Expression ofmembrane type-2and-3 matrix metalloproteinasesin eutopic endometrium of women with advanced endometriosis

in endometriosis in all women. Thus, additional factors may be present in the uterine endometrium of women who have developed the disease. The women who develop endometriosis are due to abnormalities inherent to their ectopic or eutopic endometrium. The refl uxed menstrual debris in women with endometriosis may ORIGINAL ARTICLE Korean J Obstet Gynecol 2011;54(8):441-447 http://dx.doi.org/10.5468/KJOG.2011.54.8.441 pISSN 2233-5188 · eISSN 2233-5196 EXPRESSION OF MEMBRANE TYPE-2 AND -3 MATRIX METALLOPROTEINASES IN EUTOPIC ENDOMETRIUM OF WOMEN WITH ADVANCED ENDOMETRIOSIS Hyun Kyung Chung, MD 1 , Ji Young Lee, MD 2 , Kyung A Jeong, MD 1 , Hye Won Chung, MD 1 Department of Obstetrics and Gynecology, 1 Ewha Womans University School of Medicine; 2 Konkuk University School of Medicine, Seoul, Korea

To investigate the expression of messenger RNA (mRNA) for membrane type-2 matrix metalloproteinases (MT2-MMPs) and MT3-MMP and compare their expression pattern in women with severe endometriosis and normal controls.

Quantitative competitive polymerase chain reaction was performed to evaluate the mRNA expression of MT2-MMP and MT3-MMP in endometrium from 36 women with severe endometriosis and 52 women without endometriosis throughout the menstrual cycle.

Eutopic endometrium from women with endometriosis expressed higher levels of MT3-MMP than that from normal women in secretory phase (P < 0.05). MT2-MMP expression from eutopic endometrium showed no signifi cant differences between patients with endometriosis and controls.

These results suggest that eutopic endometrium from patients with endometriosis may be more proteolytic, angiogenic and prone to growth because of greater MT3-MMP expression than endometrium from women without endometriosis. Thus, increased proteolytic and angiogenic activities may be one of the explanations of the pathogenesis of endometriosis.

Membrane type-2 matrix metalloproteinases; Membrane type-3 matrix metalloproteinases; Endometriosis WWW.KJOG.ORG442 KJOG Vol. 54, No. 8, 2011 be more prone to implant, invade and grow in peritoneum or ova- ry through the action of extracellular proteolysis and angiogenesis. Angiogenesis is facilitated by proteolysis, since endothelial cells require proteolytic activity to be able to degrade their basal membrane, to migrate and to invade the underlying extracellular matrix [6-8]. Key regulators of proteolysis belong to the family of matrix metalloproteinases (MMPs) . They represent a large family of proteolytic enzymes regulated by tumor-stromal interaction that play key roles in cancer progression, promoting proliferation, an- giogenesis and tumor metastasis [9]. In particular, the membrane- type matrix metalloproteinases (MT-MMPs) are a new subfamily of membrane-anchored MMPs, which as of today includes six mem- bers: MT1-, MT2-, MT3-, MT4-. MT5-, and MT6-MMP. Among them, MT1-, MT2-, MT3-, and MT5-MMPs are trans-membrane proteins. Their membrane-associated localization makes them particularly suited to functioning in pericellular proteolysis [10,11]. Previous re- ports showed that MT-MMPs play an important role in angiogenesis [10,12,13], especially MT1- MMP has received considerable atten- tion as being involved in tumor angiogenesis [14,15]. Several MT- MMPs have been demonstrated in whole endometrial extracts at mRNA level [16,17], and MT1- and MT2-MMP antigens have been demonstrated in various endometrial cell types [18,19]. MT-MMPs act at the cell surface where they can locally facilitate degradation of extracellular matrix, cell migration, invasion and angiogenesis. The abundance of all MT-MMP in cycling endome- trium suggests that endometrial MT-MMPs play a role in remodel- ing of cycling endometrium in preparation for implantation [20]. MT-MMPs are inhibited by tissue inhibitor of matrix metallopro- teinase-2 (TIMP-2) [16]. The endometriosis-associated increase in proteolysis and imbalance between the secretion of MMP-9 and that of its natural inhibitor, TIMP-1, revealed in the culture medium of endometrial tissue [21]. The possible changes in MT- MMPs activity in the eutopic endometrial tissue of patients with endometriosis suggest an enhanced proteolysis which could play a role in enabling this tissue to implant in ectopic locations. Therefore, the aim of the present study was to investigate whether the endometrial tissue from women with endometriosis would express a higher MT2-MMP and MT3-MMP mRNA expression consistent with higher angiogenic activity and increased growth.

1. Tissue collection Endometrial samples were obtained from 79 premenopausal women aged 29-45 years, undergoing laparoscopic surgery or hysterectomy for non-malignant lesions. Patients with pelvic inflammatory disease, adenomyosis and dysfunctional uterine bleeding were excluded. Patients have not taken the nonsteroidal anti-infl ammatory drugs, GnRH agonists and steroids for the past 6 weeks. Sufficient eutopic endometrial tissues were available from 36 patients with endometriosis stages III and IV endome- triosis diagnosed by both pathology and laparoscopic findings according to the revised American Fertility Society classifi cation of endometriosis [22]. Endometrial tissue from 52 control patients without endometriosis confi rmed by laparoscopic surgery was also collected. The study protocol was approved by the Institutional Review Board on the Use of Human Subjects in Research at Ewha Womans University and informed consent was obtained. Endometrial samples were taken using a pipette in the operating room before the laparoscopic procedure; in patients undergoing hysterectomy, the uterine cavity was opened and endometrium obtained immediately after the specimen was removed. Tissue samples were classified by histological dating according to the

of Noyes et al. [23] into two groups: proliferative phase (n=49) and secretory phase (n=30). The remaining tissue was washed in PBS solution in order to remove contaminating blood and RNA was immediately extracted. 2. RNA extraction The extraction of RNA from the tissue sample was carried out with the RNA-STAT-60 reagent (Tel-Test “B” Inc., Friendswood, TX, USA). Briefl y, tissue samples were washed three times in PBS (Gibco BRL, Grand Island, NY , USA) to remove blood contamina- tion. One hundred milligrams of tissue were homogenized in 1 mL of RNA-STAT-60 reagent. Total RNA was separated from DNA and proteins by adding chloroform and was precipitated using iso- propanol. The precipitate was washed two times in 75% ethanol, air-dried, and re-diluted in diethylpycocarbonate (DEPC)-treated dH 2O. The amount and purity of extracted RNA was quantitated by spectrophotometry in a GenQuant RNA/DNA calculator (Pharmacia Biotech Ltd., Cambridge, UK) and 10-100 μg of total RNA was routinely obtained. 3. Reverse transcription (RT) PCR Specifi c sequences of oligonucleotide primers for MT2-, and MT3- MMP were obtained from Gene Bank Database of the National Center for Biotechnology Information of the National Institutes of Health (NIH, Internet address: http://www.2.ncbi.nlm.nih.gov/cgi- bin/genbank). One corresponding set of primers for MT2-MMP WWW.KJOG.ORG 443 Hyun Kyung Chung, et al. MT2-MMP and MT3-MMP in endometriosis and MT3-MMP was found with the help from the program OLIGO 5.0 Primer Analysis Software (National Bioscience, Plymouth, MN, USA) and synthesized by Biomed, Seoul, Korea. The primer se- quences, locations on the mRNA, and sizes of the amplifi ed frag- ments are listed in Table 1. For RT-PCR, the Gen Amp RNA PCR kit (Perkin-Elmer, Foster City, CA, USA) was used. Nineteen microliters of RT-mastermix for each sample were prepared containing 5 mmol/L MgCl 2, 1X PCR buffer II, 1 mmol/L of each deoxy-NTP , 2.5 μL/L oligo (deoxythimidine)16, 20 IU ribonuclease inhibitor (all from Perkin-Elmer), 100 IU Mo- loney murine leukemia virus reverse transcriptase (Gibco BRL), and 1 μg total RNA diluted in 1 μL DEPC-treated H 2O and placed into 0.2 mL thin wall PCR tube (Applied Scientifi c, South San Francisco, CA, USA). RT was carried out in the DNA Thermal Cycler 9600 (Perkin-Elmer) using a program with the following parameters: 42°C, 15 min; 99°C, 5 min; then quenched at 4°C. After the re- action was completed, samples were stored at -20°C until the PCR. As a negative control, 1 μL DEPC-treated H2O without RNA sample was subjected to the same RT reaction. 4. Construction of the competitive and target cDNA fragment for MT2-, and MT3-MMP A 428 base pair (bp), and 532 bp fragment of native MT2-MMP, and MT3-MMP cDNA (the target) were obtained by PCR amplifi - cation of reverse-transcribed total RNA from endometrial biopsies with the regular 3’ and 5’ primers (Table 1). The PCR product was visualized by agarose gel electrophoresis stained with ethidium bromide (EtBr), cDNA was extracted from the gel, purified with an agarose gel extraction kit (Amersham Pharmacia Biotech Ltd., Piscataway, NJ, USA) and quantitated by spectrophotometry (Phar- macia Biotech Ltd., Cambridge, UK). To construct a competitive cDNA fragment, a fl oating primer with a sequence complementary to cDNA between the 3’ and 5’ primer binding sites was designed by attaching the complementary se- quence of the binding site of the original 3’- MT2-MMP, and MT3- MMP . After PCR with the regular 5’-primer and the 3’-floating primer, the PCR product was visualized by agarose gel electropho- resis stained with EtBr. cDNA extraction, purifi cation, and concen- tration determination were performed as described above. These steps resulted in cDNA fragments of 201 bp and 415 bp, each with 3’-end and 5’-end primer binding sites on their ends which were products of 227 bp and 117 bp deletion from the target cDNA, respectively. 5. Standard curve and competitive PCR for MT2-, and MT3-MMP The standard curve for MT2-MMP, and MT3-MMP was construct- ed by co-amplification of the constant amount of competitive cDNA (0.1 fmol for MT2-MMP , and 1 fmol for MT3-MMP ) with declining amounts of target cDNA (from 62.5 to 0.01525 fmol for MT2-MMP and from 500 to 0.122 fmol for MT3-MMP) obtained by serial dilution. A total of 100 μL of PCR mixture containing 1.9 mmol MgCl 2 solution, 10X PCR buffer II, 0.2 mmol/L of deoxy-NT, and 2.5 U Taq-polymerase (all from Perkin-Elmer) with correspond- ing paired primers at a concentration of 0.2 μmol/L of each primer, was placed in the Perkin-Elmer DNA Thermal Cycler 9600. PCR cycles were composed of 1 cycle of 95°C for 5 min to denature all proteins, 35 cycles of 45 sec at 94°C, 45 sec at 57°C, and 45 sec at 72°C for MT2-MMP, and 30 cycles of 45 sec at 94°C, 45 sec at 62°C, and 45 sec at 72°C for MT3-MMP. The reaction was termi- nated at 72°C for 7 min and was quenched at 4°C. One percent agarose gel electrophoresis was carried out in a H5 electrophore- sis chamber. Gels were stained with EtBr. Aliquots (25 μL) of each PCR product and dye buffer were analyzed in parallel with a 100 bp DNA ladder as a standard. After completion of electrophoresis, the gel blot was analyzed and photocopies of the blot were printed by UV densitometry (Gel- Doc and Chemidoc system, Bio-Rad Laboratories, Hercules, CA, USA). The logarithmically transformed ratios of target cDNA to competitive cDNA were plotted against the log amount of initially Table 1. Oligonucleotide primers for eutopic endometrium MT2 and MT3-MMP mRNA amplifi cation mRNA Primer 5’‐3’ Size (bp) MT2‐MMP Upstream ACC TGC ATG GAA ACA ACC TC 428 Downstream GCC CTT GAA CAC GAA CAT CT Competitor ACC TTC AGC TTC TGG TTG TTG TTT CCA TTG GGC ATC CAG 201 MT3‐MMP Upstream TCC CAA GCCAAT CAC AGT CTG G 532 Downstream AAA GGT CAG CCC CGA ATC AG Competitor A ACC CTA CAT CAC ACC CAC TC 415 WWW.KJOG.ORG444 KJOG Vol. 54, No. 8, 2011 added target cDNA in each PCR to obtain linear and reproducible standard curves. Values obtained from the regression line of the standard curve (y=b+mx) allowed us to calculate the amount of cDNA transcripts in an unknown sample: 0.1 fmol of MT2-MMP, and 1 fmol of MT3-MMP competitive cDNA were added to each unknown sample before PCR. The ratio of the densities of sample target cDNA band (428 bp, and 532 bp) to competitive cDNA (201 bp and 415 bp) were logarithmically transformed and compared the values obtained from standard curve. Quantitative competitive PCR was carried out on at least two aliquots from the RT cDNA of each patient, and the results did not differ more than ±5% and we used the average concentration for data analysis. 6. Data analysis Statistical analysis was performed by ANOVA and Post Hoc test using LSD with t-test. The statistical analysis was carried out using the SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA) with P-value < 0.05 considered statistically signifi cant.

1. RT-PCR of endometrial tissue throughout the menstrual cycle RT-PCR was employed to increase the sensitivity of detection, and the 838 bp sequence of ß-actin, 428 bp sequence of MT2-MMP , and 532 bp sequence of MT3-MMP mRNA were expressed by all eutopic endometrial samples from women with and without endometriosis in both the proliferative and secretory phase of the menstrual cycle. ß-actin mRNA expression was also measured in all the samples studied, thus confi rming the integrity of RNA and the RT-PCR process (data not shown). 2. Quantitative MT2-MMP mRNA expression in eutopic endometrial tissue from women with or without endometriosis Throughout the menstrual cycle, eutopic endometrium from pa- tients with endometriosis did not show differences of MT2-MMP mRNA expression compared to eutopic endometrium from control (Fig. 1). 3. Quantitative MT3-MMP mRNA expression in eutopic endometrial tissue from women with or without endometriosis Quantitative expression of MT3-MMP mRNA in eutopic endome- trium of patients with endometriosis was higher in secretory phase compared to that of control group. During the proliferative phase, there are no differences between eutopic endometrium with and without endometriosis ( P > 0.05) (Fig. 2).

MT-MMPs are of interest in pathogenesis of endometriosis be- cause of their specifi c features involving matrix degradation and activation of other MMPs. It is understood that the tissue destruc- Fig. 1. Quantitative and competitive polymerase chain reaction of MT2- MMP in eutopic endometrium throughout the menstrual cycle. FN, proliferative phase endometrium from normal patients; FS, proliferative phase endometrium from endometriosis patients; LN, secretory phase en- dometrium from normal patients; LS, secretory phase endometrium from endometriosis patients. Normal Endometriosis MT-MMP2 mRNA fmol/L FN FS LN LS Fig. 2. Quantitative and competitive polymerase chain reaction of MT3- MMP in eutopic endometrium throughout the menstrual cycle. FN, proliferative phase endometrium from normal patients; FS, proliferative phase endometrium from endometriosis patients; LN, secretory phase en- dometrium from normal patients; LS, secretory phase endometrium from endometriosis patients. P-value; a=0.0242 , b=0.0376, c=0.0156. NormalFN FS LN LS Endometriosis MT-MMP3 mRNA fmol/L WWW.KJOG.ORG 445 Hyun Kyung Chung, et al. MT2-MMP and MT3-MMP in endometriosis tion and invasion in endometriosis are mediated by the concerted action of various proteinases, among which the MMPs appear to play a major role. Recently, novel members of the MMP family, the MT-MMPs, have been described [24-28]. It is of particular impor- tance that at least three of these MT-MMPs, namely, MT1-, MT2-, and MT3-MMP, have been shown to be capable of not only de- grading extracellular matrix components but also activating other MMPs, such as MMP-2 and MMP-13 [24,25,29-31]. MT-MMPs (with the exclusion of MT4-MMP) activate MMP-2 (gelatinase A), an enzyme that has a key role in local invasion and dissemination of a large variety of tumors [24,32-34], through a 2-step cleavage reaction, following the formation of a membrane complex with MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) [32]. The MT-MMPs are inhibited by TIMPs family which includes four members (TIMP-1, TIMP-2, TIMP-3, and TIMP-4). For example, MT1- , MT2- , MT3- , and MT5-MMP are efficiently inhibited by TIMP-2 and TIMP-3. The effi ciency in mediating this cleavage reac- tion seems to be highest for MT1-MMP, followed by MT3-MMP, and is lower for MT2-MMP [34]. MT1-MMP also can activate procollagenase-3 (MMP-13), while recombinant forms of all 3 en- zymes can cleave a large number of ECM proteins [34]. In this study, the eutopic endometrium from women with endome- triosis expressed higher levels of MT3-MMP than that from normal women. But MT2-MMP expression from eutopic endometrium showed no signifi cant differences between patients with endome- triosis and controls. Several studies have suggested a role for MT-MMPs in the degra- dation of the extracellular matrix in malignancies and rheumatoid arthritis [35-39]. MT1-MMP may play a key role in human breast carcinoma invasion and metastasis [35]. In the other study, it was found that the expression of MT-MMP in cervical cancer cells both in vitro and in vivo was higher in invasive cervical carcinoma and lymph node metastases compared to its expression in non-invasive CIN III lesions [36]. Another study found a higher level of MT-MMP expression that MT1-MMP and MT2-MMP play an important role in the development of human urothelial carcinomas [37]. A role for MT1-MMP is not only in the matrix degradation by fi broblasts, but also in osteoclast-mediated bone resorption in RA [38]. MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT- MMP-expressing cells can penetrate and remodel type I collagen- rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases [10]. Previous data presented suggested a positive correlation between expression of MMP-2, MT1-MMP, and MT2-MMP mRNA and, pos- sibly, a role in ovarian carcinoma and endometriosis pathogenesis, mainly through tumor cell production of these enzymes [35,39]. So, we compared the patients with endometriosis and controls in expression of MT-2 and MT3-MMP. All MT-MMPs are expressed in endometrium in a cycle-dependent pattern with decreased levels during the early secretory phase. MT- MMPs may play a role in endometrial remodeling in preparation for implantation and were reduced during the receptive window [20]. And in our study also eutopic endometrium from control have tendency lower expression levels in secretory phase compare than proliferative phase. In this study, MT-3 MMP is related with proteolysis and angiogen- esis of endometriosis. It suggests that increased ability to prote- olysis and angiogenesis of endometrium is essential for surviving outside the uterus in endometriosis. In conclusion, the MT-MMPs system may play an important role in the pathogenesis of endo- metriosis.

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Davidson B, Goldberg I, Berner A, Nesland JM, Givant-Horwitz V , Bryne M, et al. Expression of membrane-type 1, 2, and 3 ma- trix metalloproteinases messenger RNA in ovarian carcinoma cells in serous effusions. Am J Clin Pathol 2001;115:517-24. 진행성 자궁내막증 여성의 자궁내막에서 membrane type-2 and -3 matrix metalloproteinases의 발현 1이화여자대학교 의학전문대학원, 2건국대학교 의학전문대학원 산부인과학교실 정현경1, 이지영2, 정경아1, 정혜원1 목적 본 연구는 진행성 자궁내막증 여성과 대조군 여성의 자궁내막에서 membrane type-2 matrix metalloproteinases (MT2-MMPs)와 MT3- MMP의 messenger RNA (mRNA)의 발현을 비교해 보고자 한다. 연구방법 36명의 진행성 자궁내막증 여성과 52명의 대조군 여성을 대상으로 생리 기간 동안 자궁내막을 채취하였으며, Quantitative competitive polymerase chain reaction을 사용하여 MT2-MMP와 MT3-MMP mRNA의 발현을 측정하였다. 결과 MT3-MMP의 경우 분비기의 진행성 자궁내막증 여성의 자궁내막에서 정상 여성보다 통계적으로 유의하게 높게 발현되었다(P＜0.05). MT2-MMP의 발현은 두 군 간에 유의한 차이를 보이지 않았다. 결론 자궁내막증 여성의 자궁내막에서 MT3-MMP의 과발현은 자궁내막증 환자의 자궁내막이 더 활발한 단백질분해와 혈관 생성의 기질을 갖 고 있음을 의미하며, 이는 자궁내막증 병인의 주된 역할 중 하나로 생각된다. 중심단어: Membrane type-2 matrix metalloproteinases, Membrane type-3 matrix metalloproteinases, 자궁내막증