Involvement of Transcription Factor 21 in the Pathogenesis of Fibrosis in Endometriosis
2019; Elsevier BV; Volume: 190; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2019.09.008
ISSN1525-2191
AutoresUmida Ganieva, Tomoko Nakamura, Satoko Osuka, Bayasula, Natsuki Nakanishi, Yukiyo Kasahara, Nobuyoshi Takasaki, Ayako Muraoka, Shotaro Hayashi, Takashi Nagai, Tomohiko Murase, Maki Goto, Akira Iwase, Fumitaka Kikkawa,
Tópico(s)Reproductive System and Pregnancy
ResumoRepeated tissue injury and repair and fibrosis play a pivotal role in endometriosis. Fibrotic tissue consists of extracellular matrix proteins, regulated by transcriptional factors promoting cell proliferation and survival. Periostin is one of the putative key extracellular matrix proteins. This study aimed to determine whether transcription factor 21 (TCF21) is involved in the development of endometriosis as an upstream regulatory gene of periostin. Formalin-fixed, paraffin-embedded tissue samples [normal endometrium of women without endometriosis; eutopic endometrium of women with endometriosis; ovarian endometriosis (OE); and deep infiltrating endometriosis (DIE)] and respective cells were analyzed. Basal, transiently stimulated, and knocked down periostin and TCF21 concentrations in stromal cells of women with or without endometriosis were examined. Periostin and TCF21 expressions were undetected in normal endometrium of women without endometriosis, weakly positive in eutopic endometrium of women with endometriosis, moderately positive in OE, and strongly positive in DIE. Type 2 helper T-cell cytokines (IL-4, IL-13, and transforming growth factor-β1) increased the mRNA expression of periostin and TCF21. These cytokines, periostin, and TCF21 colocalized in the stroma of OE and DIE. siRNA against human TCF21 gene suppressed periostin expression. Transfection of TCF21 plasmid vector into stromal cells of women without endometriosis, which originally expressed neither periostin nor TCF21, resulted in TCF21 and periostin expression. TCF21 and periostin are involved in the regulation of fibrosis in endometriosis. TCF21 may be a promising therapeutic target and biomarker in endometriosis. Repeated tissue injury and repair and fibrosis play a pivotal role in endometriosis. Fibrotic tissue consists of extracellular matrix proteins, regulated by transcriptional factors promoting cell proliferation and survival. Periostin is one of the putative key extracellular matrix proteins. This study aimed to determine whether transcription factor 21 (TCF21) is involved in the development of endometriosis as an upstream regulatory gene of periostin. Formalin-fixed, paraffin-embedded tissue samples [normal endometrium of women without endometriosis; eutopic endometrium of women with endometriosis; ovarian endometriosis (OE); and deep infiltrating endometriosis (DIE)] and respective cells were analyzed. Basal, transiently stimulated, and knocked down periostin and TCF21 concentrations in stromal cells of women with or without endometriosis were examined. Periostin and TCF21 expressions were undetected in normal endometrium of women without endometriosis, weakly positive in eutopic endometrium of women with endometriosis, moderately positive in OE, and strongly positive in DIE. Type 2 helper T-cell cytokines (IL-4, IL-13, and transforming growth factor-β1) increased the mRNA expression of periostin and TCF21. These cytokines, periostin, and TCF21 colocalized in the stroma of OE and DIE. siRNA against human TCF21 gene suppressed periostin expression. Transfection of TCF21 plasmid vector into stromal cells of women without endometriosis, which originally expressed neither periostin nor TCF21, resulted in TCF21 and periostin expression. TCF21 and periostin are involved in the regulation of fibrosis in endometriosis. TCF21 may be a promising therapeutic target and biomarker in endometriosis. Repeated tissue injury and repair, resulting in fibrosis,1Zhang Q. Duan J. Olson M. Fazleabas A. Guo S.W. Cellular changes consistent with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the progression of experimental endometriosis in baboons.Reprod Sci. 2016; 23: 1409-1421Crossref PubMed Scopus (94) Google Scholar may be one of the many pathologic mechanisms involved in endometriosis, which affects up to 15% of reproductive-age women.2Giudice L.C. Kao L.C. Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2550) Google Scholar Endometriosis is often accompanied by a profound worsening of the quality of life because of concomitant chronic pain and infertility.3Bulun S.E. Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1486) Google Scholar Although it has been hypothesized that hormonal, immunologic, and environmental changes may induce normal endometrial cells to flow retrograde into the intraperitoneal space and become endometriotic,4Sampson J.A. Metastatic or embolic endometriosis, due to the menstrual dissemination of endometrial tissue into the venous circulation.Am J Pathol. 1927; 3: 93-110.43PubMed Google Scholar the detailed description of all of the abnormalities leading to endometriosis is yet to be elucidated. Endometriosis is a benign disease going through tumor-like processes to form endometriotic lesions. Tumor-like processes include aggression,5Liu H. Lang J.H. Is abnormal eutopic endometrium the cause of endometriosis? the role of eutopic endometrium in pathogenesis of endometriosis.Med Sci Monit. 2011; 17: RA92-RA99Crossref PubMed Google Scholar evasion [epithelial-mesenchymal transition (EMT)],6Zhang Q. Dong P. Liu X. Sakuragi N. Guo S.W. Enhancer of Zeste homolog 2 (EZH2) induces epithelial-mesenchymal transition in endometriosis.Sci Rep. 2017; 7: 6804Crossref PubMed Scopus (48) Google Scholar,7Yang Y.M. Yang W.X. 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Clinical management of endometriosis.Obstet Gynecol. 2018; 131: 557-571Crossref PubMed Scopus (169) Google Scholar Extracellular matrix (ECM) proteins (fibronectin, tenascin-C, type I collagen, and laminin γ2) play an important role in endometriosis. Periostin, a 90-kDa osteoblast-specific factor-2 from the fasciclin family,17Sugiura T. Takamatsu H. Kudo A. Amann E. Expression and characterization of murine osteoblast-specific factor 2 (OSF-2) in a baculovirus expression system.Protein Expr Purif. 1995; 6: 305-311Crossref PubMed Scopus (49) Google Scholar functions as a scaffold for ECM protein assembly.18Elliott C.G. Hamilton D.W. Deconstructing fibrosis research: do pro-fibrotic signals point the way for chronic dermal wound regeneration?.J Cell Commun Signal. 2011; 5: 301-315Crossref PubMed Scopus (31) Google Scholar, 19Kii I. Nishiyama T. Li M. Matsumoto K. Saito M. Amizuka N. Kudo A. 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Endometrial stromal and epithelial cells exhibit unique aberrant molecular defects in patients with endometriosis.Reprod Sci. 2017; 25: 140-159Crossref PubMed Scopus (38) Google Scholar The gene coding for transcription factor 21, TCF21, is located on chromosome 6q23. It is a member of the basic helix-loop-helix transcription factor family and is essential for the epithelial cell differentiation. TCF21 is expressed in numerous tissues, including lung, gut, gonad, urinary tract, spleen, and kidney.33Robb L. Mifsud L. Hartley L. Biben C. Copeland N.G. Gilbert D.J. Jenkins N.A. Harvey R.P. 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Loss of β-catenin in resident cardiac fibroblasts attenuates fibrosis induced by pressure overload in mice.Nat Commun. 2017; 8: 712Crossref PubMed Scopus (104) Google Scholar,35Kanisicak O. Khalil H. Ivey M.J. Karch J. Maliken B.D. Correll R.N. Brody M.J. Lin S.C.J. Aronow B.J. Tallquist M.D. Molkentin J.D. Genetic lineage tracing defines myofibroblast origin and function in the injured heart.Nat Commun. 2016; 7: 12260Crossref PubMed Scopus (450) Google Scholar Our aim was to investigate whether TCF21 is involved in the regulation of periostin in endometriosis. We demonstrate how TCF21 regulation affects periostin expression and, therefore, the pathogenesis of endometriosis. This study provides an insight into the expression of TCF21, periostin, and cytokines, and their interaction in the samples from women with or without endometriosis, and further substantiates the value of TCF21 as a therapeutic target and putative biomarker in endometriosis. The reagents used were as follows: antiperiostin antibody (ab92460), anti–TCF21 antibody–chromatin immunoprecipitation grade (ab32981), anti–IL-4 antibody (EPR1118Y; ab62351), anti–transforming growth factor (TGF)-β1 antibody (ab92486; Abcam, Cambridge, UK), anti–IL-13 antibody (F-6; sc-390676; Santa Cruz Biotechnology, Inc., Dallas, TX), anti–β-actin antibody (Wako Pure Chemical Industries, Ltd., Osaka, Japan), clone OTI4C5 anti-DDK (Flag) monoclonal antibody (TA50011-100; OriGene Technologies, Inc., Rockville, MD), Opal Multiplex IHC Detection Kit (PerkinElmer, Waltham, MA); recombinant cytokines (PeproTech, Rocky Hill, NJ) human IL-4 (catalog number AF-200-04), human IL-13 (catalog number AF-200-13), and human TGF-β1 (catalog number AF-100-21C); Lipofectamine 3000 and Lipofectamine RNAiMAX (Invitrogen, Waltham, MA); TCF21 silencer pool (Ambion, Life Technologies, Carlsbad, CA; s13904, s13906, s224713, and s13905) and non-specific control siRNA (Invitrogen); TCF21 (https://www.ncbi.nlm.nih.gov/nuccore; accession number NM_198392) Human cDNA ORF Clone (OriGene Technologies, Inc.) and empty plasmid (OriGene Technologies, Inc.; https://www.ncbi.nlm.nih.gov/nuccore; accession number NM_004098). Green fluorescent protein– and Flag-tagged TCF21 vector plasmids were generated for the transfection into normal endometrial cells. Protein-level and cytologic localization of TCF21 and periostin were evaluated by Western blot (WB) and immunocytochemical analysis. A total of 50 formalin-fixed, paraffin-embedded (FFPE) tissue samples of women with or without endometriosis (5 μm thick sections) were used: NE, normal endometrium without endometriosis, n = 14; EE, eutopic endometrium with endometriosis, n = 11; OE, ovarian endometriosis, n = 16; and DIE, deep infiltrating endometriosis, n = 9. The ethical committee of Nagoya University Graduate School of Medicine (Nagoya, Japan) approved the experiments. Written informed consent was obtained from each patient before sampling. Stromal cells were obtained from control endometrium (n = 6) of patients undergoing surgery for fibroid or other benign gynecologic diseases and from eutopic (n = 7) and ectopic endometrium of women with OE (n = 7) and DIE (n = 3). The cells were confirmed by a pathologist to be normal endometrial [endometrial stromal cell (ESC)], eutopic (eutopic ESC), and ectopic stromal cells [chocolate cyst stromal cells (CSCs) and deep infiltrating endometriosis stromal cells (DSCs)]. The patients did not receive any hormonal treatment at least 6 months before surgery. Briefly, biopsy specimens were rinsed by phosphate-buffered saline, minced into 1-mm3 pieces, and digested with 5% dispase and collagenase (2 mg/mL; Gibco Invitrogen, Cergy Pontoise, France) for 1 hour at 37°C, with serial filtration for separation. Hypotonic lysis buffer was used to remove red blood cells. Undigested tissue was removed using 100-μm sieves. The stromal cells were further enriched by separation of epithelial cells with a 40-μm sieve. Isolated stromal cells were cultured in Dulbecco's modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (Gibco-BRL, Gaithersburg, MD), 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 0.025 mg/mL of amphotericin B, at 37°C in 5% (v/v) CO2 in air. To confirm the correct stromal phenotype, the isolated cells were checked for their positivity for vimentin (1:50 antivimentin; Cell Signaling Technology, Danvers, MA) and for their negative epithelial phenotype using cytokeratin (1:25 anticytokeratin; Cell Signaling Technology) by cell immunofluorescence. The monolayer culture cells, after the third passage, were >99% pure (the proportion of stromal cells was evaluated in five random images using ×20 magnification). Each experiment was performed in triplicate and repeated at least three times with cells isolated from separate patients. Formalin-fixed human specimens (5 μm thick) were deparaffinized, blocked in methanol/0.3% H2O2, and incubated with polyclonal primary antibodies against periostin and TCF21 (1:500; Abcam) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody. Then, all specimens were incubated with 3,3′-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark), counterstained with hematoxylin, dehydrated, and mounted. Positive and negative control slides were incorporated in each staining. Negative control slides were incubated with phosphate-buffered saline replacing the primary antibody. For semiquantification, each specimen was analyzed by counting periostin- and TCF21-positive cells in the epithelium and in the stroma, by using Zeiss ZEN 2 (blue edition) microscope and Zeiss software version 1.0en.3.1 (Carl Zeiss Microscopy GmbH, Jena, Germany). The histo score was calculated by assigning a four-point scale to the intensity of the staining in 10 random fields (0 indicates negative; 1, weak; 2, moderate; and 3, strong); the percentage of cells at each intensity level was calculated by the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)].28Shen L. Liu P. Zhang P. Zhang X. Cui J. Characterization of periostin expression in human endometrium and endometriotic lesions.Gynecol Endocrinol. 2012; 28: 815-818Crossref PubMed Scopus (11) Google Scholar The cells were lysed in a radioimmunoprecipitation buffer (10 mmol/L Tris-HCl, pH 7 to 4, 150 mmol/L NaCl, 1% Nonidet P-40, 5 mmol/L EDTA, 1% sodium deoxycholate, 0.1% SDS, 1.2% aprotinin, 5 μmol/L leupeptin, 4 μmol/L antipain, 1 mmol/L phenylmethylsulfonylfluoride, and 0.1 mmol/L Na3VO4). The cell lysates were clarified by centrifugation at 13,000 × g at 4°C for 15 minutes, for protein extraction. Equal amounts of proteins were mixed with 2× sample buffer (4% SDS), 10% β-mercaptoethanol, and 20% glycerol in 0.125 mol/L Tris, pH 6.8, containing bromophenol blue and boiled for 5 minutes. Equal amounts of proteins (30 μg) were loaded and separated by 10% SDS-PAGE for the analysis of periostin and TCF21 protein level. Blocking was done with 5% skimmed milk for 1 hour at room temperature. Then, the membranes were incubated overnight at 4°C with antiperiostin (1:1000), anti–TCF21 antibody–chromatin immunoprecipitation grade ab32981 (0.5 μg/mL), and anti–β-actin antibody (1:4000) in 5% nonfat dry milk/0.05% phosphate-buffered saline–Tween. The binding of the specific antibodies was detected using a 1:1000 dilution of horseradish peroxidase–conjugated goat anti-rabbit (antiperiostin and anti-TCF21 antibodies) and goat anti-mouse (anti–β-actin antibody) IgG secondary antibody (Dako). β-Actin was used as a loading control. The membranes were visualized by ImageQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK) after reaction with Amersham ECL WB detecting reagent (GE Healthcare). ODs were measured using ImageQuantTL (GE Healthcare). Ratios between periostin OD and β-actin OD or TCF21 OD and β-actin OD were calculated in each case. Total RNA was extracted from ESC, eutopic ESC, CSC, and DSC samples, using RNeasy Mini Kit (Qiagen, Tokyo, Japan). The RNA was also isolated from the same cells after stimulation with 20 ng/mL of cytokines, to verify whether they affect periostin and TCF21 mRNA expression (type 2 helper T cell/profibrotic: IL-4, IL-13, and TGF-β1). The RNA (10 ng) was then retrotranscribed using ReverTrace qPCR RT Master Mix (TOYOBO, Osaka, Japan), and the cDNA was used for performing real-time quantitative PCR (qPCR), which was performed in triplicate using LightCycler 480 (Roche Diagnostics, Penzberg, Germany) and KOD SYBR qPCR Mix (TOYOBO). The primers used for periostin were as follows: 5′-AATCCAAGTTGTCCCAAGCC-3′ (forward) and 5′-GCACTCTGGGCATCGTGGGA-3′ (reverse); for TCF21: 5′-TCCTGGCTAACGACAAATACGA-3′ (forward) and 5′-TTTCCCGGCCACCATAAAGG-3′ (reverse); and for Gapdh: 5′-CAGCCTCAAGATCATCAGCA-3′ (forward) and 5′-GTCTTCTGGGTGGCAGTGAT-3′ (reverse) (Thermo Fisher Scientific, Waltham, MA). Gapdh was used as internal control. Periostin and TCF21 expression levels were calculated relative to Gapdh by applying the ΔΔCT method, and the data are presented as the percentage of periostin/Gapdh and TCF21/Gapdh of eutopic ESC, CSC, and DSC relative to that of ESC. Multiplexed staining was performed using the Opal Multiplex IHC Detection Kit (PerkinElmer) following the manufacturer's protocol. Briefly, the slides were first deparaffinized in xylene and then rehydrated in ethanol, following antigen retrieval in citrate buffer (pH
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