{"paper_id":"e78754d9-c5fd-47ca-b067-40b7585659cc","body_text":"Abstract\nLysyl oxidases (LOXs) are enzymes involved in collagen deposition, extracellular membrane remodeling, and invasive/metastatic potential. Previous studies reveal an association of LOXs and endometriosis. We aimed to identify the mechanisms activated by upregulation of lysyl oxidases (LOX) in endometriotic cells and tissues. We hypothesized that LOX plays a role in endometriosis by promoting invasiveness and epithelial to mesenchymal transition (EMT).\nMethods\nThe LOX protein expression levels were measured by immunohistochemistry in lesions and endometrium on a tissue microarray (TMA) and in endometrial biopsies from patients and controls during the window of implantation (WOI). Estradiol regulation of LOX expression was determined by quantitative polymerase chain reaction (qPCR). Proliferation, invasion, and migration assays were performed in epithelial (endometrial epithelial cell), endometrial (human endometrial stromal cell), and endometriotic cell lines (ECL and 12Z). Pathway-focused multiplex qPCR was used to determine transcriptome changes due to LOX overexpression.\nResults\nLOX protein was differentially expressed in ovarian versus peritoneal lesions. During WOI, LOX levels were higher in luminal epithelium of patients with endometriosis-associated infertility compared to controls. Invasive epithelial cell lines expressed higher levels of LOX than noninvasive ones. Transfection of LOX into noninvasive epithelial cells increased their migration in an LOX inhibitor-sensitive manner. Overexpression of LOX did not fully induce EMT but the expression of genes related to fibrosis and extracellular matrix remodeling were dysregulated.\nConclusions\nThis study documents that expression of LOX is differentially regulated in endometriotic lesions and endometrium. A role for LOX in mediating proliferation, migration, and invasion of endometrial and endometriotic cells was observed, which may be implicated in the establishment and progression of endometriotic lesions.\nSimilar content being viewed by others\nReferences\nPractice Committee of the American Society for Reproductive Medicine. Definitions of infertility and recurrent pregnancy loss. Fertil Steril. 2008;89(6):1603.\nFlores I, Rivera E, Ruiz LA, Santiago OI, Vernon MW, Appleyard CB. Molecular profiling of experimental endometriosis identified gene expression patterns in common with human disease. Fertil Steril. 2007;87(5):1180–1199.\nKonno R, Fujiwara H, Netsu S, et al. Gene expression profiling of the rat endometriosis model. Am J Reprod Immunol. 2007;58(4): 330–343.\nEyster KM, Klinkova O, Kennedy V, Hansen KA. Whole genome deoxyribonucleic acid microarray analysis of gene expression in ectopic versus eutopic endometrium. Fertil Steril. 2007;88(6): 1505–1533.\nBlassioli Dentillo D, Meola J, Rosa ESJC, et al. Deregulation of LOXL1 and HTRA1 gene expression in endometriosis. Reprod Sci. 2010;17(11):1016–1023.\nCsiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001;70:1–32.\nLucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci. 2006;63(19–20): 2304–2316.\nMolnar J, Fong KS, He QP, et al. Structural and functional diversity of lysyl oxidase and the LOX-like proteins. Biochim Biophys Acta. 2003;1647(1–2):220–224.\nLazarus HM, Cruikshank WW, Narasimhan N, Kagan HM, Center DM. Induction of human monocyte motility by lysyl oxidase. Matrix Biol. 1995;14(9):727–731.\nLi W, Liu G, Chou IN, Kagan HM. Hydrogen peroxide-mediated, lysyl oxidase-dependent chemotaxis of vascular smooth muscle cells. J Cell Biochem. 2000;78(4):550–557.\nDairkee SH, Ji Y, Ben Y, Moore DH, Meng Z, Jeffrey SS. A molecular ‘signature’ of primary breast cancer cultures; patterns resembling tumor tissue. BMC Genomics. 2004;5(1):47.\nErler JT, Bennewith KL, Nicolau M, et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature. 2006;440(7088): 1222–1226.\nErler JT, Giaccia AJ. Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res. 2006;66(21):10238–10241.\nKirschmann DA, Seftor EA, Fong SF, et al. A molecular role for lysyl oxidase in breast cancer invasion. Cancer Res. 2002;62(15): 4478–4483.\nKirschmann DA, Seftor EA, Nieva DR, Mariano EA, Hendrix MJ. Differentially expressed genes associated with the metastatic phenotype in breast cancer. Breast Cancer Res Treat. 1999;55(2): 127–136.\nPayne SL, Fogelgren B, Hess AR, et al. Lysyl oxidase regulates breast cancer cell migration and adhesion through a hydrogen peroxide-mediated mechanism. Cancer Res. 2005;65(24):11429–11436.\nAkiri G, Sabo E, Dafni H, et al. Lysyl oxidase-related protein-1 promotes tumor fibrosis and tumor progression in vivo. Cancer Res. 2003;63(7):1657–1666.\nPeinado H, Del Carmen Iglesias-de la Cruz M, Olmeda D, et al. A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression. Embo J. 2005;24(19):3446–3458.\nMoreno-Bueno G, Salvador F, Martin A, et al. Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas. EMBO Mol Med. 2011;3(9):528–544.\nTalbi S, Hamilton AE, Vo KC, et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology. 2006;147(3):1097–1121.\nSavaris RF, Hamilton AE, Lessey BA, Giudice LC. Endometrial gene expression in early pregnancy: lessons from human ectopic pregnancy. Reprod Sci. 2008;15(8):797–816.\nPainter JN, Nyholt DR, Morris A, et al. High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19. Fertil Steril. 2011;95(7):2236–2240.\nRuiz LA, Dutil J, Ruiz A, et al. Single-nucleotide polymorphisms in the lysyl oxidase-like protein 4 and complement component 3 genes are associated with increased risk for endometriosis and endometriosis-associated infertility. Fertil Steril. 2011;96(2): 512–515.\nPractice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility: a committee opinion. Fertil Steril. 2012;98(3):591–598.\nBulletti C, Coccia ME, Battistoni S, Borini A. Endometriosis and infertility. J Assist Reprod Genet. 2010;27(8):441–447.\nTrinder J, Cahill DJ. Endometriosis and infertility: the debate continues. Hum Fertil (Camb). 2002;5(1 suppl):S21–S27.\nStoikos CJ, Salamonsen LA, Hannan NJ, O’Connor AE, Rombauts L, Dimitriadis E. Activin A regulates trophoblast cell adhesive properties: implications for implantation failure in women with endometriosis-associated infertility. Hum Reprod. 2010;25(7):1767–1774.\nNnoaham KE, Hummelshoj L, Webster P, et al. Impact of endometriosis on quality of life and work productivity: a multicenter study across ten countries. Fertil Steril. 2011;96(2):366–373. e8.\nGao X, Yeh YC, Outley J, Simon J, Botteman M, Spalding J. Health-related quality of life burden of women with endometriosis: a literature review. Curr Med Res Opin. 2006;22(9):1787–1797.\nFourquet J, Baez L, Figueroa M, Iriarte RI, Flores I. Quantification of the impact of endometriosis symptoms on health-related quality of life and work productivity. Fertil Steril. 2011;96(1): 107–112.\nFourquet J, Gao X, Zavala D, et al. Patients’ report on how endometriosis affects health, work, and daily life. Fertil Steril. 2010; 93(7):2424–2428.\nNoyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122(2):262–263.\nColon-Diaz M, Baez-Vega P, Garcia M, et al. HDAC1 and HDAC2 are Differentially Expressed in Endometriosis. Reprod Sci. 2012;19(5):483–492.\nBorahay MA, Lu F, Ozpolat B, et al. Mullerian inhibiting substance suppresses proliferation and induces apoptosis and autophagy in endometriosis cells in vitro. ISRN Obstet Gynecol. 2013;2013: 361489.\nZeitvogel A, Baumann R, Starzinski-Powitz A. Identification of an invasive, N-cadherin-expressing epithelial cell type in endometriosis using a new cell culture model. Am J Pathol. 2001; 159(5):1839–1852.\nBanu SK, Lee J, Starzinski-Powitz A, Arosh JA. Gene expression profiles and functional characterization of human immortalized endometriotic epithelial and stromal cells. Fertil Steril. 2008; 90(4):972–987.\nKorch C, Spillman MA, Jackson TA, et al. DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecol Oncol. 2012;127(1): 241–248.\nKniss DA, Summerfield TL. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research. Reprod Sci. 2014;21(8):1015–1019.\nKrikun G, Mor G, Alvero A, et al. A novel immortalized human endometrial stromal cell line with normal progestational response. Endocrinology. 2004;145(5):2291–2296.\nHombach-Klonisch S, Kehlen A, Fowler PA, et al. Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells. J Mol Endocrinol. 2005;34(2):517–534.\nLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408.\nJin VX, Leu YW, Liyanarachchi S, et al. Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray. Nucleic Acids Res. 2004;32(22):6627–6635.\nLi L. GADEM: a genetic algorithm guided formation of spaced dyads coupled with an EM algorithm for motif discovery. J Comput Biol. 2009;16(2):317–329.\nCarroll JS, Meyer CA, Song J, et al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet. 2006;38(11):1289–1297.\nWelboren WJ, van Driel MA, Janssen-Megens EM, et al. ChIP-Seq of ERαlpha and RNA polymerase II defines genes differentially responding to ligands. EMBO J. 2009;28(10):1418–1428.\nLiang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007 2007;2(2):329–333.\nGebäck T, Schulz MM, Koumoutsakos P, Detmar M. TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays. Biotechniques. 2009;46(4): 265–274.\nNam HJ, Park YY, Yoon G, Cho H, Lee JH. Co-treatment with hepatocyte growth factor and TGF-beta1 enhances migration of HaCaT cells through NADPH oxidase-dependent ROS generation. Exp Mol Med. 2010;42(4):270–279.\nBulun SE, Cheng YH, Pavone ME, et al. Estrogen receptor-beta, estrogen receptor-alpha, and progesterone resistance in endometriosis. Semin Reprod Med. 2010;28(1):36–43.\nBulun SE, Cheng YH, Yin P, et al. Progesterone resistance in endometriosis: link to failure to metabolize estradiol. Mol Cell Endocrinol. 2006;248(1–2):94–103.\nReynaud C, Gleyzal C, Jourdan-Le Saux C, Sommer P. Comparative functional study of the lysyl oxidase promoter in fibroblasts, Ras-transformed fibroblasts, myofibroblasts and smooth muscle cells. Cell Mol Biol (Noisy-le-grand). 1999;45(8):1237–1247.\nNishioka T, Eustace A, West C. Lysyl oxidase: from basic science to future cancer treatment. Cell Struct Funct. 2012;37(1):75–80.\nCox TR, Erler JT. Lysyl oxidase in colorectal cancer. Am J Physiol Gastrointest Liver Physiol. 2013;305(10):G659-G666.\nBaker AM, Bird D, Lang G, Cox TR, Erler JT. Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK. Oncogene. 2013;32(14):1863–1868.\nSenapati S, Barnhart K. Managing endometriosis-associated infertility. Clin Obstet Gynecol. 2011;54(4):720–726.\nVassiliadis S, Relakis K, Papageorgiou A, Athanassakis I. Endometriosis and infertility: a multi-cytokine imbalance versus ovulation, fertilization and early embryo development. Clin Dev Immunol. 2005;12(2):125–129.\nSimon C, Gutierrez A, Vidal A, et al. Outcome of patients with endometriosis in assisted reproduction: results from in-vitro fertilization and oocyte donation. Hum Reprod. 1994;9(4):725–729.\nGupta S, Goldberg JM, Aziz N, Goldberg E, Krajcir N, Agarwal A. Pathogenic mechanisms in endometriosis-associated infertility. Fertil Steril. 2008;90(2):247–257.\nGarrido N, Navarro J, Garcia-Velasco J, Remoh J, Pellice A, Simon C. The endometrium versus embryonic quality in endometriosis-related infertility. Hum Reprod Update. 2002;8(1):95–103.\nLessey BA. Implantation defects in infertile women with endometriosis. Ann N Y Acad Sci. 2002;955:265–280; discussion 293–265, 396–406.\nVan Sinderen M, Cuman C, Gamage T, et al. Localisation of the Notch family in the human endometrium of fertile and infertile women. J Mol Histol. 2014;45(6):697–706.\nCuman C, Menkhorst E, Winship A, et al. Fetal-maternal communication: the role of Notch signalling in embryo implantation. Reproduction. 2014;147(3):R75–R86.\nTanriverdi G, Denir S, Ayla S, et al. Notch signaling pathway in cumulus cells can be a novel marker to identify poor and normal responder IVF patients. J Assist Reprod Genet. 2013;30(10): 1319–1326.\nLazarus HM, Cruikshank WW, Narasimhan N, Kagan HM, Center DM. Induction of human monocyte motility by lysyl oxidase. Matrix Biol. 1995;14(9):727–731.\nErler JT, Bennewith KL, Cox TR, et al. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell. 2009;15(1):35–44.\nStaun-Ram E, Shalev E. Human trophoblast function during the implantation process. Reprod Biol Endocrinol. 2005;3:56.\nStilley JA, Birt JA, Sharpe-Timms KL. Cellular and molecular basis for endometriosis-associated infertility. Cell Tissue Res. 2012;349(3):849–862.\nHoloch KJ, Lessey BA. Endometriosis and infertility. Clin Obstet Gynecol. 2010;53(2):429–438.\nTrackman PC, Graham RJ, Bittner HK, Carnes DL, Gilles JA, Graves DT. Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1. Histochem Cell Biol. 1998;110(1):9–14.\nSethi A, Mao W, Wordinger RJ, Clark AF. Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2011;52(8):5240–5250.\nCsiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001;70:1–32.\nRojas-Cartagena C, Appleyard CB, Santiago OI, Flores I. Experimental intestinal endometriosis is characterized by increased levels of soluble TNFRSF1B and downregulation of Tnfrsf1a and Tnfrsf1b gene expression. Biol Reprod. 2005;73(6):1211–1218.\nGao Y, Xiao Q, Ma H, et al. LKB1 inhibits lung cancer progression through lysyl oxidase and extracellular matrix remodeling. Proc Natl Acad Sci U S A. 2010;107(44):18892–18897.\nBaker AM, Cox TR, Bird D, et al. The role of lysyl oxidase in SRC-dependent proliferation and metastasis of colorectal cancer. J Natl Cancer Inst. 2011;103(5):407–424.\nMoratz C, Harrison K, Kehrl JH. Regulation of chemokine-induced lymphocyte migration by RGS proteins. Methods Enzymol. 2004;389:15–32.\nHurst JH, Mendpara N, Hooks SB. Regulator of G-protein signalling expression and function in ovarian cancer cell lines. Cell Mol Biol Lett. 2009;14(1):153–174.\nHuang ZP, Ni H, Yang ZM, Wang J, Tso JK, Shen QX. Expression of regulator of G-protein signalling protein 2 (RGS2) in the mouse uterus at implantation sites. Reprod. 2003;126(3): 309–316.\nAfshar Y, Jeong JW, Roqueiro D, et al. Notch1 mediates uterine stromal differentiation and is critical for complete decidualization in the mouse. FASEB J. 2012;26(1):282–294.\nAfshar Y, Stanculescu A, Miele L, Fazleabas AT. The role of chorionic gonadotropin and Notch1 in implantation. J Assist Reprod Genet. 2007;24(7):296–302.\nAfshar Y, Miele L, Fazleabas AT. Notch1 is regulated by chorionic gonadotropin and progesterone in endometrial stromal cells and modulates decidualization in primates. Endocrinology. 2012;153(6):2884–2896.\nMitsuhashi Y, Horiuchi A, Miyamoto T, Kashima H, Suzuki A, Shiozawa T. Prognostic significance of Notch signalling molecules and their involvement in the invasiveness of endometrial carcinoma cells. Histopathology. 2012;60(5):826–837.\nHastings JM, Jackson KS, Mavrogianis PA, Fazleabas AT. The estrogen early response gene FOS is altered in a baboon model of endometriosis. Biol Reprod. 2006;75(2):176–182.\nSelam B, Kayisli UA, Akbas GE, Basar M, Arici A. Regulation of FAS ligand expression by chemokine ligand 2 in human endometrial cells. Biol Reprod. 2006;75(2):203–209.\nPortelli M, Pollacco J, Sacco K, Schembri-Wismayer P, Calleja-Agius J. Endometrial seedlings. A survival instinct? Immunomodulation and its role in the pathophysiology of endometriosis. Minerva Ginecol. 2011;63(6):563–570.\nNCBI. National Center for Biotechnology Information. Web site. http://www.ncbi.nlm.nih.gov/gene/4091. Updated April 27, 2015. Accessed May 1, 2015.\nLee J, Banu SK, Subbarao T, Starzinski-Powitz A, Arosh JA. Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases. Mol Cell Endocrinol. 2011;332(1–2):306–313.\nNCBI. National Center for Biotechnology Information. Web site. http://www.ncbi.nlm.nih.gov/gene/23230. Updated April 10, 2015. Accessed May 1, 2015.\nAuthor information\nAuthors and Affiliations\nCorresponding author\nRights and permissions\nAbout this article\nCite this article\nRuiz, L.A., Báez-Vega, P.M., Ruiz, A. et al. Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis. Reprod. Sci. 22, 1496–1508 (2015). https://doi.org/10.1177/1933719115585144\nPublished:\nIssue date:\nDOI: https://doi.org/10.1177/1933719115585144","source_license":"CC0","license_restricted":false}