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Transcription Factor NFAT5 Promotes Migration and Invasion of Rheumatoid Synoviocytes via Coagulation Factor III and CCL2 This information is current as of September 24, 2021. Saseong Lee, Jin-Sun Kong, Sungyong You, H. Moo Kwon, Seung-Ah Yoo, Chul-Soo Cho and Wan-Uk Kim J Immunol published online 23 May 2018 http://www.jimmunol.org/content/early/2018/05/22/jimmun ol.1701097 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 23, 2018, doi:10.4049/jimmunol.1701097 The Journal of Immunology

Transcription Factor NFAT5 Promotes Migration and Invasion of Rheumatoid Synoviocytes via Coagulation Factor III and CCL2

Saseong Lee,* Jin-Sun Kong,* Sungyong You,† H. Moo Kwon,‡ Seung-Ah Yoo,* Chul-Soo Cho,x and Wan-Uk Kim*,x

Fibroblast-like synoviocytes (FLSs) play a key role in the progression of rheumatoid arthritis (RA) as a primary component of invasive hypertrophied pannus. FLSs of RA patients (RA-FLSs) exhibit cancer-like features, including promigratory and proinvasive activities that largely contribute to joint cartilage and bone destruction. In this study, we hypothesized that the NF of activated T cell 5 (NFAT5), a transcription factor involving tumor invasiveness, would control the migration and invasion of RA-FLSs. Anal-

yses of transcriptomes demonstrated the significant involvement of NFAT5 in locomotion of RA-FLSs and that tissue factor (TF; also Downloaded from known as coagulation factor III) and CCL2 were the major downstream target genes of NFAT5 involving FLS migration and invasion. In cultured RA-FLSs, IL-1b and TGF-b increased TF and CCL2 expression by upregulating NFAT5 expression via p38 MAPK. Functional assays demonstrated that NFAT5- or TF-deficient RA-FLSs displayed decreased lamellipodia formation, cell migration, and invasion under IL-1b– or TGF-b–stimulated conditions. Conversely, factor VIIa, a specific activator of TF, increased migration of RA-FLSs, which was blocked by NFAT5 knockdown. Recombinant CCL2 partially restored the decrease

in migration and invasion of NFAT5-deficient RA-FLSs stimulated with IL-1b. NFAT5-knockout mouse FLSs also showed http://www.jimmunol.org/ decreased expressions of TF and CCL2 and reduced cell migration. Moreover, KRN2, a specific inhibitor of NFAT5, suppressed migration of FLSs stimulated with TGF-b. Conclusively, to our knowledge, this is the first study to provide evidence of a functional link between osmoprotective NFAT5 and TF in the migration and invasion of RA-FLSs and supports a role for NFAT5 blockade in the treatment of RA. The Journal of Immunology, 2018, 201: 000–000.

heumatoid arthritis (RA) is an autoimmune disease produce major proinflammatory cytokines, such as IL-6, and mediate characterized by tumor-like expansion of synovium and cartilage destruction via secretion of matrix metalloproteinases (2). R destruction of bone and cartilage. As a major cell type of Moreover, RA-FLSs abnormally proliferate, resist apoptotic death, by guest on September 24, 2021 the proliferating synovia, fibroblast-like synoviocytes (FLSs) play a and display abnormal invasiveness and excessive motility, culminat- crucial role in RA progression (1). FLSs of RA patients (RA-FLSs) ing in “pannus formation,” a pathologic hallmark of RA (2). The NF of activated T cell 5 (NFAT5) was originally identified as a transcription factor that regulates genes involving homeostasis against *Center for Integrative Rheumatoid Transcriptomics and Dynamics, The Catholic osmotic stress to cells (3). NFAT5 has been implicated in diabetic University of Korea, Seoul 06591, Korea; †Division of Urology, Department of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA microvascular disease in the kidney, inflammatory bowel diseases, 90048; ‡School of Nano-Bioscience and Chemical Engineering, Ulsan National In- and hypertension, which are associated with hypertonicity (4). Be- x stitute of Science and Technology, Ulsan 44919, Korea; and Division of Rheuma- yond its osmoprotective and homeostatic roles, NFAT5 also regulates tology, Department of Internal Medicine, The Catholic University of Korea, Seoul 06591, Korea the expression of a variety of proinflammatory genes, including ORCID: 0000-0003-3244-2025 (H.M.K.). cyclooxygenease-2, IL-6, and CCL2 under isotonic conditions (5). Received for publication July 31, 2017. Accepted for publication May 1, 2018. NFAT5 has a role in the progression of several tonicity-independent disorders, including microbial infection, inflammatory breast cancer, This work was supported by Grant 2015R1A3A2032927 from the National Research Foundation of Korea, which is funded by the Ministry of Education, Science, and and RA (5–7). Moreover, NFAT5 promotes a6b4 integrin–mediated Technology. cancer cell invasiveness (8), demonstrating its role in cell migration Address correspondence and reprint requests to Dr. Seung-Ah Yoo or Dr. Wan-Uk and invasion. However, whether NFAT5 controls migration and in- Kim, Center for Integrative Rheumatoid Transcriptomics and Dynamics, School of vasion of RA-FLSs has not been systematically analyzed. Medicine, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 06591, Korea (S.-A.Y.) or Division of Rheumatology, Department of Internal Med- The tissue factor (TF; also known as coagulation factor III) is a icine, School of Medicine, The Catholic University of Korea, 505 Banpo-dong, primary initiator of extrinsic pathway of blood coagulation and has a Seocho-gu, Seoul 06591, Korea (W.-U.K.). E-mail addresses: youcap78@hanmail. net (S.-A.Y.) or [email protected] (W.-U.K.) crucial role in maintaining hemostasis when blood vessels are injured The online version of this article contains supplemental material. (9). Disruption of blood vessels exposes plasma coagulation factors, including factor VII (FVII) to the subendothelial layers of the vessel Abbreviations used in this article: DAVID, Database for Annotation, Visualization and Integrated Discovery; DEG, differentially expressed gene; FLS, fibroblast-like wall (9). Thereafter, TF, which is constitutively expressed on peri- synoviocyte; FVII, factor VII; FVIIa, activated FVII; MEF, mouse embryonic fibro- vascular cells, forms a complex with FVII transforming it to activated blast; NFAT5, NF of activated T cell 5; OA, osteoarthritis; OA-FLS, FLS of OA patient; PLAT, plasminogen activator, tissue type; QRT-PCR, quantitative real-time FVII (FVIIa) and initiates the downstream coagulation protease PCR; RA, rheumatoid arthritis; RA-FLS, FLS of RA patient; siCon, control siRNA; cascade, resulting in a fibrin deposition that stops bleeding (9). In- siRNA, small interfering RNA; TF, tissue factor, coagulation factor III; WT, terestingly, TF has a role in noncoagulant pathophysiology, including wild-type. inflammation, cell migration, and apoptosis (10). In particular, TF Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 promotes cell migration by interacting with integrins and activates the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701097 2 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES intracellular actin network, resulting in lamellipodia formation (10). cells were removed. All experiments were carried out with FLSs from Coagulation factors, including FVIIa, factor Xa (FXa), and factor IIa passage 3–7. In some experiments, mouse FLSs were isolated from the joints of wild-type (WT) and NFAT5 haplo-insufficient mice as previously (FIIa), which are activated by TF in an extrinsic pathway, have D D described (15). WT and Nfat5 / mouse embryonic fibroblasts (MEFs) protease activity and activate protease-activated receptors to induce were generously gifted by Dr. H.M. Kwon (Ulsan National Institute of proinflammatory responses (11). Science and Technology, Ulsan, South Korea). RA-FLSs, mouse FLSs, and Decoding the mechanisms underlying the migration and invasion MEFs were incubated in DMEM supplemented with FBS (Thermo Fisher of RA-FLSs has been suggested to be essential to the understanding Scientific) at 37˚C in a 5% CO2 atmosphere. This study protocol was approved by the institutional review board of the Catholic Medical Center of RA pathogenesis and for the identification of therapeutic targets (XC09TIMI0070). All patients gave written informed consent to the study for RA (12, 13). However, gene expression profiles underlying protocol. hypermotility and invasiveness of RA-FLSs, reminiscent of invasive cancers, have not been systemically and comprehensively Knockdown of NFAT5, TF, p38, and p65 transcripts evaluated. RA-FLSs (1 3 105 per well) were seeded in six-well culture plates and We hypothesized that RA-FLSs are the major cell type re- grown to 80% confluence for 24 h. The cells were then transfected with sponsible for cartilage and bone destruction and that the NFAT5, an 50 nM siRNAs (Santa Cruz Biotechnology) for NFAT5, TF, p38 MAPK, or k osmoprotective transcription factor involving cancer invasiveness, NF- B p65 coated by Lipofectamine 3000 reagent (Thermo Fisher Sci- entific) in Opti-MEM medium (Thermo Fisher Scientific) following the would control such destructive phenotype of RA-FLSs. To address manufacturer’s instructions. After 6 h of transfection, the cells were placed this, we performed global gene expression profiling of RA-FLSs as in fresh culture medium. compared with the FLSs of osteoarthritis (OA) patients (OA-FLSs), used a systems approach to determine the key signaling pathways Quantitative real-time PCR Downloaded from for NFAT5-dependent regulation of FLS migration and invasion, Total RNA in the cultured cells was isolated using an RNeasy Mini kit and then carried out in vitro experiments to prove our hypothesis. (QIAGEN, Hilden, Germany), and cDNA was then synthesized using As a result, we identified novel NFAT5–TF and NFAT5–CCL2 RevertAid reverse transcriptase (Thermo Fisher Scientific). Real-time PCR was performed in the CFX96 real-time PCR system using SYBR Green pathways that are crucial for the migration and invasion of RA- PCR premix (Bio-Rad). Data were normalized to GAPDH expression in FLSs. These pathways were activated by IL-1b and TGF-b, which each sample, and relative fold-inductions were calculated using the 22DDCt are cytokines involved in RA pathogenesis. We validated the role algorithm. The following primers were used for PCR amplification of the http://www.jimmunol.org/ of these pathways in FLS invasiveness by performing in vitro target gene (forward, reverse): NFAT5,59-CCAGAAGTCATTTG- 9 9 9 F3 9 functional assays and also demonstrated the efficacy of a selec- CCTGGT-3 , and 5 -GATTCCAAGCCCACTCTTCA-3 ; ,5-GCCA- GGAGAAAGGGGAAT-39, and 59-CAGTGCAATATAGCATTTGCAGT- tive NFAT5 suppressor KRN2, 13-(2-fluorobenzyl)-berberine, in AGC-39; CCL2,59-AAGCAGAAGTGGGTTCAGGA-39, and 59- retarding promigratory capacity of FLSs. Taken together, the re- GGGGAAAGCTAGGGGAAAAT-39; plasminogen activator, tissue type sults provide evidence of a functional link between NFAT5 and (PLAT), 59-AGCGAGCCAAGGTGTTTCAA-39, and 59-CTTCCCAG- 9 9 9 coagulation factor TF and the importance of NFAT5–CCL2 CAAATCCTTCGGG-3 ; RELA,5-ATGTGGAGATCATTGAGCAGC-3 , and 59-CCTGGTCCTGTGTAGCCATT-39; MAPK14,59-TCAGTCCAT- pathway for pathological features of RA-FLSs and support a role CATTCATGCGA-39,and59-AACGTCCAACAGACCAATCA-39; GAPDH,59- for NFAT5 blockade in RA treatment. AAGGTGAAGGTCGGAGTCAA-39,and59-AATGAAGGGGTCATTGATGG-39; F3 (Mus musculus), 59-AACCCACCAACTATACCTACACT-39,and59-GTCTGT- by guest on September 24, 2021 Materials and Methods GAGGTCGCACTCG-39; Ccl2,59-TTAAAAACCTGGATCGGAACCAA-39,and 59-GCATTAGCTTCAGATTTACGGGT-39;andGapdh,59-AGGTCGGTGTGA- Reagent and Abs ACGGATTTG-39,and59-TGTAGACCATGTAGTTGAGGTCA-39 Lipofectamine 3000 reagent was purchased from Thermo Fisher Scientific (Waltham, MA). Small interfering RNAs (siRNAs) targeting human genes, ELISA for CCL2 k including NFAT5, TF, p38 MAPK, NF- B p65, and control siRNAs (siCon) CCL2 concentrations were measured in the culture supernatants of FLSs or were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). MEFs using an ELISA kit (R&D Systems). SB203580 was obtained from Sigma-Aldrich (St. Louis, MO). Alexa Fluor 488–conjugated phalloidin was obtained from Molecular Probes (Eugene, Immunofluorescence staining for TF OR). PE-conjugated anti-TF Ab was obtained from eBioscience (San Diego, CA). Recombinant mouse IL-1b, recombinant human CCL2, Frozen RA synovium was fixed with cold acetone for 10 min at 220˚C and recombinant FVII, neutralizing anti-CCL2 Ab, and anti-mouse TF Ab were blocked with 1% BSA for 30 min. For double-immunofluorescence obtained from R&D Systems (Minneapolis, MN). Anti–p38 MAPK Ab staining, tissue sections were incubated with rabbit anti-TF Ab (1:500; and anti–phospho-p38 MAPK Ab were obtained from Cell Signaling Abcam) and mouse anti-CD55 Ab (1:100; Santa Cruz Biotechnology) Technology (Beverly, MA). Anti–NF-kB p65 Abs were purchased from overnight at 4˚C. Each slide was washed three times in PBS and incubated Cell Signaling Technology and Abcam (Cambridge, U.K.). Alexa Fluor with Cy3-conjugated anti-rabbit IgG Ab and Alexa Fluor 488–conjugated 488–conjugated donkey anti-rabbit IgG Ab was obtained from Thermo anti-mouse IgG Ab (Thermo Fisher Scientific). Nuclei were stained with Fisher Scientific. Anti-CD55 Ab and anti–b-actin Ab were obtained from DAPI, and the glass slides were mounted in a ProLong Antifade solution Santa Cruz Biotechnology. Recombinant human IL-1b and RevertAid (Thermo Fisher Scientific). The stained tissues were visualized with a Reverse Transcriptase were purchased from Thermo Fisher Scientific. confocal microscope (LSM 510; Carl Zeiss, Gottingen, Germany). Recombinant human TGF-b was obtained from PeproTech (Rocky Hill, NJ). Insulin-transferrin-selenium-A, Opti-MEM medium, and FBS were Immunocytochemistry obtained from Thermo Fisher Scientific. CFX96 real-time PCR system and To visualize lamellipodia-containing RA-FLSs, immunofluorescence SYBR Green PCR premix were obtained from Bio-Rad (Richmond, CA). staining of F-actin was performed on chamber slides. After 12 h of 3 Cell culture transfection with control or NFAT5 siRNAs, RA-FLSs (3 3 10 cells per well) were seeded on eight-well chamber slides (Nalge Nunc International, FLSs were isolated from the synovial tissues of nine RA patients (seven Rochester, NY) and incubated for 12 h in DMEM supplemented with 10% women, two men; mean 6 SD age, 61.1 6 16.6) as described previously FBS. The cells were fixed with 3.7% formaldehyde in PBS for 10 min, (14). The FLSs of three OA patients (OA-FLSs, three women; mean 6 SD permeabilized with 0.1% Triton X-100 in PBS for 3 min at room tem- age, 66.3 6 5.7) were used as a control. Eight (88.9%) of the nine RA perature and incubated with Alexa Fluor 488–conjugated phalloidin patients were positive for the rheumatoid factor and/or the anti-cyclic (Molecular Probes) for 20 min at room temperature. For p65 immunoflu- citrullinated Ab. All RA patients had erosions on hand and foot x-rays. orescent staining in RA-FLSs, cells (5 3 103 cells per well) were seeded In brief, the synovial tissues were minced into small pieces ,2–3 mm in on eight-well chamber slides (Nalge Nunc International). Cells were size, then treated for 4 h with 0.1 mg/ml Type I Collagenase (Thermo transfected with control or p65 siRNAs for 24 h and then were fixed with Fisher Scientific) in a shaking water bath at 37˚C. Dissociated cells were 3.7% formaldehyde solution. After blocking with 10% normal donkey plated in 75-cm2 flasks and cultured overnight, and then the nonadherent serum for 1 h, cells were stained with anti-p65 Ab (1:100; Abcam) and The Journal of Immunology 3

Alexa 488–conjugated donkey anti-rabbit IgG (1:1000; Thermo Fisher when a gene displayed an overall p , 0.05 and when the absolute log2-fold Scientific). After washing in PBS, nuclei were stained with DAPI, and the changes were larger than the cutoff. The cutoff value was determined as the coverslips were mounted on glass slides with ProLong Antifade Kit mean absolute values of the 2.5 and 97.5 percentiles of the empirical null (Thermo Fisher Scientific). The cells were examined using a confocal distribution. In the analysis of Nfat5+/+ MEFs versus Nfat5D/D MEFs, we ar- microscope (LSM 510; Carl Zeiss) or a fluorescence microscope (Axio- bitrarily set a fold-change cutoff of 2-fold to indicate significance because no vert; Carl Zeiss). Lamellipodia-containing RA-FLSs were manually statistical analysis was possible (n = 1 for each sample). The cellular processes counted from five nonoverlapping microscopic fields obtained from each represented by the DEGs were identified as the Gene Ontology biological condition. processes having p , 0.05 (19). The DEG lists and array of cell migration/ invasion–related genes in RA-FLSs selected by using Database for Annotation, Determination of cell viability Visualization and Integrated Discovery (DAVID) software are presented at https://www.cirad-catholic.com/supplementary-figures-data. The Fisher exact Cell viability was determined by a viability assay based on conversion of test was used to determine the significance of overlap between the DEGs. In MTT (Sigma-Aldrich) to formazan. the in vitro functional experiments, data are expressed as the mean 6 SD or Western blot analysis SEM. Comparisons of the numerical data between groups were performed by paired or unpaired Mann–Whitney U test. The p values ,0.05 were considered RA-FLSs, mouse FLSs, and MEFs were lysed in radio-immunoprecipitation statistically significant. assay buffer, and the supernatants were then obtained after centrifugation at 14,000 rpm for 20 min at 4˚C. Protein concentrations were determined using the Bradford protein assay (Bio-Rad). Proteins were resolved by Results SDS-PAGE and transferred to nitrocellulose membranes after electropho- The NFAT5 signature defining promigratory properties in resis. Membranes were incubated with anti-NFAT5 Ab (1:3000; a generous RA-FLSs gift from Dr. H.M. Kwon, Ulsan National Institute of Science and Tech- nology, Ulsan, South Korea), anti-human TF Ab (1:1000; Abcam), anti- To determine the genes involved in the migration and invasion of Downloaded from mouse TF Ab (1:1000; R&D Systems), anti–p38 MAPK Ab (1:1000; Cell RA-FLSs under proinflammatory conditions, we compared the Signaling Technology), anti–NF-kB p65 Ab (1:1000; Cell Signaling b gene expression data of RA-FLSs or RA-FLSs stimulated with IL- Technology), and anti– -actin Ab (1:5000; Santa Cruz Biotechnology). b Membranes were then visualized using an enhanced chemiluminescent 1 to those of the FLSs of OA patients (OA-FLSs) as a control technique. (GSE49604). These comparisons yielded two sets of DEGs that reflect the basal difference of gene expression between RA-FLSs Cell migration and invasion assays and OA-FLSs, as well as acquired changes in the gene expression http://www.jimmunol.org/ The wound migration of RA-FLSs was measured as described previously of RA-FLSs stimulated with IL-1b, a representative proin- (7). In brief, RA-FLSs (1 3 105 per well) were plated in six-well plates; flammatory cytokine identified at a high level in RA joints. From transfected with NFAT5 siRNA, TF siRNA, or siCon; and cultured in the DEGs, 272 cell migration/invasion–related genes (194 genes DMEM supplemented with 10% FBS for 12 h. Two wound lines, in par- for RA-FLSs + IL-1b versus OA-FLSs, 124 genes for RA-FLSs allel, were made by scratching a cellular monolayer with sterile 200-ml pipette tips. After 12 h of incubation in medium supplemented with 10% versus OA-FLSs) were identified using DAVID software, (19) and FBS, cell migration was quantified by counting the cells that had moved the gene lists are downloadable at our Web site (see Materials and beyond a reference line. Methods). Boyden chamber assays to measure the migration capacity of cells was m We then asked whether the migration/invasion-related genes are

also performed using a 48-well migration chamber (8- m pore size; Neuro by guest on September 24, 2021 Probe, Gaithersburg, MD) or Transwell Inserts (8-mm pore size; Falcon; enriched in the NFAT5 signature in RA-FLSs, which is defined by BD Biosciences, San Jose, CA) according to the manufacturer’s instruc- the DEGs of RA-FLSs transfected with NFAT5 siRNA versus those tions. To evaluate the invasion of RA-FLSs, the BD BioCoat Matrigel with siCon (GSE22956). The significant overlap (20 overlapping invasion chamber assay system (8-mm pore size; BD Biosciences) was genes, p = 0.00001 for IL-1b–stimulated RA-FLSs versus OA- used according to the manufacturer’s instructions. In Boyden chamber FLSs and nine overlapping genes, p = 0.04 for unstimulated assays and Matrigel invasion assays, the nonmigrating and noninvading cells that remained on the top of the filters were removed by scrubbing RA-FLSs versus OA-FLSs) was seen between the cell migration/ with a cotton swab after 12 h of incubation, and the cells on the lower invasion–related genes and the NFAT5 signature from RA-FLSs surface of the membrane were stained with a Diff-Quik staining kit (Fig. 1A, data not shown). Such association was further validated (Sysmex, Kobe, Japan). The number of migrated cells was manually by the comparison between NFAT5-disrupted RA-FLSs and counted in three random fields. The images were obtained using a Pan- +/+ noramic MIDI slide scanner (3DHISTECH, Budapest, Hungary). NFAT5-sufficient RA-FLSs (GSE22956) or between WT (Nfat5 ) MEFs and NFAT5 knockout (Nfat5D/D) MEFs (GSE25816); sig- Flow cytometry analysis for TF nificant enrichment of cell migration and adhesion was seen in both downregulated DEGs in NFAT5 siRNA–treated RA-FLSs and the Surface expressions of TF on RA-FLSs were detected by flow cytometry. In +/+ D/D brief, cells were transfected with control or NFAT5 siRNA for 24 h and then DEGs from the comparison of Nfat5 and Nfat5 MEFs incubated in 1% FBS DMEM containing TGF-b (10 ng/ml) for 24 h. Cells (Supplemental Table I). This result suggests that NFAT5 regulates were dislodged with Accutase cell detachment solution (Innovative Cell migration and invasion of RA-FLSs. Technologies, San Diego, CA) and stained with PE-conjugated anti-TF Ab We next tried to identify effector genes that are responsible for (eBioscience). The stained samples were acquired using a FACSCanto II system (BD Biosciences). Dead cells and other cellular debris were ex- RA-FLS migration and that are also regulated by NFAT5 in RA- cluded by gating based on the forward and side scatter profile. Data FLSs. To this end, we selected 19 overlapping genes between analysis was done by using FlowJo (Tree Star, Ashland, OR) software. downregulated genes in NFAT5 siRNA–treated RA-FLSs versus siCon-treated RA-FLSs and upregulated genes in RA-FLSs or Statistical analyses RA-FLSs stimulated with IL-1b versus OA-FLSs (Fig. 1B) and Microarray data sets, including RA-FLSs versus OA-FLSs (GSE49604), found 13 out of 19 genes associated with cell migration (Fig. 1C). NFAT5 siRNA–transfected RA-FLSs and siCon-transfected RA-FLSs D D The 13 overlapping genes include ATP2A2, CCL2, CLIC3, DKK1, (GSE22956), and Nfat5+/+ MEFs versus Nfat5 / MEFs (GSE25816), can be viewed at the Gene Expression Omnibus (16). Data sets were an- EDN1, F3, JAM2, NRG1, PLAT, RRM2, TNFRSF11B, VLDLR, alyzed using the integrated statistical testing method as previously de- and WISP1 (Fig. 1C). We selected the top three genes, TF (F3), scribed (17). Briefly, for each gene, a Student t test and log2 median-ratio CCL2, and PLAT, as putative effector genes of RA-FLS migration test were performed to compute the adjusted p values using the empirical and invasion based on the extent of differential expression be- null distributions that were generated by random permutations of the b samples 1000 times. Then, the two sets of p values from the two individual tween IL-1 –stimulated RA-FLSs and OA-FLSs (see the red color tests were combined to compute the overall p values using the Stouffer gradient in Fig. 1C). Quantitative real-time PCR (QRT-PCR) method (18). The differentially expressed genes (DEGs) were selected analysis of the top three genes validated differential expression 4 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 1. Transcriptome analysis reveals that NFAT5 is involved in migration of RA-FLSs. (A) Schematic diagram showing a comparison of the DEGs in NFAT5 siRNA (siNFAT5)–transfected versus siCon-transfected RA-FLSs with the DEGs related to cell migration and/or invasion in RA-FLSs stimulated with IL- 1b (10 ng/ml) versus unstimulated OA-FLSs. Cell migration- and/or invasion-related DEGs were selected by using DAVID software. The p value indicates the significance of 20 overlapping DEGs. (B) Venn diagram depicting the number of common and distinct genes among downregulated DEGs in siNFAT5-transfected RA-FLSs, upregulated DEGs in unstimulated RA-FLSs, and upregulated DEGs in RA-FLSs stimulated with IL-1b.(C) Heat map displaying differential expression pattern of 19 genes downregulated by siNFAT5 and upregulated in RA-FLSs or IL-1b–stimulated RA-FLSs; the genes in RA-FLSs stimulated with or without IL- 1b were compared with the same OA-FLSs. Red and blue shading denotes up- and downregulation, respectively. Genes related to cell migration and invasion are marked in red to the right of heat map. Color intensity indicates fold change. (D)QRT-PCRassaysforTF, CCL2,andPLAT expressions in RA-FLSs transfected with siNFAT5 versus those with siCon. Data are the mean 6 SEM of more than three independent experiments in duplicate. **p , 0.01 versus siCon. (E) Double immunofluorescence staining of an RA synovium using anti-CD55 and anti-TF Abs. The nucleus was stained with DAPI. On H&E staining, synovial lining layer is depicted with arrows and broken lines. Colocalization of CD55 and TF is visualized in yellow in the merged image. Scale bar, 50 mm. ATP2A2, ATPase sar- coplasmic/endoplasmic reticulum Ca2+ transporting 2; CLIC3, chloride intracellular channel 3; DKK1, dickkopf WNT signaling pathway inhibitor 1; EDN1, endothelin 1; JAM2, junctional adhesion molecule 2; NRG1, neuregulin 1; RRM2, ribonucleotide reductase M2; TNFRSF11B, TNF receptor superfamily, member 11b; VLDLR, very low-density lipoprotein receptor; WISP1, WNT1 inducible signaling pathway protein 1. of TF and CCL2, but not PLAT, in NFAT5 siRNA–transfected (21–23). Moreover, intra-articular injection of IL-1b or TGF-b RA-FLSs (Fig. 1D). Moreover, TF and CCL2 contain consensus promotes arthritis progression in rodents, whereas the systemic ad- binding sites for NFAT5 in their proximal promoter regions ministration of the IL-1 receptor antagonist or intra-articular injection (Supplemental Fig. 1), suggesting that NFAT5 is involved in the of anti–TGF-b reduces experimentally induced arthritis in some transcriptional regulation of TF and CCL2. Immunofluorescence studies (24–27). Thus, we tested whether IL-1b and TGF-b can staining revealed that TF+ cells colocalized with CD55+ FLSs in trigger NFAT5–TF and NFAT5–CCL2 axes for the promigratory the lining layer of RA synovium (Fig. 1E); TF was also expressed properties of RA-FLSs, as suggested by our integrative analysis of in infiltrated leukocytes in the sublining layer as previously FLS transcriptomes. NFAT5 protein and mRNA levels in RA-FLSs reported (20). These results suggest that the NFAT5 signature is were increased by IL-1b or TGF-b treatment(Fig.2A).However,IL- associated with RA-FLS migration and that TF and CCL2 require 1b–andTGF-b–induced increases of TF expression were nearly the transcription factor NFAT5 for their full expression. completely blocked by the transfection of NFAT5 siRNA, as determined by Western blot, flow cytometry, and real-time PCR analysis b b NFAT5 regulation of IL-1 – and TGF- –induced TF and (Fig. 2B–D), suggesting that NFAT5 regulates TF expression under CCL2 expressions IL-1b–orTGF-b–stimulated conditions. CCL2 protein and mRNA IL-1b and TGF-b are highly expressed cytokines in RA joints, and levels also were markedly increased by IL-1b stimulation, which that potently induces the migration and/or invasion of RA-FLSs were significantly abrogated (72.1% reduction for mean mRNA level) The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. IL-1b and TGF-b enhance NFAT5–TF and NFAT5–CCL2 pathways. (A) NFAT5 protein and mRNA expression levels in RA-FLSs as determined by Western blot analysis and QRT-PCR, respectively. RA-FLSs were stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) for the indicated times in Western blots or for 12 h in real-time PCR in which GAPDH mRNA was used as a control. **p , 0.01 versus unstimulated RA-FLSs. (B–D) NFAT5 regulation of TF expression in RA-FLSs. RA-FLSs were stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) 24 h after transfection with NFAT5 siRNA (siNFAT5) or siCon. After 12 h, TF expression levels in FLSs were determined by Western blot analysis (B) and real-time PCR (D). Surface TF expression on siCon- or siNFAT5-treated RA-FLSs was also assessed by flow cytometry 24 h after stimulation of cells with TGF-b (10 ng/ml) (C). *p , 0.05 versus siCon-treated RA-FLSs under each condition, #p , 0.05 versus unstimulated RA-FLSs. (E) NFAT5 regulation of CCL2 expression. After 24 h of transfection with siNFAT5 or siCon, RA-FLSs were stimulated with IL-1b (10 ng/ml) and TGF-b (10 ng/ml) for 12 h and subjected to ELISA (left panel) or real-time PCR (right panel). CCL2 ELISA results were normalized relative to cell viability assessed by MTT assay. *p , 0.05 versus siCon-treated RA- FLSs under each condition, #p , 0.05 versus unstimulated RA-FLSs. (F–H) No effect of NF-kB p65 knockdown on TF expression. RA-FLSs were cultured in the presence or absence of TGF-b (10 ng/ml) 24 h after transfection with NF-kB p65 siRNA (sip65) or siNFAT5. After 12 h, NF-kB p65 expression was determined by Western blot analysis [lower panel in (F)], real-time PCR [upper panel in (F)], and immunocytochemistry (G), and TF mRNA expression was measured by real-time PCR (H). Scale bar, 25 mm. Data in (A)–(H) show representatives or mean 6 SEM of more than three independent experiments with different RA-FLSs. *p , 0.05. n.s., not significant. by NFAT5 siRNA (Fig. 2E), demonstrating that IL-1b–induced 15.0%) and TF mRNA expression (by 20.7%) in RA-FLSs stimulated CCL2 expression was, at least in part, dependent on NFAT5. How- by TNF-a (Supplemental Fig. 2A), another proinflammatory cyto- ever, NFAT5 siRNA only modestly reduced CCL2 mRNA (by kine crucial to RA pathogenesis. Additionally, in contrast to its effect 6 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES on NFAT5 expression, TGF-b only modestly upregulated CCL2 Requirement of NFAT5–TF axis for TGF-b–induced migration expression, suggesting that NFAT5 does not decisively mediate CCL2 and invasion of RA-FLSs production in TGF-b–stimulated RA-FLSs. Based on our findings that NFAT5 is crucial to TGF-b–induced TF NF-kB p65 is a transcription factor that is able to promote FLS expression, we next examined whether the NFAT5–TF axis actu- migration (28) and has been reported to upregulate TF expression in ally promotes migration and invasion of RA-FLSs under high endothelial cells and monocytes (29). Therefore, we next investigated TGF-b conditions. As a result, locomotion of RA-FLSs was sig- whether NF-kB contributes to TF upregulation in RA-FLSs. Unex- nificantly hampered by knock down of NFAT5 transcripts or TF pectedly, NF-kBp65siRNAfailedtodecreaseTF mRNA expression transcripts using siRNAs, as determined by wound migration and in RA-FLSs under both basal- and TGF-b–stimulated conditions Boyden chamber assays (Fig. 4A, 4B). Conversely, FVII, a spe- (Fig. 2H), whereas they markedly suppressed NF-kBp65mRNAand cific activator of TF, increased migration of RA-FLSs, which was protein expressions in the cytoplasm (Fig. 2F, 2G). In contrast, blocked by NFAT5 knockdown (Fig. 4C), demonstrating the NFAT5 siRNA significantly suppressed it in the same cells (Fig. 2H), essential role of NFAT5 and TF in promigratory properties of suggestingthatincontrasttoNFAT5,NF-kB p65 plays a minor role RA-FLSs. Moreover, NFAT5 siRNA or TF siRNA substantially in TF expression in RA-FLSs. inhibited the increase in TGF-b–induced migration and invasion Collectively, our data demonstrated that IL-1b and TGF-b in- of RA-FLSs (Fig. 4D, 4E). crease TF and CCL2 expressions in RA-FLSs, both of which are It has been suggested that TF is expressed in the lamellipodia dependent on NFAT5 expression. located on the migratory front region of a cell (33) and instigates

cell migration by regulating actin filament networks (34). To ex- Downloaded from p38 MAPK as a major upstream regulator of the amine whether the NFAT5–TF axis contributes to lamellipodia NFAT5–TF pathway formation in RA-FLSs, we stained F-actin and quantified cells It has been demonstrated that the p38 MAPK pathway is an upstream containing lamellipodia. The number of RA-FLSs expressing regulatorofNFAT5(30).Wealsoreported that p38 MAPK regulated lamellipodia was significantly mitigated by NFAT5 siRNA or TF LPS-induced transcriptional activity of NFAT5 in macrophages (31). siRNA at basal state (Fig. 4F). The TGF-b–induced increase in

Thus, we wanted to know whether p38 MAPK controls NFAT5–TF lamellipodia formation was also almost completely cancelled by http://www.jimmunol.org/ axis and NFAT5–CCL2 axis in RA-FLSs stimulated with IL-1b or NFAT5 siRNA or TF siRNA transfection (Fig. 4G), suggesting TGF-b.Asexpected,bothIL-1b and TGF-b increased the phos- that both NFAT5 and TF are required for lamellipodia formation in phorylation of p38 MAPK in RA-FLSs (Fig. 3A). Moreover, as RA-FLSs. shown in Supplemental Fig. 3, NFAT5 mRNA expression induced by Altogether, these data suggest that NFAT5–TF axis is essential IL-1b or TGF-b was substantially blocked by SB203580 (p38 to migratory and invasive features of RA-FLSs, which are possibly MAPK inhibitor) and SP600125 (JNK inhibitor), but not by PD98059 mediated by expediting lamellipodia formation particularly under TGF-b–stimulated conditions. (ERK1/2 inhibitor), indicating that p38 and JNK MAPK pathways areinvolvedintheIL-1b–orTGF-b–induced NFAT5 transcription in Role of NFAT5 in IL-1b–induced migration and invasion by guest on September 24, 2021 RA-FLSs. of RA-FLSs p38 MAPK is a key regulator of proinflammatory cytokine and Our next experiment was to determine whether NFAT5 and its chemokine production (32). We found that SB203580 nearly com- target genes, TF and CCL2, are involved in IL-1b–induced mi- pletely inhibited the increase in TF mRNA abundance mediated by gration and invasion of RA-FLSs. To address this issue, we in- IL-1b or TGF-b treatment (Fig. 3B). IL-1b–induced CCL2 mRNA vestigated the role of NFAT5–TF axis in IL-1b–induced migration expression in RA-FLSs was also significantly, but partially, repressed and invasion of RA-FLSs. As observed with TGF-b stimulation, by SB203580; TGF-b stimulation was not done because of its modest NFAT5 siRNA (Fig. 5D, 5E) or TF siRNA (Fig. 5A, 5B) sub- effect on CCL2 production (Fig. 3C). These observations suggest that stantially inhibited the increase in IL-1b–induced migration and p38 MAPK transmits a signal to activate NFAT5–TF and NFAT5– invasion of RA-FLSs. We also tested whether FLS migration and/ CCL2 axes in RA-FLSs upon IL-1b or TGF-b stimulation. To or invasion could be suppressed by CCL2 blockade in the presence confirm this, p38 MAPK transcripts in RA-FLSs were knocked down of IL-1b. IL-1b–induced increase in migration of RA-FLSs was usingsiRNAsforp38MAPK.p38MAPKexpressionwasmarkedly nearly completely blocked by anti-CCL2 Ab (Fig. 5C), suggesting reduced by transfection of p38 MAPK siRNA in RA-FLSs (Fig. 3D). that chemoattractant CCL2 secreted from RA-FLSs in an auto/ As expected, IL-1b–orTGF-b–induced NFAT5 and TF expression paracrine manner mediates increased migration of RA-FLSs b levels were substantially inhibited by treatment with p38 MAPK stimulated with IL-1 . Moreover, NFAT5 siRNA–induced decrease in FLS migration was partially, but significantly, recovered siRNA in RA-FLSs (Fig. 3E, 3F, 3H). However, IL-1b–induced when recombinant CCL2 was added to the upper well of a Boyden CCL2 expression was only partially, albeit significantly, suppressed chamber (Fig. 5D). IL-1b–stimulated FLS invasiveness, as de- by p38 MAPK siRNA (Fig. 3G, 3H), which is in accordance with our termined by a Matrigel invasion assay, was also reduced to basal results using SB203580 (Fig. 3C). Given that IL-1b–induced CCL2 level by NFAT5 siRNA, which was also partially restored by ad- expression was only partially dependent on NFAT5 (Fig. 2E), these dition of CCL2 (Fig. 5E). Collectively, these results, together with observations suggest that other pathways in addition to p38–NFAT5 data in Fig. 2E, indicate that IL-1b–induced FLS migration and signalarealsorequiredforCCL2upregulationinRA-FLSsstimu- invasion is, at least in part, dependent on NFAT5–CCL2 axis in b a lated with IL-1 .TNF- –induced CCL2 and TF mRNA expressions RA-FLSs. were rarely affected by p38 MAPK siRNA (Supplemental Fig. 2B), IL-1b–induced increase in lamellipodia formation in RA-FLSs suggesting that they are not regulated by the p38 pathway. Collec- was also nearly completely abrogated by NFAT5 siRNA or TF tively, these data demonstrate that p38 MAPK plays a predominant siRNA (Fig. 5F). Moreover, addition of CCL2 to NFAT5 siRNA– role in activating NFAT5–TF axis but plays a partial role in activating transfected RA-FLSs significantly recovered the IL-1b–induced NFAT5–CCL2 axis in RA-FLSs stimulated with IL-1b or TGF-b. increase in lamellipodia formation. These results demonstrate the The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. p38 MAPK is the major upstream regulator of NFAT5 under IL-1b– or TGF-b–stimulated conditions. (A) IL-1b– or TGF-b–induced upregulation of phospho-p38 MAPK. RA-FLSs were stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) for the indicated times. Expressions of p38 and phospho-p38 were determined by Western blot analysis. Data are representatives of three different RA-FLSs. (B and C) Suppression of TF and CCL2 mRNA expressions by SB203580, a p38 MAPK inhibitor. RA-FLSs were pretreated with SB203580 (20 mM) or DMSO as vehicle control for 1 h prior to stimulation with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) for 12 h. TF and CCL2 mRNA expression levels were determined by real-time PCR analysis. GAPDH mRNA was used as a control. Data are mean 6 SEM of more than three independent experiments, in duplicate, that were performed using different RA-FLSs. (D) Knockdown of p38 MAPK by siRNA in RA-FLSs. Cells were transfected with siCon or p38 siRNA (sip38) for 24 h. Reduction of p38 MAPK was confirmed by Western blot analysis (left panel) and real-time PCR analysis (right panel). (E–H) p38 siRNA inhibition of NFAT5, TF, and CCL2 expressions. RA-FLSs were transfected with siCon or sip38 for 24 h, and then the cells were stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) for 12 h (F and H)or24h(E and G). (E) NFAT5 expression was determined by Western blot analysis. TF expression levels were determined by Western blot and real-time PCR analysis (F and H), respectively, and CCL2 expression levels were measured by ELISA and real-time PCR analysis (G and H). CCL2 ELISA results were normalized relative to cell viability assessed by MTT assay. Data in (D)–(H) show representatives or mean 6 SEM of more than three independent experiments with different RA-FLSs. *p , 0.05, **p , 0.01. crucial role of both NFAT5–TF and NFAT5–CCL2 axes in FLSs than in Nfat5+/+ mouse FLSs obtained from arthritic joints lamellipodia formation in RA-FLSs stimulated with IL-1b. (Fig. 6A). Like NFAT5-deficient RA-FLSs, FLSs of Nfat5+/D mice Collectively, NFAT5 promotes the migration and invasion in had reduced migratory capacity in response to media, IL-1b or addition to lamellipodia formation through upregulation of TF and TGF-b, compared with FLSs of Nfat5+/+ mice (Fig. 6B). More- CCL2 in IL-1b–stimulated RA-FLSs. over, in comparison with Nfat5+/+ MEFs, Nfat5D/D MEFs had lower levels of TF and CCL2 expression (Fig. 6C). Collectively, these Suppression of synoviocyte migration by NFAT5 knockout or data show that NFAT5 regulates TF and CCL2 expressions in mouse NFAT5 inhibitor FLSs and MEFs, promoting the migration of mouse FLSs. To exclude possible off-target effects of NFAT5 siRNA, we isolated Recently, we identified a novel NFAT5 suppressor, KRN2, 13- primary FLSs and MEFs from Nfat5-deficient and -sufficient mice (2-fluorobenzyl)-berberine, to selectively inhibit NFAT5 expres- and then tested the aforementioned role of NFAT5 in these cells. sion in RAW 264.7 macrophages (35). We tested whether KRN2 TF and CCL2 expression levels were lower in Nfat5+/D mouse suppresses NFAT5 expression and cell migration in mouse FLSs. 8 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. TGF-b–induced migration and invasion of RA-FLSs are primarily dependent on NFAT5–TF axis. (A and B) Decrease in FLS migration by knockdown of NFAT5 or TF transcripts. After 12 h of transfection with NFAT5 siRNA (siNFAT5) or TF siRNA (siTF), RA-FLS migration was determined for 12 h by wound migration assay (A) and Boyden chamber assay (B). Scale bars, 400 mm(A), 100 mm(B). (C) NFAT5 regulation of FVII-induced FLS migration. RA-FLSs were stimulated with TGF-b (10 ng/ml) for 24 h after transfection with siNFAT5 and the stimulated cells were then cultured again in the presence or absence of FVII (5 mg/ml), a TF activator, for 12 h in a Boyden chamber. Representative images are shown in the left panel, in which the rectangular areas in the lower images are magniﬁed to the upper images. Scale bars, 200 mm (upper), 1000 mm (lower). (D and E) Suppression of TGF-b– induced migration and invasion of RA-FLSs by siNFAT5 or siTF. RA-FLSs were incubated in serum-free DMEM containing TGF-b (10 ng/ml) in the upper well of a Boyden chamber (D) or a Matrigel invasion chamber (E) 12 h after transfection with siNFAT5 or siCon; TGF-b (10 ng/ml) in DMEM containing 1% FBS was also added to the lower well. After 12 h, the migrated cells were stained violet using Diff-Quik kit and were manually counted. Representative photos are shown in the left panel, in which the rectangular areas in the lower images are magniﬁed to the upper images. Scale bars, 200 mm (upper), 1000 mm (lower) (D) and 400 mm (upper), 2000 mm(lower)(E). (F and G) Reduction of lamellipodia formation by siNFAT5 or siTF. RA-FLSs were seeded on an eight- well culture slide after 12 h of transfection with siNFAT5 or siTF and then stimulated with culture media only (DMEM containing 1% FBS) for 12 h (F) or with TGF-b (10ng/ml)for8h(G). The cells were stained with Alexa Fluor 488–conjugated phalloidin to visualize actin remodeling. White triangles indicate lamellipodia formation. Representative images of more than three different RA-FLSs are shown in the left panel. Bar graphs show the mean (6SEM) percentage of cells having lamellipodia to total cells. Scale bar, 50 mm. Data in (A)–(G)aremean6 SEM of more than three independent experiments that were performed using different RA-FLSs. *p , 0.05, **p , 0.01.

NFAT5 expression in mouse FLSs was instigated by treatment Discussion with IL-1b or TGF-b, which was markedly reduced by cotreat- The major issues in the postgenome research of RA may be the ment of KRN2 (Fig. 6D). Moreover, TGF-b–induced migration of identification of key signal transduction axes regulating distinct mouse FLSs (Nfat5+/+) was almost completely reduced by KRN2 sets of proinflammatory genes. RA-FLSs exhibit invasive char- treatment (Fig. 6E), demonstrating that KRN2 effectively re- acteristics reminiscent of cancer cells, destroying cartilage and presses the promigratory capacity of FLSs. bone, but it remains unresolved which signals decisively mediate Taken together, these data suggest that NFAT5 has a role in FLS invasive phenotypes of RA-FLSs. To our knowledge, we provide in migration in a mouse system and support a role for NFAT5 this article the first evidence that the osmoprotective transcription blockade in retarding promigratory capacity of FLSs (Fig. 7). factor NFAT5 is critical for the migration and invasion of RA-FLSs. The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 5. Both NFAT5–TF and NFAT5–CCL2 axes are required for IL-1b–induced migration and invasion of RA-FLSs. (A and B) Decrease in IL-1b– by guest on September 24, 2021 induced FLS migration and invasion by knockdown of TF transcripts. After 12 h of transfection with TF siRNA (siTF), RA-FLSs were stimulated with IL- 1b (100 pg/ml) for 12 h. The cell migration and invasion were determined by Boyden chamber assay and Matrigel invasion assay, respectively. Scale bars, 400 mm (upper), 2000 mm (lower). (C) Inhibition of IL-1b–induced RA-FLS migration by anti-CCL2 Ab. RA-FLSs were incubated in the upper wells of a Boyden chamber with serum-free DMEM containing IL-1b (100 pg/ml) in the presence or absence of anti-CCL2 neutralizing Ab for 12 h; in the lower well, 10% FBS was added. The migrated cells were stained violet using Diff-Quik kit and manually counted. Scale bars, 200 mm (upper), 1000 mm (lower). (D and E) CCL2 restoration of NFAT5 siRNA (siNFAT5)–induced decrease in the migration and invasion of RA-FLSs stimulated with IL-1b. After 12 h of transfection with siNFAT5, RA-FLSs were seeded in the upper wells of a Boyden chamber (D) or a Matrigel invasion chamber (E) and stimulated with serum-free DMEM containing IL-1b (100 pg/ml) in the absence or presence of recombinant CCL2 (100 ng/ml) for 12 h; 10% FBS (in Boyden chamber) or 1% FBS (in Matrigel invasion chamber) was added to the lower wells. The migrated cells were stained and manually counted. Scale bars, 200 mm (upper), 1000 mm (lower) (B) and 400 mm (upper), 2000 mm (lower) (C). (F) Effects of NFAT5 and its target genes TF and CCL2 on IL-1b–induced lamellipodia formation. RA-FLSs were transfected with siNFAT5 or siTF for 12 h and then stimulated with 1% FBS DMEM containing IL-1b (100 pg/ml) for 8 h; for siNFAT5-treated FLSs, recombinant CCL2 (100 ng/ml) was simultaneously added. The cells were stained with Alexa Fluor 488–conjugated phalloidin. Data show the mean (6 SEM) percentage of cells having lamellipodia to total cells. The bar graphs in (A)–(F) are mean 6 SEM of more than three independent experiments using different RA-FLSs. The images in the left panel of (A)–(E) are representatives. The rectangular areas in the lower images of (A)–(E) are magniﬁed to the corresponding upper images. *p , 0.05, **p , 0.01.

Transcriptome analysis of RA-FLSs, as compared with OA-FLSs, migration in mouse FLSs, supporting a role for NFAT5 blockade in revealed that NFAT5 was substantially involved in the locomotion the treatment of RA. Taken together, these data provide evidence that of RA-FLSs, and that TF and CCL2 as NFAT5 target genes were the NFAT5 critically controls the migration and invasion of RA-FLSs by key mediators of this process. IL-1b and TGF-b, which are the major regulating TF and CCL2 expressions. cytokines involved in RA pathogenesis (22), could increase NFAT5 Blood coagulation factors have been implicated in pathogenesis expression through activation of p38 MAPK, and thereby upregulated of chronic inflammatory diseases, including RA (11). In particular, TF and CCL2 expressions in RA-FLSs in vitro. Functional assays TF was shown to have a role in the progression of chronic arthritis demonstrated that IL-1b–orTGF-b–induced FLS migration, inva- (20, 36, 37). For example, disease severity is markedly reduced in sion, and lamellipodia formation were dependent on the NFAT5–TF TF cytoplasmic domain mutant mice with Ag-induced arthritis and/or NFAT5–CCL2 axis. Additionally, in the experiments using (36). Conversely, intra-articular injection of recombinant TF ex- FLSs and MEFs isolated from NFAT5 KO mice, NFAT5 deficiency acerbates chronic arthritis in mice (37). However, to our knowl- reduced cell migration by downregulating TF and CCL2 expression. edge, little is known about whether coagulation factors, including KRN2, a small molecule and selective inhibitor of NFAT5, sup- TF, control pathologic processes of RA-FLSs. We revealed in this pressed IL-1b–orTGF-b–induced NFAT5 expression and cell article, for the first time, to our knowledge, that NFAT5-dependent 10 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. TGF-b–induced FLS migration is inhibited by NFAT5 knockout or a specific NFAT5 inhibitor, KRN2. (A) Expression levels of NFAT5, TF, and CCL2 in mouse FLSs of Nfat5+/+ and Nfat5+/D mice as determined by Western blot analysis (upper panel) and real-time PCR (lower panel). *p , 0.05 versus Nfat5+/+ FLSs. (B) Effect of Nfat5-haploinsufficiency on the migration of mouse FLSs. Nfat5+/D FLSs and Nfat5+/+ FLSs were seeded in the upper wells of a Boyden chamber and stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) in serum-free DMEM; 10% FBS with IL-1b or 10% FBS with TGF- b was added to the lower wells. After 12 h of incubation, the migrated cells were counted. Scale bar, 2000 mm. **p , 0.01 versus Nfat5+/+ FLSs under each condition, ##p , 0.01 versus unstimulated Nfat5+/+ FLSs. (C) TF and CCL2 levels in MEFs of Nfat5+/+ versus Nfat5D/D. MEFs were stimulated with IL-1b (10 ng/ml) in DMEM containing 1% FBS or with TGF-b (10 ng/ml) in serum-free DMEM supplemented with insulin-transferrin-selenium-A (ITS-A). After 1 h, TF mRNA expression was determined by real-time PCR. After 12 h, CCL2 expression levels were determined by real-time PCR and ELISA. CCL2 ELISA results were normalized relative to cell viability assessed by MTT assay. *p , 0.05 versus Nfat5+/+ MEFs under each condition, #p , 0.05 versus unstimulated Nfat5+/+ MEFs. (D) Suppression of IL-1b– or TGF-b–induced NFAT5 expression in mouse FLSs by KRN2, a specific inhibitor of NFAT5. FLSs isolated from Nfat5+/+ mice were pretreated with KRN2 (13-[2-fluorobenzyl]-berberine, 5 mM) or DMSO as vehicle control for 1 h, and then stimulated with IL-1b (10 ng/ml) or TGF-b (10 ng/ml) for 24 h. NFAT5 expression was determined by Western blot analysis. (E) KRN2 inhibition of TGF- b–induced migration of mouse FLSs. Nfat5+/+ mouse FLSs were stimulated in the upper wells of a Boyden chamber with TGF-b (10 ng/ml) in serum-free DMEM in the presence or absence of KRN2 (5 mM); 10% FBS with TGF-b was added to the lower wells. DMSO was treated as vehicle control. After 12 h, the migrated cells were manually counted. Scale bar, 2000 mm. Data in (A)–(E) are mean 6 SEM or representatives of more than three independent experiments. ***p , 0.001.

TF is essential for increased migration and invasiveness of RA- and protease activities of TF/FVIIa complex (11), it can be postu- FLSs, thus providing an additional pathway to link between blood lated that NFAT5–TF axis not only promotes migration and invasion coagulation and chronic inflammation. Moreover, FLS migration of RA-FLSs but also triggers further proinflammatory responses and induced by FVII, a specific activator of TF, was markedly reduced bone and cartilage degradation in arthritic joints (Fig. 7). by NFAT5 knockdown, which indicates that FVII-mediated FLS Whether TGF-b regulates the NFAT5 signaling pathway re- migration is dependent on NFAT5. Considering the proinflammatory mains elusive. TGF-b is strongly expressed in the RA synovia The Journal of Immunology 11 Downloaded from

FIGURE 7. Hypothetical model for the role of NFAT5 in promoting the aggressive phenotype of RA-FLSs. Under high inflammatory conditions of RA joints, IL-1b and TGF-b can increase the expression of NFAT5 in RA-FLSs via p38 MAPK (1). Through NFAT5 activation, IL-1b (red arrow) then increases both TF and CCL2 expressions, whereas TGF-b (blue arrow) predominantly enhances TF expression while minimally affecting CCL2 levels (2). As a result, increased TF on FLS surface interacts with the intracellular actin network and organizes it to form lamellipodia (3). Additionally, extracellular factor VII can combine with TF, forming the TF/FVIIa complex (4), which synergistically facilitates the motility of RA-FLSs. Meanwhile, IL-1b– http://www.jimmunol.org/ stimulated CCL2 promotes the migration of RA-FLSs in an autocrine and paracrine manner (5). Acquisition of higher motility of RA-FLSs is accompanied by increased invasiveness, resulting in accelerated cartilage destruction (6). Although not tested in this study (indicated as broken arrow), it is possible that NFAT5 regulation of TF and CCL2 further aggravates joint inflammation by activating extravascular coagulation cascades (7) and increasing chemotaxis of inflammatory cells (8), respectively.

(38) and contributes to the RA pathologic condition by inducing Given that current therapeutic agents targeting T cells, B cells, proliferation and migration of RA-FLSs (17, 23). TGF-b activates and cytokines have had limited success, RA-FLSs are an attractive

members of the Smad family of signal transducers (39), but it also target to achieve complete remission. However, therapeutic agents by guest on September 24, 2021 uses non-Smad pathways, including p38 MAPK, to regulate a that emasculate the aggressive phenotype of RA-FLSs have not wide range of cellular functions (40). In this article, we report that been tried. Previously, we identified the novel NFAT5 inhibitor TGF-b upregulated NFAT5 and its target gene TF expressions in KRN2, 13-(2-fluorobenzyl)-berberine, through high-throughput RA-FLSs through activation of p38 MAPK. Moreover, TGF-b drug screening (35). KRN2 selectively inhibited the tran- enhanced the migratory and invasive capacity of RA-FLSs pre- scriptional activation of NFAT5 by blocking NF-kB binding to dominantly via NFAT5–TF axis, but not via NFAT5–CCL2 axis. the NFAT5 promoter region, reducing the expression of proin- Notably, NF-kB p65 siRNA failed to hamper TGF-b–induced TF flammatory genes (35). In this study, we tested the effect of KRN2 upregulation, suggesting that the NF-kB p65–TF axis is dispens- on FLS migration. NFAT5 modulated TF and CCL2 expressions able for this process. Taken together, these results are the first, to and FLS migration in a mouse system, and KRN2 effectively our knowledge, to demonstrate the importance of a novel TGF-b– inhibited promigratory capacity of mouse FLSs induced by TGF-b p38 MAPK–NFAT5–TF pathway for cell migration and invasion, as well as suppressing NFAT5 expression in FLSs stimulated with b b explaining how FLSs transmit hyperkinetic signals under high IL-1 or TGF- . Together, these observations offer new drug TGF-b stimulatory conditions, like RA joints (Fig. 7). action mechanism of KRN2, suggesting that KRN2 can be a po- IL-1b has been widely accepted as the major stimulus re- tential therapeutic agent to block FLS invasiveness. sponsible for the destructive and aggressive potential of RA-FLSs In summary, our data highlight the importance of the (22). IL-1b rapidly induces the expressions of a number of genes, osmoprotective transcription factor NFAT5 in the migration and including chemoattractant CCL2, in multiple differing cell types invasion of RA-FLSs. Through global and integrated analysis of (41). However, involvement of NFAT5–TF axis in IL-1b signaling transcriptomes in NFAT5-deficient RA-FLSs, we propose TF pathway has never been demonstrated. In the current study, IL-1b and CCL2 as the key target genes of NFAT5, representing upregulated NFAT5 and TF expressions in RA-FLSs through ac- promigratory and invasive characteristics of RA-FLSs. Upon tivation of p38 MAPK, and the in vitro activity of IL-1b was TGF-b stimulation, the noncanonical p38 MAPK pathway is substantially blocked by downregulation of NFAT5 or TF the major upstream regulator of NFAT5 expression in RA- transcripts. IL-1b also induced CCL2 secretion by RA-FLSs FLSs. We also provide an NFAT5-dictated pathway to link and thereby increased FLS migration/invasion (possibly in an blood coagulation and FLS invasiveness and functionally val- autocrine/paracrine manner), but the impact of NFAT5 on this idate the pathologic role of NFAT5–TF and NFAT5–CCL2 axes process was partial. Thus, both NFAT5–TF and NFAT5–CCL2 in RA. We anticipate that anti-NFAT5 blockades (e.g., small pathways may be required for fully promigratory and proinvasive molecules, including KRN2 or NFAT5 small hairpin RNA) will competence of RA-FLSs after IL-1b stimulation because NFAT5 provide novel candidates in the treatment of RA and other in- knockdown alone almost completely blocked IL-1b signaling for flammatory diseases in which NFAT5 and its targets, TF and FLS locomotion (Fig. 7). CCL2, play a key role. 12 NFAT5 REGULATION OF RHEUMATOID SYNOVIOCYTES

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Supplementary Figure S1. Potential binding sites for NFAT5 in promoter regions of tissue factor (F3) and CCL2. Consensus binding sites for NFAT5 are shown in the proximal

2 kb promoter regions of tissue factor (F3) (A) and CCL2 (B).

Supplementary Figure S2. Effects of NFAT5 or p38 MAPK knock down on tissue factor

(TF) and CCL2 expressions in FLS stimulated with TNFα. RA-FLSs were transfected with 50 nM of siRNAs for NFAT5 (siNFAT5, A), siRNAs for p38 (sip38, B) or control siRNA (siCon) for 24 hours. The cells were then treated with 10 ng/ml of TNFα for 12 hours.

NFAT5, p38, tissue factor (TF) and CCL2 mRNA expression levels were determined by real- time PCR analysis. GAPDH mRNA was used as a control. Data show the mean ± SEM of four independent experiments in duplicate with different RA-FLSs. * P<0.05, ** P<0.01, n.s.=not significant.

Supplemental Figure S3. MAPK regulation of NFAT5 expression in RA-FLSs stimulated with IL-1β or TGFβ. RA-FLSs were pre-treated with 20 μM SB203580 (SB, p38 inhibitor),

10 μM PD98059 (PD, ERK1/2 inhibitor) or 20 μM SP600125 (SP, JNK inhibitor) for 1 hour, and then stimulated with 10 ng/ml of IL-1β (A) or TGFβ (B) for 12 hours. NFAT5 mRNA expression levels were determined by real-time PCR analysis. GAPDH mRNA was used as a control. Data show the mean ± SEM of four independent experiments in duplicate with different RA-FLSs. * P<0.05, ** P<0.01, n.s.=not significant.

Supplementary Table S1. Gene ontology (GO) analysis of global transcript profiles in

NFAT5-deficient RA-FLSs and NFAT5 knockout mouse embryonic fibroblasts (MEFs).

Functional enrichment analysis was conducted with the downregulated differentially expressed genes (DEGs) of NFAT5 siRNA (siNFAT5)-transfected RA-FLSs versus control siRNA (siCon)-transfected RA-FLSs and DEGs of Nfat5+/+ MEFs versus Nfat5Δ/Δ MEFs, which resulted in the significantly enriched biological processes involved in cell migration and adhesion.

DEGs from Enriched biological processes Count P-Value siNFAT5-treated v.s. regulation of cell migration 8 0.028 siCon-treated RA-FLSs regulation of locomotion 8 0.05

Nfat5+/+ MEFs v.s. cell adhesion 28 6.20E-07 Nfat5 Δ/Δ MEFs cell motion 16 0.0011