{"paper_id":"adda6d51-88bc-44c6-a336-c229e18c6f1a","body_text":"WWW.KJOG.ORG 441\nReceived: 2011. 4.24.   Revised: 2011. 6.20.  Accepted: 2011. 6.23.\nCorresponding author: Hye Won Chung, MD\nDepartment of Obstetrics and Gynecology, Ewha Womans \nUniversity School of Medicine, 911-1 Mok-dong, Y angcheon-gu, \nSeoul 158-710, Korea\nTel: +82-2-2650-5568  Fax: +82-2647-9860\nE-mail: hyewon@ewha.ac.kr\nTh is is an Open Access article distributed under the terms of the Creative Commons \nAttribution Non-Commercial License (http://creativecommons.org/licenses/\nby-nc/3.0/) which permits unrestricted non-commercial use, distribution, and \nreproduction in any medium, provided the original work is properly cited.\nCopyright © 2011. Korean Society of Obstetrics and Gynecology \nEndometriosis, one of the most common gynecologic disorders, \nis broadly defi  ned as the presence of endometrial glandular and \nstromal cells outside the uterine cavity, associated with symptoms \nof dysmenorrhea, dyspaureunia, chronic pelvic pain and subfertil-\nity. But, the etiology and pathogenesis of endometriosis remain \nobscure. Theories that account for this susceptibility include genet-\nic predisposition [1], large amount of retrograde menstruation [2], \nan altered peritoneal environment [3], or an immunological sus-\nceptibility [4]. The most widely accepted theory is that the disease \nis caused by retrograde menstruation and subsequent implantation \nof endometrial glands on the surface of the abdominal cavity [2,5]. \nHowever, retrograde menstruation is frequently observed in women \nunaffected by endometriosis and such menstrual debris does not \nresult in endometriosis in all women. Thus, additional factors may be \npresent in the uterine endometrium of women who have developed \nthe disease. The women who develop endometriosis are due to \nabnormalities inherent to their ectopic or eutopic endometrium. \nThe refl  uxed menstrual debris in women with endometriosis may \nORIGINAL ARTICLE\nKorean J Obstet Gynecol 2011;54(8):441-447\nhttp://dx.doi.org/10.5468/KJOG.2011.54.8.441\npISSN 2233-5188\n · eISSN 2233-5196\nEXPRESSION OF MEMBRANE TYPE-2 AND -3 MATRIX \nMETALLOPROTEINASES IN EUTOPIC ENDOMETRIUM OF \nWOMEN WITH ADVANCED ENDOMETRIOSIS \nHyun Kyung Chung, MD\n1\n, Ji Young Lee, MD\n2\n, Kyung A Jeong, MD\n1\n, Hye Won Chung, MD\n1\nDepartment of Obstetrics and Gynecology, \n1\nEwha Womans University School of Medicine; \n2\nKonkuk University School of Medicine, Seoul, Korea\nObjective\nTo investigate the expression of messenger RNA (mRNA) for membrane type-2 matrix metalloproteinases (MT2-MMPs) and MT3-MMP \nand compare their expression pattern in women with severe endometriosis and normal controls.\nMethods \nQuantitative competitive polymerase chain reaction was performed to evaluate the mRNA expression of MT2-MMP and MT3-MMP \nin endometrium from 36 women with severe endometriosis and 52 women without endometriosis throughout the menstrual cycle. \nResults\nEutopic endometrium from women with endometriosis expressed higher levels of MT3-MMP  than that from normal women in \nsecretory phase (P < 0.05). MT2-MMP expression from eutopic endometrium showed no signifi  cant differences between patients \nwith endometriosis and controls. \nConclusion \nThese results suggest that eutopic endometrium from patients with endometriosis may be more proteolytic, angiogenic and prone \nto growth because of greater \nMT3-MMP  expression than endometrium from women without endometriosis. Thus, increased \nproteolytic and angiogenic activities may be one of the explanations of the pathogenesis of endometriosis.\nKeywords: Membrane type-2 matrix metalloproteinases; Membrane type-3 matrix metalloproteinases; Endometriosis \n\nWWW.KJOG.ORG442\nKJOG  Vol. 54, No. 8, 2011\nbe more prone to implant, invade and grow in peritoneum or ova-\nry through the action of extracellular proteolysis and angiogenesis. \nAngiogenesis is facilitated by proteolysis, since endothelial cells \nrequire proteolytic activity to be able to degrade their basal \nmembrane, to migrate and to invade the underlying extracellular \nmatrix [6-8]. Key regulators of proteolysis belong to the family of \nmatrix metalloproteinases (MMPs) . They represent a large family \nof proteolytic enzymes regulated by tumor-stromal interaction that \nplay key roles in cancer progression, promoting proliferation, an-\ngiogenesis and tumor metastasis [9]. In particular, the \nmembrane-\ntype matrix metalloproteinases (MT-MMPs) are a new subfamily of \nmembrane-anchored MMPs, which as of today includes six mem-\nbers: MT1-, MT2-, MT3-, MT4-. MT5-, and MT6-MMP. Among them, \nMT1-, MT2-, MT3-, and MT5-MMPs are trans-membrane proteins. \nTheir membrane-associated localization makes them particularly \nsuited to functioning in pericellular proteolysis [10,11]. Previous re-\nports showed that \nMT-MMPs play an important role in angiogenesis \n[10,12,13], especially MT1- MMP has received considerable atten-\ntion as being involved in tumor angiogenesis [14,15]. Several MT-\nMMPs have been demonstrated in whole endometrial extracts at \nmRNA level [16,17], and MT1- and MT2-MMP antigens have been \ndemonstrated in various endometrial cell types [18,19].\nMT-MMPs act at the cell surface where they can locally facilitate \ndegradation of extracellular matrix, cell migration, invasion and \nangiogenesis. The abundance of all \nMT-MMP in cycling endome-\ntrium suggests that endometrial MT-MMPs play a role in remodel-\ning of cycling endometrium in preparation for implantation [20]. \nMT-MMPs are inhibited by tissue inhibitor of matrix metallopro-\nteinase-2 (TIMP-2) [16]. The endometriosis-associated increase \nin proteolysis and imbalance between the secretion of MMP-9 \nand that of its natural inhibitor, TIMP-1, revealed in the culture \nmedium of endometrial tissue [21]. The possible changes in \nMT-\nMMPs activity in the eutopic endometrial tissue of patients with \nendometriosis suggest an enhanced proteolysis which could play a \nrole in enabling this tissue to implant in ectopic locations. \nTherefore, the aim of the present study was to investigate whether \nthe endometrial tissue from women with endometriosis would \nexpress a higher \nMT2-MMP  and MT3-MMP  mRNA expression \nconsistent with higher angiogenic activity and increased growth. \n \nMaterials and Methods\n1. Tissue collection\nEndometrial samples were obtained from 79 premenopausal \nwomen aged 29-45 years, undergoing laparoscopic surgery or \nhysterectomy for non-malignant lesions. Patients with pelvic \ninflammatory disease, adenomyosis and dysfunctional uterine \nbleeding were excluded. Patients have not taken the nonsteroidal \nanti-infl  ammatory drugs, GnRH agonists and steroids for the past \n6 weeks. Sufficient eutopic endometrial tissues were available \nfrom 36 patients with endometriosis stages III and IV endome-\ntriosis diagnosed by both pathology and laparoscopic findings \naccording to the revised American Fertility Society classifi  cation of \nendometriosis [22]. Endometrial tissue from 52 control patients \nwithout endometriosis confi  rmed by laparoscopic surgery was also \ncollected. The study protocol was approved by the Institutional \nReview Board on the Use of Human Subjects in Research at Ewha \nWomans University and informed consent was obtained.\nEndometrial samples were taken using a pipette in the operating \nroom before the laparoscopic procedure; in patients undergoing \nhysterectomy, the uterine cavity was opened and endometrium \nobtained immediately after the specimen was removed. Tissue \nsamples were classified by histological dating according to the \nmethod of Noyes et al. [23] into two groups: proliferative phase \n(n=49) and secretory phase (n=30). The remaining tissue was \nwashed in PBS solution in order to remove contaminating blood \nand RNA was immediately extracted. \n2. RNA extraction\nThe extraction of RNA from the tissue sample was carried out \nwith the RNA-STAT-60 reagent (Tel-Test “B” Inc., Friendswood, \nTX, USA). Briefl  y, tissue samples were washed three times in PBS \n(Gibco BRL, Grand Island, NY , USA) to remove blood contamina-\ntion. One hundred milligrams of tissue were homogenized in 1 mL \nof RNA-STAT-60 reagent. Total RNA was separated from DNA and \nproteins by adding chloroform and was precipitated using iso-\npropanol. The precipitate was washed two times in 75% ethanol, \nair-dried, and re-diluted in diethylpycocarbonate (DEPC)-treated \ndH\n2O. The amount and purity of extracted RNA was quantitated by \nspectrophotometry in a GenQuant RNA/DNA calculator (Pharmacia \nBiotech Ltd., Cambridge, UK) and 10-100 μg of total RNA was \nroutinely obtained.\n3. Reverse transcription (RT) PCR\nSpecifi  c sequences of oligonucleotide primers for \nMT2-, and MT3-\nMMP were obtained from Gene Bank Database of the National \nCenter for Biotechnology Information of the National Institutes of \nHealth (NIH, Internet address: http://www.2.ncbi.nlm.nih.gov/cgi-\nbin/genbank). One corresponding set of primers for \nMT2-MMP  \n\nWWW.KJOG.ORG 443\nHyun Kyung Chung, et al. MT2-MMP and MT3-MMP in endometriosis \nand MT3-MMP was found with the help from the program OLIGO \n5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, \nUSA) and synthesized by Biomed, Seoul, Korea. The primer se-\nquences, locations on the mRNA, and sizes of the amplifi  ed frag-\nments are listed in Table 1. \nFor RT-PCR, the Gen Amp RNA PCR kit (Perkin-Elmer, Foster City, \nCA, USA) was used. Nineteen microliters of RT-mastermix for each \nsample were prepared containing 5 mmol/L MgCl\n2, 1X PCR buffer \nII, 1 mmol/L of each deoxy-NTP , 2.5 μL/L oligo (deoxythimidine)16, \n20 IU ribonuclease inhibitor (all from Perkin-Elmer), 100 IU Mo-\nloney murine leukemia virus reverse transcriptase (Gibco BRL), and \n1 μg total RNA diluted in 1 μL DEPC-treated H\n2O and placed into \n0.2 mL thin wall PCR tube (Applied Scientifi  c, South San Francisco, \nCA, USA). RT was carried out in the DNA Thermal Cycler 9600 \n(Perkin-Elmer) using a program with the following parameters: \n42°C, 15 min; 99°C, 5 min; then quenched at 4°C. After the re-\naction was completed, samples were stored at -20°C until the \nPCR. As a negative control, 1 μL DEPC-treated H2O without RNA \nsample was subjected to the same RT reaction.\n4.  Construction of the competitive and target cDNA \nfragment for \nMT2-, and MT3-MMP \nA 428 base pair (bp), and 532 bp fragment of native MT2-MMP, \nand MT3-MMP cDNA (the target) were obtained by PCR amplifi  -\ncation of reverse-transcribed total RNA from endometrial biopsies \nwith the regular 3’ and 5’ primers (Table 1). The PCR product was \nvisualized by agarose gel electrophoresis stained with ethidium \nbromide (EtBr), cDNA was extracted from the gel, purified with \nan agarose gel extraction kit (Amersham Pharmacia Biotech Ltd., \nPiscataway, NJ, USA) and quantitated by spectrophotometry (Phar-\nmacia Biotech Ltd., Cambridge, UK). \nTo construct a competitive cDNA fragment, a fl  oating primer with \na sequence complementary to cDNA between the 3’ and 5’ primer \nbinding sites was designed by attaching the complementary se-\nquence of the binding site of the original 3’-\nMT2-MMP, and MT3-\nMMP . After PCR with the regular 5’-primer and the 3’-floating \nprimer, the PCR product was visualized by agarose gel electropho-\nresis stained with EtBr. cDNA extraction, purifi  cation, and concen-\ntration determination were performed as described above. These \nsteps resulted in cDNA fragments of 201 bp and 415 bp, each \nwith 3’-end and 5’-end primer binding sites on their ends which \nwere products of 227 bp and 117 bp deletion from the target \ncDNA, respectively.\n5.  Standard curve and competitive PCR for \nMT2-, and \nMT3-MMP \nThe standard curve for MT2-MMP, and MT3-MMP was construct-\ned by co-amplification of the constant amount of competitive \ncDNA (0.1 fmol for \nMT2-MMP , and 1 fmol for MT3-MMP ) with \ndeclining amounts of target cDNA (from 62.5 to 0.01525 fmol for \nMT2-MMP and from 500 to 0.122 fmol for MT3-MMP) obtained \nby serial dilution. A total of 100 μL of PCR mixture containing 1.9 \nmmol MgCl\n2 solution, 10X PCR buffer II, 0.2 mmol/L of deoxy-NT, \nand 2.5 U Taq-polymerase (all from Perkin-Elmer) with correspond-\ning paired primers at a concentration of 0.2 μmol/L of each primer, \nwas placed in the Perkin-Elmer DNA Thermal Cycler 9600. PCR \ncycles were composed of 1 cycle of 95°C for 5 min to denature all \nproteins, 35 cycles of 45 sec at 94°C, 45 sec at 57°C, and 45 sec \nat 72°C for \nMT2-MMP, and 30 cycles of 45 sec at 94°C, 45 sec at \n62°C, and 45 sec at 72°C for MT3-MMP. The reaction was termi-\nnated at 72°C for 7 min and was quenched at 4°C. One percent \nagarose gel electrophoresis was carried out in a H5 electrophore-\nsis chamber. Gels were stained with EtBr. Aliquots (25 μL) of each \nPCR product and dye buffer were analyzed in parallel with a 100 \nbp DNA ladder as a standard. \nAfter completion of electrophoresis, the gel blot was analyzed and \nphotocopies of the blot were printed by UV densitometry (Gel-\nDoc and Chemidoc system, Bio-Rad Laboratories, Hercules, CA, \nUSA). The logarithmically transformed ratios of target cDNA to \ncompetitive cDNA were plotted against the log amount of initially \nTable 1. Oligonucleotide primers for eutopic endometrium MT2 and MT3-MMP mRNA amplifi  cation\nmRNA Primer 5’‐3’ Size (bp)\nMT2‐MMP Upstream ACC TGC ATG GAA ACA ACC TC 428\nDownstream GCC CTT GAA CAC GAA CAT CT \nCompetitor ACC TTC AGC TTC TGG TTG TTG TTT CCA TTG GGC ATC CAG 201\nMT3‐MMP Upstream TCC CAA GCCAAT CAC AGT CTG G 532\nDownstream AAA   GGT  CAG  CCC CGA ATC AG\nCompetitor A ACC CTA CAT CAC ACC CAC TC 415\n\nWWW.KJOG.ORG444\nKJOG  Vol. 54, No. 8, 2011\nadded target cDNA in each PCR to obtain linear and reproducible \nstandard curves. Values obtained from the regression line of the \nstandard curve (y=b+mx) allowed us to calculate the amount of \ncDNA transcripts in an unknown sample: 0.1 fmol of \nMT2-MMP, \nand 1 fmol of MT3-MMP competitive cDNA were added to each \nunknown sample before PCR. The ratio of the densities of sample \ntarget cDNA band (428 bp, and 532 bp) to competitive cDNA (201 \nbp and 415 bp) were logarithmically transformed and compared \nthe values obtained from standard curve. Quantitative competitive \nPCR was carried out on at least two aliquots from the RT cDNA of \neach patient, and the results did not differ more than ±5% and \nwe used the average concentration for data analysis.\n6. Data analysis\nStatistical analysis was performed by ANOVA and Post Hoc test \nusing LSD with \nt-test. The statistical analysis was carried out using \nthe SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA) with P-value < 0.05 \nconsidered statistically signifi  cant.\nResults\n1.  RT-PCR of endometrial tissue throughout the \nmenstrual cycle\nRT-PCR was employed to increase the sensitivity of detection, and \nthe 838 bp sequence of ß-actin, 428 bp sequence of \nMT2-MMP , \nand 532 bp sequence of MT3-MMP  mRNA were expressed by \nall eutopic endometrial samples from women with and without \nendometriosis in both the proliferative and secretory phase of the \nmenstrual cycle. ß-actin mRNA expression was also measured in \nall the samples studied, thus confi  rming the integrity of RNA and \nthe RT-PCR process (data not shown).\n2.  Quantitative \nMT2-MMP mRNA expression in eutopic \nendometrial tissue from women with or without \nendometriosis\nThroughout the menstrual cycle, eutopic endometrium from pa-\ntients with endometriosis did not show differences of \nMT2-MMP \nmRNA expression compared to eutopic endometrium from control \n(Fig. 1).\n3.  Quantitative \nMT3-MMP mRNA expression in eutopic \nendometrial tissue from women with or without \nendometriosis\nQuantitative expression of \nMT3-MMP mRNA in eutopic endome-\ntrium of patients with endometriosis was higher in secretory phase \ncompared to that of control group. During the proliferative phase, \nthere are no differences between eutopic endometrium with and \nwithout endometriosis (\nP > 0.05) (Fig. 2).\nDiscussion\nMT-MMPs are of interest in pathogenesis of endometriosis be-\ncause of their specifi  c features involving matrix degradation and \nactivation of other MMPs. It is understood that the tissue destruc-\nFig. 1. Quantitative and competitive polymerase chain reaction of MT2-\nMMP  in eutopic endometrium throughout the menstrual cycle. FN, \nproliferative phase endometrium from  normal patients; FS, proliferative \nphase endometrium from  endometriosis patients; LN, secretory phase en-\ndometrium from  normal patients; LS, secretory phase endometrium from  \nendometriosis patients.\nNormal Endometriosis\nMT-MMP2 mRNA fmol/L\nFN FS LN LS\nFig. 2. Quantitative and competitive polymerase chain reaction of MT3-\nMMP  in eutopic endometrium throughout the menstrual cycle. FN, \nproliferative phase endometrium from  normal patients; FS, proliferative \nphase endometrium from  endometriosis patients; LN, secretory phase en-\ndometrium from  normal patients; LS, secretory phase endometrium from  \nendometriosis patients. \nP-value; a=0.0242 , b=0.0376,  c=0.0156.\nNormalFN FS LN LS Endometriosis\nMT-MMP3 mRNA fmol/L\n\nWWW.KJOG.ORG 445\nHyun Kyung Chung, et al. MT2-MMP and MT3-MMP in endometriosis \ntion and invasion in endometriosis are mediated by the concerted \naction of various proteinases, among which the MMPs appear to \nplay a major role. Recently, novel members of the MMP family, the \nMT-MMPs, have been described [24-28]. It is of particular impor-\ntance that at least three of these MT-MMPs, namely, MT1-, MT2-, \nand MT3-MMP, have been shown to be capable of not only de-\ngrading extracellular matrix components but also activating other \nMMPs, such as MMP-2 and MMP-13 [24,25,29-31]. \nMT-MMPs \n(with the exclusion of MT4-MMP) activate MMP-2 (gelatinase A), \nan enzyme that has a key role in local invasion and dissemination \nof a large variety of tumors [24,32-34], through a 2-step cleavage \nreaction, following the formation of a membrane complex with \nMMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) [32]. \nThe \nMT-MMPs are inhibited by TIMPs family which includes four \nmembers (TIMP-1, TIMP-2, TIMP-3, and TIMP-4). For example, \nMT1- , MT2- , MT3- , and MT5-MMP  are efficiently inhibited by \nTIMP-2 and TIMP-3. The effi  ciency in mediating this cleavage reac-\ntion seems to be highest for \nMT1-MMP, followed by MT3-MMP, \nand is lower for MT2-MMP  [34]. MT1-MMP  also can activate \nprocollagenase-3 (MMP-13), while recombinant forms of all 3 en-\nzymes can cleave a large number of ECM proteins [34].\nIn this study, the eutopic endometrium from women with endome-\ntriosis expressed higher levels of \nMT3-MMP than that from normal \nwomen. But MT2-MMP  expression from eutopic endometrium \nshowed no signifi  cant differences between patients with endome-\ntriosis and controls. \nSeveral studies have suggested a role for \nMT-MMPs in the degra-\ndation of the extracellular matrix in malignancies and rheumatoid \narthritis [35-39]. \nMT1-MMP may play a key role in human breast \ncarcinoma invasion and metastasis [35]. In the other study, it was \nfound that the expression of \nMT-MMP in cervical cancer cells both \nin vitro  and in vivo  was higher in invasive cervical carcinoma and \nlymph node metastases compared to its expression in non-invasive \nCIN III lesions [36]. Another study found a higher level of \nMT-MMP \nexpression that MT1-MMP and MT2-MMP play an important role \nin the development of human urothelial carcinomas [37]. A role for \nMT1-MMP is not only in the matrix degradation by fi  broblasts, but \nalso in osteoclast-mediated bone resorption in RA [38]. MT1-MMP \nand MT2-MMP were able to directly confer invasion-incompetent \ncells with the ability to penetrate type I collagen matrices. MT-\nMMP-expressing cells can penetrate and remodel type I collagen-\nrich tissues by using membrane-anchored metalloproteinases as \npericellular collagenases [10]. \nPrevious data presented suggested a positive correlation between \nexpression of \nMMP-2, MT1-MMP, and MT2-MMP mRNA and, pos-\nsibly, a role in ovarian carcinoma and endometriosis pathogenesis, \nmainly through tumor cell production of these enzymes [35,39]. \nSo, we compared the patients with endometriosis and controls in \nexpression of \nMT-2 and MT3-MMP. \nAll MT-MMPs are expressed in endometrium in a cycle-dependent \npattern with decreased levels during the early secretory phase. MT-\nMMPs may play a role in endometrial remodeling in preparation \nfor implantation and were reduced during the receptive window \n[20]. And in our study also eutopic endometrium from control \nhave tendency lower expression levels in secretory phase compare \nthan proliferative phase.\nIn this study, \nMT-3 MMP is related with proteolysis and angiogen-\nesis of endometriosis. It suggests that increased ability to prote-\nolysis and angiogenesis of endometrium is essential for surviving \noutside the uterus in endometriosis. In conclusion, the \nMT-MMPs \nsystem may play an important role in the pathogenesis of endo-\nmetriosis. \nReferences\n  1. Lamb K, Hoffmann RG, Nichols TR. Family trait analysis: a \ncase-control study of 43 women with endometriosis and their \nbest friends. Am J Obstet Gynecol 1986;154:596-601.\n  2. Cramer DW, Wilson E, Stillman RJ, Berger MJ, Belisle S, Schiff \nI, et al. The relation of endometriosis to menstrual characteris-\ntics, smoking, and exercise. JAMA 1986;255:1904-8.\n  3. Ramey JW, Archer DF . Peritoneal fl  uid: its relevance to the de-\nvelopment of endometriosis. 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Am J Clin Pathol 2001;115:517-24.\n진행성 자궁내막증 여성의 자궁내막에서 membrane type-2 and -3 matrix metalloproteinases의 발현 \n1이화여자대학교 의학전문대학원, 2건국대학교 의학전문대학원 산부인과학교실\n정현경1, 이지영2, 정경아1, 정혜원1\n목적\n본 연구는 진행성 자궁내막증 여성과 대조군 여성의 자궁내막에서 membrane type-2 matrix metalloproteinases (MT2-MMPs)와 MT3-\nMMP의 messenger RNA (mRNA)의 발현을 비교해 보고자 한다. \n연구방법\n36명의 진행성 자궁내막증 여성과 52명의 대조군 여성을 대상으로 생리 기간 동안 자궁내막을 채취하였으며, Quantitative competitive \npolymerase chain reaction을 사용하여 MT2-MMP와 MT3-MMP mRNA의 발현을 측정하였다. \n결과\nMT3-MMP의 경우 분비기의 진행성 자궁내막증 여성의 자궁내막에서 정상 여성보다 통계적으로 유의하게 높게 발현되었다(P＜0.05). \nMT2-MMP의 발현은 두 군 간에 유의한 차이를 보이지 않았다. \n결론\n자궁내막증 여성의 자궁내막에서 MT3-MMP의 과발현은 자궁내막증 환자의 자궁내막이 더 활발한 단백질분해와 혈관 생성의 기질을 갖\n고 있음을 의미하며, 이는 자궁내막증 병인의 주된 역할 중 하나로 생각된다.  \n중심단어: Membrane type-2 matrix metalloproteinases, Membrane type-3 matrix metalloproteinases, 자궁내막증","source_license":"CC0","license_restricted":false}