Microrna-17 Suppresses TNF-Α Signaling by Interfering with TRAF2

MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts This information is current as of October 2, 2021. Nahid Akhtar, Anil Kumar Singh and Salahuddin Ahmed J Immunol 2016; 197:2219-2228; Prepublished online 17 August 2016; doi: 10.4049/jimmunol.1600360

http://www.jimmunol.org/content/197/6/2219 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2016/08/17/jimmunol.160036 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 52 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/197/6/2219.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MicroRNA-17 Suppresses TNF-a Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts

Nahid Akhtar, Anil Kumar Singh, and Salahuddin Ahmed

TNF-a is a major cytokine implicated in rheumatoid arthritis (RA), and its expression is regulated at the transcriptional and posttranscriptional levels. However, the impact of changes in microRNA expression on posttranslational processes involved in TNF-a signaling networks is not well defined in RA. In this study, we evaluated the effect of miR-17, a member of the miR-17–92 cluster, on the TNF-a signaling pathway in human RA synovial fibroblasts (SFs). We demonstrated that miR-17 expression was significantly low in RA serum, SFs, and synovial tissues, as well as in the serum and joints of adjuvant-induced arthritis rats. RNA- sequencing analysis showed modulation of 664 genes by pre–miR-17 in human RA SFs. Ingenuity pathway analysis of RNA- sequencing data identified the ubiquitin proteasome system in the TNF-a signaling pathway as a primary target of miR-17. Downloaded from Western blot analysis confirmed the reduction in TRAF2, cIAP1, cIAP2, USP2, and PSMD13 expression by miR-17 in TNF-a– stimulated RA SFs. Immunoprecipitation assays showed that miR-17 restoration increased the K48-linked polyubiquitination of TRAF2, cIAP1, and cIAP2 in TNF-a–stimulated RA SFs. Thus, destabilization of TRAF2 by miR-17 reduced the ability of TRAF2 to associate with cIAP2, resulting in the downregulation of TNF-a–induced NF-kBp65, c-Jun, and STAT3 nuclear translocation and the production of IL-6, IL-8, MMP-1, and MMP-13 in human RA SFs. In conclusion, this study provides evidence for the role of miR-17 as a negative regulator of TNF-a signaling by modulating the protein ubiquitin processes in RA http://www.jimmunol.org/ SFs. The Journal of Immunology, 2016, 197: 2219–2228.

icroRNAs (miRNAs) are a highly conserved set of shown to regulate various target genes in specific cellular contexts, single-stranded noncoding RNAs (∼19–23 nt in length) the predicted impact of changes in miRNA expression on cellular M that are important in many developmental and physi- processes and cytokine signaling networks is difficult to predict. ological processes (1, 2), and their aberrant expression correlated Recent studies showed significant changes in the expression of with inflammatory diseases, including rheumatoid arthritis (RA) many genes by individual miRNA overexpression (10–12); how- (2–5). A key specificity determinant for miRNA target recognition ever, only a portion of differentially regulated genes were predicted is based on Watson–Crick pairing of the 59-proximal seed region direct targets, indicating that most of the changes in gene expres- by guest on October 2, 2021 (nt 2–8) in the miRNA to the seed match site in the target mRNA, sion induced by miRNA transfections are indirect (12, 13). which is located primarily in the 39UTR (6), with a small subset of Ubiquitination is a posttranslational modification process that miRNAs targeting the mRNA 59UTR and/or the coding region plays a key role in various signal-transduction cascades by priming (7–9). Although the exogenous delivery of different miRNAs was signaling proteins for degradation or stabilization through Lys- linked K48 or K63 ubiquitin chains, respectively (14). The ubiquitin

Department of Pharmaceutical Sciences, Washington State University College of proteasome system (UPS) consists of ubiquitin ligases (1–3) and Pharmacy, Spokane, WA 99210 proteomes that mediate posttranslational modifications in the cy- Received for publication March 2, 2016. Accepted for publication July 18, 2016. tokine or TLR signaling network. For instance, upon binding to a This work was supported by National Institutes of Health Grant AR063104 (to S.A.), TNF- , TNFR1 recruits adapter proteins; the protein kinase RIP1; an Arthritis Foundation Innovative Research Grant (to S.A.), and start-up funds from several ubiquitin E3 ligases, such as TRAF2, cIAP1, and cIAP2; Washington State University. and the deubiquitination (DUB) enzymes for downstream signaling N.A., A.K.S., and S.A. designed the research, analyzed and interpreted the data, and (15). However, TNF-dependent recruitment of multiple ubiquitin wrote the manuscript; N.A. and A.K.S. performed the research; and S.A. provided expertise and funding support and gave critical suggestions during drafting of the ligases and DUB enzymes implies the importance of ubiquitination manuscript. for regulating inflammation and cell death in this pathway. Being The sequences presented in this article have been submitted to the National Center two different spectrums of biological processes, namely epigenetics for Biotechnology Information Gene Expression Omnibus under accession number and posttranslational, the influence of miRNAs on the ubiquitination GSE83930. of TNF-a signaling proteins is not well known in RA. Address correspondence and reprint requests to Dr. Salahuddin Ahmed, Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, miR-17–92 is located in the locus of MIR17HG (miR-17–92 SPBS Room 411, 412 East Spokane Falls Boulevard, Spokane, WA 99210. E-mail cluster host gene), also known as C13orf25 (chromosome 13 open address: [email protected] reading frame 25). The miR-17–92 cluster transcript spans 800 nt The online version of this article contains supplemental material. and encodes six miRNAs that are transcribed from the same pro- Abbreviations used in this article: AIA, adjuvant-induced arthritis; ASK, apoptosis moter (Supplemental Fig. 1A). These six miRNAs can be grouped signaling kinase; DUB, deubiquitination; IP, immunoprecipitation; IPA, Ingenuity Pathway Analysis; miRNA, microRNA; MMP, matrix metalloproteinase; NC, nega- into four families based on their seed regions: miR-17, miR-18, tive control; NL, healthy/nondiseased; OA, osteoarthritis; RA, rheumatoid arthritis; miR-19, and miR-92. The miR-17 and miR-19 families are com- SF, synovial fibroblast; siRNA, small interfering RNA; ST, synovial tissue; UPS, posed of the pairs of miRNAs with identical seed regions: miR-19/ ubiquitin proteasome system; UTR, untranslated region. miR-20a and miR-19a/miR-19b-1 (16). As oncomirs, these miRNAs Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 are known to promote proliferation, inhibit apoptosis, and induce www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600360 2220 miR-17 SUPPRESSES TNF-a SIGNALING IN RA tumor angiogenesis (17, 18). Yet in some contexts, the miR-17 Treatment of SFs and preparation of microRNAs family was shown to negatively regulate cell proliferation (19–21) NL, OA, or RA SFs were plated in 60-mm dishes and used when .80% and inhibit cell migration and invasion in cancer (22, 23). Recent confluent. SFs were serum starved overnight and stimulated or not with studies showed that miR-20a from the same cluster regulates ap- TNF-a (20 ng/ml) for the indicated time, and cell lysates were prepared. optosis signaling kinase (ASK)1, whereas miR-19a/b were shown Human STs and the joint homogenates from the AIA study were also to regulate IL-6 and matrix metalloproteinase (MMP)-3 expres- ground to a fine powder in liquid nitrogen using a tissue pulverizer. Pul- verized tissue was used to purify total RNA containing the miRNA fraction sion in LPS-activated RA synovial fibroblasts (SFs) (24, 25). In (miRNeasy kit; QIAGEN, Valencia, CA) to study miR-17, miR-18a, miR-19a, contrast, TNF-a–induced miR-18a was reported to facilitate car- miR-19b, miR-20a, and miR-92 expression. tilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-kB signaling, with a concomitant Transient transfection upregulation of MMPs and mediators of inflammation in RA SFs RA SFs or THP-1–differentiated macrophages were transfected with pre– (26), suggesting differential effects of miRNAs from this cluster. In miR-17 (Life Technologies, Carlsbad, CA) in six-well plates or 100- or the current study, contrary to the other miRNAs in the same cluster, 150-mm dishes. RA SFs were transfected with pre-miRNAs (100 nM) of miR-17 with NC–pre-miRNAs (Life Technologies) or anti–miR-17 (150 miR-17 expression was consistently low in RA synovial tissue nM) with NC anti-miRNA using Lipofectamine RNAiMAX transfection (STs) and RA SFs, but not in osteoarthritis (OA) STs or OA SFs, reagent (Life Technologies) for 48 h and then stimulated or not with TNF-a making miR-17 more disease relevant. Thus, this study was un- (20 ng/ml) for 30 min or 24 h. Total RNA containing miRNA fraction or dertaken to determine the role of miR-17 in RA pathogenesis. cell lysate were prepared after treatment. Protein expression was determined using Western immunoblotting. Transfection efficiency was con- The results from the current study showed that miR-17 is confirmed by the significant upregulation of miR-17 expression using TaqMan sistently low in the diseased serum, STs, and SFs, as well as in a rat assays (Life Technologies). THP-1 cells were differentiated into macro- Downloaded from adjuvant-induced arthritis (AIA) model of RA. To extend these phages by treatment with PMA (300 ng/ml for 3 h) and then transfected findings, the current study was carried out to assess the effect of with pre-miRNAs (100 nM) or NC using Lipofectamine RNAiMAX transfection reagent (Life Technologies) for 48 h, followed by treatment or miR-17 overexpression on the posttranslational ubiquitination in a a not with TNF- (20 ng/ml) for 30 min. RA SFs (n = 4) were also trans- TNF- signaling. The results showed that miR-17 overexpression fected with ASK1 siRNA or NC siRNA for 48 h using Lipofectamine inhibited TRAF2 expression and its association with cIAP2, RNAiMAX transfection reagent (Life Technologies) and then stimulated thereby suppressing TNF-a signaling pathways and downstream or not with TNF-a (20 ng/ml) for 24 h. RA SFs were pretreated with the inflammatory proteins. This study provides novel insights into the selective inhibitor of ubiquitin-conjugating enzyme E1 (PYR41; 1 mM) for http://www.jimmunol.org/ a 2 h and transfected with pre–miR-17 or NC–pre-miRNA for 48 h, followed role of miR-17 in downregulating TNF- signaling by influencing by 24 h of stimulation with TNF-a. the protein-ubiquitination pathway in RA SFs. Quantitative real-time PCR analysis Materials and Methods Total RNA was reverse transcribed using a SuperScript First Strand cDNA Reagents and Abs Synthesis Kit (Life Technologies), according to the manufacturer’s protocol. Expression of miR-17 (also known as miR-17-5p), miR-18a, miR-19a, Rabbit polyclonal anti-TRAF2 (#sc-876), mouse monoclonal b-actin (#sc- miR-19b, miR-20a, and miR-92-1 was quantified using TaqMan microRNA 47778), mouse monoclonal anti–USP-14 (#sc-100630), mouse monoclonal Assays with U6snRNA as control (Life Technologies). Expression of ASK1 anti–MMP-1 (#sc-58377), rabbit polyclonal anti–MMP-13 (#sc-30073), mRNA was quantified using SYBR Green quantitative real-time PCR, and by guest on October 2, 2021 rabbit polyclonal anti–lamin A/C (#sc-20681), and rabbit polyclonal GAPDH was used as control. Quantification of the relative expression was anti–p-IkB-a (#sc-8404) Abs were purchased from Santa Cruz Biotech done using the ΔΔCt method. (Santa Cruz, CA). Rabbit monoclonal anti-cIAP1 (#7065), rabbit monoclonal anti-cIAP2 (#3031), rabbit anti-USP2 (#8036), rabbit monoclo- RNA isolation, reverse transcription, and miR-17 quantitative nal anti-RAD23A (#24555), anti-K63 polyubiquitin (#5621), anti-K48 real-time PCR in serum samples polyubiquitin (#8081), anti–STAT-3 (#9132), anti–NF-kBp65 (#8242), anti–p-c-Jun (S73) (#9164), anti–p-p38 (T180/Y182) (#4511), anti–p-JNK The Exiqon serum RNA purification protocol was followed for the total (T183/Y185) (#9251), total JNK (#8690), total p-38 (#9258), and p–STAT-3 RNA–containing small RNA fraction using a miRCURY RNA Isolation (S727) (#9134) Abs were purchased from Cell Signaling Technology Kit - Biofluids (Exiqon, Woburn, MA). Serum samples from healthy and (Beverly, MA). TRAF2 mouse monoclonal (#AM1895B) Ab for immu- RA donors, AIA rats, and naive controls were thawed on ice. A total of 200 noprecipitation (IP) was purchased from Abgent (San Diego, CA), and ml of human serum or 50 ml of rat serum from each donor was transferred anti-PSMD13 (#5937-1) Ab was purchased from Epitomics (Burlingame, into a 1.5-ml Eppendorf tube and centrifuged at 3000 3 g for 5 min at 4˚C CA). Total ASK1 (#ab131506) and p-ASK1 Thr838/845 Abs were purchased to remove debris. Serum was transferred into a new 1.5-ml Eppendorf tube, from Abcam (Cambridge, MA) and Cell Signaling Technology, respec- and 60 ml of lysis buffer was added containing 1.17 ml of carrier RNA tively. Human Cytokine Array C5 (#AAH-CYT-5) was purchased from (0.8 mg/ml) from bacteriophage MS2. Samples were incubated at room RayBiotech (Norcross, GA). SMARTpool ON-TARGET plus ASK1 small temperature for 3 min and subsequently mixed with 20 ml of protein interfering RNA (siRNA) or negative control (NC) siRNA was purchased precipitation solution. After centrifugation at 11,000 3 g, the aqueous from GE Dharmacon (Lafayette, CO). phase containing the RNA was carefully transferred into a new collection tube, and RNA was precipitated with isopropanol. The mixture was applied Isolation and culture of human healthy, OA, and RA SFs to an miRNA Mini spin column and washed several times, and RNA was eluted by the addition of 50 ml of RNase-free water. Extracted RNA was The procurement of deidentified human healthy/nondiseased (NL), OA, and processed the same day for cDNA synthesis using the Universal cDNA RA tissue (ST) was obtained under a protocol approved by the Institutional synthesis kit II and UniSp6 RNA spike-in control primer. Quantitative real- Review Board (IRB#106628). Human SFs were derived from STs of pa- m m 3 tients diagnosed with OA or RA from autopsies/amputation who underwent time PCR was performed in 10- l reactions containing 4 lof40 diluted reverse-transcription product, 5 mlof23 SYBR Green Master Mix, and total joint replacement surgery (mostly knee joints) or synovectomy. NL STs m from nonarthritis individuals were obtained at the time of autopsy or ampu- 1 l of UniRT LNA PCR primers for miR-17. Reaction mixtures were tation. ST from 18 RA patients (mean age 6 SD: 74.2 6 8.5 y), 12 OA incubated at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 s and 60˚C for 1 min, followed by melting curve stage. miRNA-93-5p was used as patients (73.8 6 12.7 y), and 8 NL subjects (60.5 6 9.8 y) was used in the reference control for sample analysis (28). A threshold cycle (Ct) was ob- current study. The deidentified human NL, OA, and RA STs were obtained served in exponential phases of the amplification, and quantification of the from the Cooperative Human Tissue Network (Columbus, OH) and the Na- DD tional Disease Research Interchange (Philadelphia, PA). Tissue specimens were relative expression levels was determined by the Ct method. washed by sterile PBS, minced, and processed as previously described (27). Bioinformatics analysis SFs were grown in RPMI 1640 containing 2 mM L-glutamine with 10% FBS, at 37˚C, in a humidified atmosphere with 5% CO2. Cells were used between Ingenuity Pathway Analysis (IPA) was used to interpret the differentially passages 5 and 10 for these studies. For some studies, RNA was prepared di- expressed genes in terms of an interaction network that might be altered as a rectly from ST from NL donors, OA and RA patients, or rat AIA or naive joints. result of RNA changes induced by miR-17 overexpression in RA SFs The Journal of Immunology 2221 compared with NC. Genes with p , 0.05 (Student t test) were selected ELISAs from mRNA Sequencing data, and a list of differentially regulated genes and their corresponding expression values were uploaded to the IPA ap- Measurement of IL-6 and IL-8 in the culture supernatants of RA SFs was plication software (Ingenuity Systems, http://www.ingenuity.com). IPA analyzed using ELISA, according to the manufacturer’s instructions (R&D was done with standard settings (Ingenuity knowledge case [gene only], Systems). direct and indirect, includes endogenous chemicals, consider only rela- tionships where confidence = experimentally observed). TargetScan 7.0, Cytokine Ab array PicTar, and miRanda algorithms were used to identify miR-17 binding We used a commercial cytokine Ab-based array designed to detect 80 sites in TRAF2, cIAP1 (BIRC2), cIAP2 (BIRC3), USP2, and PSMD13 cytokines (RayBio Human Cytokine Array C5; RayBiotech). RA SFs were mRNA. transfected with pre–miR-17 or NC–pre-miRNA for 48 h, followed by TNF-a stimulation for 24 h. Conditioned medium from four patients was RNA library preparation and sequencing pooled for each experimental condition and used in the assay. Experi- Human RA SFs from two RA patients were transfected with pre–miR-17 or ments were performed essentially as recommended by the manufacturer. NC–pre-miRNA for 48 h, and total RNA was prepared using an miRNeasy Briefly, array membranes were incubated for 1 h in 2 ml of blocking m kit (QIAGEN). Next, total RNA integrity was checked using an Agilent buffer, incubated overnight with 2 ml of the pooled (n = 4; 500 l from Technologies 2100 Bioanalyzer (Santa Clara, CA). A total of 10 ng of each) culture supernatant, and washed. Next, a mixture of 80 biotinylated high-quality RNA was used to make cDNA for amplification with the Ion Abs diluted 1:250 was added to each membrane (1 ml/array membrane) AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher and incubated for 5 h at 4˚C overnight. Membranes were washed, sand- Scientific, Grand Island, NY). The cDNA was subjected to 12 cycles of wiched Ags were detected by ECL by incubating the membranes with amplification with panel primers and barcoded with adapters, as recom- 2 ml of a peroxidase-labeled streptavidin solution (diluted 1:1000), and mended. Resulting sequencing libraries were quantified by quantitative signals were captured on x-ray films. Arrays were processed simulta- PCR using SYBR FAST Master Mix (Kapa Biosystems, Wilmington, neously for image acquisition. Each spot intensity was analyzed using GELDOC software, and the mean value was used. Normalized values MA). Sets of eight libraries were balanced and pooled, and sequencing Downloaded from beads were produced on an Ion Chef. Sequencing was performed on an Ion were calculated using the formula: X (Ny) = X(y) * P1/P(y), where P1 is P1 semiconductor sequencing chip using an Ion Proton System (Thermo the average signal density of the positive control spot on the reference Fisher Scientific). Data were collected, and primary analysis was per- array, P(y) is an average signal density of the positive control spot on array formed using Torrent Suite software version 5.0.3. Reads were mapped to “y,” X(y) is the signal density for a particular spot on array for sample “y,” the panel, and expression values were determined. R Software version R-3.2.3 and X (Ny) is the normalized value of a particular spot “X” on array for was used to generate heat map (29). Microarray data were submitted to the sample “y.” National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE83930 (http://www.ncbi.nlm.nih.gov/geo/ Immunoprecipitation assay http://www.jimmunol.org/ query/acc.cgi?token=ahgtwusgflmxnah&acc=GSE83930). RA SFs were transfected with pre–miR-17 or NC–pre-miRNA in 150-mm dishes for 48 h, starved overnight, and stimulated with TNF-a for 30 min. Luciferase reporter assay Cells were washed two times in ice-cold 13 PBS, lysed in 500 ml of RIPA A luciferase reporter construct encompassing the 39UTR of ASK1 mRNA buffer as described earlier, and used for IP assays. Clear lysate was sub- (NM_005923) with the binding sites for miR-17 and an empty control jected to protein estimation using a DC Protein Assay (Bio-Rad). A total of m reporter construct was obtained from Genecopoeia (Rockville, MD). 3 g of Ab was used per milligram of whole-cell extract (1 mg) from each ASK1 39UTR sequences were inserted downstream of the secreted Gaussia sample was subjected to IP using TRAF2 (Abgent), K63-ubiquitin (Cell Luciferase reporter gene driven by the SV40 promoter. A secreted alkaline Signaling Technologies), or K48-ubiqutin (Cell Signaling Technologies) phosphatase reporter driven by a CMV promoter cloned into the same Abs. A similar amount of nonspecific IgG control Ab (Flag M2; Sigma) vector served as the internal control for normalization. RA SFs were cotrans- was used as isotype control. Ab and whole-cell extract were incubated at 4˚C by guest on October 2, 2021 fected with ASK1 luciferase reporter construct (1 mg) with pre-miRNA on a rotor overnight, followed by incubation with protein G–Sepharose (100 nM) of miR-17 or NC–pre-miRNA (100 nM) using Lipofectamine beads for 4 h to capture Ab and protein complex. Beads were subjected to 3 2000 transfection reagent (all from Life Technologies). After 48 h, the three washes with RIPA wash buffer, followed by a final wash with 1 3 conditioned media were collected, and luciferase activity was assayed PBS. Protein beads complex was eluted by boiling in 2 SDS sample using a Secrete-Pair Dual Luminescence Assay kit (Genecopoeia). Each buffer and resolved on 4–15% Bio-Rad TGX gel using Western immu- m experiment was repeated in three independent SF donors, and each assay noblotting. For K63 and K48 IP assays, 120 l of bead volume equivalent was performed in triplicate. of Dynabeads (Invitrogen, Thermo Fisher Scientific) was subjected to cross-linking with 18 ml of K63 polyubiquitin Ab or 9 mlofK48 Preparation of nuclear extracts polyubiquitin Ab (Cell Signaling Technologies) using BS3 cross-linker (Thermo Fisher Scientific), as per the manufacturers’ instructions. Beads To study the effect of miR-17 on TNF-a–induced NF-kBp65, p–c-Jun, and were divided equally into three samples for IP assay. Protein complex p–STAT-3 activation, RA SFs were transfected with pre–miR-17 or NC– was eluted from cross-linked beads using 23 sample buffer for 15 min at pre-miRNA (100 nM) and then stimulated or not with TNF-a (20 ng/ml) 70˚C. for 30 min or 24 h. Upon termination, cells were washed with ice-cold PBS, collected by scraping, and centrifuged at 1500 3 g for 5 min at 4˚C. Induction of AIA in rats Nuclear fractions were prepared, and equal amounts of protein (15 mg) from nuclear fractions were evaluated by Western blotting to study the Female Lewis rats (Harlan Laboratories, Indianapolis, IN) weighing level of NF-kBp65, p–c-Jun, and p–STAT-3 expression. 135–160 g were injected s.c. at the base of the tail with 300 ml(5mg/ ml) of lyophilized Mycobacterium butyricum (Difco Laboratories, Western immunoblotting Detroit, MI) in sterile mineral oil. The day of adjuvant injection was considered day 0. Body weight and ankle circumferences were mea- SFs were lysed in RIPA lysis buffer with complete protease inhibitor sured on days 0, 8, and 18 in a blinded manner, as described previously mixture (Roche, Indianapolis, IN). Cell lysates/tissue homogenates/ (30). The study also included a naive (no-adjuvant) group for com- nuclear fractions were resolved on 10% SDS-PAGE gels and trans- parison. Naive and AIA rats were sacrificed on days 8 and 18. The ferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes circumferences of both hind ankles from each rat were averaged, and n were blocked with 5% nonfat dry milk powder/BSA in TBS containing is the number of rats used in each experimental group. Joints and serum 0.1% Tween-20 and probed with 1:1000 diluted polyclonal Abs or mAbs samples were collected on day 18. The animal study was approved by specific for TRAF2, cIAP1, cIAP2, USP2, RAD23A, USP14, PSMD13, k a k the Institutional Animal Care and Use Committee of Washington State K63, K48, ASK1, STAT3, p–c-Jun, p–I B- ,NF- Bp65, p-p38, p-JNK, University. total p38, total JNK, MMP-1, MMP-13, or b-actin. Culture supernatants from miR-17–transfected and TNF-a–stimulated RA SFs were concen- Statistical analyses trated and used for MMP-1 and MMP-13 expression. Immunoreactive bands were visualized using HRP-linked secondary Abs and ECL (Bio- The Student t test (unpaired two-tailed t test), followed by one-way Rad Molecular Imager ChemiDoc XRS+). Images were analyzed using ANOVA, was used to calculate statistical differences between different Gel Doc software. Each band was scanned using Image laboratory 5.1 variables. Data shown are mean 6 SE, unless stated otherwise. Compar- software, and the expression values (pixels per band) are presented as isons were performed using the GraphPad Prism 6 software package, and mean 6 SE. p , 0.05 was considered significant. 2222 miR-17 SUPPRESSES TNF-a SIGNALING IN RA

Results expressing miR-17. Among the panel of 20,803 genes, the miR-17 expression correlates inversely with RA pathogenesis expression of 15,067 genes, as shown in the representative heat map, was observed in RA SFs transfected with pre–miR-17 and miR-17–92 is a polycistronic miRNA cluster located in chromo- NC–pre-miRNA (Supplemental Fig. 2A). A total of 664 sig- some 13 (Supplemental Fig. 1A). To identify the basal expression nificantly modulated genes (301 upregulated and 363 down- levels of the miR-17–92-1 cluster in RA, we performed quantitative regulated) was used for IPA. IPA predicted the protein ubiquitin real-time PCR using RNA from SFs and STs of healthy (NL) do- pathway as a major canonical pathway affected by the dif- nors and OA and RA patients. The expression levels of all six miRNAs (miR-17 [∼69%, p , 0.01], miR-18a [∼81%, p , 0.05], ferentially regulated genes (Supplemental Fig. 2B). Interest- miR-19a [∼87%, p , 0.01], miR-20a [∼85%, p , 0.01], miR-19b ingly, IPA generated an interactome that showed connectivity k [∼79%, p , 0.01], and miR-92-1 [∼80%, p , 0.05]) were signif- among various ubiquitin ligases, NF- B family proteins, icantly decreased in RA ST compared with NL ST (Supplemental AP-1/c-Jun, and 20S and 26S proteasome system (Supplemental Fig. 1B). Interestingly, we observed that miR-17 levels were con- Fig. 2C). sistently low in ST and SFs from RA donors compared with those The heat map generated based on the list of identified ubiquitin from NL and OA donors (Fig. 1A, 1B). To further verify miR-17 ligases and other proteasome pathway proteins regulated by miR-17 expression, we used a rat AIA model in which inflammation starts overexpression showed some of the key ubiquitin and deubiquitin a around day 8 and peaks around day 18 (31). Transcriptional analysis ligases in TNF- signaling, such as TRAF2, cIAP2, USP2, of pulverized joint tissue from naive and AIA rats also showed a PSMD13, and RAD23A (Fig. 2A, 2B). This suggests that miR- a significant decrease in the expression of miR-17 on day 18 com- 17maybeintimatelyinvolvedintheregulationofUPSinTNF- signaling. The IPA results confirmed that miR-17 overexpression pared with the naive group (Fig. 1C, p , 0.05). Downloaded from Furthermore, we observed a significant decrease in miR-17 serum may significantly influence canonical pathways with high patho- levels in RA patients (Fig. 1D, p , 0.05). To further validate these logical relevance, including the protein ubiquitin pathway, a findings, we determined miR-17 levels in the serum of AIA and TNF- related weak inducer of apoptosis, and TNFR1 sig- naive animals. AIA animals exhibited signs of severe arthritis on naling (Supplemental Fig. 3A). Functional network and ca- day 18, as reflected by a significant increase in ankle circumference nonical pathway analysis of 30 selected genes associated with ∼ , TNF-a signaling and ubiquitination is shown in Fig. 3B and

( 65%), compared with the naive group (Fig. 1E, p 0.01). A lower http://www.jimmunol.org/ serum miR-17 level, together with the increase in ankle circumfer- Supplemental Fig. 4. ences,wasobservedinAIAratsonday18(Fig.1F,p , 0.01). RNA-sequencing results were confirmed by quantitative real- time PCR and Western blot analysis in RA SFs with miR-17 over- Protein ubiquitin pathway genes in RA SFs are modulated by expression. Our results showed a significant decrease in the basal miR-17: confirmation by IPA expression levels of TRAF2, cIAP1, cIAP2, USP2, and PSMD13 To determine the role of miR-17 in RA, we used a gain-of-function proteins and no change in the constitutive expression levels of model and performed RNA-sequencing analysis in RA SFs over- RAD23A and USP14 (Fig. 2C–E). by guest on October 2, 2021

FIGURE 1. Clinical significance of miR-17 expression levels in RA patients. Expression profile of miR-17 in ST (A) and in SFs (B) obtained from NL, OA, and RA donors. (C) Expression levels of miR-17 in AIA (day 18) model of human RA compared with the naive group. miRNA expression levels were evaluated using a TaqMan-based quantitative real- time PCR assay. Data are mean 6 SEM for the indicated number of animals. (A–C) U6snRNA was used as a reference control. (D) Lower miR-17 levels in the serum of human RA patients. miR-93-5p was used as a reference control. (E) AIA rats showed an increase in ankle circumferences on day 18. (F) Lower miR-17 expression was associated with RA in these AIA rats. Expression of miR-17 was determined using quantitative real-time. miR-93-5p was used as a reference control. Data are mean 6 SEM for the indicated number of serum donors or animals used per group. *p , 0.05, **p , 0.01. The Journal of Immunology 2223 Downloaded from http://www.jimmunol.org/ FIGURE 2. miR-17 regulates genes related to TNF-a signaling in RA SFs. (A) Heat map of the gene-expression data for selected genes related to TNF-a signaling and ubiquitination. Each row represents a single transcript, and each column represents a single sample. Red represents lower expression; green represents higher expression. (B) Fold change in selected genes from RNA-sequencing data (mean 6 SD; n =2).(C) Effect of miR-17 on the expression of selected genes (TRAF2, cIAP1, cIAP2, USP2, PSMD13, USP14, and RAD23A) was verified using quantitative real-time PCR. RA SFs (n = 4) were transfected with pre–miR-17 or NC–pre- miRNA for 48 h, and total RNA was prepared. GAPDH was used as a reference control. (D and E) Verification of RNA sequencing data for TRAF-2, cIAP1, cIAP2, USP2, USP14, PSMD13, and RAD23A (n = 4) by Western immunoblotting. RA SFs were transfected with pre–miR-17 or NC–pre-miRNA for 48 h, and cell lysates were prepared. Densitometry for Western blots for the indicated number of patients was performed, and values were normalized with b-actin. *p , 0.05, **p , 0.01.

miR-17 restoration modulates E2 ligases to inhibit TNF-a TNF-a signaling proteins. A modest decrease in global K48- by guest on October 2, 2021 signaling in RA SFs linked ubiquitination was also observed in TNF-a–stimulated To determine the impact of miR-17 on ubiquitination mechanisms RA SFs; however, this was not statistically significant (Fig. 3F). essential for TNF-a signaling, RA SFs were transiently transfected To further examine the effect of miR-17 on K63 and K48 with miR-17 mimic or an NC and stimulated with TNF-a for 24 h ubiquitination specific to TRAF2, cIAP1, and cIAP2 proteins, a (Fig. 3). For comparison, we also included miR-20a, another TNF- –stimulated RA SFs, with or without control or miR-17 miR-17 family member shown to have similar function (22, 32). overexpression, were immunoprecipitated with K63- and K48- Transfection with pre–miR-17 or miR-20a for 48 h resulted in a linked ubiquitinated proteins from cell lysates and probed for significant increase in the expression of both miRNAs compared TRAF2, cIAP1, and cIAP2 (Fig. 3G, 3H); however, we did not with NC-transfected samples (Fig. 3A, 3B). Consistent with our observe significant changes in K63-mediated ubiquitination of sequencing results, Western blot analysis showed that TRAF2, TRAF2, cIAP1, and cIAP2 (Fig. 3G). In contrast, Western blotting cIAP1, cIAP2, PSMD13, and USP2 expression was significantly results with K48-linked immunoprecipitated proteins showed reduced in TNF-a–stimulated RA SFs transfected with pre–miR-17 that miR-17 and miR-20a enhanced K48-mediated ubiquitination a compared with the NC (Fig. 3C). However, the expression of of TRAF2, cIAP1, and cIAP2 compared with the NC in TNF- – RAD23A and USP14 was not altered by miR-17 overexpression in stimulated RA SFs (Fig. 3H). These results suggest that miR- a TNF-a–stimulated RA SFs (Fig. 3C). We used bioinformatics 17 induces the K48-mediated ubiquitination of the key TNF- algorithms (TargetScan 7.0, PicTar, and miRanda) to determine signaling proteins TRAF2, cIAP1, and cIAP2 that may influence whether miR-17 regulates the expression of TRAF2, cIAP1, their stability and, thereby, inhibit downstream signaling events in cIAP2, USP2, and PSMD13 via the sequence spanning the 39UTR RA SFs. of their mRNA. No direct binding site of miR-17 in the 39UTR of miR-17 interferes with the association of TRAF2 and cIAP2 in TRAF2, cIAP1, cIAP2, PSMD13, or USP2 mRNA was observed TNF-a–stimulated RA SFs (data not shown), which indicates that miR-17 may impact cellular processes in RA SFs to regulate their gene expression. To further understand the impact of miR-17 on the early events in To investigate the mechanism of TRAF2 degradation by miR-17 TNF-a signaling, we transfected RA SFs with NC–pre-miRNA, in response to TNF-a stimulation, we analyzed K63- and K48- pre–miR-17, or pre–miR-20a, followed by TNF-a stimulation for linked ubiquitination patterns in miR-17–transfected RA SFs. 30 min. Cell lysates were immunoprecipitated with TRAF2 Ab Surprisingly, our results showed that miR-17 and miR-20a in- and analyzed for its association with the signaling partners cIAP1, creased global K63 ubiquitination in TNF-a–stimulated RA SFs cIAP2, and RAD23A in RA SFs. We found that miR-17 prefer- (Fig. 3D, 3E), which suggests that miR-17 and miR-20a may entially inhibited the association of cIAP2 with TRAF2 in TNF- enhance total K63 ubiquitination processes to stabilize certain a–stimulated RA SFs but elicited no effect on RAD23A (Fig. 4A). 2224 miR-17 SUPPRESSES TNF-a SIGNALING IN RA Downloaded from http://www.jimmunol.org/

FIGURE 3. miR-17 regulates ubiquitination and DUB processes to inhibit TNF-a signaling in RA SFs. (A and B) Exogenous delivery of miR-17 and miR-20a in RA SFs (n = 4) showing a signiﬁcant increase in the expression of both miRNAs after 48 h of transfection. (C) Effect of miR-17 on TRAF2, cIAP1, cIAP2, USP2, USP-14, PSMD13, and RAD23A in TNF-a–stimulated RA SFs. RA SFs were transfected with pre–miR-17 for 48 h, followed by stimulation with TNF-a for 24 h. Cell lysates (n = 4) were prepared and assayed for their expression by Western immunoblotting. Densitometry for the Western blots for the indicated number of patients was performed, and values were normalized with b-actin. (D) RA SFs were transfected with pre–miR-17/ pre–miR-20a/NC–pre-miRNA for 48 h, followed by TNF-a stimulation for 30 min. Total cell lysates (n = 3) were used to determine the effect on global expression of K63-linked (upper panel) and K48-linked (lower panel) polyubiquitination. (E and F) Densitometry analysis for K63 and K48 band intensity values normalized to b-actin. RA SFs (n = 3) were transfected with pre–miR-17/pre–miR-20a/NC–pre-miRNA for 48 h, followed by 30 min of TNF-a stimulation, immunoprecipitated for K63 polyubiquitin (G) or K48 polyubiquitin (H), and probed for the expression of TRAF2, cIAP1, and cIAP2. *p , 0.05, **p , 0.01. by guest on October 2, 2021

To investigate whether this effect of miR-17 is specific to RA expression in TNF-a–stimulated RA SFs (Fig. 5A, 5B). ASK1 (a SFs, THP-1–differentiated macrophages were transfected with serine-threonine kinase) regulates downstream p38 and JNK NC–pre-miRNA, pre–miR-17, or pre–miR-20a, followed by TNF-a pathways and was shown to play a critical role in RA pathogenesis stimulation for 30 min. Cell lysates were immunoprecipitated with via TNF-a signaling (33, 34). Activation of ASK1 is tightly reg- TRAF2 and probed for the same panel of proteins shown in Fig. 4A ulated by the phosphorylation of threonine residue (Thr838 and and 4B. In agreement with the findings in RA SFs, we found a Thr845 of human and mouse ASK1, respectively) (33, 34). A decrease in the association of TRAF2 with cIAP2 in macrophages higher expression of total ASK1 and ASK1 Thr838 was found in (Fig. 4C). Similar to RA SFs, no early impact of miR-17 over- RA SFs compared with NL SFs (Fig. 6A–C). Interestingly, we expression was observed on the expression of these proteins, as found that miR-17 binds to the ASK1 39UTR to regulate its ex- seen in the analyzed inputs (Fig. 4B, 4D). To study the impact of pression in RA SFs, which may downregulate p-p38 and p-JNK miR-17 on the ubiquitination pathways, a chemical inhibitor of (Fig. 6D–F). Interestingly, ASK1 knockdown also inhibited TNF- ubiquitin-conjugating enzyme E1 (PYR41) with no effect on E2 or a–induced IL-6 and IL-8 production in RA SFs (Fig. 6G). miR-17 E3 ubiquitin ligases was used. RA SFs were pretreated with PYR41 was shown to target the STAT3 39UTR and regulate STAT3 ex- for 2 h and then transfected with miR-17 (Supplemental Fig. 2D, pression, thereby leading to a loss of suppressive function in 2E). Our results showed that inhibiting the ubiquitin E1 ligase had myeloid-derived suppressor cells (35, 36). Consistent with the no effect on TNF-a–induced IL-6 and IL-8 production, whereas previous findings, an overexpression of miR-17 reduced the nu- miR-17 decreased production in RA SFs (Supplemental Fig. 2D, clear translocation of p-STAT3 in TNF-a–stimulated RA SFs 2E). These findings suggest that miR-17 might interfere with the (Fig. 5A, 5B). Furthermore, we found that miR-17 also inhibited UPS to impact the stability and efficiency of ubiquitin E3 ligase TNF-a–induced IkB-a phosphorylation compared with NC TRAF2 to associate with cIAP1/cIAP2 complex and participate in (Fig. 5A, 5B). In accordance with these findings, our results showed TNF-a signaling. that miR-17 moderately reduced TNF-a–induced activation and nuclear translocation of transcription factors NF-kBp65 and p–c-Jun Impact of miR-17 on MAPK/NF-kB/STAT3 pathways in TNF- in RA SFs (Fig. 5A, 5B). a–stimulated RA SFs a RA SFs were transfected with pre–miR-17 and then stimulated miR-17 inhibits TNF- –induced IL-6, IL-8, and MMP with TNF-a for 30 min or 24 h to assess the regulation of sig- production in RA SFs naling proteins and soluble proteins, respectively. We observed To understand the impact of TNF-a signaling inhibition by miR- that miR-17 overexpression led to a decrease in p-p38 and p-JNK 17, we determined the effect of miR-17 overexpression on the The Journal of Immunology 2225 Downloaded from http://www.jimmunol.org/

FIGURE 4. miR-17 interferes with the association of TRAF2-cIAP2 in TNF-a–stimulated RA SFs. (A)RASFs(n = 4) were transfected with pre–miR-17/ pre–miR-20a/NC–pre-miRNA for 48 h, followed by TNF-a-stimulation for 30 min. Cell lysates were immunoprecipitated with TRAF2 or IgG and probed for changes in the expression of cIAP1, cIAP2, and RAD23A. Densitometric analysis of cIAP1 and cIAP2 band intensity. (B) RA SFs were transfected with miR-17 for 48 h, followed by stimulation with TNF-a for 30 min. Cell lysates (n = 4) were assayed for the expression of TRAF2, cIAP1, cIAP2, RAD23A, and b-actin by Western immunoblotting. (C) THP-1–differentiated macrophages (n = 3) were transfected with pre–miR-17/pre–miR-20a/NC–pre-miRNA by guest on October 2, 2021 for 48 h, followed by TNF-a stimulation for 30 min. Total cell lysates were immunoprecipitated with TRAF2 or IgG and probed for changes in the expression of cIAP1 and cIAP2. Densitometric analysis of cIAP1 and cIAP2 band intensity is shown. (D) THP-1–differentiated macrophages were transfected with miR-17 for 48 h, followed by stimulation with TNF-a for 30 min. Cell lysates (n = 3) were assayed for the expression of TRAF2, cIAP1, cIAP2, and b-actin by Western immunoblotting. *p , 0.05, **p , 0.01. production of 80 cytokines, chemokines, and growth factors using TNF-a signaling events in RA (Fig. 6H). We found that miR-17 cytokine array. Densitometry analysis of the array membrane results expression is very low in RA, and its restoration in vitro may obtained from the conditioned media of RA SFs treated with NC– induce K48-linked ubiquitination of TRAF2 and cIAP2 and pre-miRNA or miR-17 in the presence of TNF-a showed a marked may reduce their association, which results in a reduction in inhibition of several proinflammatory cytokines and chemokines TNF-a–induced inflammatory mediators in RA SFs. Importantly, known to contribute to RA pathogenesis, including G-CSF (∼24%), this study validated the IPA data that suggest miR-17 may influence GM-CSF (∼19%), GRO (∼17%), GRO-a (∼31%), CCL23 (∼21%), posttranslational modifications independent of its 39UTR binding. IL-3 (∼16%), IL-6 (∼15%), IL-7 (∼26%), IL-8 (∼19%), MCP-1 These findings provide an opportunity for further understanding of (∼15%), M-CSF (∼25%), MDC (∼24%), MIG (∼24%), MIP-1b the impact of miRNA on cellular and posttranslational mechanisms, (∼25%), and TNF-b (∼22%) (Fig. 5C). To confirm this array result, such as the protein ubiquitination pathway, that are important in we used the same conditioned media to quantitate IL-6 and IL-8 the cytokine signaling networks for RA pathogenesis. production by ELISA. Consistent with the array results, miR-17 TRAF2, an E3 ubiquitin ligase, is a critical upstream component overexpression inhibited TNF-a–induced IL-6 and IL-8 produc- in the TNF-a signaling that activates MAPK and NF-kB path- tion in RA SFs (Fig. 5D, 5E). In contrast, the inhibition of endog- ways through the recruitment of cIAP1 and cIAP2 to the TNFR enous miR-17 expression showed a significant increase in IL-6 signaling complex (37, 38). TRAF2 is tightly regulated by posttrans- and IL-8 production in TNF-a–stimulated RA SFs (Fig. 5F, 5G). lational modifications, such as autophosphorylation, ubiquitination, or Extending these findings, our results also showed that over- DUB (39). Among these, ubiquitination plays an important role expression of miR-17 inhibited TNF-a–induced MMP-1 and MMP-13 in diverse cellular events and signaling; however, this mechanism production in RA SFs (Fig. 5H, 5I), suggesting a protective role in proinflammatory cytokine signaling networks has not been for miR-17 in cartilage degradation. well studied in RA SFs (40, 41). Our results showing that miR-17 reduced K48-linked ubiquitination in RA SFs further confirms Discussion the IPA findings of the influence on protein ubiquitin pathways. In the current study, we identified the role of miR-17 in modulating The UPS is indispensable for TNF-a signal transduction and posttranslational protein ubiquitination pathways to downregulate balances the cellular expression and activity of proteins by 2226 miR-17 SUPPRESSES TNF-a SIGNALING IN RA Downloaded from http://www.jimmunol.org/

FIGURE 5. Impact of miR-17 on MAPK/NF-kB/STAT3 pathways and downstream mediators in TNF-a–stimulated RA SFs. (A and B)EffectofmiR-17on p38, JNK, and IkB-a phosphorylation and nuclear translocation of NF-kBp65, p–c-Jun, and p-STAT3 in TNF-a–stimulated RA SFs. RA SFs (n = 3) were transfected with pre–miR-17 for 48 h and then stimulated or not with TNF-a for 30 min. Total cell lysates were probed for p38, JNK, and IkB-a, and nuclear fractions of NF-kBp65, p–c-Jun, and p-STAT3 were prepared and assayed using Western immunoblotting. (B) Densitometric analysis of Western blots (n =3)is shown. Band intensities were normalized to their total respective forms, b-actin, or lamin A/C (for nuclear fraction). (C) Human cytokine Ab array (Ray Biotech) was used to measure the secretion of 80 cytokines in the pooled conditioned medium (n = 4) from miR-17 or NC-miRNA overexpressed and TNF-a (20ng/ml)24hstimulatedRASFs.(D and E) RA SFs (n = 4) were transfected with pre–miR-17 for 48 h and then stimulated with TNF-a for24hto

determine IL-6 and IL-8 production using quantitative ELISA. Data are mean 6 SEM for the indicated number of SF donors. (F and G) RA SFs (n =4) by guest on October 2, 2021 were transfected with anti–miR-17 for 48 h and stimulated with TNF-a for 24 h to determine IL-6 and IL-8 production using quantitative ELISA. Data are mean 6 SEM for the indicated number of SF donors. (H and I) RA SFs were transfected with pre–miR-17 or NC for 48 h, followed by TNF-a stimulation for 24 h. Conditioned medium (n = 3) was concentrated using Amicon Ultra Centrifugal filters (Millipore) and analyzed for MMP-1 and MMP-13 protein expression using Western immunoblotting. Densitometric analysis of MMP-1 and MMP-13 (n = 3) is shown. *p , 0.05, **p , 0.01. ubiquitination and degradation (42). In contrast, DUB removes phages also confirmed that the proteasomal degradation of TRAF2 ubiquitin conjugates by activating ATP hydrolysis, thereby res- and cIAP2 by miR-17 further reduced their association and, cuing proteins from degradation (43). Among the different DUB possibly, downstream signaling events. enzymes (USP14, USP2, and PSMD13) modulated by miR-17 in In classical TNF-a signaling, TNFR2 forms a complex with RNA-sequencing data, our results in RA SF lysates confirmed TRAF2 and cIAP1/2 to further recruit and activate RIPK1, which that miR-17 suppressed the expression of PSMD13 and USP2 in further activates TAK1 kinase activity, leading to activation of the TNF-a–stimulated RA SFs. These results suggest that miR-17 NF-kB and MAPK pathways (15). As a result of the lack of affects the stability of TRAF2 and/or cIAP2 proteins, in part, TRAF2/cIAP2 association and efficient signaling activation by by regulating DUB activity of PSMD13, an important proteome miR-17, we observed inhibition of the activation of TNF-a– component, and USP2, a ubiquitin-specific protease important in induced p-p38, p-JNK, and p–IkB-a and a consequent reduction TNF-a–induced RA SFs. USP2 was shown to target multiple sub- in the nuclear translocation of p–c-Jun and NF-kBp65. The inhi- strates for protein stability (e.g., p53, cyclin D1) (44), and its mRNA bition of p–c-Jun and NF-kB transcription factors also caused a may be modulated by TNF-a in a cell-specific manner (45). Further significant reduction in TNF-a–induced MMP-1 and MMP-13 investigation is needed to understand the mechanism involved in production in RA SFs. We showed recently that a similar reduc- downregulation of USP2 by miR-17. In agreement with our results, tion in TRAF6 and TAK1 association by epigallocatechin-3- the miR-17–92 cluster was shown to target E3 ubiquitin ligases, gallate inhibited IL-1b signaling and downstream mediators of thereby affecting PTEN subcellular localization through mono- inflammation in RA SFs (47). It is worth noting that MMP-1 and ubiquitination of limb-innervating lateral motor neurons (46). MMP-13 have no 39UTR binding regions for miR-17, which Our findings from IP assays showed that miR-17 and miR-20a further suggests that this might be an indirect effect of miR-17 on enhanced K48-mediated ubiquitination of TRAF2, cIAP1, and tissue remodeling mediated in RA. Our results showed that miR-17 cIAP2, suggesting that these miRNAs induce K48-mediated pro- targets the ASK1 39UTR to regulate ASK1 expression, which may teasomal degradation of key TNF-a signaling proteins TRAF2, result in a reduction in p38 and JNK phosphorylation in RA SFs. A cIAP1, and cIAP2, inhibiting their expression and downstream decrease in TNF-a–induced IL-6 and IL-8 production was observed signaling. Further validation studies in THP-1–activated macro- in response to miR-17 overexpression, as well as after ASK1 The Journal of Immunology 2227 Downloaded from http://www.jimmunol.org/

FIGURE 6. miR-17 may directly target the ASK1 39UTR to regulate its expression in RA SFs. (A–C) Expression of total ASK1 and p-ASK Thr838 protein was evaluated in NL, OA, and RA SFs by Western immunoblotting. (D) Bioinformatics analysis of the predicted binding sites for miR-17 in the ASK1 mRNA 39UTR and their cross-species conservation. (E) Inhibitory effect of miR-17 on ASK1 39UTR luciferase reporter activity in RA SFs. ASK1 reporter vectors were transfected in RA SFs (n = 3) with pre–miR-17 or NC–pre-miRNA. The experiment was performed in triplicate. (F) The effect of pre– miR-17 transfection on ASK1 mRNA and protein expression at the basal level and in TNF-a–stimulated RA SFs (n = 3) was assessed using quantitative real-time PCR and Western immunoblotting, respectively. GAPDH or b-actin was used as a loading control. (G) The effect of ASK-1 siRNA on TNF-a– induced production of IL-6 and IL-8 in RA SFs was analyzed using ELISA. RA SFs (n = 4) were transfected with ASK1 siRNA for 48 h and stimulated with TNF-a for 24 h. Conditioned medium was analyzed for IL-6 and IL-8 production. Data are mean 6 SEM for the indicated number of patients using H a different donors. ( ) Schematic representation of the effect of miR-17 on TNF- signaling proteins in RA SFs. Our data suggest that miR-17 modulation of by guest on October 2, 2021 the UPS is an important mechanism that inhibits SF–mediated inflammation and tissue destruction in RA. *p , 0.05, **p , 0.01. knockdown in RA SFs. Importantly, in addition to directly targeting 39UTR (6). In the current study, in silico prediction analysis ASK1, miR-17 may influence the association of TRAF2 and cIAP2 showed no direct binding site for miR-17 in the 39UTRs of in TNF-a–stimulated RA SFs. We hypothesize that miR-17 may TRAF2, cIAP1, cIAP2, PSMD13, and USP2 mRNAs, indicating regulate TNF-a signaling at multiple steps in RA, and its influence that miR-17 may not target the respective mRNA 39UTRs of on TRAF2 and cIAP2 may be the earliest and most prominent in these genes to regulate their expression in RA SFs. A recently the TNF-a signaling pathway. In addition, recent studies showed described model to examine indirect effect of miRNA transfection that miR-17 targets the STAT3 39UTR to regulate its expression in postulates that the transfected miRNAs may compensate endog- myeloid-derived suppressor cells and in other cell types (35, 36), enous miRNAs for the available RNA-induced silencing complex suggesting that STAT3 regulation may be a direct downstream and, consequently, alter the regulation of their downstream targets effect of miR-17 through 39UTR binding. (51). Recent studies showed that, among all of the genes that were The miR-17–92 cluster was shown to influence acquired and differentially expressed with the miRNA modulation, ,20% were innate immune responses (48); however, individual members of this predicted miRNA targets (12), suggesting that the underlying co- cluster showed different biological activities. This is evident from ordinated changes in the overall patterns of gene expression involve our results whereby miR-17 mediated the suppression of TRAF2/ the modulation of centralized hub genes (52). These regulatory cIAP2 association. Furthermore, miR-17 was shown to promote genes (i.e., hub genes) have the potential to control a group of osteogenic differentiation in the inflammatory microenvironment, downstream genes to force differential gene-expression outcomes and miR-20a promoted osteogenesis of human myeloid-derived (53). Our finding is an important step toward understanding the suppressor cells via BMP signaling (49, 50). In contrast, miR-18a impact of miRNAs on the essential cellular processes and TNF-a enhanced MMP-1, IL-6, and IL-8 expression in TNF-a–stimulated signaling network in RA SFs, a cell type that has not been targeted RA SFs (26). Another study showed the suppression of miR-19a and for therapeutic approaches. Further studies are required to extend miR-19b in RA SFs by TLR ligands and identified TLR2 as a direct these in vitro findings in animal models of human RA to under- target of the miR-19 family (25). These findings suggest that in- stand the synovial and systemic benefit of therapeutically restoring dividual miRNAs in a cluster might have completely different miR-17 expression levels in RA. influences, depending on the level of expression and the cytokine signaling pathways involved that contribute to RA pathogenesis. Acknowledgments A key determinant for miRNA target recognition is the seed We thank Karen Porter and Sadiq Umar for help with the AIA animal match site in the target mRNA, which is located primarily in the study. 2228 miR-17 SUPPRESSES TNF-a SIGNALING IN RA

Disclosures 27. Ahmed, S., A. Pakozdi, and A. E. Koch. 2006. Regulation of interleukin-1beta- induced chemokine production and matrix metalloproteinase 2 activation by The authors have no financial conflicts of interest. epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts. Arthritis Rheum. 54: 2393–2401. 28. Mestdagh, P., P. Van Vlierberghe, A. De Weer, D. Muth, F. Westermann, References F. Speleman, and J. Vandesompele. 2009. A novel and universal method for Genome Biol. 1. Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. microRNA RT-qPCR data normalization. 10: R64. Cell 116: 281–297. 29. Khomtchouk, B. B., D. J. Van Booven, and C. Wahlestedt. 2014. Heatmap- 2. Akhtar, N., and T. M. Haqqi. 2013. MicroRNAs: tiny regulators with the great Generator: high performance RNAseq and microarray visualization software potential for gene regulation. In microRNAs in Toxicology and Medicine. S. C. suite to examine differential gene expression levels using an R and C++ hybrid Sahu, ed. Wiley-Blackwell, New York, p. 287–300. computational pipeline. Source Code Biol. Med. 9: 30. 3. Murata, K., H. Yoshitomi, S. Tanida, M. Ishikawa, K. Nishitani, H. Ito, and 30. Ahmed, S., H. Marotte, K. Kwan, J. H. Ruth, P. L. Campbell, B. J. Rabquer, T. Nakamura. 2010. Plasma and synovial fluid microRNAs as potential bio- A. Pakozdi, and A. E. Koch. 2008. Epigallocatechin-3-gallate inhibits IL-6 markers of rheumatoid arthritis and osteoarthritis. Arthritis Res. Ther. 12: R86. synthesis and suppresses transsignaling by enhancing soluble gp130 produc- 4. Salehi, E., R. Eftekhari, M. Oraei, A. Gharib, and K. Bidad. 2015. MicroRNAs in tion. Proc. Natl. Acad. Sci. USA 105: 14692–14697. rheumatoid arthritis. Clin. Rheumatol. 34: 615–628. 31. Joe, B., and R. L. Wilder. 1999. Animal models of rheumatoid arthritis. Mol. 5. Mogilyansky, E., and I. Rigoutsos. 2013. The miR-17/92 cluster: a com- Med. Today 5: 367–369. prehensive update on its genomics, genetics, functions and increasingly 32. Yu, Z., N. E. Willmarth, J. Zhou, S. Katiyar, M. Wang, Y. Liu, P. A. McCue, important and numerous roles in health and disease. Cell Death Differ. 20: A. A. Quong, M. P. Lisanti, and R. G. Pestell. 2010. microRNA 17/20 inhibits 1603–1614. cellular invasion and tumor metastasis in breast cancer by heterotypic signaling. 6. Lewis, B. P., C. B. Burge, and D. P. Bartel. 2005. Conserved seed pairing, often Proc. Natl. Acad. Sci. USA 107: 8231–8236. flanked by adenosines, indicates that thousands of human genes are microRNA 33. Hayakawa, T., A. Matsuzawa, T. Noguchi, K. Takeda, and H. Ichijo. 2006. The targets. Cell 120: 15–20. ASK1-MAP kinase pathways in immune and stress responses. Microbes Infect. 7. Akhtar, N., M. S. Makki, and T. M. Haqqi. 2015. MicroRNA-602 and 8: 1098–1107. microRNA-608 regulate sonic hedgehog expression via target sites in the coding 34. Matsuzawa, A., K. Saegusa, T. Noguchi, C. Sadamitsu, H. Nishitoh, S. Nagai, S. Koyasu, K. Matsumoto, K. Takeda, and H. Ichijo. 2005. ROS-dependent region in human chondrocytes. Arthritis Rheumatol. 67: 423–434. Downloaded from 8. Lee, I., S. S. Ajay, J. I. Yook, H. S. Kim, S. H. Hong, N. H. Kim, activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4- S. M. Dhanasekaran, A. M. Chinnaiyan, and B. D. Athey. 2009. New class of mediated innate immunity. Nat. Immunol. 6: 587–592. microRNA targets containing simultaneous 59-UTR and 39-UTR interaction 35. Zhang, M., Q. Liu, S. Mi, X. Liang, Z. Zhang, X. Su, J. Liu, Y. Chen, M. Wang, sites. Genome Res. 19: 1175–1183. Y. Zhang, et al. 2011. Both miR-17-5p and miR-20a alleviate suppressive po- 9. Brummer,€ A., and J. Hausser. 2014. MicroRNA binding sites in the coding re- tential of myeloid-derived suppressor cells by modulating STAT3 expression. gion of mRNAs: extending the repertoire of post-transcriptional gene regulation. J. Immunol. 186: 4716–4724. BioEssays 36: 617–626. 36. Kumar, R., S. K. Sahu, M. Kumar, K. Jana, P. Gupta, U. D. Gupta, M. Kundu, 10. Baek, D., J. Villeń, C. Shin, F. D. Camargo, S. P. Gygi, and D. P. Bartel. 2008. and J. Basu. 2015. MicroRNA 17-5p regulates autophagy in Mycobacterium The impact of microRNAs on protein output. Nature 455: 64–71. tuberculosis-infected macrophages by targeting Mcl-1 and STAT3. Cell Micro- http://www.jimmunol.org/ 11. Selbach, M., B. Schwanhaüsser, N. Thierfelder, Z. Fang, R. Khanin, and biol. 18: 679–691. N. Rajewsky. 2008. Widespread changes in protein synthesis induced by 37. Rothe, M., M. G. Pan, W. J. Henzel, T. M. Ayres, and D. V. Goeddel. 1995. The microRNAs. Nature 455: 58–63. TNFR2-TRAF signaling complex contains two novel proteins related to bacu- 12. Shahab, S. W., L. V. Matyunina, C. G. Hill, L. Wang, R. Mezencev, L. D. Walker, loviral inhibitor of apoptosis proteins. Cell 83: 1243–1252. and J. F. McDonald. 2012. The effects of microRNA transfections on global 38. Liu, Z. G., H. Hsu, D. V. Goeddel, and M. Karin. 1996. Dissection of TNF re- patterns of gene expression in ovarian cancer cells are functionally coordinated. ceptor 1 effector functions: JNK activation is not linked to apoptosis while NF- BMC Med. Genomics 5: 33. kappaB activation prevents cell death. Cell 87: 565–576. 13. Lim, L. P., N. C. Lau, P. Garrett-Engele, A. Grimson, J. M. Schelter, J. Castle, 39. Brown, K. D., B. S. Hostager, and G. A. Bishop. 2002. Regulation of TRAF2 D. P. Bartel, P. S. Linsley, and J. M. Johnson. 2005. Microarray analysis shows signaling by self-induced degradation. J. Biol. Chem. 277: 19433–19438. that some microRNAs downregulate large numbers of target mRNAs. Nature 40. Chen, Z. J. 2012. Ubiquitination in signaling to and activation of IKK. Immunol. 433: 769–773. Rev. 246: 95–106. 14. Tseng, P. H., A. Matsuzawa, W. Zhang, T. Mino, D. A. Vignali, and M. Karin. 41. van Wijk, S. J., and H. T. Timmers. 2010. The family of ubiquitin-conjugating by guest on October 2, 2021 2010. Different modes of ubiquitination of the adaptor TRAF3 selectively ac- enzymes (E2s): deciding between life and death of proteins. FASEB J. 24: tivate the expression of type I interferons and proinflammatory cytokines. Nat. 981–993. Immunol. 11: 70–75. 42. Wullaert, A., K. Heyninck, S. Janssens, and R. Beyaert. 2006. Ubiquitin: tool and 15. Gyrd-Hansen, M., and P. Meier. 2010. IAPs: from caspase inhibitors to modu- target for intracellular NF-kappaB inhibitors. Trends Immunol. 27: 533–540. lators of NF-kappaB, inflammation and cancer. Nat. Rev. Cancer 10: 561–574. 43. Phillips, A. H., Y. Zhang, C. N. Cunningham, L. Zhou, W. F. Forrest, P. S. Liu, 16. Mendell, J. T. 2008. miRiad roles for the miR-17-92 cluster in development and M. Steffek, J. Lee, C. Tam, E. Helgason, et al. 2013. Conformational dynamics disease. Cell 133: 217–222. control ubiquitin-deubiquitinase interactions and influence in vivo signaling. 17. Olive, V., I. Jiang, and L. He. 2010. mir-17-92, a cluster of miRNAs in the midst Proc. Natl. Acad. Sci. USA 110: 11379–11384. of the cancer network. Int. J. Biochem. Cell Biol. 42: 1348–1354. 44. Davis, M. I., and A. Simeonov. 2015. Ubiquitin-specific proteases as druggable 18. Grillari, J., M. Hackl, and R. Grillari-Voglauer. 2010. miR-17-92 cluster: ups and targets. Drug Target Rev. 2: 60–64. downs in cancer and aging. Biogerontology 11: 501–506. 45. Haimerl, F., A. Erhardt, G. Sass, and G. Tiegs. 2009. Down-regulation of the 19. Poliseno, L., L. Pitto, M. Simili, L. Mariani, L. Riccardi, A. Ciucci, M. Rizzo, de-ubiquitinating enzyme ubiquitin-specific protease 2 contributes to tumor M. Evangelista, A. Mercatanti, P. P. Pandolfi, and G. Rainaldi. 2008. The proto- necrosis factor-alpha-induced hepatocyte survival. J. Biol. Chem. 284: 495– oncogene LRF is under post-transcriptional control of MiR-20a: implications for 504. senescence. PLoS One 3: e2542. 46. Tung, Y. T., Y. L. Lu, K. C. Peng, Y. P. Yen, M. Chang, J. Li, H. Jung, S. Thams, 20. Yu, Z., C. Wang, M. Wang, Z. Li, M. C. Casimiro, M. Liu, K. Wu, J. Whittle, Y. P. Huang, J. H. Hung, and J. A. Chen. 2015. Mir-17∼92 governs motor neuron X. Ju, T. Hyslop, et al. 2008. A cyclin D1/microRNA 17/20 regulatory feedback subtype survival by mediating nuclear PTEN. Cell Rep. 11: 1305–1318. loop in control of breast cancer cell proliferation. J. Cell Biol. 182: 509–517. 47. Singh, A. K., S. Umar, S. Riegsecker, M. Chourasia, and S. Ahmed. 2016. 21. Hossain, A., M. T. Kuo, and G. F. Saunders. 2006. Mir-17-5p regulates breast Regulation of transforming growth factor b-activated kinase activation by cancer cell proliferation by inhibiting translation of AIB1 mRNA. Mol. Cell. epigallocatechin-3-gallate in rheumatoid arthritis synovial fibroblasts: suppres- Biol. 26: 8191–8201. sion of K(63)-linked autoubiquitination of tumor necrosis factor receptor- 22. Chang, C. C., Y. J. Yang, Y. J. Li, S. T. Chen, B. R. Lin, T. S. Wu, S. K. Lin, associated factor 6. Arthritis Rheumatol. 68: 347–358. M. Y. Kuo, and C. T. Tan. 2013. MicroRNA-17/20a functions to inhibit cell 48. Tsitsiou, E., and M. A. Lindsay. 2009. microRNAs and the immune response. migration and can be used a prognostic marker in oral squamous cell carcinoma. Curr. Opin. Pharmacol. 9: 514–520. Oral Oncol. 49: 923–931. 49. Liu, Y., W. Liu, C. Hu, Z. Xue, G. Wang, B. Ding, H. Luo, L. Tang, X. Kong, 23. Lin, Y. H., C. J. Liao, Y. H. Huang, M. H. Wu, H. C. Chi, S. M. Wu, C. Y. Chen, X. Chen, et al. 2011. MiR-17 modulates osteogenic differentiation through a Y. H. Tseng, C. Y. Tsai, I. H. Chung, et al. 2013. Thyroid hormone receptor coherent feed-forward loop in mesenchymal stem cells isolated from periodontal represses miR-17 expression to enhance tumor metastasis in human hepatoma ligaments of patients with periodontitis. Stem Cells 29: 1804–1816. cells. Oncogene 32: 4509–4518. 50. Zhang,J.F.,W.M.Fu,M.L.He,W.D.Xie,Q.Lv,G.Wan,G.Li,H.Wang,G.Lu, 24. Philippe, L., G. Alsaleh, A. Pichot, E. Ostermann, G. Zuber, B. Frisch, J. Sibilia, X. Hu, et al. 2011. MiRNA-20a promotes osteogenic differentiation of human S. Pfeffer, S. Bahram, D. Wachsmann, and P. Georgel. 2013. MiR-20a regulates mesenchymal stem cells by co-regulating BMP signaling. RNA Biol. 8: 829–838. ASK1 expression and TLR4-dependent cytokine release in rheumatoid 51. Khan, A. A., D. Betel, M. L. Miller, C. Sander, C. S. Leslie, and D. S. Marks. fibroblast-like synoviocytes. Ann. Rheum. Dis. 72: 1071–1079. 2009. Transfection of small RNAs globally perturbs gene regulation by endog- 25. Philippe, L., G. Alsaleh, G. Suffert, A. Meyer, P. Georgel, J. Sibilia, enous microRNAs. Nat. Biotechnol. 27: 549–555. D. Wachsmann, and S. Pfeffer. 2012. TLR2 expression is regulated by microRNA 52. Shalgi, R., D. Lieber, M. Oren, and Y. Pilpel. 2007. Global and local architecture miR-19 in rheumatoid fibroblast-like synoviocytes. J. Immunol. 188: 454–461. of the mammalian microRNA-transcription factor regulatory network. PLOS 26. Trenkmann, M., M. Brock, R. E. Gay, B. A. Michel, S. Gay, and L. C. Huber. Comput. Biol. 3: e131. 2013. Tumor necrosis factor a-induced microRNA-18a activates rheumatoid 53. Seo, C. H., J. R. Kim, M. S. Kim, and K. H. Cho. 2009. Hub genes with positive arthritis synovial fibroblasts through a feedback loop in NF-kB signaling. Ar- feedbacks function as master switches in developmental gene regulatory net- thritis Rheum. 65: 916–927. works. Bioinformatics 25: 1898–1904.