Targeting Serine/Threonine Kinase 40 Chemokine Production in Keratinocytes Via and Modulates Inflammatory Cytokine and Microrna

MicroRNA-31 Is Overexpressed in Psoriasis and Modulates Inflammatory Cytokine and Chemokine Production in Keratinocytes via Targeting Serine/Threonine Kinase 40 This information is current as of September 26, 2021. Ning Xu, Florian Meisgen, Lynn M. Butler, Gangwen Han, Xiao-Jing Wang, Cecilia Söderberg-Nauclér, Mona Ståhle, Andor Pivarcsi and Enikö Sonkoly J Immunol published online 10 December 2012 http://www.jimmunol.org/content/early/2012/12/10/jimmun Downloaded from ol.1202695

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 10, 2012, doi:10.4049/jimmunol.1202695 The Journal of Immunology

MicroRNA-31 Is Overexpressed in Psoriasis and Modulates Inﬂammatory Cytokine and Chemokine Production in Keratinocytes via Targeting Serine/Threonine Kinase 40

Ning Xu,* Florian Meisgen,* Lynn M. Butler,† Gangwen Han,‡ Xiao-Jing Wang,‡ Cecilia So¨derberg-Naucle´r,† Mona Sta˚hle,* Andor Pivarcsi,* and Eniko¨ Sonkoly*

Psoriasis is characterized by a specific microRNA expression profile, distinct from that of healthy skin. MiR-31 is one of the most highly overexpressed microRNAs in psoriasis skin; however, its biological role in the disease has not been studied. In this study, we show that miR-31 is markedly overexpressed in psoriasis keratinocytes. Specific inhibition of miR-31 suppressed NF-kB–driven promoter luciferase activity and the basal and TNF-a–induced production of IL-1b, CXCL1/growth-related oncogene-a, CXCL5/

epithelial-derived neutrophil-activating peptide 78, and CXCL8/IL-8 in human primary keratinocytes. Moreover, interference with Downloaded from endogenous miR-31 decreased the ability of keratinocytes to activate endothelial cells and attract leukocytes. By microarray expression profiling, we identified genes regulated by miR-31 in keratinocytes. Among these genes, we identified serine/threonine kinase 40 (STK40), a negative regulator of NF-kB signaling, as a direct target for miR-31. Silencing of STK40 rescued the suppressive effect of miR-31 inhibition on cytokine/chemokine expression, indicating that miR-31 regulates cytokine/chemokine expression via targeting STK40 in keratinocytes. Finally, we demonstrated that TGF-b1, a cytokine highly expressed in psoriasis

epidermis, upregulated miR-31 expression in keratinocytes in vitro and in vivo. Collectively, our findings suggest that overexpres- http://www.jimmunol.org/ sion of miR-31 contributes to skin inflammation in psoriasis lesions by regulating the production of inflammatory mediators and leukocyte chemotaxis to the skin. Our data indicate that inhibition of miR-31 may be a potential therapeutic option in psoriasis. The Journal of Immunology, 2013, 190: 000–000.

soriasis is a common chronic inflammatory skin disease, epidermis (1). There is a close interdependence between kerati- which affects 2–3% of the population. It is a lifelong nocytes and immune cells in psoriatic skin: the cytokines and P disease with spontaneous remissions and exacerbations chemokines secreted by keratinocytes, such as IL-1b,TNF-a, that are severely detrimental to the patients’ quality of life (1). CXCL1/growth-related oncogene-a, CXCL5/epithelial-derived Psoriasis skin lesions are typically characterized by keratinocyte neutrophil-activating peptide 78, and CXCL8/IL-8 activate and by guest on September 26, 2021 hyperproliferation and aberrant differentiation, increased vascu- attract immune cells to migrate into epidermis and dermis; immune larity in the dermis, and infiltration of inflammatory cells, such as cell–derived cytokines, in turn, act on keratinocytes to increase the macrophages, neutrophils, and lymphocytes into the dermis and expression of inflammatory genes, promote keratinocyte proliferation, and impair keratinocyte differentiation (reviewed in Ref. 1). microRNAs (miRNAs) are ∼ 22nt long single-stranded noncoding *Molecular Dermatology Research Group, Unit of Dermatology and Venereology, Department of Medicine, Karolinska Institute, SE-17176 Stockholm, Sweden; RNAs that mediate posttranscriptional silencing by binding with †Cell and Molecular Immunology Group, Department of Medicine, Karolinska In- ‡ partial complementarity to the 39-untranslated region (39-UTR) of stitute, SE-17176 Stockholm, Sweden; and Department of Pathology, University of ∼ Colorado Denver, Aurora, CO 80045 target mRNA (2). miRNAs are estimated to regulate 60% of all protein-coding genes in humans and have been shown to participate Received for publication September 26, 2012. Accepted for publication November 5, 2012. in the regulation of almost every cellular process investigated to date This work was supported by the Swedish Research Council (Vetenskapsra˚det), the (3). We and others have previously identified a distinct miRNA Swedish Society of Medicine (Svenska La¨karesa¨llskapet), the Swedish Psoriasis expression profile in psoriasis skin compared with healthy skin (4– Association (Psoriasisfo¨rbundet), the European Skin Research Foundation, the 8). Several of these deregulated miRNAs have been shown to act on Welander and Finsens Foundation, the Tore Nilssons Foundation, the Lars Hierta Memorial Foundation, the Sigurt and Elsa Golje Memorial Foundation, the Centre of cellular processes crucial for psoriasis. For example, miR-203 (7), Excellence for Research on Inflammation and Cardiovascular Disease, the Karolinska miR-125b (9), miR-424 (4), and miR-99a (6) regulate keratinocyte Institute, and the Stockholm County Council. N.X. was supported by the Swedish Society for Medical Research. A.P. was supported by the LEO Pharma Research proliferation and differentiation, whereas miR-21 suppresses T cell Foundation. E.S. was supported by the Stockholm County Council. apoptosis (10). Although being in the infancy, these studies reveal The datasets presented in this article have been submitted to the National Center for important roles for miRNAs in the biology of psoriasis. Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih. In this study, we identify a function for miR-31 in the context of gov/geo/) under accession number GSE41905. psoriasis. We show that specific inhibition of miR-31, a miRNA Address correspondence and reprint requests to Dr. Ning Xu, Molecular Dermatology overexpressed in psoriasis keratinocytes, suppresses NF-kBsig- Research Group, Centre for Molecular Medicine, L8:02, Department of Medicine, Karolinska Institute, SE-17176 Stockholm, Sweden. E-mail address: [email protected] naling and the production of IL-1b, CXCL1, CXCL5, and CXCL8/ The online version of this article contains supplemental material. IL-8. Serine/threonine kinase 40 (STK40), a negative modulator of Abbreviations used in this article: GSEA, gene set enrichment analysis; K5, keratin 5; NF-kB signaling, was identified as a direct target for miR-31. LNA, locked nucleic acid; miRNA, microRNA; qRT-PCR, quantitative real-time Furthermore, we identify TGF-b1, a cytokine highly expressed in PCR; siRNA, small interfering RNA; STK40, serine/threonine kinase 40; Treg, psoriasis epidermis, as a potent regulator of miR-31 expression in regulatory T cell; 39-UTR, 39-untranslated region; WT, wild-type. keratinocytes in vitro and in vivo. Taken together, our results Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 suggest that suppressing miR-31 in psoriasis skin may alleviate

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202695 2 miR-31 IN PSORIASIS inflammation by interfering with the cross-talk between kerati- 2100 Bioanalyzer and Nanodrop ND-1000. One hundred nanograms of nocytes and immune cells. total RNA was used to prepare cDNA following the Affymetrix 39IVT Express Kit labeling protocol. Standardized array processing procedures recommended by Affymetrix including hybridization, fluidics processing, Materials and Methods and scanning were used. Genes showing at least 1.2-fold regulation, and Patients p , 0.05 was considered to be differentially expressed. Gene set enrichment analysis (GSEA) was performed using public software from Broad Four-millimeter punch biopsies were taken, after informed consent, from Institute (14, 15). The data discussed in this publication have been de- nonlesional (n = 20) and lesional skin (n = 43) of patients with moderate or posited in the National Center for Biotechnology Information’s Gene severe chronic plaque psoriasis and from noninflamed, nonirritated skin of Expression Omnibus (16) and are accessible through GEO Series ac- healthy individuals (n = 35). The psoriasis patients had not received sys- cession number GSE41905 (http://www.ncbi.nlm.nih.gov/geo/query/acc. temic immunosuppressive treatment or psoralen1ultraviolet A/solarium/ cgi?acc=GSE41905). UV for at least 1 mo and topical therapy for at least 2 wk before skin biopsy. ELISA RNA extraction and quantitative real-time PCR The culture medium was collected from transfected keratinocytes at the Total RNA was extracted from tissues and cells using miRNeasy Mini kit indicated time points and stored at 280˚C. The protein levels of CXCL1, (Qiagen). Skin biopsies from human and K5.TGF-b1 mice (11, 12) were CXCL5, and CXCL8/IL-8 were analyzed by ELISA (R&D Systems) fol- homogenized in liquid nitrogen using a Mikro-Dismembrator U (Braun lowing the manufacturer’s instructions. Biotech) prior to RNA extraction. Epidermal cells were isolated from punch skin biopsies by dispase treatment and trypsin digestion as described Leukocyte chemotaxis in Ref. 13. CD45-negative cells were subsequently isolated using MACS separation columns (Miltenyi Biotec), according to the manufacturer’s Human leukocytes were isolated from 0.2% EDTA anticoagulated whole instructions. Quantification of miR-31 by TaqMan Real-Time PCR was blood collected by venipuncture from healthy donors. Erythrocytes were Downloaded from performed as described previously (7). Its expression was normalized removed using dextran sedimentation (1:1 mixture of blood: 6% dextran/0.9% between different samples based on the values of U48 RNA expression in NaCl), followed by one or two rounds of hypotonic lysis using ddH2O. human and snoRNA 251 in mouse. The primary miRNA transcripts were Purified leukocytes were suspended in EpiLife serum-free keratinocyte 5 quantified by TaqMan Pri-miRNA assay (Hs03302684_pri; Applied Bio- growth medium, and 3 3 10 cells were added to the inner chamber of systems) following the manufacturer’s instructions. To quantify mRNAs, a BD Falcon Cell Culture Insert. Leukocytes migrate through a 3-mm 500 ng total RNA was reverse transcribed using the RevertAid First Strand porosity polyethylene terephthalate membrane toward the outer chamber cDNA Synthesis Kit (Fermentas). The mRNAs of IL-1b, CXCL1, CXCL5, containing culture medium from keratinocytes, which were transfected CXCL8/IL-8, STK40, ICAM-1, VCAM-1, and E-selectin were quantified with anti–miR-31 or anti–miR-Ctrl for 72 h. After incubation for 3 h at 37˚C http://www.jimmunol.org/ by TaqMan gene expression assays (Applied Biosystems). Target gene in 5% CO2, the migrating cells in the medium of the outer chamber were expression was normalized based on the expression of the internal positive quantified by CyQUANT GR dye (Life Technologies) staining. control 18S RNA: 59-CGGCTACCACATCCAAGGAA-39 (forward), 59- Endothelial cell activation GCTGGAATTACCGCGGCT-39 (reverse), and 59-FAM-TGCTGGCAC- CAGACTTGCCCTC-TAMRA-39 (probe). HUVECs were isolated as previously described (17) and maintained in Medium 199 (Invitrogen) containing 20% FCS, 28 mg/ml gentamicin, 2.5 In situ hybridization mg/ml amphotericin B, 1 ng/ml epidermal growth factor, and 1 mg/ml In situ hybridization was performed on frozen sections (10 mm in thickness) hydrocortisone (all from Sigma-Aldrich). Second-passage HUVECs were of skin biopsy specimens as described previously (9). Briefly, after incu- treated with keratinocytes culture medium for 4 h and then harvested. bated in acetylation solution (0.06 M HCl, 1.3% trietanolamin, and 0.6% Luciferase reporter assays by guest on September 26, 2021 acetic anhydride in diethyl pyrocarbonate-treated water) for 10 min at room temperature, sections were incubated in permeabilization buffer (1% Renilla luciferase reporter plasmids containing synthetic sequence repeats of Triton X-100) for 30 min at room temperature, washed, and prehybri- that are fully complementary to miR-31 (miR-31 sensor) or 39-UTR of the dized for 1 h at 50˚C. Hybridization with digoxigenin-labeled miRCURY STK40 gene-cloned downstream of the reporter gene and empty luciferase locked nucleic acid (LNA) probes (Exiqon) was performed over night at vector were obtained from SwitchGear Genomics. The mutations were gen- 50˚C. Slides were then washed four times with 23 SSC buffer followed by erated with the predicted target site of STK40 39-UTR using the QuickChange one time with 0.13 SSC buffer at 67˚C. The probe binding was detected XL site-directed mutagenesis kit (Stratagene), according to the manu- by incubating the sections with alkaline phophatase–conjugated sheep anti- facturer’s instructions. NF-kB reporter plasmid pGL4.32[luc2P/NF-kB- digoxigenin Fab fragments (1:2500 [Roche]) for 1 h at room temperature. RE/Hygro] Vector was obtained from Promega, which contains five copies Sections were visualized by adding BM purple alkaline phophatase sub- of an NF-kB response element that drives transcription of the luciferase re- strate (Roche), according to the manufacturer’s instructions. porter gene luc2P (Photinus pyralis). Human primary keratinocytes growing in 24-well plates were cotransfected with the luciferase reporters (25 ng/ml) Cells culture and treatments together with 10 nM anti–miR-31 or anti–miR-Ctrl using FuGENE HD Human adult epidermal keratinocytes used in miR-31 function study were transfection reagent (Promega). Luciferase activity was analyzed 24 h purchased from Cascade Biologics and cultured in EpiLife serum-free posttransfection using LightSwitch Luciferase Assay reagent (SwitchGear) keratinocyte growth medium including Human Keratinocyte Growth or Dual-Luciferase Reporter Assay System (Promega). Supplement at a final Ca2+ concentration of 0.06 mM (Cascade Biologics) Immunohistochemistry at 37˚C in 5% CO2. To study the biological effects of miR-31 on keratinocytes, third passage keratinocytes at 30–50% confluence were trans- STK40 protein expression was analyzed in both frozen and formalin-fixed fected with 10 nM miRIDIAN miR-31 hairpin inhibitor or miRNA hairpin paraffin embedded skin sections (7 mm in thickness) using rabbit anti- inhibitor negative control number 1 (ThermoFisher Scientific), 30 nM human STK40 Ab (1:200) (Sigma-Aldrich) and the avidin-biotin-peroxidase Silencer select predesigned small interfering RNA (siRNA) for STK40 complex staining system (Vector Laboratories) following the manu- (siRNA ID: s38327), or siRNA-negative control number 1 (Ambion) using facturer’s instructions. Replacement of the primary Ab with rabbit Ig Lipofectamine 2000 (Invitrogen). Keratinocytes were treated with TNF-a fraction (DakoCytomation) in the stainingprocesswasusedasnegative (50 ng/ml; Immunotools) or TGF-b1 (3 ng/ml; R&D Systems) at the in- control (Supplemental Fig. 3). dicated time points. Three-dimensional epidermal equivalents were pur- Statistics chased from MatTek. TGF-b1 (10 ng/ml; R&D systems) was added into the culture medium. Seventy-two hours later, the total RNA was extracted Statistical significance for experiments was determined by Mann–Whitney with miRNeasy Mini kit (Qiagen). U test or Student t test. Correlation between the expression of different genes in the same samples was made using Pearson’s correlation test on log- Gene expression microarray transformed data. p , 0.05 was considered to be statistically significant. Expression profiling of primary human keratinocytes transfected with 10 nM Study approval anti–miR-31 or anti–miR-Ctrl for 48 h (in triplicates) was performed using Affymetrix Genechip system at the Microarray core facility of Karolinska The clinical materials were taken after patients’ consent and the study was Institute. In brief, total RNA was extracted using the miRNeasy Mini Kit approved by the Stockholm Regional Ethics Committee and conducted (Qiagen), and RNA quality and quantity were determined using Agilent according to the Declaration of Helsinki’s principles. All procedures in- The Journal of Immunology 3 volving mice were approved by Institutional Animal Care and Use Com- expression of mature miR-31 in these skin biopsies, suggesting that mittee at Colorado School of Public Health (University of Colorado the upregulation of miR-31 in psoriasis skin may occur at the Denver, Aurora, CO). transcriptional level, rather than during processing of the primary transcript. Results To investigate which cell type(s) in the skin expresses miR-31, miR-31 is upregulated in psoriasis keratinocytes we surveyed miR-31 expression in a panel of isolated primary Earlier expression profiling data from our and other groups showed human cell types. qRT-PCR results showed that miR-31 was mainly that miR-31 is one of the miRNAs overexpressed in psoriasis skin expressed by keratinocytes, fibroblasts and melanocytes isolated (5, 7, 8). To confirm these data, we measured mature miR-31 from the skin (Supplemental Fig. 1). To identify which cell type(s) expression in skin biopsies from healthy donors (n = 14), nonle- in the skin are primarily responsible for the increased expression sional (n = 10), and lesional skin (n = 25) from psoriasis patients of miR-31 in psoriasis, we performed in situ hybridization on skin using quantitative real-time PCR (qRT-PCR) (Fig. 1A). miR-31 sections from healthy individuals (n = 11), nonlesional (n = 6), and expression was found to be dramatically increased in psoriasis lesional skin (n = 11) from psoriasis patients using miR-31–specific lesional skin compared with healthy skin (33-fold change; p =4.13 LNA-modified probes (Fig. 1C). The expression of miR-31 was 2 10 7) and compared with psoriasis nonlesional skin (14-fold change; low in healthy skin and restricted to the basal cell layer of the 2 p =8.13 10 7). In addition, we found that the expression of pri- epidermis. In contrast, miR-31 expression was higher in psoriasis mary miR-31 transcript (pri-miR-31), from which the mature miR- nonlesional skin and appeared at both basal and suprabasal cell 31 is processed, was also upregulated in psoriasis lesional skin (n = layers. In lesional skin from the same psoriasis patients, miR-31

9) compared with healthy skin (n = 7) (Fig. 1B). The expression of expression was further upregulated and mainly detected in the Downloaded from pri-miR-31 correlated positively (R = 0.7659; p = 0.0014) with the suprabasal layers. http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. The expression of miR-31 in healthy and psoriasis skin. (A) MiR-31 expression was analyzed in healthy (n = 14), psoriasis nonlesional (n = 10), and lesional skin samples (n = 25) using qRT-PCR. Results for individual patients and mean are shown. (B) Expression of primary miR-31 transcript (pri-miR-31) was analyzed in healthy (n = 7) and psoriasis lesional skin (n =9)byqRT-PCR.(C) In situ hybridization was performed on healthy skin (upper panel, n = 11), psoriasis nonlesional skin (middle panel, n = 6), and lesional skin sections (lower panel, n = 11) using miR-31–speciﬁc LNA probe (left panel) or scrambled probe (right panel). Blue-purple color indicates miR-31 expression. Scale bar, 50 mm. (D) miR-31 expression was analyzed on RNA from epidermal keratinocytes (CD45neg) isolated from healthy skin (n = 10), psoriasis nonlesional (n = 4), and lesional skin (n = 7) using qRT- PCR. **p , 0.01, ***p , 0.001; Mann–Whitney U test. 4 miR-31 IN PSORIASIS

To quantify the change of miR-31 level in epidermal cells, we the expression of the host gene of miR-31, encoding a long measured miR-31 expression in sorted epidermal CD45 (common noncoding RNA, LOC554202 (18), was not affected by miR-31 leukocyte Ag)-negative cells from healthy (n =10),psoriasis inhibition, neither that of the adjacent IFN ε (IFNE1) gene nonlesional (n = 4), and lesional skin (n = 7) (Fig. 1D). In line with (Supplemental Fig. 2C). the results of in situ hybridization, qRT-PCR analysis revealed Interestingly, we found that several mediators of key importance a 13-fold (p = 0.0001) higher miR-31 expression in CD45neg cells in psoriasis pathogenesis, such as IL-1b,CXCL1,CXCL5,and sorted from psoriasis lesional skin compared with those obtained CXCL8/IL-8, were downregulated by anti–miR-31 in keratinocytes from healthy skin and a 9-fold (p = 0.0061) increase compared (Supplemental Table I), and this was validated by qRT-PCR on with psoriasis nonlesional skin. Collectively, our results demon- keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl for strate that miR-31 is upregulated in keratinocytes in psoriasis skin 24, 48, 72, and 96 h (Fig. 2A). Consistently, the amount of these lesions. chemokines secreted into the culture medium was decreased by anti–miR-31, shown by ELISA (Fig. 2B). Notably, IL-1b was not miR-31 regulates the production of inflammatory mediators in detectable in the culture medium, because the cultured human ker- keratinocytes atinocytes produce but do not process the IL-1b precursor into its To study the biological role of miR-31 in keratinocytes, we biologically active form because of the lack of IL-1 convertase (19). transfected miR-31 hairpin inhibitor (anti–miR-31) into primary Next, we aimed to test whether inhibition of miR-31 can effi- human keratinocytes to inhibit endogenous miR-31. Inhibition of ciently suppress the production of IL-1b, CXCL1, CXCL5, and miR-31 was confirmed by qRT-PCR analysis of miR-31 expres- CXCL8/IL-8 under inflammatory conditions. To this end, we mea- sion and by luciferase assay using a synthetic miR-31-target as sured the effect of miR-31 inhibition on mRNA and protein levels Downloaded from sensor (Supplemental Fig. 2A, 2B). We performed a global tran- of these cytokine/chemokines in keratinocytes treated with the scriptome analysis of keratinocytes upon suppression of endoge- proinflammatory cytokine TNF-a (Fig. 2C, 2D). Consistent with nous miR-31 using Affymetrix arrays, which identified 234 genes earlier reports, TNF-a induced the expression of IL-1b, CXCL1, that were significantly changed (Supplemental Table I). Of note, CXCL5, and CXCL8/IL-8. Inhibition of endogenous miR-31 http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Inhibition of miR-31 suppresses the production of cytokines/chemokines by keratinocytes. The expression and secretion of CXCL1, CXCL8, CXCL5, and IL-1b were analyzed in keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl for 24–96 h by qRT-PCR (A) and ELISA (B) or analyzed in keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl for 48 h and then treated with TNF-a for 6 h by qRT-PCR (C) and ELISA (D). *p , 0.05, **p , 0.01, ***p , 0.001; Student t test. The Journal of Immunology 5 suppressed TNF-a–induced cytokine/chemokine production both miR-31 regulates the NF-kB pathway in keratinocytes at mRNA (Fig. 2C) and protein level (Fig. 2D). The NF-kB pathway is one of the signaling pathways commonly miR-31 regulates the endothelial cell–activating and regulating the expression of IL-1b, CXCL1, CXCL5, and CXCL8/ leukocyte-attracting capacity of keratinocytes IL-8. We therefore investigated whether other target genes of the NF-kB pathway are also regulated by miR-31 in keratinocytes. To Because CXCL1, CXCL5, and CXCL8 have the ability to activate answer this question, we evaluated the enrichment for known endothelial cells and recruit polymorphonuclear leukocytes into target genes of the NF-kB pathway (summarized on the Web site: inflamed tissues (20), we examined whether the capacity of kera- http://bioinfo.lifl.fr/NF-KB/) in our microarray data of keratino- tinocytes to attract leukocytes was affected by miR-31. The first cytes transfected with anti–miR-31 or anti–miR-Ctrl (Fig. 4A). step of recruitment of leukocytes to the skin is the attachment of Results of the GSEA (14, 15) revealed that NF-kB pathway target circulating cells to vascular endothelial cells, which is mediated genes were significantly enriched among the genes downregulated by cell adhesion molecules, such as ICAM-1, VCAM-1, and E- by anti–miR-31, and a negative enrichment score curve was gen- selectin (21). It has been shown that the expression of these cell erated by GSEA (p , 0.001). These data indicated that inhibition adhesion molecules is increased on dermal vessels of psoriatic skin of miR-31 might affect the activity of the NF-kB signal trans- (22–25). To test the effect of miR-31 on endothelial cell-activating duction pathway. capacity of keratinocytes, we measured the expressions of ICAM- To test this hypothesis, we determined the effect of miR-31 1, VCAM-1, and E-selectin in primary HUVECs incubated with inhibitor on NF-kB–dependent promoter luciferase reporter gene culture medium from miR-31 inhibitor–treated keratinocytes. qRT- activity in human primary keratinocytes (Fig. 4B). Results of lu- PCR results demonstrated that inhibition of endogenous miR-31 in Downloaded from ciferase assays demonstrated that inhibition of miR-31 suppressed keratinocytes decreased their capacity to induce the expression of both the basal and TNF-a–induced NF-kB–dependent promoter these cell adhesion molecules in endothelial cells (Fig. 3A). luciferase activity, indicating that miR-31 regulates the activity of Next, we considered whether altered expression of miR-31 in NF-kB pathway in keratinocytes. keratinocytes could affect their capacity to attract leukocytes. To address this question, we performed migration assays with pe- miR-31 targets STK40, a negative regulator of the NF-kB

ripheral blood leukocytes using conditioned supernatant from miR- pathway http://www.jimmunol.org/ 31 inhibitor–treated keratinocytes (Fig. 3B). The results revealed Next, we aimed to identify the molecular mechanism by which that supernatant from keratinocytes with inhibited miR-31 ex- miR-31 modulates the NF-kB pathway. miRNAs exert biological pression attracted less leukocytes compared with medium from functions by regulating their target genes. To predict target genes control treated cells. Taken together, these data show that inhibi- of miR-31, we used three different public algorithms, TargetScan tion of miR-31 in keratinocytes results in decreased endothelial cell activation and leukocyte chemotaxis. by guest on September 26, 2021

FIGURE 4. miR-31 regulates the NF-kB pathway in keratinocytes. (A) Microarray analysis was performed in independent biological triplicates for primary human keratinocytes transfected with either anti–miR-31 or anti– FIGURE 3. miR-31 regulates the endothelial cell–activating and leuko- miR-Ctrl. Genes represented in the profile data set were ranked by fold cyte-attracting capacity of keratinocytes. (A) HUVECs were treated with change (anti–miR-31/anti–miR-Ctrl). GSEA evaluated enrichment within the medium from cultured keratinocytes transfected with anti–miR-31 or anti– profile data set for the reported target genes of NF-kB signaling pathway. miR-Ctrl, and the expression of endothelial cell activation markers (ICAM-1, Vertical bars along the x axis of the GSEA plot denote the positions of NF-kB VCAM-1, and E-selectin) was analyzed by qRT-PCR. (B) Human leukocytes target genes within the ranked list. NES, normalized enrichment score. (B) chemotaxis toward the unused medium (med) or the medium from cultured Keratinocytes were transfected with NF-kB luciferase reporter plasmid keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl for 54 h and then 48 h after the transfection of anti–miR-31 or anti–miR-Ctrl. Eighteen hours treated with or not with TNF-a for 18 h. The migrating cells were quantified later, the cells were treated with or not with TNF-a for 5 h, and luciferase by CyQUANT GR dye staining. *p , 0.05, **p , 0.01; Student t test. activity was measured. **p , 0.01, ***p , 0.001; Student t test. 6 miR-31 IN PSORIASIS

(26), miRanda (27), and PicTar (28). Within the ranked gene list The gene showing highest up-regulation upon miR-31 inhibition from microarray analysis of keratinocytes transfected with either was STK40 (Supplemental Table I), which had been identified as anti–miR-31 or anti–miR-Ctrl, the enrichment of these predicted a suppressor of NF-kB–mediated transcription (29). The regulation miR-31 targets was evaluated with GSEA (Fig. 5A). Most pre- of STK40 expression by miR-31 has been previously observed in dicted target genes were found to be upregulated upon inhibition ovarian cancer cells (30). STK40 contains two conserved potential of miR-31, and GSEA generated positive enrichment score curves, binding sites for miR-31 in its 39-UTR (Fig. 5B), and the dere- irrespective of which prediction algorithm was used, indicating pression of STK40 transcript upon inhibition of miR-31 in human that the microarray analysis was specific and sensitive for detecting primary keratinocytes was confirmed by qRT-PCR analysis (Fig. direct target genes for miR-31. 5C). To determine whether STK40 is a bona fide target of miR-31, Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. MiR-31 targets STK40, a negative regulator of the NF-kB signaling pathway. (A) GSEA evaluated the enrichment of predicted miR-31 targets, as determined by the given algorithms (TargetScan, miRanda, and PicTar), within the microarray profiling data set of keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl. (B) Nucleotide resolution of the predicted miR-31 binding site in 39-UTR of STK40 mRNA: seed sequence (green letters); and mutated miR-31 binding sites (red letters). (C) The miR-31 mediated regulation of STK40 was verified by qRT-PCR in keratinocytes transfected with anti–miR-31 or anti–miR-Ctrl for 24–96 h. (D) Keratinocytes were transfected with luciferase reporter plasmid containing WT or mutant (Mut) STK40 39- UTR or empty vector (Vector) together with anti–miR-31 or anti–miR-Ctrl. **p , 0.01, ***p , 0.001; Student t test. (E) Human skin from the same donors was used for the detection of miR-31 by in situ hybridization (left panel) and STK40 expression by immunohistochemistry (right panel). Red-brown color indicates STK40 expression. Scale bar, 50mm. The Journal of Immunology 7 we performed 39-UTR luciferase reporter assays with luciferase signal of STK40 was detected also in the basal and spinous layers reporter gene constructs containing the full-length 39-UTR of of the epidermis. In psoriasis nonlesional skin, the strong signal of STK40 mRNA in human primary keratinocytes (Fig. 5D). The STK40 in granular layers was similar as in healthy skin, whereas wild-type (WT) STK40 39-UTR luciferase activity was increased the expression of STK40 in basal and spinous layers was slightly 8-fold (p = 0.004) by anti–miR-31 in comparison with anti–miR- decreased. In psoriasis lesional skin, the STK40 signal was absent Ctrl. Mutation of one of the predicted target sites (Mut1) markedly in the lower spinous layers but strongly expressed in the upper decreased luciferase activity upon miR-31 inhibition, although the spinous layers, which is opposite to the pattern of miR-31 ex- luciferase activity was still affected by anti–miR-31. Mutation of pression in psoriasis lesional skin. No STK40-positive leukocytes target site #2 (Mut 2) or both target sites (Mut1+2), however, were observed in the dermis. The reciprocal expression of miR-31 completely abolished the effect of miR-31 inhibition on reporter and STK40 further supports that STK40 is regulated by miR-31 in gene expression. These data demonstrate that miR-31 directly psoriasis skin. regulates STK40 expression through binding to the two predicted target sites in its 39-UTR. miR-31 regulates cytokine/chemokine expression via targeting Next, we analyzed the expression of STK40 in healthy (n = 11), STK40 psoriasis nonlesional (n = 9), and lesional skin (n = 11) by im- To determine whether the observed effects of miR-31 on kerati- munohistochemistry. miR-31 expression in the same donors was nocyte cytokine/chemokine production are, at least partially, me- detected by in situ hybridization (Fig. 5E, Supplemental Fig. 3). diated through STK40, we analyzed the effects of silencing of STK40 expression was mainly detected in the cytoplasm and the STK40 expression by siRNA in keratinocytes (Fig. 6). STK40 plasma membrane of epidermal keratinocytes. In healthy skin, expression in keratinocytes was significantly increased by anti– Downloaded from STK40 was strongly expressed in the granular layers and a weaker miR-31, which was prevented by simultaneous transfection with http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. MiR-31 regulates cytokine/chemokine expression via targeting STK40. Kera- tinocytes were cotransfected with siRNA and anti-miR for 48 h and then treated with TNF-a for 6 h. STK40 expression was detected by qRT-PCR (A). The expression and secretion of IL-1b, CXCL1, CXCL5, and CXCL8/IL-8 were analyzed by qRT-PCR (B) and ELISA (C). (D) Keratinocytes were transfected with an NF-kB reporter plasmid 48 h after the transfection of anti-miR and siRNA. Eighteen hours later, the cells were treated with TNF-a for 5 h, and the luciferase activity was measured. *p , 0.05, **p , 0.01, ***p , 0.001; Student t test. 8 miR-31 IN PSORIASIS

STK40 siRNAs (Fig. 6A). In line with our previous data, the basal a multilayered artificial epidermis built with human primary ker- and TNF-a–induced expression of IL-1b, CXCL1, CXCL5, and atinocytes on an air-liquid interphase (Fig. 7A). CXCL8/IL-8 was decreased by anti–miR-31 in keratinocytes. To investigate whether miR-31 is regulated by TGF-b1invivo, However, silencing of STK40 rescued the suppressive effect of we measured miR-31 expression in the skin of K5.TGF-b1 trans- anti–miR-31 on IL-1b, CXCL1, CXCL5, CXCL8/IL-8, which was genic mice, which overexpress this cytokine in an epidermis- shown both at mRNA level by qRT-PCR (Fig. 6B), and at protein specific manner and develop a psoriasis-like skin disorder (11, 12). level by ELISA (Fig. 6C). These data indicate that the effect of qRT-PCR results showed that the expression of miR-31 was in- miR-31 on the production of inflammatory mediators requires creased 35-fold (p = 0.01) in the skin of K5.TGF-b1 mice com- STK40. In line with these results, knockdown of STK40 expres- pared with WT littermates (Fig. 7B). Treatment with etanercept, sion rescued the suppressive effect of anti–miR-31 on NF-kB– which blocks TNF-a activity and is one of the current therapies dependent promoter luciferase activty (Fig. 6D). These data in- for psoriasis, efficiently alleviated the psoriasis phenotype of K5. dicate that the regulation of keratinocyte cytokine/chemokine TGF-b1 mice (11) and resulted in a 4-fold (p = 0.005) reduction production by miR-31 is, at least partially, mediated through of miR-31 expression compared with the K5.TGF-b1 mice treated STK40. with normal saline (Fig. 7B). In line with this, in situ hybridization showed that miR-31 was upregulated in the hyperplastic epidermis b miR-31 is upregulated by TGF- 1 in keratinocytes both in vitro of K5.TGF-b1 mice compared with WT littermates, whereas and in vivo treatment with etanercept reduced epidermal hyperplasia and To understand the mechanism of miR-31 overexpression in pso- miR-31 expression (Fig. 7C). Taken together, our results suggest riasis keratinocytes, we systematically surveyed the effect of that increased levels of TGF-b1 in psoriatic skin may contribute to Downloaded from cytokines, growth factors, and cell differentiation on miR-31 ex- the upregulation of miR-31 in psoriasis. pression in keratinocytes. Cells were treated with cytokines rele- vant for psoriasis pathology (TNF-a, IL-22, TGF-b1, IL-6, IL-20, Discussion IFN-g, and GM-CSF), growth factors (epidermal growth factor In this study, we show that miR-31 is upregulated in psoriasis and and keratinocyte growth factor) and keratinocyte differentiation- identify it as a novel regulator of NF-kB activity. We demonstrate driving factors (1.5 mM CaCl2,12-O-tetradecanoylphorbol-13- that inhibition of endogenous miR-31 in keratinocytes suppresses http://www.jimmunol.org/ acetate, and cell confluence) andmiR-31expressionwasana- the production of inflammatory mediators and the capability of lyzed by qRT-PCR. miR-31 was not significantly changed by keratinocytes to activate endothelial cells and to attract leuko- these factors (data not shown) with the exception of TGF-b1, cytes. Moreover, we identify STK40, a negative regulator of the a cytokine previously reported to be upregulated in the epidermis NF-kB pathway, as a novel target for miR-31 in keratinocytes and and serum of psoriasis patients (31). TGF-b1 significantly induced demonstrate that silencing of STK40 can rescue the effect of miR- miR-31 expression in keratinocyte cultures (Fig. 7A). Further- 31 on inflammatory mediators. Finally, we identify TGF-b1as more, upregulation of miR-31 by TGF-b1 was also observed in a regulator of miR-31 expression in vitro and in vivo. Our results three-dimensional reconstructed epidermal equivalents, which is thus suggest a model in which TGF-b1 induces miR-31 in pso- by guest on September 26, 2021

FIGURE 7. miR-31 is upregulated by TGF-b1 in keratinocytes in vitro and in vivo. (A) miR-31 expression was analyzed in keratinocytes in monolayer cell culture (two-dimensional) and three-dimensional epidermal equivalents treated with TGF-b1 (3 or 10 ng/ml, respectively) for 24 or 72 h. (B) miR-31 expression was analyzed in skins of WT mice (n = 3) and K5.TGF-b1 transgenic mice treated with normal saline (control, n = 3) or etanercept (n =3)by qRT-PCR. *p , 0.05, **p , 0.01, ***p , 0.001; Student t test. (C) In situ hybridization was performed on skin from WT mice (left panel) and K5.TGF-b1 transgenic mice treated with normal saline (middle panel) or Etanercept (right panel) using an miR-31–specific LNA probe (upper panel) or scrambled probe (middle panel). Blue-purple color indicates miR-31 expression. H&E staining (bottom panel) was performed for the same biopsies. Scale bar, 50 mm. The Journal of Immunology 9 riasis keratinocytes that leads to increased NF-kB activity partially (reviewed in Ref. 1). In accordance with known functions of through suppression of STK40. In turn, inflammatory cytokine/ CXCL1, CXCL5, and CXCL8/IL-8, inhibition of endogenous chemokines are induced, which contributes to endothelial cell miR-31 in keratinocytes impaired the capability of conditioned activation, leukocyte attraction and clinically, skin inflammation supernatant to activate endothelial cells or to attract leukocytes, (Fig. 8). suggesting that miR-31 may be involved in the cross-talk between miR-31 is widely expressed and plays diverse roles in different keratinocytes and immune cells in psoriasis. Because miR-31 tissue and cell types. This miRNA has been shown to positively expression is not confined to keratinocytes, its deregulation in regulate corneal epithelial glycogen metabolism (32), to inhibit other cell types may also contribute to psoriatic skin inflammation. fibrogenesis and pulmonary fibrosis (33), to regulate lymphatic It was previously shown that low levels of miR-31 in human vascular lineage–specific differentiation (34), and to reduce TNF- natural regulatory T cells (Tregs) contributes to high expression of induced expression of E-selectin on human endothelial cells as FOXP3, which is a master transcription factor crucial for Treg a feedback control of inflammation (35). In pathological con- function and identified as the direct target of miR-31 (40). Pso- ditions, miR-31 has been mainly studied in cancer and was riasis has been associated with impaired immune suppressive ca- identified as a critical and pleiotropical regulator of tumor me- pacity of Tregs (41) and their enhanced propensity to differentiate tastasis and growth (reviewed in Ref. 36). In mouse skin, miR-31 into inflammatory IL-17A–producing cells (42). Thus, it would be has been shown to control hair cycle–associated gene expression interesting to further investigate the effect of miR-31 on the programs (37). However, its role in skin diseases and in particular, function and differentiation of Tregs in the context of psoriasis. psoriasis, was unknown. We found that inhibition of miR-31 in Our findings show that miR-31 regulates the activity of NF-kB keratinocytes decrease the production of IL-1b, CXCL1, CXCL5, pathway in keratinocytes, which is a key signaling pathway reg- Downloaded from and CXCL8, suggesting that miR-31 has an immunomodulatory ulating cytokine/chemokine expression (43) and which is activated function in keratinocytes. Keratinocytes in psoriasis lesions in psoriasis lesional skin (1, 44, 45). Importantly, activation of NF- overexpress several proinflammatory cytokines and chemokines, kB in both keratinocytes and lymphocytes is required to develop which contribute to the maintenance of inflammation in psoriasis the psoriasis-like phenotype in transgenic mice, indicating the skin lesions and represent a crucial element in psoriasis patho- importance of this pathway in psoriasis (46). In this study, we

genesis (1). IL-1b is a proinflammatory cytokine, which is es- demonstrate that in keratinocytes miR-31 directly targets STK40, http://www.jimmunol.org/ sential for Th17 differentiation (38) and potentiates immune cell which was previously shown to inhibit TNF-induced NF-kB ac- activation by regulating T cell–targeting chemokine production in tivation (29). Interestingly, the regulation of STK40 expression by keratinocytes (39). Keratinocyte-derived CXCL1, CXCL5, and miR-31 was also observed in ovarian cancer cells (30). In the skin, CXCL8/IL-8 can potently activate endothelial cells and are che- STK40 showed a reciprocal expression pattern with miR-31, moattractants for multiple subsets of leukocytes, especially neu- which further supports a functional miRNA:mRNA interaction trophil granulocytes, by binding to the cognate receptors CXCR1 in vivo. Derepression of STK40 through miR-31 may, at least and CXCR2 (20). These chemokines play prominent roles in re- partially, explain the reduced activity of NF-kB signaling by anti– cruitment and retention of inflammatory cells into psoriasis skin, miR-31 in keratinocytes. miR-31 was recently shown to down- as well as regulation of the formation of new blood vessels regulate noncanonical pathway of NF-kB activation via targeting by guest on September 26, 2021 NF-kB–inducing kinase in leukemic T cells (47). On the contrary, the expression of NF-kB–inducing kinase was not significantly altered by miR-31 inhibition in human primary keratinocytes, as shown in our microarray profiling data. The seemingly paradoxi- cal findings with regard to the regulatory role of miR-31 in the NF-kB signaling pathway indicate that the biological function of miR-31 is highly cell-type dependent. By a systematic screen using cytokines, growth factors, and factors modulating cell differentiation, we identified TGF-b1as a potent regulator of miR-31 expression in primary keratinocytes, in reconstructed human epidermal equivalents, and in a transgenic mouse model. TGF-b1 is a cytokine with increased levels in the epidermis and serum of psoriasis patients (9, 29, 30), and its in- volvement in psoriasis pathogenesis is supported by the finding that mice overexpressing TGF-b1 driven by a keratin 5 (K5) promoter in keratinocytes (K5.TGF-b1), exhibit a psoriasis-like phenotype (11, 12). The infiltration of inflammatory cells observed in K5.TGF-b1 mice may partially be due to the upregulation of miR-31 by TGF-b1 in keratinocytes, leading to increased production of chemoattractants. Importantly, TGF-b1, along with IL-1b, is critical for the differentiation of Th17 cells (38), which play essential pathogenic role in psoriasis. TGF-b1–induced miR- FIGURE 8. Proposed mechanism by which miR-31 modulates IL-1b, 31 can lead to increased IL-1b levels and thus can contribute to CXCL1, CXCL5, and CXCL8/IL-8 production by keratinocytes in psori- the amplification of Th17-type inflammation in psoriasis. Notably, asis skin. TGF-b1, which is highly expressed in psoriatic skin, upregulates we found that in keratinocytes miR-31 was not induced by TNF-a, the expression of miR-31 in keratinocytes. miR-31 directly suppresses STK40, a suppressor of NF-kB signaling activation induced by TNF-a. although this cytokine has been shown to upregulate miR-31 ex- The activation of NF-kB signaling contributes to the elevated expression pression in endothelial cells (27), further underlining that miR-31 and secretion of IL-1b, CXCL1, CXCL5, and CXCL8/IL-8, which pro- is regulated in a cell-type dependent manner. mote vascular endothelial cell activation and attract leukocytes via che- Taken together, we show that miR-31 is overexpressed in pso- motaxis into the skin. riasis keratinocytes and identify STK40, a negative regulator of 10 miR-31 IN PSORIASIS

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