). In the present study, we reported that MLN4924 inhibited MLN4924 differentially regulated LPS- and IFN-b–induced IFN-b expression both in vitro and in vivo, with a neddylation- signaling. With the coaction of decreased expression of IFN-b independent manner. MLN4924 greatly inhibited LPS-, poly(I:C)-, and enhanced activation of the JAK/STAT pathway, MLN4924 and SeV-induced IFN-b production in multiple cell types (Fig. 1), had no effects on the expression of IFN-b–inducible genes and whereas NEDD8 knockdown could not attenuate IFN-b production subsequent antiviral immune responses. (Fig. 4). Additionally, NEDP1 overexpression could not inhibit IRF3 5D–induced IFN-b activation (Fig. 4E). These data suggested that MLN4924 inhibits IFN-b production in vivo neddylation inhibition could not regulate TLR3/4- and RIG-I– To further investigate whether MLN4924 attenuates IFN-b pro- induced IFN-b expression. MLN4924 exerted its inhibitory duction in vivo, IFN-b secretion in blood was measured in mice effects with a neddylation-independent mechanism. Furthermore, i.p. injected with MLN4924 for 1 h, followed by LPS or poly(I:C) we found that MLN4924 inhibited IRF3 transcriptional activation challenge. As shown in Fig. 6, mice administered with MLN4924 (Fig. 2B–D) and binding to IFN-b promoter (Fig. 3D). exhibited a signiﬁcant decrease in the level of IFN-b in the serum, Although we found that MLN4924 attenuated IFN-b expression, indicating that MLN4924 inhibits IFN-b production in vivo. the expression of several IFN-b–inducible genes (such as ISG15, IFIT1, and IFIT2) could not be inhibited following MLN4924 Discussion treatment (Fig. 5A). Additionally, MLN4924 also had no effects MLN4924, as a selective NAE inhibitor, regulates multiple signal on VSV replication (Fig. 5B, 5C). Secreted IFN-b triggers the pathways by inhibiting neddylation of target molecules (12). For JAK/STAT pathway and initiates the transcription of various example, MLN4924 blocks neddylation-dependent ubiquitylation antiviral genes, including ISG15, IFIT1, and IFIT2 (6, 27). The of Ku and then inhibits release of Ku from DNA-damage sites STATs that are activated in response to type I IFNs include (30). MLN4924 increases the size of the nucleolus and activates STAT1, STAT2, and others. Smurf1 could inhibit STAT1 phos- p53 through the ribosomal–Mdm2 pathway (31). MLN4924 sup- phorylation (28), and neddylation is crucial for the activation of presses AKT and mTOR signaling via upregulation of REDD1 in Smurf1 (29). We also found that MLN4924 enhanced the phos- human myeloma cells (32). MLN4924 inhibits Vpx/Vpr-induced phorylation of STAT1 (Fig. 5D). MLN4924-mediated enhancement SAMHD1 degradation by inhibiting the neddylation of E3 ubiq- of STAT1 phosphorylation was greatly inhibited in Smurf1 knock- uitin ligase and blocks macaque SIV replication in myeloid cells down macrophages (Fig. 5F). MLN4924 also inhibited LPS-induced 6 MLN4924 INHIBITS IFN-b PRODUCTION Downloaded from http://www.jimmunol.org/

FIGURE 5. MLN4924 has no effects on VSV replication. ( ) RT-PCR analyses of ISG15, IFIT1, IFIT2, or IFN-a expression in peritoneal macrophages by guest on September 28, 2021 pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS or poly(I:C) for 2 h. (B and C) HeLa cells (1 3 105) were pretreated with DMSO or MLN4924 (100 and 500 nM) for 2 h and then infected with VSV-GFP (multiplicity of infection of 0.1) for 16 h, and then imaged by microscopy (B)or analyzed by RT-PCR (C). Original magniﬁcation 340. (D) Mouse peritoneal macrophages were pretreated with DMSO or MLN4924 for 2 h and then stimulated with IFN-b (20 ng/ml) for indicated time period. Phosphorylated STAT1 was examined by Western blot analysis. (E) Western blot analysis of Smurf1 expression in mouse peritoneal macrophages transfected with scramble siRNA or Smurf1 siRNA for 48 h. (F) Mouse peritoneal macrophages were transfected with scramble siRNA or Smurf1 siRNA for 48 h and then pretreated with DMSO or MLN4924 for 2 h and then stimulated with IFN-b (20 ng/ml) for 1 h. Phosphorylated STAT1 was examined by Western blot analysis. (G) Mouse peritoneal macrophages were transfected with scramble siRNA or Smurf1 siRNA for 48 h and then pretreated with DMSO or MLN4924 for 2 h and then stimulated with LPS for 2 h. ISG15 expression was examined by RT-PCR. Data are shown as means 6 SD (n = 3) of one representative experiment. **p , 0.01, :p . 0.05.

ISG15 expression in Smurf1 knockdown macrophages (Fig. 5G). Thus, activation of the JAK/STAT pathway, MLN4924 had no effects on the MLN4924 may promote STAT1 phosphorylation by inhibiting neddy- expression of IFN-b–inducible genes and subsequent antiviral im- lation of Smurf1, and then promote activation of the JAK/STAT path- mune responses. However, the exact mechanisms of neddylation and way. With the coaction of decreased expression of IFN-b and enhanced MLN4924inJAK/STATpathwayneedtobefurtherinvestigated.

FIGURE 6. MLN4924 inhibits IFN-b production in vivo. Female C57BL/6J mice (6 wk old, 3 mice per group) were pretreated with DMSO or 10 mg/kg MLN4924 i.p. administration for 2 h, and then treated with PBS, 1.8 mg/kg LPS, or 1 mg/kg poly(I:C) i.p. administration for 1 h. IFN-b in the serum was detected by ELISA. The Journal of Immunology 7

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