Role of Periostin and Interleukin-4 in Recurrence of Pterygia

Cornea Role of Periostin and Interleukin-4 in Recurrence of Pterygia

Chuan-Hui Kuo,1 Dai Miyazaki,1 Keiko Yakura,1 Kaoru Araki-Sasaki,2 and Yoshitsugu Inoue1

PURPOSE. To identify the candidate genes for pterygia recur- mitomycin treatment, radiation, and amniotic membrane trans- rence from a pterygia transcriptome and to analyze their tran- plantation, recurrences still occur.1 scriptional regulation and functional relationships. We have determined the minimum gene set that would ETHODS accurately differentiate primary from recurrent pterygia using a M . Transcriptional networks for pterygia recurrence 2 were constructed using network analysis that was applied to mathematical classification algorithm. We found that only 184 genes that showed a significant twofold change in the three genes—periostin, tissue inhibitor of metalloproteinases-2 (TIMP-2), and l-3-phosphoserine phosphatase homolog whole genome. Of the identified recurrence-related candidate 2 genes in the major networks, periostin and IL-4 were analyzed (PSPHL)—differentiate primary from recurrent pterygia. We have also reported on the numerous transcripts that are for transcriptional relationships using pterygia-derived fibro- 2 blasts. Immunohistochemical analysis was used to study ptery- related to pterygia recurrence. However, the functional in- gia tissue. Effector candidate molecule for recurrence periostin volvement in the genome or their significance in recurrence was analyzed for cell adhesive function. remains speculative. We have used knowledge-based analysis to understand the global interactions of the genes in the con- RESULTS. The pterygia transcriptome was divided into four text of their function and regulation. To simplify the complex major biological networks with high significance scores (P Ͻ Ϫ17 biological phenomena played by genes, network analysis has 10 ). The classifier with the highest accuracy using the been reported to be a very efficient method.3 We have applied support vector machine algorithm was periostin, which was the Ingenuity Pathways Knowledge Base (Ingenuity Systems, successfully linked to the network of cell cycle, connective Redwood City, CA, http://www.ingenuity.com), a biological tissue development and function, and cell morphology. Analy- network database created from known protein-protein interac- ses using pterygia-derived fibroblasts showed that periostin tions and the activation of signaling pathways, and found that was required for cell adhesion that was mediated by a pre- the complex phenomena of pterygia recurrences can be simply sumed pterygia-related extracellular matrix protein, fibronec- summarized into four highly significant molecular networks. In tin. Periostin was found to be transcriptionally induced by IL-4. this analyses, periostin and TIMP-2, which we reported as The IL-4-stimulated periostin induction was suppressed by significant classifier genes in the support vector machine MAP kinase/ERK kinase 1 inhibitor, indicating an involvement (SVM)-based classification algorithm,2 were statistically linked of the MAP kinase pathway. Pathologically, IL-4 was transcrip- to the generated networks. Previously, TIMP-2 was shown to tionally elevated in recurrent pterygia tissue and was localized be involved in tissue remodeling processes of pterygia.1 Al- to perivascular tissues and endothelial cells in the stroma of the though periostin showed the highest classification accuracies subconjunctiva of pterygia. of primary and recurrent pterygia,2 its functional characteris- CONCLUSIONS. Periostin is induced by IL-4 and is involved in the tics are not yet well understood, and its involvement in the fibronectin-mediated pterygia fibroblast adhesion. These find- pathogenesis of pterygia has not been reported. ings indicate that periostin probably promotes the recurrence Thus, the purpose of this study was to determine the mo- of pterygia. (Invest Ophthalmol Vis Sci. 2010;51:139–143) lecular function of periostin in pterygia pathology and the DOI:10.1167/iovs.09-4022 causative transcriptional event for periostin induction based on the generated networks. We shall show the function of peri- pterygium is an excessive fibrovascular proliferation of ostin in the recurrence of pterygia and relate it to the role of Adegenerated bulbar conjunctival tissue on the ocular sur- IL-4 as its inductive signal. face. At present, surgery is the only method for treating pterygia, and recurrences are a major complication of the surgical treatment of pterygia. Although numerous approaches have been attempted to reduce recurrences, such as adjunctive MATERIALS AND METHODS Patient Backgrounds and Specimen Collection

From the 1Division of Ophthalmology and Visual Science, Tottori Pterygia heads were collected from 21 eyes of 21 Asian patients University Faculty of Medicine, Tottori, Japan; and 2Ideta Eye Hospital, undergoing pterygium excision with conjunctival autografting at the Kumamoto, Japan. Tottori University Hospital (Yonago, Japan) and the Ideta Eye Hospital Supported by Grant-in-Aid for Scientiﬁc Research (C) 21592258 (Kumamoto, Japan). Of these, 11 eyes of 11 patients showed primary from the Ministry of Education, Culture, Sports, Science and Technol- pterygia and 10 eyes of 10 patients showed recurrent pterygia. Patients ogy. were 11 men and 10 women whose mean age was 68 Ϯ 2 years (range, Submitted for publication May 21, 2009; revised July 1, 2009; 52–85 years). Patients with primary cicatricial ocular surface disease, accepted July 21, 2009. history of ocular allergic symptoms, and inﬂammatory ocular surface Disclosure: C.-H. Kuo, None; D. Miyazaki, None; K. Yakura, None; K. Araki-Sasaki, None; Y. Inoue, None disease of unknown etiology were excluded. The study protocol con- Corresponding author: Dai Miyazaki, Division of Ophthalmology formed to the tenets of the Declaration of Helsinki, and the procedures and Visual Science, Tottori University Faculty of Medicine, 36-1 Nishi- used were approved by the Tottori University Ethics Committee. cho, Yonago Tottori 683-8504, Japan; [email protected]. Signed informed consent was obtained from all patients.

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Transcriptional Analysis Molecular Probes Inc., Eugene, OR) according to the manufacturer’s protocol. Briefly, the solution (CyQUANT GR; Molecular Probes Inc.) Pterygia head tissues collected from primary and recurrence cases was added to each well, and the fluorescence of the samples was were transcriptionally analyzed using an oligo microarray (AceGene, measured at 520 nm with excitation at 480 nm fluorescence micro- Hitachi, Japan) corresponding to 30,336 human genes.2 Genes that plate reader (FL600; BioTek Instruments, Winooski, VT). Cell counts were differentially induced or suppressed in the primary and recurrent were calculated based on the calibration curve of fluorescence of pterygia cases were extracted from the whole genome using t tests. diluted cell suspension. This set of genes was analyzed using a bioinformatics approach. To gain insight into the transcriptional network of molecular events Evaluation of Periostin Expression by in terms of the mechanism of recurrence, we applied commercially available analysis software (Ingenuity Pathway Analysis 3.0, based on Flow Cytometry the Ingenuity Pathways Knowledge Base; Ingenuity Systems, Redwood Pterygia fibroblasts were cultured with recombinant IL-4 (PeproTech, City, CA; http://www.ingenuity.com). The resultant networks were Rocky Hill, NJ) at the selected concentration for 48 hours. To inhibit evaluated by the significance score, which is expressed as the negative MAP kinase activity, a highly selective inhibitor of MAP kinase/ERK logarithm of that P-value. The obtained score in each instance indi- kinase1 (MEK1), PD98059 (10 ␮M; New England Biolabs, Beverly, MA), cated the likelihood that the assembly of a set of focus genes in a was added to the media. Then the serum-starved fibroblasts were network could be explained by random chance alone. collected by enzyme treatment (Accutase; Innovative Cell Technolo- gies) and incubated for 20 minutes at 37°C in a tube rotator. The Real-Time RT-PCR fibroblasts were stained by incubation with antiperiostin antibody (1 ␮g/mL) or control IgG in 0.1% BSA/PBS for 30 minutes at 4°C. After Total RNA was isolated from the treated fibroblasts using a purification kit washing, the cells were incubated with Alexa Fluor 488-antirabbit IgG (RNeasy Mini Kit; Qiagen, Hilden, Germany) according to the manufac- (Molecular Probes, Inc.) for 20 minutes at 4°C. The expression of turer’s protocol. One microgram of RNA samples was reverse transcribed periostin on the surfaces of the fibroblasts was analyzed (FACSCalibur; using random hexamers and superscript III (Invitrogen, Carlsbad, CA). Becton-Dickinson, San Jose, CA). The cDNAs were amplified and quantified (LightCycler; Roche, Mann- heim, Germany) with a one-step quantification of RNA kit (QuantiTect Statistical Analysis SYBR Green PCR kit; Qiagen). Sequences of the used real-time PCR primer pairs were as follows: IL-4: forward, 5Ј-CACCGAGTTGACCGTAACAG-3Ј; Data are presented as the mean Ϯ SEM. Statistical analyses were reverse, 5Ј- GCCCTGCAGAAGGTTTCC-3Ј; periostin: forward, 5Ј-TTGAG- performed by unpaired Student’s t-tests (two tailed) or ANOVA analy- ACGCTGGAAGGAAAT-3Ј; reverse, 5Ј-AGATCCGTGAAGGTGGTTTG-3Ј; sis. IL-13: forward, 5Ј-TGGAGGACTTCTAGGAAAACGA-3Ј; reverse, 5Ј-CC- CCTTTGCTCACCAGTCT-3Ј; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward, 5Ј-AGCCACATCGCTCAGACAC-3Ј; reverse, 5Ј-GC- RESULTS Ј CCAATACGACCAAATCC-3 . Construction of Network Model of To ensure equal loading and amplification, all products were nor- malized to GAPDH transcript as an internal control. Pterygia Recurrence To understand the global interactions of the pterygia-related Establishment of Pterygia Tissue–Derived genes in terms of their function and regulation, we applied Fibroblast Line network analysis on the previously determined pterygia transcriptome.2 We hypothesized this would allow us to determine Head tissues of pterygia were collected from four eyes of four patients pterygia recurrence-related biological phenomena in a simpli- without a history of pterygia surgery and were undergoing primary fier form. To construct the network model, differentially in- excision with conjunctival autografting at the Tottori University Hos- duced or suppressed genes in the primary and recurrent ptery- pital. Tissues were minced using a sterilized forceps and scissors and gia cases were extracted from the whole genome. We focused digested in 12.5 mg/mL collagenase (Nitta Gelatin, Osaka, Japan) at on a gene set consisting of 184 genes that had a twofold change 37°C in a humidified atmosphere of 5% CO for 1 hour. The digested 2 and that had been shown to contain valid transcriptional infor- tissue was filtered and washed by centrifugation at 1500 rpm for 5 mation to generate an efficient support vector machine classi- minutes and was cultured in Dulbecco’s modified Eagle’s medium fier.2 (DMEM; Gibco-BRL, Carlsbad, CA) supplemented with 15% FBS (vol/ When this set of genes was analyzed (Ingenuity Pathway vol) at 37°C in 5% CO . The pterygia-derived conjunctival fibroblasts 2 Knowledge Base of known signaling networks; Ingenuity Sys- were cultured to at least the third passage before use. tems), four major biological networks with high significance scores (P Ͻ 10Ϫ17) were successfully generated (Table 1). Of Fibroblast Adhesion Assay these, periostin was placed in one of the major networks An adhesion assay was performed as described.4 Briefly, flat-bottom (network 4), which was annotated as cell cycle, connective cell culture plates (Corning Costar, Cambridge, MA) were coated with tissue development and function, and cell morphology. 0to40␮g/mL purified human fibronectin (Gibco-BRL) in PBS at 4°C The most significant network (network 1) was annotated as overnight. The plates were washed three times with PBS and blocked cell-to-cell signaling and interaction, hematologic system devel- with 10 ␮g/mL heat-denatured bovine serum albumin (BSA; 85°C for opment and function, and cell cycling. Interestingly, this net- 15 minutes; Sigma, St. Louis, MO) for 1 hour at 37°C. After blocking, work predicted vascular endothelial growth factor as the most the plates were washed with DMEM. linked gene, suggesting a role of network 1 in neovasculariza- Fibroblasts, detached by enzyme treatment (Accutase; Innovative tion processes. Another major network was network 2, which Cell Technologies, San Diego, CA), were incubated in DMEM for 30 was annotated to function as immune response, cell-to-cell minutes at 37°C in a tube rotator. The cells were then incubated in signaling and interaction, and hematologic system develop- 0.1% BSA/PBS with antiperiostin (rabbit polyclonal antibody; 1:500; ment and function and predicted IL-4 as the most linked gene. Biovendor, Brno, Czech Republic) or control IgG for 30 minutes at 4°C. Another SVM classifier, TIMP-2, was listed in network 3, to- After washing, the fibroblasts were seeded on fibronectin-coated plates gether with membrane metalloproteinase 7 (MMP7). Top func- (5 ϫ 105 cells/well) in DMEM containing 1 ␮g/mL antiperiostin or tion of the network 3 was annotated as cell death, immuno- control antibody and were incubated at 37°C for 1 hour. The medium logic diseases, and DNA replication/recombination/repair. Of was aspirated, and the plates were washed with PBS three times. The the previously reported prognosis determinant candidate number of attached fibroblast was determined by dye (CyQUANT GR; genes, PSPHL was not found in the generated networks.

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TABLE 1. Transcriptional Network of Pterygia Recurrence

Score Network Focus Genes Predicted Genes (؊log [P]) Functions

1 ACVR1B, ADRB2, AKAP5, AKAP11, CDKN3, CDKN1A, CKLFSF3, CXCL12, 30 Cell-to-cell signaling and ATP1B1, BTK, CA2, CD97, CHRM1, DLG4, E2F1, F2, GABRB3, GABRD, interaction, CYP3A5, DMTF1, GABRG3, GRK5, GRP, IL6, KIF2C, MAPK1, Prlpe, SMTN, hematologic system MMP1, NPAT, PPBP, THPO SRC, ST5, VEGF development and function, cell cycle 2 ACP5, CD1E, CD79B, CR2, IGHM, MS4A1, ALDH1A2, ARG2, CCL24, CCL26, 19 Immune response, cell- NCLN, PKNOX1, PLAC8, POU2F2, SATB1, CD1B, CD1C, CEBPB, CLEC7A, to-cell signaling and TK2, USP33 CTLA2A, CYSLTR1, IL4, IL13, INSR, interaction, ISG20, LTA4H, MAOA, Retnla, hematologic system SPRR2B, STAT4, SYNGR2, TFEC, development and Trim30 function 3 AP1S1, APOC2, ARHGEF7, DNASE1L3, AP1B1, AP1M1, ARL6IP, CIDEC, 19 Cell death, EHD1, EIF4F1, GSTM1, HIST1H1C, COL14A1, H2AFZ, HIPK1, HMGN2, immunologic MMP7, SNAP29, TFAM, TIMP2, TLE4 KRT15, MT1L, MYC, MYO9B, diseases, DNA PDRG1, PLF2, PRKRIR, SLK, replication, TAGLN2, TMSB4x, TNF, TP53, recombination, and UBE2S repair 4 BF, CEACAM7, DDX11, GDF2, GRIP1, ABCG2, ANXA3, C12orf8, CSPG3, 17 Cell cycle, connective LRMP, PIK3R1, POSTN, RAB27A, EGF, ESR1, FNTA, FSTL1, GJB2, tissue development RNASEL, SWAP70, TRPC7 HBP1, HRAS, HSPB8, Ins1, and function, cell MYOD1, PFKFB1, PFKM, PPIB, morphology RB1, RPS6KB2, SH2D3A, SYT3, TOM1L1, TRPC6

Focus genes denote input genes assigned to the calculated networks. Predicted genes are genes predicted for involvement in the calculated networks. The most significant gene in the network was underlined. Significant support vector machine classifiers periostin (POSTN) and TIMP-2 are shown in boldface italic.

Increased Expression of IL-4 in the pterygia epithelia. In primary cases, IL-4 was barely de- Recurrent Pterygia tected. Of all the genes either assigned or predicted in the networks, Induction of Periostin by IL-4 periostin was the SVM classifier of recurrence with the highest accuracy.2 Periostin has recently been implicated as an adhe- To determine the roles played by IL-4 in the constructed sion molecule or a tissue remodeling mediator in Th2-type networks, we tested whether IL-4 could induce the recurrence cytokine-mediated diseases,5 though its roles on the ocular classifier, periostin, using pterygia-derived fibroblast cells. Re- surface remains unexplored. A representative Th2-type cyto- combinant IL-4-stimulated fibroblasts were examined for peri- kine, IL-4, which was predicted in the calculated network 2, is ostin mRNA using real-time PCR. Our results showed that IL-4 known to induce adhesion molecules or tissue matrix modu- induced periostin in a dose-dependent manner (Fig. 2A). 6 For the IL-4-activated genes, ERK1/2-mediated MAP kinase lators in colon carcinoma cells. Therefore, we hypothesized 7 that IL-4 may contribute to the recurrence process by the cascade is generally used. To examine an involvement of the induction of gene(s) in the observed networks. ERK1/2 pathway in the transcriptional activation of periostin, Initially, we used real-time PCR to determine whether the IL-4-stimulated-fibroblasts were treated with PD98059, a MEK1 inhibitor. PD98059 significantly inhibited the transcriptional mRNA of IL-4 was transcriptionally induced in pterygia tissues. Ͻ Analysis of all pterygia cases showed significant induction of activation of periostin by IL-4 (at 100 pg/mL and 1 ng/mL; P 0.05), indicating an involvement of the MEK1/ERK1 pathway IL-4 compared with normal controls (pterygia tissue, 23.5 Ϯ in periostin induction. 9.3 relative copies; normal conjunctiva, 0.7 Ϯ 0.5 relative We next examined whether the transcriptional activation of copies/GAPDH; P Ͻ 0.05). Because tissue remodeling is a periostin may actually translate into periostin expression. Flow critical phenomenon in pterygia recurrence, we next exam- cytometric analysis confirmed the expression of periostin by ined whether IL-4 may indeed be induced after recurrence. In IL-4 (Fig. 2B) compared with control-treated fibroblasts (Fig. recurrence cases, the IL-4 mRNA levels were made worse than 2C). Consistent with the transcriptional outcome, PD98059 the primary cases (Fig. 1). In network 2, another Th2 type treatment inhibited periostin expression (Fig. 2D). cytokine, IL-13, was detected. Therefore, we also examined the IL-13 mRNA levels. In contrast to IL-4, IL-13 was reduced in Pterygia Fibroblast Adhesion Mediated recurrent cases though the reduction was not statistically significant (primary pterygia, 2.3 Ϯ 2.1 relative copies; recurrent by Periostin pterygia, 0.07 Ϯ 0.06 relative copies/GAPDH), further support- Finally, to understand the role of periostin in pterygia recur- ing an important role of IL-4 in network 2. rence, we examined the functional role of periostin using Immunohistochemical analysis was performed to confirm pterygia-derived fibroblasts. Because pterygial tissues express that IL-4 was translated in pterygia tissues. Consistent with the large amounts of fibronectin, one of the cell matrix ligands of transcriptional analysis, IL-4 was prominently expressed in the periostin, we tested whether periostin might promote fi- recurrent tissue and localized mainly in the perivascular tissues bronectin-mediated cell adhesion using a cell adhesion assay. and endothelial cells in the stroma of the subconjunctiva (Fig. Our results showed that fibronectin significantly promoted 1B). Inflammatory cells, including macrophages or lympho- fibroblast adhesion in a dose-dependent manner (Fig. 3). When cytes, also stained positive for IL-4 and were found focally in fibroblasts were treated with antiperiostin, fibronectin-mediat-

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plays an important pivotal role in inducing ECM deposition and subepithelial fibrosis and in exacerbating airway hyperrespon- siveness, eosinophil infiltration, and mast cell activation.5,9–12 However, we observed IL-13 transcripts were greatly reduced, though the reduction was not statistically significant. Because IL-13 is known to compete for IL-4 binding,13 this may poten- tiate IL-4 binding. An infiltration of mast cells has been recog- nized in pterygia tissue; however, a reasonable explanation for the infiltration has not been presented.14–16 Our findings have revealed a previously unrecognized, but presumably crucial, pathway for pterygia fibrosis. A recent comprehensive analyses of pterygia transcriptome identified fibronectin as the top induced gene in the genome, followed by other ECM-related genes (e.g., collagen type III and versican).17 However, IL-4 induction has been unnoticed and hidden in our transcriptome and in others, probably because of its relatively low constitutive levels. Successful identification by network model prediction provided us with an important perspective in pterygia pathogenesis. From an epidemiologic standpoint, exposure to ultraviolet light (UV) is one of most important factors for the development of pterygia. Indeed, UV exposure induces IL-4 expression in

FIGURE 1. Differential expression of IL-4 in primary and recurrent pterygia tissues. (A) Recurrent tissues shows significantly more periostin transcript (P Ͻ 0.05; n ϭ 11 eyes for primary tissue; n ϭ 10 eyes for recurrent tissue). (B) Localization of IL-4 in primary and recurrent pterygia by immunohistochemistry. Upregulated IL-4 expression (brown) in epithelium and perivascular tissues of recurrent pterygia (right) compared with primary pterygia (left). Asterisks: inflammatory cells stained positive. Ep, conjunctival epithelium. Original magnifica- tion, ϫ1000.

ed-fibroblast adhesion was significantly inhibited. Thus, periostin was shown to mediate fibroblast adhesion in a fibronectin- rich environment, including pterygia tissue. This critical function of periostin in the pathogenesis of pterygia may promote recurrence.

DISCUSSION In pterygial tissue, extracellular matrix protein and metalloproteinases (MMPs), including collagen types III and IV, laminin, fibronectin, and MMPs 8, 9, and 13, are abundantly expressed.8 During the recurrence process, the deposition of such ECM proteins continues and is accompanied by tissue-remodeling processes. These changes eventually lead to marked subepithe- FIGURE 2. Periostin induction of pterygia fibroblast by IL-4. (A) IL-4 lial fibrosis. treatment induced periostin mRNA in a dose-dependent manner at a The results of this study demonstrated a new pathway, range of 0 to 1 ng/mL IL-4. Treatment with MEK 1 inhibitor PD98059 ␮ Ͻ ϭ leading to pterygia recurrence, that involves IL-4 and periostin. (10 M) significantly suppressed IL-4 induction. *P 0.05; n 3. (B–D) Periostin protein expression by IL-4 was examined by flow Using knowledge-based bioinformatics, we identified an IL-4 cytometric analysis. Periostin was induced on pterygia fibroblasts by network of immune responses and cell-to-cell signaling, pre- IL-4 (1 ng/mL) (B). No detectable expression of periostin was observed sumably serving as a tissue fibrotic factor in pterygia pathogen- on nonstimulated fibroblasts (C). PD98059 (10 ␮M) inhibited the esis. For example, in Th2-type diseases, including bronchial expression of periostin (D). Data are representative of repeated asthma, subepithelial fibrosis is a cardinal feature. IL-4 or IL-13 experiments.

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