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Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-κB activator
Biochemical and Biophysical Research Communications 410 (2011) 247–251
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-jB activator Shanshan Yu a,1, Bo Gao a,1, Zhijian Duan a, Wei Xu a, Sidong Xiong a,b,⇑ a b
Institute for Immunobiology, Department of Immunology, Shanghai Medical College of Fudan University, Shanghai 200032, PR China Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou 215006, PR China
a r t i c l e
i n f o
Article history: Received 15 May 2011 Available online 27 May 2011 Keywords: TRIM22 NF-jB Pro-inflammatory cytokine PDTC
a b s t r a c t Increasing evidence suggests that TRIM family proteins may play important roles in the regulation of innate immune signaling pathways. Here we report TRIM22 is involved in the activation of NF-jB. It was found that overexpression of TRIM22 could dose-dependently activate NF-jB as demonstrated by reporter gene assay and electrophoretic mobility shift assay, but had no effect on the activity of other transcription factors, including NF-AT, AP-1, C/EBP and IRFs. Further study showed that both the N-terminal RING domain and C-terminal SPRY domain were crucial for TRIM22-mediated NF-jB activation. Moreover, our results revealed that TRIM22 overexpression could significantly induce the secretion of pro-inflammatory cytokines by human macrophage cell line U937 in an NF-jB-dependent manner. These data suggested that TRIM22 was a positive regulator of NF-jB-mediated transcription. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Tripartite motif (TRIM) family proteins are involved in diverse cell processes, including apoptosis, differentiation and transcriptional regulation [1]. Recent studies show that some TRIM proteins, such as TRIM5a, TRIM19, TRIM22 and TRIM28, have antiviral activity [2–6]. Interestingly, there is growing evidence that some TRIM proteins, especially those induced by interferons (IFNs), play an important role in regulating signaling pathways involved in innate immune response [7]. For example, TRIM25 is essential for RIG-I-mediated antiviral activity [8]; TRIM21 is involved in the regulation of some pro-inflammatory cytokines through modulating multiple interferon regulatory factors (IRFs) [9,10]; and TRIM19 has been reported to negatively regulate NF-jB activity through recruiting NF-jB to nuclear bodies [11]. It is well established that regulation of immune signaling is crucial for antiviral and inflammatory responses. Several transcription factors are reported to be critical for regulating immune signaling, including nuclear factor-jB (NF-jB), activator protein-1 (AP-1), nuclear factor of activated T cells (NF-AT), CCAAT/enhancer binding protein (C/EBP) and IRFs [12–16]. Of these transcription factors, NF-jB receives particular attentions. Many studies have shown that NF-jB plays important roles in immune regulation and inflammation through the induction of a large set of downstream genes, including cytokines, chemokines, adhesion molecules and effectors [17].
TRIM22 was originally identified as an IFN-inducible protein in Daudi cells and found to possess antiviral activity against HIV [4]. Our previous data demonstrated that TRIM22 was one of the most strongly induced TRIM family molecules in response to IFNs stimulation in HepG2 cells and could inhibit HBV replication efficiently in a RING-domain-dependent manner [5]. Additionally, TRIM22 was identified as a RING finger E3 ubiquitin ligase in our previous study [18], and its E3 ligase activity was reported to be crucial for its antiviral activity against encephalomyocarditis virus [19]. Furthermore, TRIM22 was also revealed to be implicated in some inflammatory diseases, such as systemic lupus erythematosus (SLE) [20]. In consideration of the importance of TRIM22 in both antiviral responses and inflammatory diseases, we sought to investigate whether the IFN-inducible TRIM22 could contribute to the regulation of immune signaling in this study. We found that TRIM22 could dose-dependently activate NF-jB, but not other transcription factors, such as AP-1, NF-AT, C/EBP and IRFs. Both N terminal RING domain and C terminal SPRY domain was crucial for the TRIM22-mediated activation of NF-jB. Further study showed that TRIM22 expression could induce the production of pro-inflammatory cytokines in U937, which could be blocked by NF-jB inhibitor PDTC.
2. Materials and methods 2.1. Plasmids
⇑ Corresponding author. Address: Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou 215006, PR China. Fax: +86 512 65113488. E-mail address: [email protected] (S. Xiong). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.124
Plasmids expressing myc-tagged TRIM22 and its mutants were described in our previous study [5,18]. The reporter plasmids pNF-jB-Luc, pAP-1-Luc, pNF-AT-Luc, pC/EBP-Luc and pIRF-E-Luc containing firefly luciferase gene were obtained from Stratagene
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S. Yu et al. / Biochemical and Biophysical Research Communications 410 (2011) 247–251
(Stratagene, La Jolla, CA). The plasmid pRL-SV40 containing Renilla luciferase gene was purchased from Promega (Promega, Madison, WI). 2.2. Cell culture and transfection Human embryonic kidney cells HEK293 were maintained in DMEM (Gibco, UK) supplemented with 10% fetal calf serum (FCS, Gibco, UK) and 100 U/ml penicillin and 100 lg/ml streptomycin. U937 cells were maintained in RPMI 1640 medium (Gibco, UK) supplemented with 10% FCS and 100 U/ml penicillin and 100 lg/ ml streptomycin. 293 cells were transfected with indicated plasmids by the calcium phosphate precipitation method. For the transfection of U937 cells, 1 106 cells were resuspended in 100 ll of Cell Line Nucleofector Solution C (Amaxa GmbH, Köln, Germany) and nucleofected with 2 lg of indicated plasmids. 2.3. Luciferase assays Luciferase reporter plasmids were transfected into cells together with plasmids encoding TRIM22 or its mutants. pRL-SV40 was co-transfected in each experiment as an internal control for transfection. At 24 h after transfection, the cells were harvested with the addition of cell lysis buffer and the luciferase assay was performed according to the manufacturer’s instructions (Promega, Madison, WI). The luciferase activity was calculated as percentage of the empty pcDNA vector and the values were normalized to the activity of Renilla luciferase in the cell lysates. 2.4. Electrophoretic mobility shift assay (EMSA) Nuclear protein extracts were made as described previously [21]. A total of 8 lg of nuclear proteins was preincubated on ice with 2 lg of poly(deoxyinosine–deoxycytosine) as an unspecific competitor and 1 lg of sonificated sperm DNA in band shift buffer (50 mM Tris, 150 mM KCl, 5% glycerol, 10 mM MgCl2 and 0.1% NP-40, 5 mM EDTA, 2.5 mM DTT) for 15 min. Biotin-labeled oligonucleotides containing the NF-jB binding sequence (50 GGGGACTTTCCC-30 ) were then added in a total volume of 20 ll, incubated on ice for 20 min, and loaded onto 6% native polyacrylamide gels in 0.5 Tris–borate–EDTA buffer. The gels were blotted on nylon membrane, and the blot was cross-linked by UV irradiation. Biotin-labeled probe was detected by a Lightshift Chemiluminescent EMSA kit (Pierce, Rockford, IL) according to the manufacturer’s recommendations. 2.5. Immunofluorescence microscopy 293 cells were transfected with plasmids expressing myctagged TRIM22 and its mutants. At 48 h after transfection, cells on coverslips were fixed in 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100, 1% BSA in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h at room temperature. Cells were then incubated with anti-myc antibody (9E10, 1:500 dilution) (Santa Cruz, CA) overnight at 4 °C, followed by appropriate secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (eBiosicences, San Diego, CA). After stained with 40 ,6-diamidino-2phenylindole (DAPI) (Santa Cruz, CA), cells were examined under a confocal laser-scanning microscope (Leica, Germany). 2.6. ELISA The concentrations of TNF-a, IL-6, IL-1b and IL-10 in the cell culture supernatants of U937 were determined by commercially available ELISA Kits (eBiosicences, San Diego, CA).
2.7. Statistics The data represent the means ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test. A P < 0.05 was considered to be significant. 3. Results 3.1. TRIM22 selectively activates NF-jB To investigate the possible role of TRIM22 in the regulation of immune signaling, we examined the effect of TRIM22 expression on the activity of several transcription factors, including NF-jB, AP-1, NF-AT, C/EBP and IRFs, by reporter gene assay. As shown in Fig. 1A, overexpression of TRIM22 could activate NF-jB in a dose-dependent manner, while had slight or no effect on the activity of other transcription factors, suggesting that TRIM22 could selectively activate NF-jB. To further investigate the effect of TRIM22 on NF-jB activity, we performed EMSA by incubating nuclear extracts from empty vector or TRIM22 expression plasmid (pTRIM22)-transfected cells with biotin-labeled NF-jB consensus oligonucleotide. The EMSA results also showed that TRIM22 could dose-dependently activate NF-jB (Fig. 1B). 3.2. The contribution of N-terminal RING domain and C-terminal SPRY domain to the TRIM22-mediated NF-jB activation TRIM22 contains a cluster of a RING-finger domain, a B box/ coiled coil domain (B Box/CCD) and a SPRY domain [1]. Our previous investigation demonstrated that TRIM22 was a RING finger E3 ligase [18], and both its RING and SPRY domain were crucial for its anti-HBV activity [5]. We next investigated the role of RING or SPRY domain in TRIM22-mediated activation of NF-jB. Results showed that its RING domain deletion mutant (TRIM22-DRING) and SPRY domain deletion mutant (TRIM22-DSPRY) lost the ability to activate NF-jB (Fig. 2B). We also investigated the intracellular localization of wild-type or mutant TRIM22 in 293 cells using immunofluorescence staining. It was found that wild-type TRIM22 and TRIM22-DRING were predominantly localized in the nucleus, while TRIM22-DSPRY was localized exclusively in the cytoplasm, consistent with what we reported in HepG2 cells [5]. The conserved cysteine residue at position 15 is critical for RING domain-mediated biological functions [4,5]. To further investigate the role of RING domain in the TRIM22-mediated NF-jB activation, the 15th cysteine of RING domain was substituted into the alanine (TRIM22-C15A) (Fig. 2A). Just as the TRIM22-DRING, the TRIM22C15A was also localized in the nuclei of 293 cells (Fig. 2C) and lost the ability to activate NF-jB (Fig. 2B), further demonstrating the crucial role of the nuclear-located RING domain in TRIM22-mediated NF-jB activation. 3.3. TRIM22 induces the secretion of pro-inflammatory cytokines It is well documented that NF-jB plays a central role in the inflammatory processes by augmenting the expression of inflammatory genes. We therefore investigated whether TRIM22 itself could induce the production of pro-inflammatory cytokines by macrophages. We transfected human macrophage cell line U937 with plasmids expressing TRIM22 or its mutants. Posttransfection (12 h), cells were differentiated with phorbol myristate acetate (PMA) for another 24 h. Cell supernatants were then assayed for the pro-inflammatory cytokines, including TNF-a, IL-6 and IL-1b. As shown in Fig. 3, we found that the secretion of TNF-a, IL-6 and IL-1b from pTRIM22-transfected U937 cells was significantly higher than that from the control groups
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Fig. 1. TRIM22 activates NF-jB, but not other transcription factors. (A) Indicated amounts of pTRIM22 and 0.1 lg of pRL-SV40 were transfected into 293 cells together with 0.1 lg of pNF-jB-Luc, pAP-1-Luc, pNF-AT-Luc, pC/EBP-Luc and pIRF-E-Luc, respectively, 24 h post-transfection, firefly luciferase activities were measured and normalized to Renilla luciferase activities. Results were representative of three experiments. (B) EMSA was performed with the nuclear protein extracted from empty vector or pTRIM22transfected 293 cells and biotin-labeled NF-jB probe.
Fig. 2. The contribution of RING and SPRY domain to TRIM22-mediated activation of NF-jB. (A) Schematic diagram of wild-type or mutant TRIM22. (B) TRIM22 plasmids were co-transfected with pNF-jB-Luc into 293 cells. Post-transfection (24 h), the luciferase activity was measured. Results were representative of three experiments. (C) The myc-tagged plasmids expressing wild-type TRIM22, deletion mutants and point mutants were transfected into 293 cells. Post-transfection (24 h), intracellular localization of TRIM22 or its mutants were detected by indirect immunofluorescence staining using anti-myc antibody and FITC anti-mouse antibody and examined under a confocal laserscanning microscopy. The nuclei were counterstained with DAPI.
(P < 0.05), while the secretion of those pro-inflammatory cytokines from TRIM-DSPRY-, TRIM22-DRING- or TRIM22-C15Atransfected U937 cells was comparable to that from the control group. Additionally, we also tested the effect of TRIM22 expression on the production of IL-10, which has been shown to be regulated by a variety of transcription factors, including Jun proteins, NF-AT and IRF4 [22–24]. It was found TRIM22 expression did not affect the production of IL-10 (Fig. 3). These data suggested that overexpression of TRIM22, but not its mutants, could induce the production of pro-inflammatory cytokines from macrophages.
3.4. TRIM22 induces the expression of pro-inflammatory cytokines, which is dependent on NF-jB activation To further investigate whether the induction of pro-inflammatory cytokines by TRIM22 was dependent on its effect on NF-jB activity, PDTC, an inhibitor of IjBa, was used. PDTC inhibits the dissociation of NF-jB/IjBa complex, thereby suppressing the activation of NF-jB. U937 cells transfected with pTRIM22 were treated with or without 20 lM PDTC for 1 h. Cells were then differentiated with 20 nM PMA, and the production of pro-inflammatory cytokines was assayed. Results showed that pTRIM22-transfected
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Fig. 3. TRIM22 stimulates U937 cells to secret pro-inflammatory cytokines. Plasmids expressing TRIM22, deletion mutants and point mutants were transfected into U937 cells. Post-transfection (12 h), PMA was added to the cell supernatants to a final concentration of 20 nM. Post-differentiation (24 h), the expression of TNF-a (A), IL-6 (B), IL-1b (C) and IL-10 (D) from differentiated U937 were measured by ELISA. Results were representative of three experiments.
Fig. 4. The induction of pro-inflammatory cytokines by TRIM22 is dependent on its effect on NF-jB activity. U937 cells were transfected with pTRIM22 or empty vector. Post-transfection (12 h), cells were incubated with or without 20 lM PDTC for 1 h and then differentiated with 20 nM PMA. Post-differentiation (24 h), the expression of TNF-a, IL-6 and IL-1b from differentiated U937 were measured by ELISA. Results were representative of three experiments.
U937 cells secreted much less TNF-a, IL-6 and IL-1b upon PDTC treatment than those without PDTC treatment (Fig. 4). 4. Discussion Members of TRIM family proteins are regarded as a part of innate immune system to counter intracellular pathogens. In our previous investigations, we found TRIM22 was strongly induced by IFNs, and played an important role in antiviral innate immunity against HBV [5]. Additionally, TRIM22 was also implicated in the antiviral immunity against several other viruses, and in the inflammatory diseases, such as SLE [19,20]. However, the precise mechanisms by which TRIM22 performs its biological functions remain largely unknown. In this paper, we found that TRIM22 was involved in immune signaling by regulating NF-jB activity,
which may be useful for deciphering the molecular mechanisms of TRIM22-mediated biological functions. It is well established that transcription factors, such as NF-jB, AP-1, NF-AT, C/EBP and IRFs, are involved in the regulation of immune signaling. Recent studies show that some TRIM family proteins play important roles in innate immune regulation via controlling the activity of these transcription factors. For example, the NF-jB activity can be regulated by TRIM19, TRIM21, TRIM23 and TRIM30a [10,25–27]; the transcriptional activity of AP-1 has been reported to be inhibited by TRIM45 [28]; TRIM19 is implicated in the regulation of NF-AT and C/EBP activity [29,30]; and TRIM21 is also reported to be a regulator of IRF3 and IRF8 [9,10]. To investigate whether TRIM22 was involved in the regulation of immune signaling, we examined the effect of TRIM22 expression on the activity of NF-jB, AP-1, NF-AT, C/EBP and IRFs. Results showed that TRIM22 could dose-dependently induce the activation of NF-jB, but not other transcription factors. TRIM22 is a RING finger protein, and the conserved 15th and 18th cysteines of its RING domain are especially important for its biological functions [4,5]. In this study, we found that once its RING domain deleted or the 15th cysteine mutated into alanine, TRIM22 would lose its ability to activate NF-jB. Interestingly, we and other groups demonstrated that RING domain was also crucial for TRIM22-mediated antiviral activity and its E3 ubiquitin ligase activity [5,19]. In our previous study, C-terminal SPRY domain was demonstrated to be important for TRIM22-mediated antiHBV activity. Additionally, the SPRY domain of TRIM5a was also revealed to be crucial for TRIM5a-mediated anti-HIV activity [2]. We therefore tested the contribution of SPRY domain to TRIM22mediated activation of NF-jB. Our results showed that deletion of SPRY domain, which was required for the nuclear localization of TRIM22, would lead to the inability of TRIM22 to activate NFjB. Combined with our previous study [18], we speculate that TRIM22 might regulate the activity of NF-jB via inducing
S. Yu et al. / Biochemical and Biophysical Research Communications 410 (2011) 247–251
ubiquitination and degradation of the inhibitors of NF-jB in the nucleus. The transcription factor NF-jB has been shown to have effects on activation of a number of cytokine genes, particularly those associated with inflammatory and metabolic responses [31–33]. In the present study, we also demonstrated that overexpression of TRIM22 in human macrophage cell line U937 resulted in enhanced expression of pro-inflammatory cytokines, including TNF-a, IL-6 and IL-1b. By contrast, overexpression of TRIM22 had no effect on the production of IL-10. By using NF-jB inhibitor PDTC, we further revealed that the induction of pro-inflammatory cytokines by TRIM22 was dependent on its effect on NF-jB activity. As the pro-inflammatory cytokines have been implicated in antiviral immunity [34–36], we speculate that the up-regulation of the pro-inflammatory cytokines by TRIM22 might be one of the mechanisms underlying its antiviral activity, which deserves further studies. Acknowledgments This work was supported by grants of National Natural Science Foundation of China (30890141, 81072428, 30872355), Shanghai STCSM Grant (09JC1401800), Doctoral Fund of Youth Scholars of Ministry of Education of China (2009071120060), Major State Basic Research Development Program of China (2007CB512401), Jiangsu provincial Basic Research Program (Innovation Scholars Climbing Program, SBK201010093) and Program for Outstanding Medical Academic Leader of Shanghai (LJ06011). References [1] A. Reymond, G. Meroni, A. Fantozzi, et al., The tripartite motif family identifies cell compartments, EMBO J. 20 (2001) 2140–2151. [2] M. Stremlau, C.M. Owens, M.J. Perron, et al., The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys, Nature 427 (2004) 848–853. [3] M.K. Chelbi-Alix, F. Quignon, L. Pelicano, et al., Resistance to virus Infection conferred by the interferon-induced promyelocytic leukemia protein, J. Virol. 72 (1998) 1043–1051. [4] S.D. Barr, J.R. Smiley, F.D. Bushman, et al., The interferon response inhibits HIV particle production by induction of TRIM22, PLoS Pathog. 4 (2008) e1000007. [5] B. Gao, Z. Duan, W. Xu, et al., Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain, Hepatology 50 (2009) 424–433. [6] D. Wolf, K. Hug, S.P. Goff, TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells, Proc. Natl. Acad. Sci. USA 105 (2008) 12521–12526. [7] F.W. McNab, R. Rajsbaum, J.P. Stoye, A. O’Garra, Tripartite-motif proteins and innate immune regulation, Curr. Opin. Immunol. 23 (2011) 46–56. [8] M.U. Gack, Y.C. Shin, C.H. Joo, et al., TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity, Nature 446 (2007) 916–920. [9] K. Yang, H.X. Shi, X.Y. Liu, et al., TRIM21 is essential to sustain IFN regulatory factor 3 activation during antiviral response, J. Immunol. 182 (2009) 3782– 3792. [10] H.J. Kong, D.E. Anderson, C.H. Lee, et al., Cutting edge: autoantigen Ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression in macrophages, J. Immunol. 179 (2007) 26–30. [11] W.S. Wu, Z.X. Xu, W.N. Hittelman, et al., Promyelocytic leukemia protein sensitizes tumor necrosis factor alpha-induced apoptosis by inhibiting the NFkappaB survival pathway, J. Biol. Chem. 278 (2003) 12294–12304.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Identification of tripartite motif-containing 22 (TRIM22) as a novel NF-jB activator Shanshan Yu a,1, Bo Gao a,1, Zhijian Duan a, Wei Xu a, Sidong Xiong a,b,⇑ a b
Institute for Immunobiology, Department of Immunology, Shanghai Medical College of Fudan University, Shanghai 200032, PR China Jiangsu Key Laboratory of Infection and Immunity, Institute of Biology and Medical Sciences, Soochow University, Suzhou 215006, PR China
a r t i c l e
i n f o
Article history: Received 15 May 2011 Available online 27 May 2011 Keywords: TRIM22 NF-jB Pro-inflammatory cytokine PDTC
a b s t r a c t Increasing evidence suggests that TRIM family proteins may play important roles in the regulation of innate immune signaling pathways. Here we report TRIM22 is involved in the activation of NF-jB. It was found that overexpression of TRIM22 could dose-dependently activate NF-jB as demonstrated by reporter gene assay and electrophoretic mobility shift assay, but had no effect on the activity of other transcription factors, including NF-AT, AP-1, C/EBP and IRFs. Further study showed that both the N-terminal RING domain and C-terminal SPRY domain were crucial for TRIM22-mediated NF-jB activation. Moreover, our results revealed that TRIM22 overexpression could significantly induce the secretion of pro-inflammatory cytokines by human macrophage cell line U937 in an NF-jB-dependent manner. These data suggested that TRIM22 was a positive regulator of NF-jB-mediated transcription. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Tripartite motif (TRIM) family proteins are involved in diverse cell processes, including apoptosis, differentiation and transcriptional regulation [1]. Recent studies show that some TRIM proteins, such as TRIM5a, TRIM19, TRIM22 and TRIM28, have antiviral activity [2–6]. Interestingly, there is growing evidence that some TRIM proteins, especially those induced by interferons (IFNs), play an important role in regulating signaling pathways involved in innate immune response [7]. For example, TRIM25 is essential for RIG-I-mediated antiviral activity [8]; TRIM21 is involved in the regulation of some pro-inflammatory cytokines through modulating multiple interferon regulatory factors (IRFs) [9,10]; and TRIM19 has been reported to negatively regulate NF-jB activity through recruiting NF-jB to nuclear bodies [11]. It is well established that regulation of immune signaling is crucial for antiviral and inflammatory responses. Several transcription factors are reported to be critical for regulating immune signaling, including nuclear factor-jB (NF-jB), activator protein-1 (AP-1), nuclear factor of activated T cells (NF-AT), CCAAT/enhancer binding protein (C/EBP) and IRFs [12–16]. Of these transcription factors, NF-jB receives particular attentions. Many studies have shown that NF-jB plays important roles in immune regulation and inflammation through the induction of a large set of downstream genes, including cytokines, chemokines, adhesion molecules and effectors [17].
TRIM22 was originally identified as an IFN-inducible protein in Daudi cells and found to possess antiviral activity against HIV [4]. Our previous data demonstrated that TRIM22 was one of the most strongly induced TRIM family molecules in response to IFNs stimulation in HepG2 cells and could inhibit HBV replication efficiently in a RING-domain-dependent manner [5]. Additionally, TRIM22 was identified as a RING finger E3 ubiquitin ligase in our previous study [18], and its E3 ligase activity was reported to be crucial for its antiviral activity against encephalomyocarditis virus [19]. Furthermore, TRIM22 was also revealed to be implicated in some inflammatory diseases, such as systemic lupus erythematosus (SLE) [20]. In consideration of the importance of TRIM22 in both antiviral responses and inflammatory diseases, we sought to investigate whether the IFN-inducible TRIM22 could contribute to the regulation of immune signaling in this study. We found that TRIM22 could dose-dependently activate NF-jB, but not other transcription factors, such as AP-1, NF-AT, C/EBP and IRFs. Both N terminal RING domain and C terminal SPRY domain was crucial for the TRIM22-mediated activation of NF-jB. Further study showed that TRIM22 expression could induce the production of pro-inflammatory cytokines in U937, which could be blocked by NF-jB inhibitor PDTC.
2. Materials and methods 2.1. Plasmids
⇑ Corresponding author. Address: Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou 215006, PR China. Fax: +86 512 65113488. E-mail address: [email protected] (S. Xiong). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.05.124
Plasmids expressing myc-tagged TRIM22 and its mutants were described in our previous study [5,18]. The reporter plasmids pNF-jB-Luc, pAP-1-Luc, pNF-AT-Luc, pC/EBP-Luc and pIRF-E-Luc containing firefly luciferase gene were obtained from Stratagene
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(Stratagene, La Jolla, CA). The plasmid pRL-SV40 containing Renilla luciferase gene was purchased from Promega (Promega, Madison, WI). 2.2. Cell culture and transfection Human embryonic kidney cells HEK293 were maintained in DMEM (Gibco, UK) supplemented with 10% fetal calf serum (FCS, Gibco, UK) and 100 U/ml penicillin and 100 lg/ml streptomycin. U937 cells were maintained in RPMI 1640 medium (Gibco, UK) supplemented with 10% FCS and 100 U/ml penicillin and 100 lg/ ml streptomycin. 293 cells were transfected with indicated plasmids by the calcium phosphate precipitation method. For the transfection of U937 cells, 1 106 cells were resuspended in 100 ll of Cell Line Nucleofector Solution C (Amaxa GmbH, Köln, Germany) and nucleofected with 2 lg of indicated plasmids. 2.3. Luciferase assays Luciferase reporter plasmids were transfected into cells together with plasmids encoding TRIM22 or its mutants. pRL-SV40 was co-transfected in each experiment as an internal control for transfection. At 24 h after transfection, the cells were harvested with the addition of cell lysis buffer and the luciferase assay was performed according to the manufacturer’s instructions (Promega, Madison, WI). The luciferase activity was calculated as percentage of the empty pcDNA vector and the values were normalized to the activity of Renilla luciferase in the cell lysates. 2.4. Electrophoretic mobility shift assay (EMSA) Nuclear protein extracts were made as described previously [21]. A total of 8 lg of nuclear proteins was preincubated on ice with 2 lg of poly(deoxyinosine–deoxycytosine) as an unspecific competitor and 1 lg of sonificated sperm DNA in band shift buffer (50 mM Tris, 150 mM KCl, 5% glycerol, 10 mM MgCl2 and 0.1% NP-40, 5 mM EDTA, 2.5 mM DTT) for 15 min. Biotin-labeled oligonucleotides containing the NF-jB binding sequence (50 GGGGACTTTCCC-30 ) were then added in a total volume of 20 ll, incubated on ice for 20 min, and loaded onto 6% native polyacrylamide gels in 0.5 Tris–borate–EDTA buffer. The gels were blotted on nylon membrane, and the blot was cross-linked by UV irradiation. Biotin-labeled probe was detected by a Lightshift Chemiluminescent EMSA kit (Pierce, Rockford, IL) according to the manufacturer’s recommendations. 2.5. Immunofluorescence microscopy 293 cells were transfected with plasmids expressing myctagged TRIM22 and its mutants. At 48 h after transfection, cells on coverslips were fixed in 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100, 1% BSA in PBS for 10 min, and blocked with 1% BSA in PBS for 1 h at room temperature. Cells were then incubated with anti-myc antibody (9E10, 1:500 dilution) (Santa Cruz, CA) overnight at 4 °C, followed by appropriate secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (eBiosicences, San Diego, CA). After stained with 40 ,6-diamidino-2phenylindole (DAPI) (Santa Cruz, CA), cells were examined under a confocal laser-scanning microscope (Leica, Germany). 2.6. ELISA The concentrations of TNF-a, IL-6, IL-1b and IL-10 in the cell culture supernatants of U937 were determined by commercially available ELISA Kits (eBiosicences, San Diego, CA).
2.7. Statistics The data represent the means ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using Student’s t test. A P < 0.05 was considered to be significant. 3. Results 3.1. TRIM22 selectively activates NF-jB To investigate the possible role of TRIM22 in the regulation of immune signaling, we examined the effect of TRIM22 expression on the activity of several transcription factors, including NF-jB, AP-1, NF-AT, C/EBP and IRFs, by reporter gene assay. As shown in Fig. 1A, overexpression of TRIM22 could activate NF-jB in a dose-dependent manner, while had slight or no effect on the activity of other transcription factors, suggesting that TRIM22 could selectively activate NF-jB. To further investigate the effect of TRIM22 on NF-jB activity, we performed EMSA by incubating nuclear extracts from empty vector or TRIM22 expression plasmid (pTRIM22)-transfected cells with biotin-labeled NF-jB consensus oligonucleotide. The EMSA results also showed that TRIM22 could dose-dependently activate NF-jB (Fig. 1B). 3.2. The contribution of N-terminal RING domain and C-terminal SPRY domain to the TRIM22-mediated NF-jB activation TRIM22 contains a cluster of a RING-finger domain, a B box/ coiled coil domain (B Box/CCD) and a SPRY domain [1]. Our previous investigation demonstrated that TRIM22 was a RING finger E3 ligase [18], and both its RING and SPRY domain were crucial for its anti-HBV activity [5]. We next investigated the role of RING or SPRY domain in TRIM22-mediated activation of NF-jB. Results showed that its RING domain deletion mutant (TRIM22-DRING) and SPRY domain deletion mutant (TRIM22-DSPRY) lost the ability to activate NF-jB (Fig. 2B). We also investigated the intracellular localization of wild-type or mutant TRIM22 in 293 cells using immunofluorescence staining. It was found that wild-type TRIM22 and TRIM22-DRING were predominantly localized in the nucleus, while TRIM22-DSPRY was localized exclusively in the cytoplasm, consistent with what we reported in HepG2 cells [5]. The conserved cysteine residue at position 15 is critical for RING domain-mediated biological functions [4,5]. To further investigate the role of RING domain in the TRIM22-mediated NF-jB activation, the 15th cysteine of RING domain was substituted into the alanine (TRIM22-C15A) (Fig. 2A). Just as the TRIM22-DRING, the TRIM22C15A was also localized in the nuclei of 293 cells (Fig. 2C) and lost the ability to activate NF-jB (Fig. 2B), further demonstrating the crucial role of the nuclear-located RING domain in TRIM22-mediated NF-jB activation. 3.3. TRIM22 induces the secretion of pro-inflammatory cytokines It is well documented that NF-jB plays a central role in the inflammatory processes by augmenting the expression of inflammatory genes. We therefore investigated whether TRIM22 itself could induce the production of pro-inflammatory cytokines by macrophages. We transfected human macrophage cell line U937 with plasmids expressing TRIM22 or its mutants. Posttransfection (12 h), cells were differentiated with phorbol myristate acetate (PMA) for another 24 h. Cell supernatants were then assayed for the pro-inflammatory cytokines, including TNF-a, IL-6 and IL-1b. As shown in Fig. 3, we found that the secretion of TNF-a, IL-6 and IL-1b from pTRIM22-transfected U937 cells was significantly higher than that from the control groups
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Fig. 1. TRIM22 activates NF-jB, but not other transcription factors. (A) Indicated amounts of pTRIM22 and 0.1 lg of pRL-SV40 were transfected into 293 cells together with 0.1 lg of pNF-jB-Luc, pAP-1-Luc, pNF-AT-Luc, pC/EBP-Luc and pIRF-E-Luc, respectively, 24 h post-transfection, firefly luciferase activities were measured and normalized to Renilla luciferase activities. Results were representative of three experiments. (B) EMSA was performed with the nuclear protein extracted from empty vector or pTRIM22transfected 293 cells and biotin-labeled NF-jB probe.
Fig. 2. The contribution of RING and SPRY domain to TRIM22-mediated activation of NF-jB. (A) Schematic diagram of wild-type or mutant TRIM22. (B) TRIM22 plasmids were co-transfected with pNF-jB-Luc into 293 cells. Post-transfection (24 h), the luciferase activity was measured. Results were representative of three experiments. (C) The myc-tagged plasmids expressing wild-type TRIM22, deletion mutants and point mutants were transfected into 293 cells. Post-transfection (24 h), intracellular localization of TRIM22 or its mutants were detected by indirect immunofluorescence staining using anti-myc antibody and FITC anti-mouse antibody and examined under a confocal laserscanning microscopy. The nuclei were counterstained with DAPI.
(P < 0.05), while the secretion of those pro-inflammatory cytokines from TRIM-DSPRY-, TRIM22-DRING- or TRIM22-C15Atransfected U937 cells was comparable to that from the control group. Additionally, we also tested the effect of TRIM22 expression on the production of IL-10, which has been shown to be regulated by a variety of transcription factors, including Jun proteins, NF-AT and IRF4 [22–24]. It was found TRIM22 expression did not affect the production of IL-10 (Fig. 3). These data suggested that overexpression of TRIM22, but not its mutants, could induce the production of pro-inflammatory cytokines from macrophages.
3.4. TRIM22 induces the expression of pro-inflammatory cytokines, which is dependent on NF-jB activation To further investigate whether the induction of pro-inflammatory cytokines by TRIM22 was dependent on its effect on NF-jB activity, PDTC, an inhibitor of IjBa, was used. PDTC inhibits the dissociation of NF-jB/IjBa complex, thereby suppressing the activation of NF-jB. U937 cells transfected with pTRIM22 were treated with or without 20 lM PDTC for 1 h. Cells were then differentiated with 20 nM PMA, and the production of pro-inflammatory cytokines was assayed. Results showed that pTRIM22-transfected
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Fig. 3. TRIM22 stimulates U937 cells to secret pro-inflammatory cytokines. Plasmids expressing TRIM22, deletion mutants and point mutants were transfected into U937 cells. Post-transfection (12 h), PMA was added to the cell supernatants to a final concentration of 20 nM. Post-differentiation (24 h), the expression of TNF-a (A), IL-6 (B), IL-1b (C) and IL-10 (D) from differentiated U937 were measured by ELISA. Results were representative of three experiments.
Fig. 4. The induction of pro-inflammatory cytokines by TRIM22 is dependent on its effect on NF-jB activity. U937 cells were transfected with pTRIM22 or empty vector. Post-transfection (12 h), cells were incubated with or without 20 lM PDTC for 1 h and then differentiated with 20 nM PMA. Post-differentiation (24 h), the expression of TNF-a, IL-6 and IL-1b from differentiated U937 were measured by ELISA. Results were representative of three experiments.
U937 cells secreted much less TNF-a, IL-6 and IL-1b upon PDTC treatment than those without PDTC treatment (Fig. 4). 4. Discussion Members of TRIM family proteins are regarded as a part of innate immune system to counter intracellular pathogens. In our previous investigations, we found TRIM22 was strongly induced by IFNs, and played an important role in antiviral innate immunity against HBV [5]. Additionally, TRIM22 was also implicated in the antiviral immunity against several other viruses, and in the inflammatory diseases, such as SLE [19,20]. However, the precise mechanisms by which TRIM22 performs its biological functions remain largely unknown. In this paper, we found that TRIM22 was involved in immune signaling by regulating NF-jB activity,
which may be useful for deciphering the molecular mechanisms of TRIM22-mediated biological functions. It is well established that transcription factors, such as NF-jB, AP-1, NF-AT, C/EBP and IRFs, are involved in the regulation of immune signaling. Recent studies show that some TRIM family proteins play important roles in innate immune regulation via controlling the activity of these transcription factors. For example, the NF-jB activity can be regulated by TRIM19, TRIM21, TRIM23 and TRIM30a [10,25–27]; the transcriptional activity of AP-1 has been reported to be inhibited by TRIM45 [28]; TRIM19 is implicated in the regulation of NF-AT and C/EBP activity [29,30]; and TRIM21 is also reported to be a regulator of IRF3 and IRF8 [9,10]. To investigate whether TRIM22 was involved in the regulation of immune signaling, we examined the effect of TRIM22 expression on the activity of NF-jB, AP-1, NF-AT, C/EBP and IRFs. Results showed that TRIM22 could dose-dependently induce the activation of NF-jB, but not other transcription factors. TRIM22 is a RING finger protein, and the conserved 15th and 18th cysteines of its RING domain are especially important for its biological functions [4,5]. In this study, we found that once its RING domain deleted or the 15th cysteine mutated into alanine, TRIM22 would lose its ability to activate NF-jB. Interestingly, we and other groups demonstrated that RING domain was also crucial for TRIM22-mediated antiviral activity and its E3 ubiquitin ligase activity [5,19]. In our previous study, C-terminal SPRY domain was demonstrated to be important for TRIM22-mediated antiHBV activity. Additionally, the SPRY domain of TRIM5a was also revealed to be crucial for TRIM5a-mediated anti-HIV activity [2]. We therefore tested the contribution of SPRY domain to TRIM22mediated activation of NF-jB. Our results showed that deletion of SPRY domain, which was required for the nuclear localization of TRIM22, would lead to the inability of TRIM22 to activate NFjB. Combined with our previous study [18], we speculate that TRIM22 might regulate the activity of NF-jB via inducing
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ubiquitination and degradation of the inhibitors of NF-jB in the nucleus. The transcription factor NF-jB has been shown to have effects on activation of a number of cytokine genes, particularly those associated with inflammatory and metabolic responses [31–33]. In the present study, we also demonstrated that overexpression of TRIM22 in human macrophage cell line U937 resulted in enhanced expression of pro-inflammatory cytokines, including TNF-a, IL-6 and IL-1b. By contrast, overexpression of TRIM22 had no effect on the production of IL-10. By using NF-jB inhibitor PDTC, we further revealed that the induction of pro-inflammatory cytokines by TRIM22 was dependent on its effect on NF-jB activity. As the pro-inflammatory cytokines have been implicated in antiviral immunity [34–36], we speculate that the up-regulation of the pro-inflammatory cytokines by TRIM22 might be one of the mechanisms underlying its antiviral activity, which deserves further studies. Acknowledgments This work was supported by grants of National Natural Science Foundation of China (30890141, 81072428, 30872355), Shanghai STCSM Grant (09JC1401800), Doctoral Fund of Youth Scholars of Ministry of Education of China (2009071120060), Major State Basic Research Development Program of China (2007CB512401), Jiangsu provincial Basic Research Program (Innovation Scholars Climbing Program, SBK201010093) and Program for Outstanding Medical Academic Leader of Shanghai (LJ06011). References [1] A. Reymond, G. Meroni, A. Fantozzi, et al., The tripartite motif family identifies cell compartments, EMBO J. 20 (2001) 2140–2151. [2] M. Stremlau, C.M. Owens, M.J. Perron, et al., The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys, Nature 427 (2004) 848–853. [3] M.K. Chelbi-Alix, F. Quignon, L. Pelicano, et al., Resistance to virus Infection conferred by the interferon-induced promyelocytic leukemia protein, J. Virol. 72 (1998) 1043–1051. [4] S.D. Barr, J.R. Smiley, F.D. Bushman, et al., The interferon response inhibits HIV particle production by induction of TRIM22, PLoS Pathog. 4 (2008) e1000007. [5] B. Gao, Z. Duan, W. Xu, et al., Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain, Hepatology 50 (2009) 424–433. [6] D. Wolf, K. Hug, S.P. Goff, TRIM28 mediates primer binding site-targeted silencing of Lys1,2 tRNA-utilizing retroviruses in embryonic cells, Proc. Natl. Acad. Sci. USA 105 (2008) 12521–12526. [7] F.W. McNab, R. Rajsbaum, J.P. Stoye, A. O’Garra, Tripartite-motif proteins and innate immune regulation, Curr. Opin. Immunol. 23 (2011) 46–56. [8] M.U. Gack, Y.C. Shin, C.H. Joo, et al., TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity, Nature 446 (2007) 916–920. [9] K. Yang, H.X. Shi, X.Y. Liu, et al., TRIM21 is essential to sustain IFN regulatory factor 3 activation during antiviral response, J. Immunol. 182 (2009) 3782– 3792. [10] H.J. Kong, D.E. Anderson, C.H. Lee, et al., Cutting edge: autoantigen Ro52 is an interferon inducible E3 ligase that ubiquitinates IRF-8 and enhances cytokine expression in macrophages, J. Immunol. 179 (2007) 26–30. [11] W.S. Wu, Z.X. Xu, W.N. Hittelman, et al., Promyelocytic leukemia protein sensitizes tumor necrosis factor alpha-induced apoptosis by inhibiting the NFkappaB survival pathway, J. Biol. Chem. 278 (2003) 12294–12304.
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