Downregulation of Mir-200A-3P, Targeting Ctbp2, Is Involved in The

Downregulation of miR-200a-3p, Targeting CtBP2, Is Involved in the Hypoproduction of IL-2 in Systemic Lupus Erythematosus− Derived T Cells This information is current as of September 28, 2021. Eri Katsuyama, Minglu Yan, Katsue Sunahori Watanabe, Syun Matsushima, Yuriko Yamamura, Sumie Hiramatsu, Keiji Ohashi, Haruki Watanabe, Takayuki Katsuyama, Sonia Zeggar, Nobuya Yoshida, Vaishali R. Moulton, George C. Tsokos, Ken-Ei Sada and Jun Wada Downloaded from J Immunol published online 24 April 2017 http://www.jimmunol.org/content/early/2017/04/22/jimmun ol.1601705 http://www.jimmunol.org/

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 24, 2017, doi:10.4049/jimmunol.1601705 The Journal of Immunology

Downregulation of miR-200a-3p, Targeting CtBP2, Is Involved in the Hypoproduction of IL-2 in Systemic Lupus Erythematosus–Derived T Cells

Eri Katsuyama,* Minglu Yan,* Katsue Sunahori Watanabe,* Syun Matsushima,* Yuriko Yamamura,* Sumie Hiramatsu,* Keiji Ohashi,* Haruki Watanabe,* Takayuki Katsuyama,*,† Sonia Zeggar,* Nobuya Yoshida,† Vaishali R. Moulton,† George C. Tsokos,† Ken-Ei Sada,* and Jun Wada*

Systemic lupus erythematosus (SLE) damages multiple organs by producing various autoantibodies. In this study, we report that decreased microRNA (miR)-200a-3p causes IL-2 hypoproduction through zinc finger E-box binding homeobox (ZEB)1 and Downloaded from C-terminal binding protein 2 (CtBP2) in a lupus-prone mouse. First, we performed RNA sequencing to identify candidate microRNAs and mRNAs involved in the pathogenesis of SLE. We found that miR-200a-3p was significantly downregulated, whereas its putative targets, ZEB2 and CtBP2, were upregulated in CD4+ T cells from MRL/lpr-Tnfrsf6lpr mice compared with C57BL/6J mice. ZEB1 and ZEB2 comprise the ZEB family and suppress various genes, including IL-2 by recruiting CtBP2. IL-2 plays a critical role in immune tolerance, and insufficient IL-2 production upon stimulation has been recognized in SLE pathogenesis. Therefore, we hypothesized that decreased miR-200a-3p causes IL-2 deficit through the ZEB1–CtBP2 and/or ZEB2–CtBP2 complex in SLE CD4+ T cells. Overexpres- http://www.jimmunol.org/ sion of miR-200a-3p induced IL-2 production by downregulating ZEB1, ZEB2, and CtBP2 in EL4 cell lines. We further revealed that miR-200a-3p promotes IL-2 expression by reducing the binding of suppressive ZEB1–CtBP2 and ZEB2–CtBP2 complexes on negative regulatory element A in the IL-2 promoter in EL4 cells. Interestingly, the ZEB1–CtBP2 complex on negative regulatory element A was significantly upregulated after PMA/ionomycin stimulation in lupus CD4+ T cells. Our studies have revealed a new epigenetic pathway in the control of IL-2 production in SLE whereby low levels of miR-200a-3p accumulate the binding of the ZEB1–CtBP2 complex to the IL-2 promoter and suppress IL-2 production. The Journal of Immunology, 2017, 198: 000–000.

ystemic lupus erythematosus (SLE) induces multiple organ creased IL-2 production is a well-known characteristic of both

damage associated with the overproduction of various human and murine SLE (2, 3). Reduced IL-2 production is related by guest on September 28, 2021 S autoantibodies and inflammatory cell infiltration (1). De- to the impairment of regulatory T cells (Tregs) in SLE patients and in a murine model of SLE (4, 5), suggesting that the dysregulation of IL-2 contributes to the autoimmune phenomena in SLE. *Department of Nephrology, Rheumatology, Endocrinology, and Metabolism, The mechanisms involved in the IL-2 defect in SLE have been Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700 8558, Japan; and †Division of Rheumatology, Department of elucidated in part by the discovery of various transcription factors Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, that bind to the IL-2 promoter. In SLE, phosphorylated cAMP re- MA 02215 sponse element modulator, an IL-2 suppressor, is aberrantly up- ORCIDs: 0000-0002-6960-9151 (M.Y.); 0000-0002-9566-244X (Y.Y.); 0000-0001- regulated compared with the IL-2 activator phosphorylated CREB, 7837-086X (H.W.); 0000-0002-8230-3528 (S.Z.); 0000-0001-9589-2360 (G.C.T.); 0000-0003-1020-0818 (K.-E.S.). in contrast to healthy controls under stimulation (6, 7). This imbalance Received for publication October 5, 2016. Accepted for publication March 24, 2017. is further exacerbated by increased levels of calcium calmodulin This work was supported by Japan Society for the Promotion of Science Grant kinase IV (8) and serine/threonine protein phosphatase 2A (9). 16K19602. Zinc finger E-box binding homeobox (ZEB)1 and ZEB2 are All authors made contributions to the conception and design of the study, as well as final transcription factors belonging to the ZEB family and close ho- approval of the version to be published. E.K., K.S.W., and J.W. conceived and designed the mologs characterized by two clusters of zinc fingers separated by a study; E.K., M.Y., S.M., S.H., Y.Y., K.O., H.W., T.K., S.Z., N.Y., and V.R.M. acquired the data; and E.K., K.-E.S., K.S.W., G.C.T., and J.W. analyzed and interpreted the data. homeodomain (10). ZEB1 and ZEB2 bind to various promoters by The sequences presented in this article have been submitted to the Gene Expres- their zinc finger motif and play a vital role in the development of sion Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number the vertebrate embryo; these proteins are crucial for the matura- GSE87219. tion of the neural duct and cranial organs (10). Recently, ZEB1 Address correspondence and reprint requests to Dr. Katsue Sunahori Watanabe, and ZEB2 have been recognized as important players in the in- Department of Nephrology, Rheumatology, Endocrinology, and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical duction of the epithelial–mesenchymal transition (EMT), which Sciences, 2-5-1 Shikata-cho, Kitaku, Okayama 700 8558, Japan. E-mail address: shift the phenotype of tumor cells to induce invasion into the [email protected] surrounding tissues and metastasis (11–13). Besides, ZEB1 targets Abbreviations used in this article: Aldh1a1, aldehyde dehydrogenase family 1, sub- and suppresses IL-2 by recruiting a corepressor, C-terminal binding family A1; B6, C57BL/6J; ChIP, chromatin immunoprecipitation; CtBP2, C-terminal binding protein 2; EMT, epithelial–mesenchymal transition; Iono, ionomycin; miR/ protein 2 (CtBP2) (14–17). ZEB1 was identified as a negative reg- miRNA, microRNA; MRL/lpr, MRL/lpr-Tnfrsf6lpr; NRE, negative regulatory ele- ulator of IL-2 (Nil-2-a) that binds to a negative regulatory element ment; SLE, systemic lupus erythematosus; Treg, regulatory T cell; UTR, untranslated (NRE)-A in the IL-2 promoter (14). ZEB1 and ZEB2 need CtBP2 region; ZEB, zinc finger E-box binding homeobox. that directly binds to the Pro-Leu-Asp-Leu-Ser (PLDLS) sequence in Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 ZEB1 and ZEB2 and is essential for them to exert their suppressive

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601705 2 DOWNREGULATION OF miR-200A-3p DECREASES IL-2 IN SLE activity (18). However, despite the powerful IL-2 suppression T cells were stimulated with plate-bound anti-CD3 Abs (2 mg/ml; BioLegend) ability of ZEB1, ZEB2, and CtBP2, the function of the ZEB family and anti-CD28 Abs (2 mg/ml; BioLegend). Cells were stimulated with PMA/ and CtBP2 in autoimmune diseases is unknown. Iono for 6 h or stimulated with anti-CD3/CD28 Abs for 24 h. MicroRNAs (miRNAs) are small noncoding RNAs that bind to the RNA isolation and real-time RT-PCR 39 untranslated region (UTR) of target genes and degrade mRNA or Total cellular RNAs were extracted with an miRNeasy mini kit (Qiagen). arrest translation (19). Recent studies have shown growing evidence cDNAs were reverse transcribed from mRNAs with a high-capacity cDNA that various miRNAs control SLE pathogenesis (20). However, only RT kit (Thermo Fisher Scientific) and cDNAs from miRNAs with a TaqMan two reports have been published on miRNAs that regulate IL-2 in miRNA reverse transcription kit (Thermo Fisher Scientific). Real-time SLE. The downregulation of miRNA (miR)-31 in peripheral blood PCRs for miR-200a-3p (000502), miR-181a-5p (000480), miR-101a-3p T cells has been reported in patients with SLE, which reduces IL-2 (002253), miR-466p-3p (464896_mat), sno-202 (001232), and sno-234 (001234) were performed using TaqMan primer/probes with TaqMan miRNA by decreasing RhoA (21). It was also reported that the under- assays (Thermo Fisher Scientific) and normalized to sno-202 and/or sno-234 expression of miR-155 in juvenile SLE PBMCs is related to a lower by the DDCt method. Real-time PCRs for IL-2 (Mm00434256_m1), ZEB1 production of IL-2 via the increased expression of the catalytic (Mm00495564_m1), ZEB2 (Mm00497193_m1), CtBP2 (Mm00515572_m1), subunit of serine/threonine protein phosphatase 2A (22). and GAPDH (Mm99999915_g1) were performed using ABI TaqMan gene expression assays (Applied Biosystems) and normalized to GAPDH by the In the present study, we found that miR-200a-3p, an miR-200 DDCt method. family miRNA that was previously reported as a regulator of cancer metastasis by directly suppressing promoter activity of Transfection of miRNA mimics ZEB1 and ZEB2 genes, was significantly downregulated in the The transfection of miRNA mimics (mirVana; Invitrogen) was performed

lpr Downloaded from MRL/lpr-Tnfrsf6 (MRL/lpr) lupus-prone mouse compared with with Lipofectamine RNAiMAX (Invitrogen) by the reverse transfection control mouse. We demonstrated that miR-200a-3p plays a critical procedure. Cells were transfected with miR-200a-3p mimic or its negative role in the regulation of the IL-2 expression through ZEB1, ZEB2, control at a final concentration of 10 nM and harvested 24 h later. and CtBP2 in EL4 T cells. Furthermore, we showed that the Transfection of plasmid and luciferase assay ZEB1–CtBP2 complex failed to dissociate from the IL-2 gene after PMA/ionomycin (Iono) stimulation in mouse primary CD4+ EL4 cells were cotransfected with 0.5 ng of pRL-TK Renilla plasmid (Promega) and 100 ng of IL-2 promoter firefly reporter plasmids [pIL-2 T cells, which explains the mechanism of the IL-2 defect in SLE. (-585)-Luc] (a gift from Dr. A. Rao, Addgene plasmid no. 12194) using http://www.jimmunol.org/ Our study presents novel data in the epigenetic control of IL-2 Neon electroporation system (Thermo Fisher Scientific) in serum-free production in systemic autoimmunity. medium with one pulse with a voltage of 1080 V and width of 50 ms. Twenty-four hours after transfection, 106 cells/ml were stimulated with 10 ng/ml PMA and 1 mM Iono. Twenty-four hours after stimulation, the Materials and Methods cells were lysed and assayed for luciferase activity using a Dual-Luciferase Mice reporter assay system (Promega) in accordance with the manufacturer’s instructions. Cotransfection of a Renilla luciferase plasmid (pRL-TK) was Genetic lupus-prone female MRL/lpr and their control C57BL/6J (B6) performed to normalize the assays. mice were purchased from The Jackson Laboratory and Charles River Laboratories, respectively. The sample of female MRL/MpJ at 16 wk old ELISA by guest on September 28, 2021 was procured and studied at the Beth Israel Deaconess laboratories (In- stitutional Animal Care and Use Committee approval 088-2015). At 8 and The amount of IL-2 protein secreted in the supernatants from cell cultures 14–16 wk of age, MRL mice and B6 mice were sacrificed, and their spleen was measured by ELISA in accordance with the manufacturers’ instructions tissues were collected. The experiments were approved by the Animal (mouse IL-2 ELISA Ready-SET-Go!; eBioscience). The absorbance was Care and Use Committee of the Department of Animal Resources, Ad- determined using a microplate reader set at 450 nm. vanced Science Research Center, Okayama University (OKU-2015569 and EMSA OKU-2013092). Nuclear extracts were prepared from EL4 cells using NXTRACT (Sigma- Isolation of mouse primary T cells Aldrich). For binding assays (20 ml final volume), 80 mg of total nuclear Mouse primary CD4+ T cells were isolated using a CD4 isolation kit II extract from EL4 cells was incubated with 1 mgof39 biotin-labeled (Miltenyi Biotec) by negative selection. A purity rate of .96.6% for oligonucleotide in the presence of 2 mlof103 binding buffer (100 mM + m m m isolated CD4 T cells was confirmed by flow cytometry. Tris, 500 mM KCl, 10 mM DTT [pH 7.5]), 1 l of 50% glycerol, 1 g/ l poly(deoxyinosinic-deoxycytidylic) acid,1% Nonidet P-40, and 10 mM miRNA profiling using a next-generation sequencer ZnSO4 at room temperature for 20 min. The oligonucleotides used in the + binding and competition assays were as follows: murine NRE-A sequence, Total RNA, including miRNA, was purified from CD4 T cells of MRL 59-CTGCCACACAGGTAAAGTCTT-39; mutant NRE-A sequence, 59- lupus-prone mice and B6 control mice using an miRNeasy mini kit CTGCCACACATTTAAAGTCTT-39 (mutation underlined). Both 39 biotin- (Qiagen). The quality of the total RNA was confirmed by spectropho- labeled and unlabeled dsNRE-A and dsNRE-A mutant oligonucleotides tometry using an Agilent 2100 Bioanalyzer (Agilent Technologies). Small (Sigma-Aldrich) were prepared by annealing the single stranded comple- RNAs were then subjected to the Illumina TruSeq small RNA sample mentary oligonucleotides. All competitors were used at 200-fold excess. For preparation protocol (Illumina). mRNAs were subjected to the Illumina supershift experiments, the nuclear extracts were incubated with anti-ZEB1 TruSeq RNA sample preparation protocol. Sequencing was performed Ab (1:100) (TCF8/ZEB1 no. 3396; Cell Signaling Technology), anti-ZEB2 using Illumina HiSeq. In each group, the read numbers of miRNA/mRNA Ab (1:25) (anti-Smad interacting protein no. 138222; Abcam), and anti- were counted, after which the miRNA/mRNA list was drafted by com- CtBP2 Ab (1:25) (no. 13256; Cell Signaling Technology). The binding paring the downregulated miRNA with the concomitantly upregulated mixtures were loaded onto a 6% native acrylamide gel (Thermo Fisher mRNA and comparing the upregulated miRNA with the concomitantly Scientific) in Tris/borate/EDTA buffer and electrophoresed for 50 min at downregulated mRNA. All raw and processed data are freely accessible in room temperature under a constant 100 V. The gels were then transferred to a the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under nylon membrane at 4˚C for 50 min under a constant 100 V and exposed to the accession number GSE87219. UV light to crosslink for 15 min. The DNA binding activity was detected Cells, cell culture, and stimulation using a LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific). The image was obtained using an LAS-3000 IR. The EL4 mouse T cell line was obtained from the American Type Culture Collection and cultured in DMEM (Life Technologies) supplemented with 10% Chromatin immunoprecipitation assay FBS (Life Technologies), 100 U/ml penicillin, and 100 mg/ml streptomycin. A Chromatin immunoprecipitation (ChIP) analysis was performed in accor- total of 106 cells/ml (EL4 cells or MRL- and B6-derived CD4+ Tcells)were dance with the manufacturer’s instructions (no. 9005S; Cell Signaling stimulated with PMA (10 ng/ml; Sigma-Aldrich) and Iono (1 mM; Sigma- Technology). Briefly, cells were cross-linked with 1% formaldehyde. After Aldrich) at 37˚C in a 5% CO2 incubator. For anti-CD3/CD28 stimulation, EL4 the cross-linked cells were lysed, the chromatin was harvested and frag- The Journal of Immunology 3 mented with enzymatic digestion using micrococcal nuclease. Nuclear miRNA and mRNA of CD4+ T cells isolated from the spleens of extracts were further treated with an ultrasound sonicator with six sets of MRL/lpr mice and B6 mice. A total of 45 miRNAs were down- 20-s pulses. Immunoprecipitations were performed with ZEB1 (TCF8/ regulated, and 69 miRNAs were upregulated in MRL/lpr mice ZEB1 no. 3396; Cell Signaling Technology), ZEB2 (anti-Smad interacting protein no. 138222; Abcam), and CtBP2 (no. 13256; Cell Signaling compared with the control mice when cut-off values were defined Technology) Abs. After DNA-protein cross-links were reversed, the DNA as ,0.5 and .2, respectively. In the present study we focused on was purified, and quantitative real-time PCR was performed with the primer the miRNAs downregulated in MRL mice compared with B6 mice 9 9 pairs as follows: (forward) 5 -TCCAGAGAGTCATCAGAAGAGG-3 and (Table I). Among these, we selected miRNAs that were down- (reverse) 59-TGGGGGTGTCACGATGTTTTAC-39. regulated (,0.5) and were associated with significant upregulation Statistical analyses of their target mRNAs demonstrated by RNA sequencing. During All results are shown as the mean 6 SEM of data from at least three separate the selection process, miR-200a-3p (target mRNAs with signi- experiments, each performed with more than triplicate samples. The dif- ficance: oxidized low-density lipoprotein receptor 1, Tmem56, ferences between the groups were analyzed for statistical significance using aldehyde dehydrogenase family 1, subfamily A1 [Aldh1a1]), miR- ANOVA with a Tukey–Kramer post hoc test or an unpaired t test, when 181a-5p (Clec4e), miR-101a-3p (Abcd2), and miR-466p-3p appropriate, to determine the differences. All statistical analyses were per- (Nrg1, Tex15) were identified. By quantitative real-time PCR, formed using the JMP 9.0 software package (SAS Institute, Cary, NC). only miR-200a-3p was reproducibly downregulated in MRL/lpr mice compared with B6 mice (Fig. 1B). Next, we assessed Results + whether the B6 mouse is appropriate as the control of MRL/lpr miR-200a-3p is downregulated in murine lupus CD4 T cells and whether the genetic background affected the expression of

To identify new candidate miRNAs involved in the pathogenesis of miR-200a-3p. Age- and sex-matched MRL/MPJ (Fas+/+) mice that Downloaded from SLE, we performed Illumina HiSeq to record the expression of present late and mild autoimmune phenotype (23, 24) were in-

Table I. miRNAs downregulated in CD4+ T cells derived from MRL/lpr mice compared with B6 mice

miRNAs Total Reads B6 Reads MRL Reads MRL/B6 B6/MRL Fold Change http://www.jimmunol.org/ mmu-miR-200a-3pa 182 179 3 5.322 0.100 0.019 mmu-miR-3096a-5p 711 679 32 20.188 1.068 0.053 mmu-let-7e-5p 929 860 69 25.569 2.302 0.090 mmu-miR-181a-5pa 974,831 878,003 96,828 26,104.366 3,230.720 0.124 mmu-miR-181c-5p 133,138 116,759 16,379 3,471.423 546.495 0.157 mmu-miR-125a-5p 2,266 1,966 300 58.452 10.010 0.171 mmu-miR-466d-3p 107 90 17 2.676 0.567 0.212 mmu-miR-150-5p 429,018 359,766 69,252 10,696.391 2,310.632 0.216 mmu-miR-669c-5p 980 817 163 24.291 5.439 0.224 mmu-miR-181a-1-3p 5,120 4,225 895 125.616 29.862 0.238 by guest on September 28, 2021 mmu-miR-28a-5p 3,246 2,673 573 79.472 19.118 0.241 mmu-miR-669o-5p 144 118 26 3.508 0.868 0.247 mmu-miR-467e-5p 654 532 122 15.817 4.071 0.257 mmu-miR-423-3p 37,287 30,079 7,208 894.294 240.499 0.269 mmu-miR-342-3p 33,135 25,958 7,177 771.771 239.465 0.310 mmu-miR-128-1-5p 491 382 109 11.357 3.637 0.320 mmu-let-7f-5p 828,120 638,551 189,569 18,985.094 6,325.076 0.333 mmu-let-7c-5p 40,279 30,987 9,292 921.291 310.033 0.337 mmu-miR-1843b-5p 858 655 203 19.474 6.773 0.348 mmu-miR-669a-3p 269 203 66 6.035 2.202 0.365 mmu-miR-669o-3p 269 203 66 6.035 2.202 0.365 mmu-miR-423-5p 14,740 11,087 3,653 329.633 121.884 0.370 mmu-miR-30e-3p 11,550 8,672 2,878 257.832 96.026 0.372 mmu-miR-28a-3p 1,565 1,167 398 34.697 13.279 0.383 mmu-miR-466b-3pa 440 328 112 9.752 3.737 0.383 mmu-miR-466c-3pa 440 328 112 9.752 3.737 0.383 mmu-miR-466p-3pa 440 328 112 9.752 3.737 0.383 mmu-miR-181b-5p 13,960 10,334 3,626 307.246 120.984 0.394 mmu-miR-1964-3p 606 447 159 13.290 5.305 0.399 mmu-miR-181c-3p 1,601 1,176 425 34.964 14.180 0.406 mmu-let-7b-5p 6,005 4,410 1,595 131.116 53.218 0.406 mmu-miR-874-3p 191 140 51 4.162 1.702 0.409 mmu-miR-1843a-5p 6,388 4,656 1,732 138.430 57.789 0.417 mmu-miR-466a-3p 187 136 51 4.043 1.702 0.421 mmu-miR-466e-3p 187 136 51 4.043 1.702 0.421 mmu-let-7g-5p 177,601 128,494 49,107 3,820.322 1,638.483 0.429 mmu-miR-99b-5p 2,892 2,092 800 62.198 26.692 0.429 mmu-miR-10b-5p 825 596 229 17.720 7.641 0.431 mmu-let-7a-5p 151,325 107,294 44,031 3,190.014 1,469.119 0.461 mmu-miR-101a-3pa 9,187 6,454 2,733 191.887 91.188 0.475 mmu-miR-30d-5p 16,187 11,355 4,832 337.601 161.222 0.478 mmu-miR-26b-5p 25,261 17,619 7,642 523.840 254.980 0.487 mmu-miR-744-5p 2,885 2,003 882 59.552 29.428 0.494 mmu-miR-101b-3p 2,899 2,010 889 59.760 29.662 0.496 mmu-miR-3068-3p 286 198 88 5.887 2.936 0.499 The candidate miRNAs are sorted by fold change. amiRNAs analyzed in Fig. 1A. 4 DOWNREGULATION OF miR-200A-3p DECREASES IL-2 IN SLE cluded to control for the genetic background. As shown in Fig. 1C, Gain of function of miR-200a-3p decreases ZEB1/ZEB2/CtBP2 the levels of miR-200a-3p were still significantly decreased in expression and increases IL-2 production MRL/lpr mice compared with MRL/MPJ mice, and MRL/MPJ To determine the gain effect of miR-200a-3p, miR-200a-3p mimic mice also presented lower levels of miR-200a-3p compared with or control mimic was transfected into EL4 mouse T cell lines by B6 mice. As shown in Fig. 1D, the levels of miR-200a-3p were lipofection. The transfection efficacy of miR-200a-3p was con- already decreased in MRL/lpr mice compared with B6 mice at firmed by quantitative PCR in each experiment (Fig. 3A). As younger age (8 wk old). miR-200a-3p levels increased at older age shown in Fig. 3B, the mRNA levels of ZEB1, ZEB2, and CtBP2 in both mice; however, the expression was still significantly lower were significantly decreased upon overexpression of miR-200a- in MRL/lpr mice. These results suggest that decreased miR-200a- 3p, demonstrating that miR-200a-3p negatively regulates the ex- 3p is seen in MRL/MPJ mice independent of the Fas mutation, and pressions of ZEB1, ZEB2, and CtBP2. the trend starts prior to the onset of disease. We also assessed whether miR-200a-3p influences IL-2 ex- ZEB2 and CtBP2 are the candidate targets of miR-200a-3p pression, because it has been reported that ZEB1 suppresses IL-2 We next investigated the target genes of miR-200a-3p that were up- by recruiting CtBP2 (17). EL4 T cells transfected with miR-200a- regulated in MRL/lpr compared with B6 mice. We retrieved the 3p mimic or control mimic were stimulated with PMA (10 ng/ml) predicted and validated target genes of miR-200a-3p using TargetS- and Iono (1 mM). IL-2 mRNA levels were measured by quanti- can, and their mRNA expressions were evaluated by RNA sequencing tative PCR after 6 h of PMA/Iono stimulation, and IL-2 protein data. As the candidate target genes of miR-200a-3p, oxidized low- levels in the supernatants were measured by ELISA after 24 h of density lipoprotein receptor 1, Aldh1a1, ZEB2, CtBP2, and Kruppel-€ stimulation. As shown in Fig. 3C (left) and 3D (left), IL-2 ex- Downloaded from like factor 12 were selected, because they demonstrated upregulation pression was significantly upregulated by the overexpression of with statistically significant differences or have been reported to be miR200a-3p at both the mRNA and cytokine levels. IL-2 ex- functionally related to the regulation of the immune system. Among pression displayed the same change when cells were stimulated them, Aldh1a1, ZEB2, and CtBP2 were reproducibly upregulated with anti-CD3/CD28 Abs (Fig. 3C, right, 3D, right), although the (Fig. 2 and data not shown). Becaise CtBP2 and ZEBs are tran- peak of IL-2 expression was later compared with the stimulation

scription factors and form the complex coordinating the expression of with PMA/Iono (peak at 6 h versus 24 h). In the following ex- http://www.jimmunol.org/ various genes, we focused on CtBP2 and ZEB2. The mRNA experiments, PMA/Iono stimulation was chosen considering that it pression of ZEB1 was rather reduced by RNA sequencing (Fig. 2), evokes higher levels of IL-2 and at earlier time points. We then and we further conﬁrmed the mRNA expression by quantitative RT- performed luciferase reporter assays to determine whether miR- PCR. Because the target sequence for miR-200a-3p was conﬁrmed in 200a-3p regulated IL-2 promoter activity. We used pIL-2(-585)- the 39-UTR of both the ZEB1 and ZEB2 genes, and because both Luc, which includes the murine IL-2 promoter 2585 to 234 rela- ZEB1 and ZEB2 recruit CtBP2 for its suppressive activity, we in- tive to the transcription start site (25). EL4 T cells were cotrans- vestigated both ZEB1 and ZEB2 in the subsequent experiments. fected with IL-2 promoter luciferase construct and miR-200a-3p by guest on September 28, 2021

FIGURE 1. The downregulation of miR-200a-3p in CD4+ T cells of MRL mice. (A) The expression of miR-200a- 3p, miR-181a-5p, miR-101a-3p, and miR- 466p-3p (combination of miR-466b-3p, miR-466c-3p, and miR-466p-3p) was evaluated by TaqMan quantitative PCR. (B) The miR-200a-3p expression was evaluated in MRL mice compared with B6 mice (n = 5 per B6 group and n =4 per MRL group, 16-wk-old female). (C) The miR-200a-3p expression was evaluated in MRL mice compared with B6 and MRL/MPJ mice (n = 3 per group, 16-wk-old female). (D) The miR-200a- 3p expression was evaluated in 8-wk-old mice compared with 16-wk-old mice (n = 3 per group, female) (mean 6 SEM). ***p , 0.0001. The Journal of Immunology 5

It has been reported that ZEB1 binds to NRE-A 100 bp from the transcription start site of the IL-2 gene and suppresses IL-2 production (16) (Fig. 4A). Additionally, ZEB1 and ZEB2 have been reported to recognize 59-CACCT-39 in various promoters by their zinc finger cluster, which also applies to the NRE-A sequence (26). To determine whether ZEB1, ZEB2, and CtBP2 bind to NRE-A under the regulation of miR-200a-3p, EMSA was first performed in an EL4 cell line overexpressing miR- 200a-3p. Nuclear extracts isolated from the EL4 cells with 6 h FIGURE 2. Upregulation the ZEB2 and CtBP2 expression in the CD4+ stimulation of PMA/Iono were incubated with 39 biotin-labeled T cells of MRL/lpr mice. The expression of ZEB2 and CtBP2 in CD4+ dsNRE-A oligonucleotide in EMSA. The protein–NRE-A mix- T cells of mice spleen was evaluated by TaqMan quantitative PCR (n =9 tures were then subjected to a native PAGE. As shown in lanes per group, 16-wk-old female) (mean 6 SEM). ***p , 0.0001. 1–6 of Fig. 4B, the specific band position of protein–NRE-A probe complex was primarily examined as positive control mimic or control mimic and stimulated with PMA/Iono for 24 h that band. The protein–NRE-A probe complex (lane 1) was abol- has been set as the peak of IL-2 according to the pre-examined time ished by 200-fold excess of unlabeled NRE-A probe (lane 2), course. As shown in Fig. 3E, the overexpression of miR-200a-3p whereas an equal amount of unlabeled mutant–NRE-A probe significantly increased the IL-2 promoter activity after 24 h of failed to achieve such effects (lane 3). A supershift assay was stimulation. These results indicate that miR-200a-3p promotes IL-2 performed using the Abs against the transcription factors and Downloaded from production through the activation of the IL-2 promoter and suggest showedthatZEB1(filledarrowinFig.4B,lane4)andCtBP2 that miR-200a-3p modulates the ZEB1, ZEB2, and CtBP2 expres- with a faint band, but not ZEB2, were composed of a complex of sion and activity for the transcriptional regulation of IL-2 production. proteins and oligonucleotide. Furthermore, the overexpression of miR-200a-3p mimic reduced miR-200a-3p recruits ZEB1, ZEB2, and CtBP2 directly to the the protein–NRE-A complex compared with negative control IL-2 promoter mimic (open arrows in Fig. 4B, lanes 7 and 8), demonstrating that http://www.jimmunol.org/ We next investigated whether miR-200a-3p has an effect on the miR-200a-3p reduces the binding of ZEB1 and CtBP2 on NRE-A, direct bindings of ZEB1, ZEB2, and CtBP2 to the IL-2 promoter. which results in a reduction in the suppressive activity of the by guest on September 28, 2021

FIGURE 3. miR-200a-3p mimic–induced IL-2 production under stimulation in EL4 T cell lines as a gain-of-function analysis. (A) The transfection efficacy of miR-200a-3p mimic was confirmed by TaqMan quantitative PCR. miR-200a-3p was transfected by lipofection, and the expression was compared with the negative control mimic after 24 h transfection. (B) The mRNA levels of ZEB1, ZEB2, and CtBP2 after the overexpression of miR-200a- 3p were confirmed by TaqMan quantitative PCR. (C) The mRNA level of IL-2 after the overexpression of miR-200a-3p was evaluated by TaqMan quantitative PCR. EL4 cells were transfected with either miR-200a-3p mimic or negative control mimic. After 24 h transfection, 106 cells/ml were stimulated with PMA (10 ng/ml) and Ionomycin (1 mM), or 2 3 106 cells/ml were stimulated with plate-bound anti-CD3 (2 mg/ml) and anti-CD28 (2 mg/ml) Abs. After stimulation, IL-2 mRNA level was evaluated by TaqMan quantitative PCR. Left; PMA/Iono stim for 6 h. Right; anti-CD3/CD28 Abs stim for 24 h. (D) The IL-2 cytokine level in the supernatant of the cells was evaluated by ELISA. After 24 h stimulation, the supernatant of the culture medium were harvested. Left; PMA/Iono stim. Right; anti-CD3/CD28 Abs stim. (E) The IL-2 promoter activity was evaluated by a luciferase assay. EL4 cells were cotransfected with miR-200a-3p mimic or negative control mimic and with the construct driven by the IL-2 promoter (2585 to 234 relative to transcription start site) or empty vector by electroporation. The luciferase activity was measured after 24 h stimulation (mean 6 SEM). Data are shown from six (A and B) and three (C–E) independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. NC, negative control. 6 DOWNREGULATION OF miR-200A-3p DECREASES IL-2 IN SLE

FIGURE 4. Reduction in the recruitment of ZEB1 and CtBP2 on NRE-A of IL-2 promoter by overexpression of miR-200a-3p. (A) A negative regulatory element (NRE- A) is located at 2110 to 2101 from the transcription start site of IL-2. The C-terminal zinc ﬁnger cluster of ZEB1 binds to NRE-A and suppresses IL-2 production. The underlined sequences are conserved. (B) Binding activity of the ZEB1–CtBP2 and ZEB2–CtBP2 (ZEB1/ ZEB2–CtBP2) complexes with NRE-A was determined by EMSA using EL4 T cells. As positive controls, EL4 cells without mimic were stimulated with PMA/Iono for 6 h and their nuclear proteins were subsequently extracted. The positive control nuclear proteins were used in lanes 1–6. miR-200a-3p mimic or negative control mimic was transfected into the EL4 T cells and similarly stimulated, and the nuclear protein was applied Downloaded from (lanes 7 and 8). Lanes 1–3 show the competition effects. The speciﬁc NRE-A protein band was competed with 200-fold excess unlabeled NRE probe (lane 2) but failed to compete with 200-fold excess unlabeled mutant probe (lane 3). Lanes 4–6 are for the supershift bands: anti- ZEB1, ZEB2, or CtBP2 Abs were added, respectively.

The open arrows in lanes 7 and 8 show NRE-A protein http://www.jimmunol.org/ binding with miR-200a-3p mimic or negative control mimic, respectively.

ZEB1–CtBP2 complex on the IL-2 promoter. To further confirm the NRE-A, whereas the complex dissociates from NRE-A under this notion, we performed a ChIP assay. EL4 cells transfected with stimulation to produce sufficient IL-2 in the control mice (Fig. 5C, by guest on September 28, 2021 miR-200a-3p mimics or control mimics were stimulated for 6 h 6 h versus 9 h). The binding ability of ZEB2 was also accumulated and then advanced to the crosslink step. The ChIP assay showed after stimulation in MRL/lpr-derived CD4+ T cells; however, the significantly reduced recruitment of ZEB1, ZEB2, and CtBP2 on enrichment was rather low in MRL/lpr mice compared with B6 NRE-A under conditions of overexpression of miR-200a-3p cells (Fig. 5C). These results demonstrated that the dysregulation of (Fig. 5A), indicating that ZEB2 is also able to bind to NRE-A ZEB1–CtBP2 complexes binding to NRE-A upon the stimulation is and is negatively regulated by miR-200a-3p. crucially associated with IL-2 hypoproduction in MRL/lpr mice. Taken together with the gain-of-function findings in EL4 cells, these The ZEB1–CtBP2 complexes fail to dissociate from the NRE in results suggest that miR-200a-3p regulates the ZEB1/ZEB2–CtBP2 the IL-2 promoter after stimulation in lupus-prone mice complexes on NRE-A upon stimulation in murine T cells and in- To clarify whether ZEB1–CtBP2 and ZEB2–CtBP2 (ZEB1/ZEB2– fluences IL-2 production (Fig. 6A). In the lupus model, the ZEB1– CtBP2) complexes on NRE-A influence the IL-2 production after CtBP2 complex is critical in suppressing IL-2 and ZEB2 may be stimulation in lupus-derived T cells, we performed ChIP assays only partially regulated by miR-200a-3p (Fig. 6B). using CD4+ T cells derived from the spleen of MRL/lpr mice and B6 mice. At first, IL-2 hypoproduction was confirmed anew in Discussion MRL/lpr-derived CD4+ T cells compared with B6 (Fig. 5B). In the present study, we found that miR-200a-3p positively reg- Regarding the dynamics of the ZEB1 on NRE-A, it has been ulates IL-2 production in murine T cells through the recruitment reported that the binding activity reached its peak at 6 h after of ZEB1–CtBP2 and ZEB2–CtBP2 (ZEB1/ZEB2–CtBP2) com- PMA/Iono stimulation, after which the binding abruptly declines plexes to the IL-2 gene promoter. Also in humans, the serum and at 9 h of stimulation in EL4 cells (16). This is consistent with the urinary levels of miR-200a, miR-200b, and miR-200c have been time course of IL-2 transcription initiation after PMA/Iono stim- reported to be significantly decreased in active SLE patients ulation, as can be seen in Fig. 3C. Therefore, the time course was compared with healthy subjects. Additionally, the miR-200a ex- set as 0, 6, and 9 h. As expected, the dynamics of the binding of pression correlated inversely with proteinuria and SLEDAI (27). ZEB1, ZEB2, and CtBP2 to NRE-A reached a peak at 6 h and This report supports our data and suggests that miR-200a may also significantly decreased at 9 h after PMA/Ion stimulation in B6 play an important role in patients with SLE. mice (Fig. 5C). Alternatively, the kinetics was impaired in MRL/lpr It has been determined that all miR-200 family members neg- mice, and the binding levels for ZEB1 and CtBP2 but not ZEB2 atively regulate ZEB1 and ZEB2 and suppress EMT by binding to were significantly higher throughout the time course of the ex- the 39-UTR of ZEB1 and ZEB2, whereas ZEB1 and ZEB2 bind to periment after stimulation (Fig. 5C). the promoter region of the miR-200 family members and decrease These results indicate that the ZEB1–CtBP2 complexes in MRL/lpr the expression of the miR-200 family in various cancer models mice are unresponsive to the stimulation and fail to dissociate from (28–34). miR-200 family members are divided into two groups The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ FIGURE 5. The impaired dynamics of ZEB1, ZEB2, and CtBP2 on NRE-A regulated by miR-200a-3p. (A) The complex between NRE-A and ZEB1– CtBP2 and ZEB2–CtBP2 (ZEB1/ZEB2–CtBP2) complex after miR-200a-3p overexpression was confirmed by a ChIP assay. EL4 cells were transfected either with miR-200a-3p mimic or negative control mimic and stimulated with PMA/Iono. After 6 h stimulation, the cells were harvested and directly advanced to the crosslink step. Quantitative PCR was performed using an NRE-A primer. The ChIP results are presented as a percentage of 2% input DNA. (B) The cytokine levels of IL-2 were evaluated by ELISA. MRL- or B6-derived CD4+ T cells were stimulated with PMA/Iono for 9 h and each supernatant was subsequently harvested. (C) The ZEB1/ZEB2–CtBP2 complex on NRE-A of MRL and B6 mice under stimulation was evaluated by a ChIP assay. Primary CD4+ T cells from MRL or B6 mice were stimulated with PMA/Iono for 6 and 9 h. The cells were promptly advanced to the crosslink step. Quantitative PCR was performed using an NRE-A primer. The ChIP results are presented as a percentage of 2% input DNA (mean 6 SEM). Data are shown from three independent experiments (A), three independent experiments with n = 3 per group (B), and n = 8 per MRL group, n = 40 per B6 group, 14-wk-old female (C). *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 28, 2021 according to their seed sequences: the first one is miR-141 and CtBP2 on NRE-A after stimulation. It was previously reported that miR-200a; the second one is miR-200b, miR-200c, and miR-429. c-Rel, an activator of IL-2, failed to access near the NRE-A site These two had quite similar seed sequences: miR-141 and miR- with anti-CD3/CD28 stimulation, whereas the activator was able to 200a recognize 59-AACACU-39, and miR-200b, miR-200c, and bind with PMA/Iono stimulation (43). This fact may suggest that miR-429 recognize 59-AAUACU-39 (33). Eight miR-200 binding anti-CD3/CD28 stimulation is not sufficient for dissociating sup- sites are included in 39-UTR of ZEB1 and nine binding site in the pressive factors on the NRE-A, and therefore we selected PMA/ 39-UTR of ZEB2. This ZEB/miR-200 double-negative feedback Iono stimulation to assess the NRE-A binding ability. As expected, loop controls ZEB factors, the strong EMT inducers of cancer ZEB1 and CtBP2, except ZEB2, were significantly accumulated on metastasis. The ZEB/miR-200 loop also might be related with the NRE in MRL/lpr mice compared with B6 mice after stimulation senescence (35–37) and apoptosis (38–41), which promote the (Fig. 5C). CtBP2 was consistently upregulated in MRL/lpr mice at development of cancer. the mRNA and nuclear protein levels in MRL/lpr CD4+ Tcells ZEB1 was first reported as Nil-2-a to bind to NRE-A and (Figs. 2, 4B, 5C). CtBP2 is critically regulated by miR-200a-3p, suppress IL-2 in Jurkat T cells. ZEB1 is also associated with the which has the essential role for suppressing ability of ZEB1 and differential production of IL-2 in Th1/2 EL4 cells. Although there ZEB2 against IL-2. is no previous report on the mechanism whereby ZEB2 influences It is still unclear which factors regulate the transcriptional ac- IL-2 production, it has been reported that ZEB2 also binds to 59- tivities and maturation of miR-200a-3p. As mentioned before, ZEB1 CACCT-39 in the NRE-A sequence through its zinc finger motif and ZEB2 are known to directly regulate the miR-200 family. In- (42). Our study has revealed the impaired balance of miR-200a-3p terestingly, EBV negatively regulates miR-200a and miR-200b in and ZEB1/ZEB2 in murine T cells and demonstrated the func- gastric cancer (44). EBV is considered as a trigger for the onset of tional impact of miR-200a-3p on IL-2 production. SLE; therefore, an miR-200a-3p–mediated pathway might be a There is some discrepancy between gain-of-function analysis in candidate for the development of SLE. EL4 cells and mouse primary CD4+ T cells in the point that ZEB1 It is also intriguing that the miR-200a-3p may act on Tregs. If mRNA and the ZEB2–CtBP2 complex were not fully regulated in miR-200a-3p can regulate Tregs by increasing IL-2, then targeting MRL mice with decreased level of miR-200a-3p. These results Tregs may be a useful therapeutic option for patients with SLE. Our suggest the presence of other regulators in vivo and that the miR- study revealed the critical role of miR-200a-3p in the regulation of 200a-3p/ZEB1/ZEB2 loop in MRL mice might also be regulated by IL-2 through ZEB1, ZEB2, and CtBP2. An IL-2 defect in SLE is other miRNAs or transcription factors or suppressors. linked to a reduction in the numbers of Tregs and an increase in the Because IL-2 production under stimulation in SLE is markedly numbers of autoreactive T cells. Low-dose IL-2 therapy has shown impaired, we were intrigued by the effects of the ZEB1, ZEB2, and promising results in correcting reduced numbers of Tregs in SLE 8 DOWNREGULATION OF miR-200A-3p DECREASES IL-2 IN SLE

FIGURE 6. Model of IL-2 regulation by miR-200a-3p in murine T cells through ZEB1–CtBP2 and ZEB2–CtBP2 complex. (A) miR-200a-3p negatively regulates the recruitment of suppressive transcription factors (ZEB1 or ZEB2 and CtBP2) on NRE- A and promotes IL-2 production in murine T cells. (B) After PMA/Iono stimulation, the ZEB1–CtBP2 complexes dissociate from NRE-A in normal CD4+ T cells and produce

IL-2. In contrast, the complexes hardly dis- Downloaded from sociate from NRE-A in primary murine lupus CD4+ T cells after stimulation and subsequently fail to produce IL-2. ZEB, ZEB1 or ZEB2. http://www.jimmunol.org/

(5). Therefore, investigating the mechanism of IL-2 defect will interleukin-2 selectively corrects regulatory T cell defects in patients with systemic lupus erythematosus. Ann. Rheum. Dis. 75: 1407–1415. by guest on September 28, 2021 clarify the central pathogenesis of SLE. 6. Juang, Y. T., Y. Wang, E. E. Solomou, Y. Li, C. Mawrin, K. Tenbrock, In conclusion, we have demonstrated that miR-200a-3p posi- V. C. Kyttaris, and G. C. Tsokos. 2005. Systemic lupus erythematosus serum IgG tively regulates IL-2 through the ZEB1/ZEB2–CtBP2 complexes increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV. J. Clin. Invest. 115: 996–1005. binding to the NRE-A site of the IL-2 promoter in murine T cells. 7. Solomou, E. E., Y. T. Juang, M. F. Gourley, G. M. Kammer, and G. C. Tsokos. The ability of the ZEB1–CtBP2 complexes to dissociate from the 2001. 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Corrections

Katsuyama, E., M. Yan, K. S. Watanabe, S. Matsushima, Y. Yamamura, S. Hiramatsu, K. Ohashi, H. Watanabe, T. Katsuyama, S. Zeggar, N. Yoshida, V. R. Moulton, G. C. Tsokos, K.-E. Sada, and J. Wada. 2017. Downregulation of miR-200a-3p, targeting CtBP2 complex, is involved in the hypoproduction of IL-2 in systemic lupus erythematosus–derived T cells. J. Immunol. 198: 4268–4276.

The fourth author was omitted from the author list as originally published. The corrected author list is shown below. The afﬁliations were correct as published and are shown below for reference.

Eri Katsuyama,* Minglu Yan,* Katsue Sunahori Watanabe,* Mariko Narazaki,* Syun Matsushima,* Yuriko Yamamura,* Sumie Hiramatsu,* Keiji Ohashi,* Haruki Watanabe,* Takayuki Katsuyama,*,† Sonia Zeggar,* Nobuya Yoshida,† Vaishali R. Moulton,† George C. Tsokos,† Ken-Ei Sada,* and Jun Wada*

*Department of Nephrology, Rheumatology, Endocrinology, and Metabolism, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700 8558, Japan; and †Division of Rheumatology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800810