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Interleukin-32α modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase Cɛ-dependent sumoylation
The International Journal of Biochemistry & Cell Biology 55 (2014) 136–143
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Interleukin-32␣ modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase C-dependent sumoylation Yun Sun Park a , Jeong-Woo Kang a , Dong Hun Lee a , Man Sub Kim a , Yesol Bak a , Young Yang b , Hee-Gu Lee c , Jintae Hong d , Do-Young Yoon a,∗ a
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea Research Center for Women’s Disease, Department of Life Systems, Sookmyung Women’s University, Seoul 140-742, Republic of Korea c Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea d College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju 361-463, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 17 July 2014 Accepted 21 August 2014 Available online 29 August 2014 Keywords: Interleukin 32␣ Promyelocytic leukemia zinc finger protein Small ubiquitin-like modifier-2
a b s t r a c t Interleukin-32 (IL-32) is a proinflammatory cytokine. However, there is growing evidence that IL-32 also plays a mediatory role intracellularly. In this study, we present evidence that IL-32␣ modifies and inhibits promyelocytic leukemia zinc finger (PLZF), a sequence-specific transcriptional regulator that regulates the expression of a subset of interferon (IFN)-stimulated genes (ISGs). We screened IL-32␣-interacting proteins in a human spleen cDNA library using the yeast two-hybrid assay, and investigated the functional relevance of the interaction between IL-32␣ and PLZF. We demonstrated that IL-32␣ interacts with protein kinase C (PKC)␦ and PKC in a phorbol 12-myristate 13-acetate (PMA) dependent way, and that PKC regulates the interaction of IL-32␣ with PLZF. We verified the involvement of PKC in the interaction between these proteins by using various PKC inhibitors. PLZF is known to be modified by small ubiquitin-like modifier (SUMO)-1, but it is unclear whether SUMO-2 conjugation of PLZF occurs. We showed that IL-32␣ inhibited SUMO-2-conjugation of PLZF. Further, we demonstrated that sumoylated PLZF decreased when IL-32␣ was co-expressed. PKC affected the sumoylation of PLZF only in the presence of IL-32␣ because PKC inhibitor treatment did not reduce PLZF sumoylation in the absence of IL-32␣. We finally investigated whether IL-32␣-mediated inhibition of PLZF sumoylation affected the transcriptional activity of PLZF, and demonstrated that the inhibition of sumoylation of PLZF by IL-32␣ down-regulated ISGs induced by PLZF. Together, our data suggest that IL-32␣ associates with PLZF and PKC, and then inhibits PLZF sumoylation, resulting in suppression of the transcriptional activity of PLZF. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Human interleukin-32 (IL-32) is a proinflammatory cytokine expressed in the activated natural killer cells and T-cells (Cheon et al., 2011; Dinarello and Kim, 2006). Although several variants such as ␣,  and ␥ isoforms have been known to induce inflammation, the precise role of each isoform remains elusive. IL-32 induces expression of other inflammatory cytokines such as TNF-␣, IL-1, IL-6, and IL-8 (Heinhuis et al., 2011; Imaeda et al., 2011; Kang
Abbreviations: IL-32, interleukin-32; PLZF, promyelocytic leukemia zinc finger gene; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SUMO-2, small ubiquitin-like modifier-2; ISGs, interferon-stimulated genes. ∗ Corresponding author at: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, 1 Hwayang-dong, Gwangjingu, Seoul 143-701, Republic of Korea. Tel.: +82 2 444 4218. E-mail address: [email protected] (D.-Y. Yoon). http://dx.doi.org/10.1016/j.biocel.2014.08.018 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
et al., 2009, 2012; Kim et al., 2005; Netea et al., 2005; Nold-Petry et al., 2009). IL-32 is also up regulated in various diseases, including chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and Mycobacterium tuberculosis infection (Bai et al., 2011; Calabrese et al., 2008; Joosten et al., 2006; Mun et al., 2009; Netea et al., 2006; Shioya et al., 2007). IL-32 has several isoforms such as ␣, , ␥, ␦, , and . Among these isoforms, IL-32 is abundant in various cells and IL-32␥ has been shown to display the strongest biological activity (Choi et al., 2009). All isoforms have RGD and DDX motifs. The RGD tri-peptide motif is found in extracellular matrix proteins such as fibronectin, and binds to integrin. The DDX motif is a RGD-binding site that is found on cytoskeletal proteins such as integrin (Heinhuis et al., 2012). Previous reports have indicated that IL-32 is involved in the immune response through the MAPK, NF-kappa B and AP-1 signaling pathways (Joosten et al., 2013; Nakayama et al., 2013; Pan et al., 2011).
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Fig. 1. Screening for IL-32␣-interacting proteins (IL-32␣ IPs) using a yeast two-hybrid system. IL-32␣ IPs were screened from a human spleen cDNA library using a yeast two-hybrid system. Candidate colonies were reconfirmed and grouped (right) according to their identities after sequencing, and listed in the same order as in the right figure.
Protein kinase C (PKC) regulates the functions of various proteins by phosphorylation. The PKC protein family has about ten isozymes, and is divided into the three subfamilies of classical, novel, and atypical according to the second messenger (Chang et al., 1997). PKC is activated by phorbol 12-myristate 13-aceate (PMA). The promyelocytic leukemia zinc finger (PLZF) protein belongs to the POK (POZ and Krüppel)/ZBTB (zinc finger and BTB) protein family (Dhordain et al., 2000; Lee and Maeda, 2012). PLZF is a sequence-specific DNA-binding protein that represses the transcriptional activity of target genes such as cyclin A and the IL-3 receptor ␣ chain (Bailey et al., 1999; Kang et al., 2003; Rho et al., 2010, 2006; Yeyati et al., 1999). PLZF is critical for development of the myeloid lineage (Alonzo et al., 2010; Doulatov et al., 2009; Girard et al., 2013; Ward et al., 2001). IFN-mediated signaling is known to be induced by the transcriptional activity of PLZF. PLZF modulates the expression of a large family of IFN-stimulated genes (ISGs) (Xu et al., 2009). Three types of SUMO proteins are found in vertebrates: SUMO1, -2, and -3. SUMO-2 and -3 have 95% sequence conservation. However, SUMO-1 has only 50% similarity to the other SUMOs and has different functions and properties (Herrmann et al., 2007; Kamitani et al., 1998a,b; Saitoh and Hinchey, 2000; Tatham et al., 2001). Post-translational modification by sumoylation is critical for protein function (Watts, 2013). The SUMO system is relevant to cellular processes such as gene expression, cell-cycle progression, DNA replication, and DNA repair. SUMO modification occurs in lysine residues in target genes and sumoylated sites are highly conserved (Bergink and Jentsch, 2009; Geiss-Friedlander and Melchior, 2007; Ulrich, 2009). Modification of PLZF by SUMO-1 conjugation is well known. SUMO-1 modification is required for transcriptional repression by PLZF, and this modification increases the DNA binding activity of PLZF (Chao et al., 2007; Kang et al., 2003). ZBTB1, which belongs to the POK (POZ and Krüppel)/ZBTB (zing finger and BTB) protein family like PLZF, is modulated by SUMO-2 conjugation (Matic et al., 2010). Although SUMO-1 modification of PLZF has been well characterized, it is unclear whether PLZF is also modified by SUMO-2. We previously reported that IL-32␣ interacts with PMA-stimulated PKC and that pan PKC inhibitor treatment
represses these interactions (Kang et al., 2012). In this study, we further verified our previous results and demonstrated that IL-32␣ interacts with PLZF and PKC, resulting in suppression of the transcriptional activity of PLZF. 2. Materials and methods 2.1. Reagents and cell culture The human promyelomonocytic THP-1 and human embryonic kidney 293 cells were purchased from American Type Culture Collection (ATCC; Rockville, MD). The THP-1 cells were grown in RPMI 1640 (WelGENE, Daegu, Korea) supplemented with 2 mM lglutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (Hyclone, Logan, UT). HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Hyclone). Phorbol 12-myristate 13-acetate was purchased from Sigma (St. Louis, MO). MAPK inhibitors (PD98059, SB203580, and SP100625) and PKC inhibitors (Gö6850, Gö6976, Ro-31-8220, and rottlerin) were purchased from Calbiochem (San Diego, CA). IL-32␣-expressing THP-1 stable cell lines were previously established (Kang et al., 2012). 2.2. Construction of expression vectors IL-32␣ cDNA was subcloned into pcDNA3.1 + 6× myc vector using EcoRI and XhoI. cDNAs for PKC␦ and PKC were subcloned into the pcDNA3.1 + 5× FLAG vector using EcoRI and XhoI (Kang et al., 2012). SUMO-2 and PLZF cDNAs were PCR-amplified from a human spleen cDNA library (Clontech, Palo Alto, CA). Primers sets were as follows: PLZF: sense 5 -CGC GAA TTC ATG GAT CTG ACA AAA-3 , antisense 5 -ATT CTC GAG CAC ATA GCA CAG GTA3 , SUMO-2: sense 5 -GCT GAA TTC ATG GCC GAC GAA AAG CCC-3 , antisense 5 -GAA CTC GAG CTA ACC TCC CGT CTG CTG-3 . The entire amplified PLZF gene was cloned into the pcDNA3.1 + 5× FLAG vector using EcoRI and XhoI. SUMO-2 cDNA was cloned into the pCS3MT 6× myc vector using EcoRI and XhoI.
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2.3. Yeast two-hybrid screening cDNA of IL-32␣ was cloned into the pHybLex/ZT vector (Invitrogen), a bait plasmid containing the LexA DNA binding domain. The MATCHMAKER GAL4 Two-Hybrid System 3 and Human Spleen cDNA Library were purchased from BD Biosciences (Palo Alto, CA). Yeast two-hybrid screening was performed according to the manufacturer’s protocols. Briefly, IL-32 bait fusion plasmids were cloned into pHybLex/ZT vector and amplified in E.coli using zeomycin selection. Bait plasmids were introduced into yeast strain L40 that was precultured in SD-U (-Uracil) medium and transformed clones were selected on SD-UT (-Uracil, -Tryptophan). Prey plasmid (human spleen cDNA library containing pACT2 plasmid) was introduced into the bait-transformed yeast that was precultured in SD-UT medium and selected on SD-UTLH (-Uracil, -Tryptophan, -Leucine, -Histidine). The selected colonies were streaked on SDUTLH-X-GAL plates for a second round of selection. The positive blue colonies were harvested and plasmids were extracted using ZymoprepTM II Yeast Plasmid Miniprep Kit (ZYMO Research, Irvine, CA). Extracted plasmids were amplified by introducing them into E. coli followed by purification for sequencing. The selected plasmid DNAs were identified by sequencing (Xenotech, Daejon, South Korea) and subjected to BLAST searches. 2.4. Transfection THP-1 promonocytic cells were transfected with pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 using the NeonTM transfection system (Invitrogen) according to the manufacturer’s instructions. HEK293 cells were transfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 or ubiquitin using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 2.5. Western blotting and immunoprecipitation HEK293, THP-1 EV and THP-1-IL-32␣ cells were cotransfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 or ubiquitin and then lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, 20 mM -glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Western blotting was performed using anti-myc tag antibody (Millipore, Bedford, MA); anti-FLAG-tag antibody (Sigma); anti-PKC ␦/ antibody (Santa Cruz Biotechnology); anti-IL-32 antibody KU32-52 (Kang et al., 2009; Kim et al., 2008); anti-PLZF antibody (Santa Cruz Biotechnology); anti-RSAD2 antibody (Santa Cruz biotechnology); and anti-PLSCR1 antibody (R&D). For immunoprecipitation, cell lysates were mixed with 1 g of anti-myc antibody, 1 g of anti-PKC antibody, and 1 g of anti-FLAG antibody for 1 h and then pulled down using 35 l of protein G-agarose beads (KPL, Gaithersburg, MD). 2.6. Reverse-transcription polymerase chain reaction (RT-PCR) and real-time qPCR analyses THP-1 EV and THP-1-IL-32␣ were treated with 10 nM PMA for the indicated times and then, total RNA was extracted for RT-PCR. Total RNA was extracted using the Easy-Blue total RNA extraction kit (iNtRon Biotechnology, Seoul, Korea). For cDNA synthesis, reverse transcription was performed with 2 g of total RNA, oligo dT, dNTPs, DTT, buffer and Superscript M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA). cDNA was analyzed by qPCR using iQ SYBR Green Supermix (both from BioRad, Hercules, CA) according to the manufacturer’s instructions with a relative quantification protocol using the Chromo 4 real time PCR detector (Bio-Rad, Hercules, CA). All target genes were normalized to expression of the housekeeping gene GAPDH. The primers sets were as
Fig. 2. IL-32␣ interacts with PMA-activated PKC␦ and PKC. HEK293 cells were cotransfected with 6× myc-tagged IL-32␣ and 5× FLAG-tagged PKC␦, or PKC. After overnight incubation, cells were pre-treated with 5 M of the pan-PKC inhibitor, Gö6850, for 2 h, then with PMA (50 nM) for a further 3 h. Immunoprecipitation was carried out with 1 g of myc-tag antibody. Immunoprecipitation was performed with 1 g of myc antibody, and the pulled-down PKC␦/ were detected with FLAG antibody. Expression level of each gene was determined by western blotting with 10 g of whole cell lysates (WCL) (A and B).
follows: CXCL10: sense 5 -ACT GTA CGC TGT ACC TGC ATC AGC A3 , antisense 5 -ACA CGT GGA CAA AAT TGG CTT GCA G-3 ;IFIT2: sense 5 -GCA TTG CCA AAT TGG GTG CTG CT-3 , antisense 5 -AGA GCA TGG AGG CTG GCA AGA A-3 ;PLSCR1: sense 5 -GCG CCA CAG CCT CCA TTA AAC TGT-3 , antisense 5 -ATG GCC CAC AGC AAT TTC GGG T-3 ;RSAD2: sense 5 -GCC AAG GAA AGA AGA ACC ATG TGG A-3 , antisense 5 -TCT CAC CCT CAA TTA AGA GGC ACT G-3 . 2.7. Sumoylation assay We used a commercially available sumoylation assay kit (Abcam, San Francisco, CA) according to the manufacturer’s instructions. Briefly, HEK293 cells were cotransfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 and were then treated with PMA for 8 h. The HEK293 cells were lysed in 50 mM HEPES, pH7.5, 150 mM NaCl, 5% glycerol, 20 mM -glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Extracted whole cells lysates were incubated with anti-FLAG-tag antibody (Sigma) for 60 min and then SUMO detection antibody was added. After incubation, signal reporter solution was added. Finally, color developing solution was added and absorbance was read on a microplate reader at 450 nm. We calculated sumoylation of the PLZF protein as follows. %Sumoylation =
OD (SUMO-2 transfected sample − negative control) × 100% OD(SUMO-2 untransfected sample − negative control)
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2.8. Statistical analysis Data are presented as means ± SDs of results from at least three independent experiments. Statistical significance was assessed with Student’s t-test. A p-value < 0.05 was considered to be statistically significant.
3. Results 3.1. IL-32˛ interacts with PLZF in a PKC-dependent manner A human spleen cDNA library was screened for IL-32␣interacting proteins using a lexA-based yeast two-hybrid system (Fig. 1). Bait IL-32␣ was expressed as a LexA fusion, whereas the spleen cDNA library was fused to the GAL 4 activation domain as a prey protein. Positive interactions with IL-32␣ were selected by cell growth and blue color on selective media. We selected twelve candidates, including PLZF, as binding partners of IL-32␣. Based on the screening data, we investigated the physiological relevance of the IL-32␣ interaction with PLZF. To determine the relationship between PMA signaling and the interaction between IL-32␣ and PLZF, we first verified our previous findings that IL-32␣ interacts with PKC␦ and PKC upon PMA stimulation (Kang et al., 2012) (Fig. 2). We then confirmed the yeast screening data by immunoprecipitation. IL-32␣ co-immunoprecipitated with PLZF, and this interaction was PKC dependent because pull-down of PLZF was inhibited by the pan-PKC inhibitor, Gö6850 (Fig. 3A). We then investigated whether the interaction of IL-32␣ with PLZF was PKC-dependent (Figs. 3B and C). The classical PKC inhibitor, Gö6976, did not affect the interaction of IL-32␣ with PLZF in IL-32␣ expressing THP-1 cells (Fig. 3C). However, as shown in Fig. 4, the interaction between IL-32␣ and PLZF was totally inhibited by a pan-PKC inhibitor (Gö6850) and a PKC-specific inhibitor (Ro-31-8220), but was affected to a lesser extent by a PKC␦-specific inhibitor (Rottlerin) and MAPK inhibitors (PD98059 and SB203580). These data suggest that PKC is mostly involved in the IL-32␣ interaction with PLZF.
3.2. IL-32˛ inhibits PLZF sumoylation upon PMA stimulation
Fig. 3. IL-32␣ interacts with PMA-activated PKC and PLZF. HEK293 cells were co-transfected with myc-tagged IL-32␣ expression vector and FLAG-tagged PLZF. Immunoprecipitation was performed in the same way as in Fig. 2(A). HEK293 cells were co-transfected with FLAG-tagged PLZF and PKC. After overnight incubation, cells were treated with 5 M of pan-PKC inhibitor, Gö6850, for 2 h, then PMA (50 nM) for a further 3 h. Immunoprecipitation was carried out with 1 g of PKC antibody (B). THP-1-IL-32␣ was transfected with FLAG-tagged PLZF expression vector. After
We next investigated whether the interaction of IL-32␣ with PLZF affected PLZF function. PLZF is a transcriptional regulator and a critical factor for myeloid lineage development. The transcriptional activity of PLZF is modulated by post-translational protein modifications, such as phosphorylation and sumoylation (Ball et al., 1999; Chao et al., 2007; Costoya et al., 2008; Kang et al., 2003, 2008). SUMO-1 competes with ubiquitin on the lysine 242 residue of PLZF, and which regulates the protein stability as well as transcriptional activity of PLZF (Kang et al., 2008). Because we did not detect any change in the phosphorylation status of PLZF (data not shown), we examined whether IL-32␣ affected the sumoylation pattern of PLZF. Interestingly, we found SUMO-2 modification of PLZF, and observed that PLZF sumoylation was increased by PMA treatment in the absence of IL-32␣, but the sumoylation of PLZF was suppressed when IL-32␣ was co-expressed. The effect of IL32␣ on PLZF sumoylation was dependent on PKC activation by PMA treatment. These modifications disappeared in response to Gö6850 treatment (Figs. 5A and B). We evaluated the extent of sumoylation of PLZF by quantitative sumoylation analysis, and found that IL-32␣ significantly suppressed PLZF sumoylation (Fig. 5C).
overnight incubation, cells were pretreated with 5 M Gö6850 or 10 M Gö6976 for 2 h, and then further treated with 50 nM PMA for 3 h. Immunoprecipitation was performed with 1 g of anti-myc antibody (C).
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Fig. 4. Interaction between PLZF and IL-32␣ requires PKC activation. HEK293 cells were co-transfected with myc-tagged IL-32␣ expression vector and FLAG-tagged PLZF. After overnight incubation, cells were pretreated with the pan PKC inhibitor, Gö6850 (6850; 5 M); ERK inhibitor, PD98059 (PD; 25 M); p38 inhibitor, SB203580 (SB; 10 M); classical PKC inhibitor, Gö6976 (6976; 10 M); PKC inhibitor, Ro31-8220 (Ro31; 10 M); and PKC ␦ inhibitor Rottlerin (Rott; 10 M) for 2 h. Cells were then treated with 50 nM PMA for a further 3 h. Immunoprecipitation was performed with 1 g of myc antibody, and the pulled-down PLZF was detected with FLAG antibody.
3.3. Down-regulation of PLZF sumoylation by IL-32˛ inhibits the PLZF-mediated transcription of IFN-stimulated genes We next investigated the physiological outcome of IL-32␣mediated down-regulation of PLZF sumoylation using RT-qPCR. As described earlier, PLZF regulates the expression of IFN-stimulated genes (ISGs) such as CXCR10, IFIT2, PLSCR1 and RSAD2 (Xu et al., 2009). We generated an IL-32␣-expressing stable cell line and vector control cell line using THP-1 promonocytic myeloid cells. IL-32␣ interacted with PLZF in a PKC-dependent manner (Fig. 3C). When THP-1-IL-32␣ cells were stimulated with PMA, we found that PLZF-regulated CXCL10 gene expression was decreased in comparison with that of empty vector (EV) THP-1 cells (Fig. 6A). We demonstrated that expression levels of other genes regulated by PLZF, such as IFIT2, PLSCR1, and RSAD2, were also down-regulated (Figs. 6B–D). We also confirmed the expression levels of RSAD2 and PLSCR1 by western blot analyses. Consistent with the gene expression level results in real time PCR experiments, protein levels of PLSCR1 and RSAD2 were also down-regulated in THP-1-IL-32␣ cells in response to PMA treatment (Fig. 6E), although the level of IFIT2 did not alter significantly (data not shown). We hypothesized that PKC inhibition might modulate gene expression of PLSCR1, one of the ISGs, in the presence of IL-32␣. To evaluate this, we pretreated IL-32␣ expressing THP-1 cells and THP-1-EV cells with the pan PKC inhibitor Gö6850, and PLSCR1 expression was recovered up to the level observed in PMA treated THP-1-EV cells. In contrast, pretreatment of cells with the classical PKC inhibitor, Gö6976, did not affect the expression level of PLSCR1 (Fig. 6F). Therefore, our data suggest that IL-32␣ down-regulates ISGs by acting as an intracellular inflammatory mediator via binding to PLZF and inhibiting PLZF sumoylation. 4. Discussion IL-32 is a well-known cytokine that plays a critical role in a variety of inflammatory diseases. The receptors for IL-32 remain to be identified, though intracellular functions of IL-32 have been reported. Recent reports have highlighted the identification of IL32-interacting proteins. We recently showed that IL-32␣ interacts with STAT3 and PKC ␦/ to regulate IL-6 production (Kang et al., 2012). Moreover, we have shown that IL-32 isoforms interact with each other; in particular, interaction of IL-32 with IL-32␦ inhibits the effect of IL-32 on IL-10 production (Kang et al., 2014, 2013).
Fig. 5. IL-32␣ inhibits SUMO-2 conjugation with PLZF. HEK293 cells were co-transfected with FLAG-tagged PLZF expression vector. myc-tagged SUMO-2 expression vector and with or without myc-tagged IL-32␣ expression vector. After overnight incubation, cells were treated with 50 nM PMA for the indicated times. Immunoprecipitation was carried out with 1 g of FLAG antibody, and then SUMO-2 conjugated PLZF was detected with myc antibody (A). HEK293 cells were cotransfected using the same conditions as described in A. After overnight incubation, cells were pretreated with the pan-PKC inhibitor Gö6850 (6850; 10 M) for 2 h before 50 nM PMA treatment for 8 h. Immunoprecipitation was performed with 1 g of FLAG antibody (B). HEK293 cells were co-transfected with FLAG-tagged PLZF expression vector, with or without myc-tagged IL-32␣ expression vector. After overnight incubation, cells were treated with 50 nM PMA for 8 h, and then cell extracts were harvested for an in vivo sumoylation ELISA as described in Section 2 (C). Asterisks indicate significant attenuation of expression following IL-32 expression in HEK293 cells (*P < 0.05).
We demonstrated in a previous study and the current study that IL-32␣ interacts with IL-32 isoforms using a yeast two hybrid system (Kang et al., 2014, and Fig. 1). Many reports have indicated that IL-32 may play diverse roles in cells through interactions with other proteins. However, the underlying functions and interacting protein partners of IL-32 have remained elusive. In this study, we provided evidence that potential interacting protein and cellular function of IL-32␣. PLZF has been shown to regulate a subset of ISGs, namely CXCL10, IFIT2, PLSCR1, and RSAD2 by inducing an
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Fig. 6. IL-32␣ down-regulates expression of genes induced by PLZF. THP-1 EV cells and THP-IL-32␣ cells were transfected with FLAG-tagged PLZF vector. After 24 h incubation, cells were treated with 10 nM PMA in a time-dependent manner. Total RNAs were extracted and gene expression levels induced by PLZF were assessed by real-time PCR: CXCL10 (A), IFIT2 (B), PLSCR1 (C), and RSAD2 (D). Asterisks indicate significant attenuation of expression following THP-1 EV (*P < 0.05). RSAD2 and PLSCR1 protein levels were detected with 80 g of whole cell lysates by western blotting (E). THP-1 EV cell and THP-1-IL-32␣ cells were transfected with FLAG-tagged PLZF vector. Cells were pretreated with the pan-PKC inhibitor Gö6850 (6850; 10 M) or the classical PKC inhibitor Gö6976 (6976; 10 M) for 2 h, and then treated with 10 nM PMA for 24 h (F). CXCL10 represents chemokine (C-X-C motif) ligand 10; IFIT2 represents interferon-induced protein with tetratricopeptide repeats 2; PLSCR1 represents phospholipid scramblase 1; RSAD2 represents radical S-adenosyl methionine domain-containing 2.
interferon-mediated antiviral response in embryonic fibroblasts (MEFs) derived from plzf knockout mice (Xu et al., 2009). In this study, we reported for the first time that IL-32␣ interacts with PLZF upon PMA stimulation, and thereby regulates the transcription of IFN-stimulated genes (ISGs). ISGs have been reported to regulate both innate and adaptive immunity and affect antiviral response and immunomodulatory activities of numerous immune cells including macrophages, natural killer (NK) cells, dendritic cells, and T cells. Type I interferons stimulations regulate transcriptional levels of ISGs through Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal transduction (Borden et al., 2007; Chang et al., 2004; Haque and Williams, 1998; Stark et al., 1998; Tenoever et al., 2007; van Boxel-Dezaire et al., 2006). IL-32 has been shown to activate the NF-B and p38 signaling pathways (Yousif et al., 2013). We showed that the interaction between IL-32␣ and PLZF was mediated mainly via the PKC signaling pathway and partially by MAPKs, NF-B, or other PKCs, except for PKC. IL-32␣ was identified as a key signaling agent for diverging signaling pathways, involving PKCs. These results
validate our previous finding that IL-32␣ up regulates IL-6 production through PKC phosphorylation (Kang et al., 2012). These results indicate that IL-32␣ functions as a modulator of signal transduction in the immune response and provide new insights into the PLZF signaling pathway. We found that IL-32␣ altered post-translational modification of PLZF. Increased intracellular ROS levels due to serum starvation have been shown to induce ubiquitin conjugation and decrease SUMO-1 conjugation of PLZF. These modifications regulate PLZF biological function through competition for lysine 242 in the RD2 domain of PLZF (Chao et al., 2007; Kang et al., 2003, 2008). We confirmed that IL-32␣ interacts equally with PLZF K242 mutant as well as with wild-type PLZF in HEK293 cells (data not shown). Ubiquitin induces degradation, while sumoylation stabilizes the protein, allowing it to function as a transcriptional repressor (Comerford et al., 2003; Huang et al., 2003; Kang et al., 2008; Stelter and Ulrich, 2003). We detected ubiquitin-conjugated PLZF by immunoprecipitation. Levels of ubiquitin-conjugated PLZF increased markedly 8 h after PMA stimulation in HEK293 cells in the absence of IL-32␣,
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while the ubiquitin conjugated PLZF appeared at 4 h and expression of this protein was sustained up to 8 h in HEK293 cells in the presence of IL-32␣ (data not show). We showed that there was no difference in protein expression levels of PLZF, despite IL-32␣induced ubiquitin modification of PLZF. These results suggest that IL-32␣ does not affect PLZF stabilization. SUMO-2 and -3 conjugation of various proteins regulate the transcriptional activities of target genes (Rytinki and Palvimo, 2008; Stindt et al., 2011). SUMO-1 and ubiquitin conjugation of PLZF have been described in previous reports, and we demonstrated here for the first time that PLZF is also modified by SUMO-2. Our results indicate that IL-32␣ inhibits SUMO-2 conjugation of PLZF and then, down-regulates the function of PLZF as a transcriptional regulator. Our findings provide new mechanistic insights into IL-32␣’s role as an intracellular mediator, and indicate that IL-32␣ has a broader function in immune responses than previously recognized. Conflict of interest The authors declare that no financial or other conflict of interest exists in relation to the content of this article. Acknowledgement This research was supported by the basic program (2012R1A2A2A 02008751, and partially 2013-A423-0061) of the National Research Foundation of Korea (NRF). References Alonzo ES, Gottschalk RA, Das J, Egawa T, Hobbs RM, Pandolfi PP, et al. Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3. J Immunol 2010;184(3):1268–79. Bai X, Ovrutsky AR, Kartalija M, Chmura K, Kamali A, Honda JR, et al. IL-32 expression in the airway epithelial cells of patients with Mycobacterium avium complex lung disease. Int Immunol 2011;23(11):679–91. Bailey P, Downes M, Lau P, Harris J, Chen SL, Hamamori Y, et al. The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD. Mol Endocrinol 1999;13(7):1155–68. Ball HJ, Melnick A, Shaknovich R, Kohanski RA, Licht JD. The promyelocytic leukemia zinc finger (PLZF) protein binds DNA in a high molecular weight complex associated with cdc2 kinase. Nucleic Acids Res 1999;27(20):4106–13. Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 2009;458(7237):461–7. Borden EC, Sen GC, Uze G, Silverman RH, Ransohoff RM, Foster GR, et al. Interferons at age 50: past, current and future impact on biomedicine. Nat Rev Drug Discov 2007;6(12):975–90. Calabrese F, Baraldo S, Bazzan E, Lunardi F, Rea F, Maestrelli P, et al. IL-32: a novel proinflammatory cytokine in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;178(9):894–901. Chang EY, Szallasi Z, Acs P, Raizada V, Wolfe PC, Fewtrell C, et al. Functional effects of overexpression of protein kinase C-alpha: -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3. J Immunol 1997;159(6):2624–32. Chang HM, Paulson M, Holko M, Rice CM, Williams BR, Marie I, et al. Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity. Proc Natl Acad Sci USA 2004;101(26):9578–83. Chao TT, Chang CC, Shih HM. SUMO modification modulates the transrepression activity of PLZF. Biochem Biophys Res Commun 2007;358(2):475–82. Cheon S, Lee JH, Park S, Bang SI, Lee WJ, Yoon DY, et al. Overexpression of IL-32alpha increases natural killer cell-mediated killing through up-regulation of Fas and UL16-binding protein 2 (ULBP2) expression in human chronic myeloid leukemia cells. J Biol Chem 2011;286(14):12049–55. Choi JD, Bae SY, Hong JW, Azam T, Dinarello CA, Her E, et al. Identification of the most active interleukin-32 isoform. Immunology 2009;126(4):535–42. Comerford KM, Leonard MO, Karhausen J, Carey R, Colgan SP, Taylor CT. Small ubiquitin-related modifier-1 modification mediates resolution of CREBdependent responses to hypoxia. Proc Natl Acad Sci USA 2003;100(3):986–91. Costoya JA, Hobbs RM, Pandolfi PP. Cyclin-dependent kinase antagonizes promyelocytic leukemia zinc-finger through phosphorylation. Oncogene 2008;27(27):3789–96. Dhordain P, Albagli O, Honore N, Guidez F, Lantoine D, Schmid M, et al. Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins: LAZ3 (BCL6) and PLZF. Oncogene 2000;19(54):6240–50. Dinarello CA, Kim SH. IL-32, a novel cytokine with a possible role in disease. Ann Rheum Dis 2006;65(Suppl. 3):iii61–4.
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Interleukin-32␣ modulates promyelocytic leukemia zinc finger gene activity by inhibiting protein kinase C-dependent sumoylation Yun Sun Park a , Jeong-Woo Kang a , Dong Hun Lee a , Man Sub Kim a , Yesol Bak a , Young Yang b , Hee-Gu Lee c , Jintae Hong d , Do-Young Yoon a,∗ a
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea Research Center for Women’s Disease, Department of Life Systems, Sookmyung Women’s University, Seoul 140-742, Republic of Korea c Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea d College of Pharmacy and Medical Research Center, Chungbuk National University, Cheongju 361-463, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 December 2013 Received in revised form 17 July 2014 Accepted 21 August 2014 Available online 29 August 2014 Keywords: Interleukin 32␣ Promyelocytic leukemia zinc finger protein Small ubiquitin-like modifier-2
a b s t r a c t Interleukin-32 (IL-32) is a proinflammatory cytokine. However, there is growing evidence that IL-32 also plays a mediatory role intracellularly. In this study, we present evidence that IL-32␣ modifies and inhibits promyelocytic leukemia zinc finger (PLZF), a sequence-specific transcriptional regulator that regulates the expression of a subset of interferon (IFN)-stimulated genes (ISGs). We screened IL-32␣-interacting proteins in a human spleen cDNA library using the yeast two-hybrid assay, and investigated the functional relevance of the interaction between IL-32␣ and PLZF. We demonstrated that IL-32␣ interacts with protein kinase C (PKC)␦ and PKC in a phorbol 12-myristate 13-acetate (PMA) dependent way, and that PKC regulates the interaction of IL-32␣ with PLZF. We verified the involvement of PKC in the interaction between these proteins by using various PKC inhibitors. PLZF is known to be modified by small ubiquitin-like modifier (SUMO)-1, but it is unclear whether SUMO-2 conjugation of PLZF occurs. We showed that IL-32␣ inhibited SUMO-2-conjugation of PLZF. Further, we demonstrated that sumoylated PLZF decreased when IL-32␣ was co-expressed. PKC affected the sumoylation of PLZF only in the presence of IL-32␣ because PKC inhibitor treatment did not reduce PLZF sumoylation in the absence of IL-32␣. We finally investigated whether IL-32␣-mediated inhibition of PLZF sumoylation affected the transcriptional activity of PLZF, and demonstrated that the inhibition of sumoylation of PLZF by IL-32␣ down-regulated ISGs induced by PLZF. Together, our data suggest that IL-32␣ associates with PLZF and PKC, and then inhibits PLZF sumoylation, resulting in suppression of the transcriptional activity of PLZF. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Human interleukin-32 (IL-32) is a proinflammatory cytokine expressed in the activated natural killer cells and T-cells (Cheon et al., 2011; Dinarello and Kim, 2006). Although several variants such as ␣,  and ␥ isoforms have been known to induce inflammation, the precise role of each isoform remains elusive. IL-32 induces expression of other inflammatory cytokines such as TNF-␣, IL-1, IL-6, and IL-8 (Heinhuis et al., 2011; Imaeda et al., 2011; Kang
Abbreviations: IL-32, interleukin-32; PLZF, promyelocytic leukemia zinc finger gene; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SUMO-2, small ubiquitin-like modifier-2; ISGs, interferon-stimulated genes. ∗ Corresponding author at: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, 1 Hwayang-dong, Gwangjingu, Seoul 143-701, Republic of Korea. Tel.: +82 2 444 4218. E-mail address: [email protected] (D.-Y. Yoon). http://dx.doi.org/10.1016/j.biocel.2014.08.018 1357-2725/© 2014 Elsevier Ltd. All rights reserved.
et al., 2009, 2012; Kim et al., 2005; Netea et al., 2005; Nold-Petry et al., 2009). IL-32 is also up regulated in various diseases, including chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and Mycobacterium tuberculosis infection (Bai et al., 2011; Calabrese et al., 2008; Joosten et al., 2006; Mun et al., 2009; Netea et al., 2006; Shioya et al., 2007). IL-32 has several isoforms such as ␣, , ␥, ␦, , and . Among these isoforms, IL-32 is abundant in various cells and IL-32␥ has been shown to display the strongest biological activity (Choi et al., 2009). All isoforms have RGD and DDX motifs. The RGD tri-peptide motif is found in extracellular matrix proteins such as fibronectin, and binds to integrin. The DDX motif is a RGD-binding site that is found on cytoskeletal proteins such as integrin (Heinhuis et al., 2012). Previous reports have indicated that IL-32 is involved in the immune response through the MAPK, NF-kappa B and AP-1 signaling pathways (Joosten et al., 2013; Nakayama et al., 2013; Pan et al., 2011).
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Fig. 1. Screening for IL-32␣-interacting proteins (IL-32␣ IPs) using a yeast two-hybrid system. IL-32␣ IPs were screened from a human spleen cDNA library using a yeast two-hybrid system. Candidate colonies were reconfirmed and grouped (right) according to their identities after sequencing, and listed in the same order as in the right figure.
Protein kinase C (PKC) regulates the functions of various proteins by phosphorylation. The PKC protein family has about ten isozymes, and is divided into the three subfamilies of classical, novel, and atypical according to the second messenger (Chang et al., 1997). PKC is activated by phorbol 12-myristate 13-aceate (PMA). The promyelocytic leukemia zinc finger (PLZF) protein belongs to the POK (POZ and Krüppel)/ZBTB (zinc finger and BTB) protein family (Dhordain et al., 2000; Lee and Maeda, 2012). PLZF is a sequence-specific DNA-binding protein that represses the transcriptional activity of target genes such as cyclin A and the IL-3 receptor ␣ chain (Bailey et al., 1999; Kang et al., 2003; Rho et al., 2010, 2006; Yeyati et al., 1999). PLZF is critical for development of the myeloid lineage (Alonzo et al., 2010; Doulatov et al., 2009; Girard et al., 2013; Ward et al., 2001). IFN-mediated signaling is known to be induced by the transcriptional activity of PLZF. PLZF modulates the expression of a large family of IFN-stimulated genes (ISGs) (Xu et al., 2009). Three types of SUMO proteins are found in vertebrates: SUMO1, -2, and -3. SUMO-2 and -3 have 95% sequence conservation. However, SUMO-1 has only 50% similarity to the other SUMOs and has different functions and properties (Herrmann et al., 2007; Kamitani et al., 1998a,b; Saitoh and Hinchey, 2000; Tatham et al., 2001). Post-translational modification by sumoylation is critical for protein function (Watts, 2013). The SUMO system is relevant to cellular processes such as gene expression, cell-cycle progression, DNA replication, and DNA repair. SUMO modification occurs in lysine residues in target genes and sumoylated sites are highly conserved (Bergink and Jentsch, 2009; Geiss-Friedlander and Melchior, 2007; Ulrich, 2009). Modification of PLZF by SUMO-1 conjugation is well known. SUMO-1 modification is required for transcriptional repression by PLZF, and this modification increases the DNA binding activity of PLZF (Chao et al., 2007; Kang et al., 2003). ZBTB1, which belongs to the POK (POZ and Krüppel)/ZBTB (zing finger and BTB) protein family like PLZF, is modulated by SUMO-2 conjugation (Matic et al., 2010). Although SUMO-1 modification of PLZF has been well characterized, it is unclear whether PLZF is also modified by SUMO-2. We previously reported that IL-32␣ interacts with PMA-stimulated PKC and that pan PKC inhibitor treatment
represses these interactions (Kang et al., 2012). In this study, we further verified our previous results and demonstrated that IL-32␣ interacts with PLZF and PKC, resulting in suppression of the transcriptional activity of PLZF. 2. Materials and methods 2.1. Reagents and cell culture The human promyelomonocytic THP-1 and human embryonic kidney 293 cells were purchased from American Type Culture Collection (ATCC; Rockville, MD). The THP-1 cells were grown in RPMI 1640 (WelGENE, Daegu, Korea) supplemented with 2 mM lglutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% fetal bovine serum (Hyclone, Logan, UT). HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Hyclone). Phorbol 12-myristate 13-acetate was purchased from Sigma (St. Louis, MO). MAPK inhibitors (PD98059, SB203580, and SP100625) and PKC inhibitors (Gö6850, Gö6976, Ro-31-8220, and rottlerin) were purchased from Calbiochem (San Diego, CA). IL-32␣-expressing THP-1 stable cell lines were previously established (Kang et al., 2012). 2.2. Construction of expression vectors IL-32␣ cDNA was subcloned into pcDNA3.1 + 6× myc vector using EcoRI and XhoI. cDNAs for PKC␦ and PKC were subcloned into the pcDNA3.1 + 5× FLAG vector using EcoRI and XhoI (Kang et al., 2012). SUMO-2 and PLZF cDNAs were PCR-amplified from a human spleen cDNA library (Clontech, Palo Alto, CA). Primers sets were as follows: PLZF: sense 5 -CGC GAA TTC ATG GAT CTG ACA AAA-3 , antisense 5 -ATT CTC GAG CAC ATA GCA CAG GTA3 , SUMO-2: sense 5 -GCT GAA TTC ATG GCC GAC GAA AAG CCC-3 , antisense 5 -GAA CTC GAG CTA ACC TCC CGT CTG CTG-3 . The entire amplified PLZF gene was cloned into the pcDNA3.1 + 5× FLAG vector using EcoRI and XhoI. SUMO-2 cDNA was cloned into the pCS3MT 6× myc vector using EcoRI and XhoI.
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2.3. Yeast two-hybrid screening cDNA of IL-32␣ was cloned into the pHybLex/ZT vector (Invitrogen), a bait plasmid containing the LexA DNA binding domain. The MATCHMAKER GAL4 Two-Hybrid System 3 and Human Spleen cDNA Library were purchased from BD Biosciences (Palo Alto, CA). Yeast two-hybrid screening was performed according to the manufacturer’s protocols. Briefly, IL-32 bait fusion plasmids were cloned into pHybLex/ZT vector and amplified in E.coli using zeomycin selection. Bait plasmids were introduced into yeast strain L40 that was precultured in SD-U (-Uracil) medium and transformed clones were selected on SD-UT (-Uracil, -Tryptophan). Prey plasmid (human spleen cDNA library containing pACT2 plasmid) was introduced into the bait-transformed yeast that was precultured in SD-UT medium and selected on SD-UTLH (-Uracil, -Tryptophan, -Leucine, -Histidine). The selected colonies were streaked on SDUTLH-X-GAL plates for a second round of selection. The positive blue colonies were harvested and plasmids were extracted using ZymoprepTM II Yeast Plasmid Miniprep Kit (ZYMO Research, Irvine, CA). Extracted plasmids were amplified by introducing them into E. coli followed by purification for sequencing. The selected plasmid DNAs were identified by sequencing (Xenotech, Daejon, South Korea) and subjected to BLAST searches. 2.4. Transfection THP-1 promonocytic cells were transfected with pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 using the NeonTM transfection system (Invitrogen) according to the manufacturer’s instructions. HEK293 cells were transfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 or ubiquitin using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 2.5. Western blotting and immunoprecipitation HEK293, THP-1 EV and THP-1-IL-32␣ cells were cotransfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 or ubiquitin and then lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, 20 mM -glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Western blotting was performed using anti-myc tag antibody (Millipore, Bedford, MA); anti-FLAG-tag antibody (Sigma); anti-PKC ␦/ antibody (Santa Cruz Biotechnology); anti-IL-32 antibody KU32-52 (Kang et al., 2009; Kim et al., 2008); anti-PLZF antibody (Santa Cruz Biotechnology); anti-RSAD2 antibody (Santa Cruz biotechnology); and anti-PLSCR1 antibody (R&D). For immunoprecipitation, cell lysates were mixed with 1 g of anti-myc antibody, 1 g of anti-PKC antibody, and 1 g of anti-FLAG antibody for 1 h and then pulled down using 35 l of protein G-agarose beads (KPL, Gaithersburg, MD). 2.6. Reverse-transcription polymerase chain reaction (RT-PCR) and real-time qPCR analyses THP-1 EV and THP-1-IL-32␣ were treated with 10 nM PMA for the indicated times and then, total RNA was extracted for RT-PCR. Total RNA was extracted using the Easy-Blue total RNA extraction kit (iNtRon Biotechnology, Seoul, Korea). For cDNA synthesis, reverse transcription was performed with 2 g of total RNA, oligo dT, dNTPs, DTT, buffer and Superscript M-MuLV reverse transcriptase (New England Biolabs, Beverly, MA). cDNA was analyzed by qPCR using iQ SYBR Green Supermix (both from BioRad, Hercules, CA) according to the manufacturer’s instructions with a relative quantification protocol using the Chromo 4 real time PCR detector (Bio-Rad, Hercules, CA). All target genes were normalized to expression of the housekeeping gene GAPDH. The primers sets were as
Fig. 2. IL-32␣ interacts with PMA-activated PKC␦ and PKC. HEK293 cells were cotransfected with 6× myc-tagged IL-32␣ and 5× FLAG-tagged PKC␦, or PKC. After overnight incubation, cells were pre-treated with 5 M of the pan-PKC inhibitor, Gö6850, for 2 h, then with PMA (50 nM) for a further 3 h. Immunoprecipitation was carried out with 1 g of myc-tag antibody. Immunoprecipitation was performed with 1 g of myc antibody, and the pulled-down PKC␦/ were detected with FLAG antibody. Expression level of each gene was determined by western blotting with 10 g of whole cell lysates (WCL) (A and B).
follows: CXCL10: sense 5 -ACT GTA CGC TGT ACC TGC ATC AGC A3 , antisense 5 -ACA CGT GGA CAA AAT TGG CTT GCA G-3 ;IFIT2: sense 5 -GCA TTG CCA AAT TGG GTG CTG CT-3 , antisense 5 -AGA GCA TGG AGG CTG GCA AGA A-3 ;PLSCR1: sense 5 -GCG CCA CAG CCT CCA TTA AAC TGT-3 , antisense 5 -ATG GCC CAC AGC AAT TTC GGG T-3 ;RSAD2: sense 5 -GCC AAG GAA AGA AGA ACC ATG TGG A-3 , antisense 5 -TCT CAC CCT CAA TTA AGA GGC ACT G-3 . 2.7. Sumoylation assay We used a commercially available sumoylation assay kit (Abcam, San Francisco, CA) according to the manufacturer’s instructions. Briefly, HEK293 cells were cotransfected with pcDNA3.1 + 6× myc-IL-32␣, pcDNA3.1 + 5× FLAG-PLZF and pCS3MT-SUMO-2 and were then treated with PMA for 8 h. The HEK293 cells were lysed in 50 mM HEPES, pH7.5, 150 mM NaCl, 5% glycerol, 20 mM -glycerophosphate, 1% NP-40, 0.5% TX-100, and 1 mM EDTA. Extracted whole cells lysates were incubated with anti-FLAG-tag antibody (Sigma) for 60 min and then SUMO detection antibody was added. After incubation, signal reporter solution was added. Finally, color developing solution was added and absorbance was read on a microplate reader at 450 nm. We calculated sumoylation of the PLZF protein as follows. %Sumoylation =
OD (SUMO-2 transfected sample − negative control) × 100% OD(SUMO-2 untransfected sample − negative control)
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2.8. Statistical analysis Data are presented as means ± SDs of results from at least three independent experiments. Statistical significance was assessed with Student’s t-test. A p-value < 0.05 was considered to be statistically significant.
3. Results 3.1. IL-32˛ interacts with PLZF in a PKC-dependent manner A human spleen cDNA library was screened for IL-32␣interacting proteins using a lexA-based yeast two-hybrid system (Fig. 1). Bait IL-32␣ was expressed as a LexA fusion, whereas the spleen cDNA library was fused to the GAL 4 activation domain as a prey protein. Positive interactions with IL-32␣ were selected by cell growth and blue color on selective media. We selected twelve candidates, including PLZF, as binding partners of IL-32␣. Based on the screening data, we investigated the physiological relevance of the IL-32␣ interaction with PLZF. To determine the relationship between PMA signaling and the interaction between IL-32␣ and PLZF, we first verified our previous findings that IL-32␣ interacts with PKC␦ and PKC upon PMA stimulation (Kang et al., 2012) (Fig. 2). We then confirmed the yeast screening data by immunoprecipitation. IL-32␣ co-immunoprecipitated with PLZF, and this interaction was PKC dependent because pull-down of PLZF was inhibited by the pan-PKC inhibitor, Gö6850 (Fig. 3A). We then investigated whether the interaction of IL-32␣ with PLZF was PKC-dependent (Figs. 3B and C). The classical PKC inhibitor, Gö6976, did not affect the interaction of IL-32␣ with PLZF in IL-32␣ expressing THP-1 cells (Fig. 3C). However, as shown in Fig. 4, the interaction between IL-32␣ and PLZF was totally inhibited by a pan-PKC inhibitor (Gö6850) and a PKC-specific inhibitor (Ro-31-8220), but was affected to a lesser extent by a PKC␦-specific inhibitor (Rottlerin) and MAPK inhibitors (PD98059 and SB203580). These data suggest that PKC is mostly involved in the IL-32␣ interaction with PLZF.
3.2. IL-32˛ inhibits PLZF sumoylation upon PMA stimulation
Fig. 3. IL-32␣ interacts with PMA-activated PKC and PLZF. HEK293 cells were co-transfected with myc-tagged IL-32␣ expression vector and FLAG-tagged PLZF. Immunoprecipitation was performed in the same way as in Fig. 2(A). HEK293 cells were co-transfected with FLAG-tagged PLZF and PKC. After overnight incubation, cells were treated with 5 M of pan-PKC inhibitor, Gö6850, for 2 h, then PMA (50 nM) for a further 3 h. Immunoprecipitation was carried out with 1 g of PKC antibody (B). THP-1-IL-32␣ was transfected with FLAG-tagged PLZF expression vector. After
We next investigated whether the interaction of IL-32␣ with PLZF affected PLZF function. PLZF is a transcriptional regulator and a critical factor for myeloid lineage development. The transcriptional activity of PLZF is modulated by post-translational protein modifications, such as phosphorylation and sumoylation (Ball et al., 1999; Chao et al., 2007; Costoya et al., 2008; Kang et al., 2003, 2008). SUMO-1 competes with ubiquitin on the lysine 242 residue of PLZF, and which regulates the protein stability as well as transcriptional activity of PLZF (Kang et al., 2008). Because we did not detect any change in the phosphorylation status of PLZF (data not shown), we examined whether IL-32␣ affected the sumoylation pattern of PLZF. Interestingly, we found SUMO-2 modification of PLZF, and observed that PLZF sumoylation was increased by PMA treatment in the absence of IL-32␣, but the sumoylation of PLZF was suppressed when IL-32␣ was co-expressed. The effect of IL32␣ on PLZF sumoylation was dependent on PKC activation by PMA treatment. These modifications disappeared in response to Gö6850 treatment (Figs. 5A and B). We evaluated the extent of sumoylation of PLZF by quantitative sumoylation analysis, and found that IL-32␣ significantly suppressed PLZF sumoylation (Fig. 5C).
overnight incubation, cells were pretreated with 5 M Gö6850 or 10 M Gö6976 for 2 h, and then further treated with 50 nM PMA for 3 h. Immunoprecipitation was performed with 1 g of anti-myc antibody (C).
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Fig. 4. Interaction between PLZF and IL-32␣ requires PKC activation. HEK293 cells were co-transfected with myc-tagged IL-32␣ expression vector and FLAG-tagged PLZF. After overnight incubation, cells were pretreated with the pan PKC inhibitor, Gö6850 (6850; 5 M); ERK inhibitor, PD98059 (PD; 25 M); p38 inhibitor, SB203580 (SB; 10 M); classical PKC inhibitor, Gö6976 (6976; 10 M); PKC inhibitor, Ro31-8220 (Ro31; 10 M); and PKC ␦ inhibitor Rottlerin (Rott; 10 M) for 2 h. Cells were then treated with 50 nM PMA for a further 3 h. Immunoprecipitation was performed with 1 g of myc antibody, and the pulled-down PLZF was detected with FLAG antibody.
3.3. Down-regulation of PLZF sumoylation by IL-32˛ inhibits the PLZF-mediated transcription of IFN-stimulated genes We next investigated the physiological outcome of IL-32␣mediated down-regulation of PLZF sumoylation using RT-qPCR. As described earlier, PLZF regulates the expression of IFN-stimulated genes (ISGs) such as CXCR10, IFIT2, PLSCR1 and RSAD2 (Xu et al., 2009). We generated an IL-32␣-expressing stable cell line and vector control cell line using THP-1 promonocytic myeloid cells. IL-32␣ interacted with PLZF in a PKC-dependent manner (Fig. 3C). When THP-1-IL-32␣ cells were stimulated with PMA, we found that PLZF-regulated CXCL10 gene expression was decreased in comparison with that of empty vector (EV) THP-1 cells (Fig. 6A). We demonstrated that expression levels of other genes regulated by PLZF, such as IFIT2, PLSCR1, and RSAD2, were also down-regulated (Figs. 6B–D). We also confirmed the expression levels of RSAD2 and PLSCR1 by western blot analyses. Consistent with the gene expression level results in real time PCR experiments, protein levels of PLSCR1 and RSAD2 were also down-regulated in THP-1-IL-32␣ cells in response to PMA treatment (Fig. 6E), although the level of IFIT2 did not alter significantly (data not shown). We hypothesized that PKC inhibition might modulate gene expression of PLSCR1, one of the ISGs, in the presence of IL-32␣. To evaluate this, we pretreated IL-32␣ expressing THP-1 cells and THP-1-EV cells with the pan PKC inhibitor Gö6850, and PLSCR1 expression was recovered up to the level observed in PMA treated THP-1-EV cells. In contrast, pretreatment of cells with the classical PKC inhibitor, Gö6976, did not affect the expression level of PLSCR1 (Fig. 6F). Therefore, our data suggest that IL-32␣ down-regulates ISGs by acting as an intracellular inflammatory mediator via binding to PLZF and inhibiting PLZF sumoylation. 4. Discussion IL-32 is a well-known cytokine that plays a critical role in a variety of inflammatory diseases. The receptors for IL-32 remain to be identified, though intracellular functions of IL-32 have been reported. Recent reports have highlighted the identification of IL32-interacting proteins. We recently showed that IL-32␣ interacts with STAT3 and PKC ␦/ to regulate IL-6 production (Kang et al., 2012). Moreover, we have shown that IL-32 isoforms interact with each other; in particular, interaction of IL-32 with IL-32␦ inhibits the effect of IL-32 on IL-10 production (Kang et al., 2014, 2013).
Fig. 5. IL-32␣ inhibits SUMO-2 conjugation with PLZF. HEK293 cells were co-transfected with FLAG-tagged PLZF expression vector. myc-tagged SUMO-2 expression vector and with or without myc-tagged IL-32␣ expression vector. After overnight incubation, cells were treated with 50 nM PMA for the indicated times. Immunoprecipitation was carried out with 1 g of FLAG antibody, and then SUMO-2 conjugated PLZF was detected with myc antibody (A). HEK293 cells were cotransfected using the same conditions as described in A. After overnight incubation, cells were pretreated with the pan-PKC inhibitor Gö6850 (6850; 10 M) for 2 h before 50 nM PMA treatment for 8 h. Immunoprecipitation was performed with 1 g of FLAG antibody (B). HEK293 cells were co-transfected with FLAG-tagged PLZF expression vector, with or without myc-tagged IL-32␣ expression vector. After overnight incubation, cells were treated with 50 nM PMA for 8 h, and then cell extracts were harvested for an in vivo sumoylation ELISA as described in Section 2 (C). Asterisks indicate significant attenuation of expression following IL-32 expression in HEK293 cells (*P < 0.05).
We demonstrated in a previous study and the current study that IL-32␣ interacts with IL-32 isoforms using a yeast two hybrid system (Kang et al., 2014, and Fig. 1). Many reports have indicated that IL-32 may play diverse roles in cells through interactions with other proteins. However, the underlying functions and interacting protein partners of IL-32 have remained elusive. In this study, we provided evidence that potential interacting protein and cellular function of IL-32␣. PLZF has been shown to regulate a subset of ISGs, namely CXCL10, IFIT2, PLSCR1, and RSAD2 by inducing an
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Fig. 6. IL-32␣ down-regulates expression of genes induced by PLZF. THP-1 EV cells and THP-IL-32␣ cells were transfected with FLAG-tagged PLZF vector. After 24 h incubation, cells were treated with 10 nM PMA in a time-dependent manner. Total RNAs were extracted and gene expression levels induced by PLZF were assessed by real-time PCR: CXCL10 (A), IFIT2 (B), PLSCR1 (C), and RSAD2 (D). Asterisks indicate significant attenuation of expression following THP-1 EV (*P < 0.05). RSAD2 and PLSCR1 protein levels were detected with 80 g of whole cell lysates by western blotting (E). THP-1 EV cell and THP-1-IL-32␣ cells were transfected with FLAG-tagged PLZF vector. Cells were pretreated with the pan-PKC inhibitor Gö6850 (6850; 10 M) or the classical PKC inhibitor Gö6976 (6976; 10 M) for 2 h, and then treated with 10 nM PMA for 24 h (F). CXCL10 represents chemokine (C-X-C motif) ligand 10; IFIT2 represents interferon-induced protein with tetratricopeptide repeats 2; PLSCR1 represents phospholipid scramblase 1; RSAD2 represents radical S-adenosyl methionine domain-containing 2.
interferon-mediated antiviral response in embryonic fibroblasts (MEFs) derived from plzf knockout mice (Xu et al., 2009). In this study, we reported for the first time that IL-32␣ interacts with PLZF upon PMA stimulation, and thereby regulates the transcription of IFN-stimulated genes (ISGs). ISGs have been reported to regulate both innate and adaptive immunity and affect antiviral response and immunomodulatory activities of numerous immune cells including macrophages, natural killer (NK) cells, dendritic cells, and T cells. Type I interferons stimulations regulate transcriptional levels of ISGs through Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal transduction (Borden et al., 2007; Chang et al., 2004; Haque and Williams, 1998; Stark et al., 1998; Tenoever et al., 2007; van Boxel-Dezaire et al., 2006). IL-32 has been shown to activate the NF-B and p38 signaling pathways (Yousif et al., 2013). We showed that the interaction between IL-32␣ and PLZF was mediated mainly via the PKC signaling pathway and partially by MAPKs, NF-B, or other PKCs, except for PKC. IL-32␣ was identified as a key signaling agent for diverging signaling pathways, involving PKCs. These results
validate our previous finding that IL-32␣ up regulates IL-6 production through PKC phosphorylation (Kang et al., 2012). These results indicate that IL-32␣ functions as a modulator of signal transduction in the immune response and provide new insights into the PLZF signaling pathway. We found that IL-32␣ altered post-translational modification of PLZF. Increased intracellular ROS levels due to serum starvation have been shown to induce ubiquitin conjugation and decrease SUMO-1 conjugation of PLZF. These modifications regulate PLZF biological function through competition for lysine 242 in the RD2 domain of PLZF (Chao et al., 2007; Kang et al., 2003, 2008). We confirmed that IL-32␣ interacts equally with PLZF K242 mutant as well as with wild-type PLZF in HEK293 cells (data not shown). Ubiquitin induces degradation, while sumoylation stabilizes the protein, allowing it to function as a transcriptional repressor (Comerford et al., 2003; Huang et al., 2003; Kang et al., 2008; Stelter and Ulrich, 2003). We detected ubiquitin-conjugated PLZF by immunoprecipitation. Levels of ubiquitin-conjugated PLZF increased markedly 8 h after PMA stimulation in HEK293 cells in the absence of IL-32␣,
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while the ubiquitin conjugated PLZF appeared at 4 h and expression of this protein was sustained up to 8 h in HEK293 cells in the presence of IL-32␣ (data not show). We showed that there was no difference in protein expression levels of PLZF, despite IL-32␣induced ubiquitin modification of PLZF. These results suggest that IL-32␣ does not affect PLZF stabilization. SUMO-2 and -3 conjugation of various proteins regulate the transcriptional activities of target genes (Rytinki and Palvimo, 2008; Stindt et al., 2011). SUMO-1 and ubiquitin conjugation of PLZF have been described in previous reports, and we demonstrated here for the first time that PLZF is also modified by SUMO-2. Our results indicate that IL-32␣ inhibits SUMO-2 conjugation of PLZF and then, down-regulates the function of PLZF as a transcriptional regulator. Our findings provide new mechanistic insights into IL-32␣’s role as an intracellular mediator, and indicate that IL-32␣ has a broader function in immune responses than previously recognized. Conflict of interest The authors declare that no financial or other conflict of interest exists in relation to the content of this article. Acknowledgement This research was supported by the basic program (2012R1A2A2A 02008751, and partially 2013-A423-0061) of the National Research Foundation of Korea (NRF). References Alonzo ES, Gottschalk RA, Das J, Egawa T, Hobbs RM, Pandolfi PP, et al. Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3. J Immunol 2010;184(3):1268–79. Bai X, Ovrutsky AR, Kartalija M, Chmura K, Kamali A, Honda JR, et al. IL-32 expression in the airway epithelial cells of patients with Mycobacterium avium complex lung disease. Int Immunol 2011;23(11):679–91. Bailey P, Downes M, Lau P, Harris J, Chen SL, Hamamori Y, et al. The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD. Mol Endocrinol 1999;13(7):1155–68. Ball HJ, Melnick A, Shaknovich R, Kohanski RA, Licht JD. The promyelocytic leukemia zinc finger (PLZF) protein binds DNA in a high molecular weight complex associated with cdc2 kinase. Nucleic Acids Res 1999;27(20):4106–13. Bergink S, Jentsch S. Principles of ubiquitin and SUMO modifications in DNA repair. Nature 2009;458(7237):461–7. Borden EC, Sen GC, Uze G, Silverman RH, Ransohoff RM, Foster GR, et al. Interferons at age 50: past, current and future impact on biomedicine. Nat Rev Drug Discov 2007;6(12):975–90. Calabrese F, Baraldo S, Bazzan E, Lunardi F, Rea F, Maestrelli P, et al. IL-32: a novel proinflammatory cytokine in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;178(9):894–901. Chang EY, Szallasi Z, Acs P, Raizada V, Wolfe PC, Fewtrell C, et al. Functional effects of overexpression of protein kinase C-alpha: -beta, -delta, -epsilon, and -eta in the mast cell line RBL-2H3. J Immunol 1997;159(6):2624–32. Chang HM, Paulson M, Holko M, Rice CM, Williams BR, Marie I, et al. Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity. Proc Natl Acad Sci USA 2004;101(26):9578–83. Chao TT, Chang CC, Shih HM. SUMO modification modulates the transrepression activity of PLZF. Biochem Biophys Res Commun 2007;358(2):475–82. Cheon S, Lee JH, Park S, Bang SI, Lee WJ, Yoon DY, et al. Overexpression of IL-32alpha increases natural killer cell-mediated killing through up-regulation of Fas and UL16-binding protein 2 (ULBP2) expression in human chronic myeloid leukemia cells. J Biol Chem 2011;286(14):12049–55. Choi JD, Bae SY, Hong JW, Azam T, Dinarello CA, Her E, et al. Identification of the most active interleukin-32 isoform. Immunology 2009;126(4):535–42. Comerford KM, Leonard MO, Karhausen J, Carey R, Colgan SP, Taylor CT. Small ubiquitin-related modifier-1 modification mediates resolution of CREBdependent responses to hypoxia. Proc Natl Acad Sci USA 2003;100(3):986–91. Costoya JA, Hobbs RM, Pandolfi PP. Cyclin-dependent kinase antagonizes promyelocytic leukemia zinc-finger through phosphorylation. Oncogene 2008;27(27):3789–96. Dhordain P, Albagli O, Honore N, Guidez F, Lantoine D, Schmid M, et al. Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins: LAZ3 (BCL6) and PLZF. Oncogene 2000;19(54):6240–50. Dinarello CA, Kim SH. IL-32, a novel cytokine with a possible role in disease. Ann Rheum Dis 2006;65(Suppl. 3):iii61–4.
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