Dysregulation of Lysyl Oxidase Expression in Lesions and Endometrium of Women With Endometriosis

Lysyl 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).

The 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.

LOX 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.

This 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. Similar content being viewed by others

Practice Committee of the American Society for Reproductive Medicine. Definitions of infertility and recurrent pregnancy loss. Fertil Steril. 2008;89(6):1603. Flores 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. Konno R, Fujiwara H, Netsu S, et al. Gene expression profiling of the rat endometriosis model. Am J Reprod Immunol. 2007;58(4): 330–343. Eyster 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. Blassioli Dentillo D, Meola J, Rosa ESJC, et al. Deregulation of LOXL1 and HTRA1 gene expression in endometriosis. Reprod Sci. 2010;17(11):1016–1023. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001;70:1–32. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci. 2006;63(19–20): 2304–2316. Molnar 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. Lazarus HM, Cruikshank WW, Narasimhan N, Kagan HM, Center DM. Induction of human monocyte motility by lysyl oxidase. Matrix Biol. 1995;14(9):727–731. Li 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. Dairkee 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. Erler JT, Bennewith KL, Nicolau M, et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature. 2006;440(7088): 1222–1226. Erler JT, Giaccia AJ. Lysyl oxidase mediates hypoxic control of metastasis. Cancer Res. 2006;66(21):10238–10241. Kirschmann DA, Seftor EA, Fong SF, et al. A molecular role for lysyl oxidase in breast cancer invasion. Cancer Res. 2002;62(15): 4478–4483. Kirschmann 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. Payne 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. Akiri 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. Peinado 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. Moreno-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. Talbi 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. Savaris 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. Painter 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. Ruiz 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. Practice Committee of the American Society for Reproductive Medicine. Endometriosis and infertility: a committee opinion. Fertil Steril. 2012;98(3):591–598. Bulletti C, Coccia ME, Battistoni S, Borini A. Endometriosis and infertility. J Assist Reprod Genet. 2010;27(8):441–447. Trinder J, Cahill DJ. Endometriosis and infertility: the debate continues. Hum Fertil (Camb). 2002;5(1 suppl):S21–S27. Stoikos 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. Nnoaham 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. Gao 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. Fourquet 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. Fourquet 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. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122(2):262–263. Colon-Diaz M, Baez-Vega P, Garcia M, et al. HDAC1 and HDAC2 are Differentially Expressed in Endometriosis. Reprod Sci. 2012;19(5):483–492. Borahay 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. Zeitvogel 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. Banu 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. Korch 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. Kniss 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. Krikun 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. Hombach-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. Livak 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. Jin 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. Li 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. Carroll JS, Meyer CA, Song J, et al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet. 2006;38(11):1289–1297. Welboren 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. Liang 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. Gebäck 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. Nam 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. Bulun 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. Bulun 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. Reynaud 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. Nishioka T, Eustace A, West C. Lysyl oxidase: from basic science to future cancer treatment. Cell Struct Funct. 2012;37(1):75–80. Cox TR, Erler JT. Lysyl oxidase in colorectal cancer. Am J Physiol Gastrointest Liver Physiol. 2013;305(10):G659-G666. Baker 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. Senapati S, Barnhart K. Managing endometriosis-associated infertility. Clin Obstet Gynecol. 2011;54(4):720–726. Vassiliadis 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. Simon 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. Gupta S, Goldberg JM, Aziz N, Goldberg E, Krajcir N, Agarwal A. Pathogenic mechanisms in endometriosis-associated infertility. Fertil Steril. 2008;90(2):247–257. Garrido 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. Lessey BA. Implantation defects in infertile women with endometriosis. Ann N Y Acad Sci. 2002;955:265–280; discussion 293–265, 396–406. Van 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. Cuman C, Menkhorst E, Winship A, et al. Fetal-maternal communication: the role of Notch signalling in embryo implantation. Reproduction. 2014;147(3):R75–R86. Tanriverdi 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. Lazarus HM, Cruikshank WW, Narasimhan N, Kagan HM, Center DM. Induction of human monocyte motility by lysyl oxidase. Matrix Biol. 1995;14(9):727–731. Erler 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. Staun-Ram E, Shalev E. Human trophoblast function during the implantation process. Reprod Biol Endocrinol. 2005;3:56. Stilley JA, Birt JA, Sharpe-Timms KL. Cellular and molecular basis for endometriosis-associated infertility. Cell Tissue Res. 2012;349(3):849–862. Holoch KJ, Lessey BA. Endometriosis and infertility. Clin Obstet Gynecol. 2010;53(2):429–438. Trackman 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. Sethi 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. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001;70:1–32. Rojas-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. Gao 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. Baker 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. Moratz C, Harrison K, Kehrl JH. Regulation of chemokine-induced lymphocyte migration by RGS proteins. Methods Enzymol. 2004;389:15–32. Hurst 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. Huang 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. Afshar 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. Afshar 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. Afshar 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. Mitsuhashi 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. Hastings 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. Selam 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. Portelli 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. NCBI. National Center for Biotechnology Information. Web site. http://www.ncbi.nlm.nih.gov/gene/4091. Updated April 27, 2015. Accessed May 1, 2015. Lee 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. NCBI. National Center for Biotechnology Information. Web site. http://www.ncbi.nlm.nih.gov/gene/23230. Updated April 10, 2015. Accessed May 1, 2015. Author information Authors and Affiliations Corresponding author Rights and permissions About this article Cite this article Ruiz, 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 Published: Issue date: DOI: https://doi.org/10.1177/1933719115585144