{"paper_id":"da732799-84e8-4e47-bd1c-2baae4f791e7","body_text":"CD82 gene suppression in endometrial stromal\ncells leads to increase of the cell invasiveness in\nthe endometriotic milieu\nMing-Qing Li1, Xiao-Fan Hou 1, Shi-Jian Lv 1, Yu-Han Meng 1, Xiao-Qiu Wang 1,\nChuan-Ling Tang1 and Da-Jin Li1,2\n1Laboratory for Reproductive Immunology, Fudan University Shanghai Medical College, Hospital and Institute of Obstetrics and Gynecology,\nIBS, Shanghai 200011, People’s Republic of China\n2Department of Obstetrics and Gynecology, Hainan Medical College, Affiliated Hospital, Haikou 570102, People’s Republic of China\n(Correspondence should be addressed to D-J Li at Laboratory for Reproductive Immunology, Fudan University Shanghai Medical College,\nHospital and Institute of Obstetrics and Gynecology, IBS; Email: djli@shmu.edu.cn)\nAbstract\nTetraspanin CD82 is a wide-spectrum tumor metastasis suppressor that inhibits motility and invasiveness of cancer\ncells. Endometriosis is a benign gynecological disorder, but appears malignant behaviors including invasion, ectopic\nimplantation and recurrence. This study is to elucidate the role of CD82 expression regulation in the pathogenesis of\nendometriosis. The short interfering RNA silence was established to analyze the roles of CD82, chemokine CCL2, and its\nreceptor CCR2 in the invasiveness of endometrial stromal cells (ESCs). We have found that the mRNA and protein\nlevels of CD82 in the primary normal ESCs from endometrium without endometriosis are significantly higher than that of\nthe primary ESCs from eutopic endometrium and ectopic tissue. CD82 inhibits the invasiveness of ESCs by\ndownregulating CCL2 secretion and CCR2 expression via mitogen-activated protein kinase (MAPK) and integrinb1 signal\npathway, and in turn upregulating the expression of TIMP1 and TIMP2 in an autocrine manner. The combination of\n2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with 17b-estradiol can promote the invasion of ESCs via suppressing CD82\nexpression and stimulating CCL2 secretion and CCR2 expression, and the enhanced interaction of CCL2–CCR2 recruits\nmore macrophages into the ectopic milieu in a paracrine manner, which further downregulates CD82 expression in the\nectopic ESCs. Our study has demonstrated for the first time that the abnormal lower CD82 expression in ESCs induced\nby TCDD and estrogen may be an important molecular basis of endometriosis pathogenesis through enhancing the CCL2\nsecretion and CCR2 expression and the invasion of ESCs via MAPK and integrin b1 signal pathway.\nJournal of Molecular Endocrinology (2011) 47, 195–208\nIntroduction\nEndometriosis is a very frequent benign gynecological\ndisorder in fertile women, but the pathogenesis\nstill remains controversial despite extensive research;\nhowever, Sampson’s theory of implantation of endo-\nmetrial cells and fragments refluxed during the\nmenstrual period is generally accepted among these\nhypotheses (Sampson 1925). Retrograde menstruation\nis, however, a physiological process that takes places\nalmost in all menstruation cycles, and a growing body of\nevidence suggests that the primary defect in endo-\nmetriosis can be located in the eutopic endometrium.\nAbnormalities inherent to the eutopic endometrium\nthat are not found in the endometrium of women\nwithout endometriosis might therefore contribute to\nectopic growth outside the uterine cavity ( Ulukus et al.\n2006). Different characteristics of eutopic endome-\ntrium of women with endometriosis, such as aberrant\nproduction of cytokine, growth, adhesion, and angio-\ngenic factors as well as specific cancer-related\nmolecules, are believed to contribute to the occurrence\nand maintenance of this disease.\nThe CD82 metastasis suppressor gene is implicated in\nbiological processes of tumor invasion, metastases,\ngrowth of metastatic tumors, cell motility, and adhesion\n(Takaoka et al .1 9 9 8, Yang et al .2 0 0 1). The growing\nevidence shows that CD82 inhibits cell motility through\nregulating the associated protein such as integrin\n(Mannion et al .1 9 9 6, Sugiura & Berditchevski 1999 ,\nSridhar & Miranti 2006 ), epidermal growth factor\nreceptor (EGFR;Odintsovaet al.2 0 0 0), and Duffy antigen\nreceptor for chemokines (DARC; Bandyopadhyay et al .\n2006). The expression of CD82 is involved in decidual\ntransformation from human endometrial stromal cells\n(ESCs;Gellersenet al.2 0 0 7). Moreover, our previous work\nhas confirmed that CD82 in decidual stromal cells\ncontrols the trophoblasts invasiveness by suppressing\nintegrinb1/mitogen-activated protein kinase (MAPK)/\nERK1/2 signal pathway in human early pregnancy ( Li\net al.2 0 1 0). Interestingly, the decidualized ESCs support\ntrophoblasts invasion by paracrine signals, such as\n195\nJournal of Molecular Endocrinology (2011) 47, 195–208 DOI: 10.1530/JME-10-0165\n0952–5041/11/047–195 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nHB-EGF, IL1, and LIF, which also can induce CD82\nin ESCs for controlling trophoblasts invasion (Gonzalez\net al . 2011 ). Since similarity between cancer and\nendometriosis is valid, it appears feasible to hypothesize\nthat the regulation of CD82 expression occurs in the\neutopic endometrium that predisposes to invasion,\nimplantation, adhesion, survival, and growth of ESC in\nthe ectopic milieu.\nAn increased number of active macrophages have\nbeen found in peritoneal fluid of patients with\nendometriosis. A series of research has shown that\nchemokines produced in the endometriotic milieu may\ncontribute to a feed-forward cascade of events, which\naccentuates the recruitment of leukocytes into the\nperitoneal cavity of patients with endometriosis\n(Akoum et al . 2000 ). CCL2 is a specific factor that\nchemoattracts and activates monocytes and macro-\nphages that is a major ligand of receptor CCR2. The\nmonocyte/macrophage system is considered to play an\nimportant role in the maintenance of humoral and cell-\nmediated immunity. It has been reported that the\nactivity of CCL2 is elevated in the peritoneal cavity of\npatients with endometriosis ( Akoum et al . 1996 a,b).\nSeveral investigators reported a relationship of CCL2\nlevels in serum (Akoum et al. 1996a,b, Kim et al. 2008)o r\nperitoneal fluid ( Arici et al . 1997 , Kim et al . 2008 )t o\nendometriosis, but the published results are conflict-\ning. Moreover, Garcia-Velasco et al . (1999) have found\nthat integrin b1 can stimulate the CCL2 secretion\nduring the process of ESCs adhesion to extracellular\nmatrix (ECM), therefore, it can be speculated that\nCD82 may regulate the biological function of ESCs by\nthe effect of integrinb1 on CCL2 secretion.\nThe initial phase of endometriosis is an invasion event\nthat requires ECM breakdown and repair of tissues, such\nas an increased activity of these enzymes (MMP1, MMP2,\nand MMP9; Bruner-Tran et al . 2002 , Wu et al . 2005 ).\nIndeed, MMPs and TIMPs levels have been correlated\nto the development and progression of endometriosis\n(Wu et al. 2005, Kang et al. 2008). In addition, integrins\nmediate the cell–cell and cell–matrix interaction, and\nregulate various cellular functions including motility,\nmigration, death, metastasis, and proliferation ( Hynes\n1992), and are also related to the progression of uterine\nadenomyosis (Klemmt et al. 2007).\nRecently, evidence has begun to accumulate that\n2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure\npromotes occurrence of endometriosis ( Birnbaum &\nCummings 2002 , Rier & Foster 2003 ). Research work\non primates has shown that exposure to TCDD is\nassociated with an increased prevalence and severity of\nendometriosis (Rier & Foster 2002 ). Within either the\nendocrine or immune system, exposure to TCDD affects\nindividual cell behavior by initially binding to the aryl\nhydrocarbon receptor (AhR) that rapidly forms a\nheterodimeric complex with AhR nuclear translocator\n(ARNT; Mimura & Fujii-Kuriyama 2003 ). The TCDD/\nAhR/ARNT complex associates with dioxin response\nelements to act as a signal transducer and transcription\nfactor for target genes, including cytochromes P450 1A1\n(CYP1A1), P450 1B1 (CYP1B1) (Kress & Greenlee 1997),\nand the upregulation of these genes is involved in cell\nproliferation, differentiation and inflammation. Since\nendometriosis is an estrogen-dependent disease (Rizner\n2009), and the inflammatory milieu in the peritoneal\ncavity of women with endometriosis has been extensively\ncharacterized, altered metabolism of estradiol by TCDD\nor other dioxin-like halogenated aromatic hydrocar-\nbons, and pro-inflammatory effects of TCDD may be\ninvolved in the pathogenesis of endometriosis. Our\nprevious research has demonstrated that the com-\nbination of 17 b-estradiol (E\n2) with TCDD upregulates\nCXCR1 expression in ESCs, and promotes secretion of\nIL8, a ligand of CXCR1, in co-culture of ESC–HPMC\n(HMrSV5, a human peritoneal mesothelial cell line)\ncells (Shi et al. 2006). Moreover, combination of E\n2 with\nTCDD increases the secretion of RANTES and MIP-1 a,\npromotes the invasiveness of ESCs and increases the\nexpression of MMP2 and MMP9 in ESCs (Yu et al. 2008).\nIn this study, we first evaluated the expression of\nCD82 in the primary ESCs from the normal endome-\ntrium, or eutopic and ectopic ESCs with endometriosis,\nand then observed the effects of TCDD and E\n2 on CD82\nexpression and invasiveness of the ESCs. To better\nunderstand the role of CD82 in the progression of\nendometriosis, we investigated the effects of CD82\nexpression on CCL2 secretion, CCR2 and the invasion-\nrelated molecules expression, and their potential pro-\ninvasion activity to ESCs.\nMaterials and methods\nTissue collection, cell isolation, and culture\nAll tissue samples were obtained with informed consent\nin accordance with the requirements of the research\nethics committee in Hospital of Obstetrics and Gynecol-\nogy, Fudan University Shanghai Medical College.\nSamples of endometriotic peritoneal surface lesion\n(nZ6) and ovarian lesion ( nZ6) were obtained from\nwomen age 21–49 years undergoing laparoscopy for\npain or other benign indications. The patients with\nendometriosis were classified according to the revised\nAmerican Fertility Society (AFS) classification: five in\nStage 1 and seven in Stage 2. Endometrial tissues were\nobtained from fertile women (age 22–48 years) with\n(nZ12) or without ( nZ6) endometriosis as control.\nThe samples were obtained by pipelle biopsy during\ndiagnostic laparoscopy or by uterine curettage for\nbenign indications. The absence of visible endome-\ntriosis as the control was confirmed by the surgeon\nM-QL I and others . CD82 suppression in the endometriotic milieu196\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nperforming the operation. None of the women had\nreceived hormonal medication in the 3 months before\nthe surgical procedure. All the samples were obtained in\nthe proliferative phase of the cycle, which was confirmed\nhistologically according to established criteria.\nAll the tissues were collected under sterile conditions\nand transported to the laboratory on ice in DMEM/F-12\n(Gibco) with 10% FCS (Hyclone, Logan, UT, USA). The\nendometriotic tissue was dissected away from the\nadjacent tissue, and diagnosis was confirmed by histo-\nlogical examination. The endometriotic and endo-\nmetrial tissues were digested with collagenase type IV\n(0\n.1%; Sigma) for 30 min at 378C with constant agitation\nfor recovering ESCs. The tissue pieces were filtrated\nthrough sterile gauzes (pore diameter sizes: 200 mesh)\nto remove debris. Following gentle centrifugation, the\nsupernatant was discarded, and the cells were resus-\npended in DMEM/F-12. The ESCs were separated from\nepithelial cells by passing them over sterile gauzes (pore\ndiameter sizes: 400 mesh). The filtrated suspension was\nlayered over Ficoll, and centrifuged at 800g for 20 min to\nfurther remove leukocytes and erythrocytes, and the\nmiddle layer was collected and then washed with\nD-Hanks solution. The ESCs were placed in a culture\nflask, and allowed to adhere for 20 min. The adherent\nstromal cells were cultured as monolayer in flasks with\nD M E M / F - 1 2c o n t a i n i n g1 0 %F C Sa n d2 0 m m o l / l\nHEPES and incubated in 5% CO\n2 at 37 8C. This method\nsupplied a 95% purity of ESCs.\nHuman monocyte U937 cell line (purchased from\nBank of Cell, Chinese Academy of Sciences, Shanghai,\nChina) was maintained in RPMI 1640 medium (Life\nTechnologies) with 10% bovine calf serum and\n20 mmol/l HEPES at 37 8C in a humidified, CO\n2-\ncontrolled (5%) incubator.\nQuantitative real-time PCR\nThe total RNA was extracted from the normal ( nZ6),\neutopic ( nZ6), and ectopic ( nZ6) ESCs with Tri\nreagent (Molecular Research Center, USA). The cDNA\nwas generated with oligo (dT) 18 primers using Revert\nAid First, Strand cDNA Synthesis Kit (Fementas Life\nScience, Glen Burnie, MD, USA). Triplicate samples\ncontaining cDNA prepared as mentioned-above, Taq-\nman universal PCR master mix (Applied Biosystems,\nFoster City, CA, USA), specific primers and fluorescent\ndye-labeled Taqman MGB probes for CD82 and\nGAPDH were mixed, and analyzed on an ABI7000\nthermal cycler (Applied Biosystems). The primers were\ndesigned and synthesized by TaKaRa Biotechnology\nCo., Ltd (Dalian, China). The primer pairs for cDNA\namplification were as follows: 5\n0-CTG GGG CTG TAC\nTTT GCT TTC-30 (forward) and 50-CAG AAG CCC TTC\nCTC ACA GAA-3 0 (reverse) for human CD82;5 0-GGG\nGAG CCA AAA GGG TCA TCA TCT-3 0 (forward) and\n50-GAG GGG CCA TCC ACA GTC TTC T-3 0 (reverse)\nfor human glyceraldehyde-3-phosphate dehydrogenase\n(GAPDH). The cycling conditions consisted of a\ndenaturation step at 95 8C for 10 min, 40 cycles at\n95 8C for 15 s, a 60 s annealing step at 62 8C, and finally\na holding temperature of 15 8C. To determine the\namount of gene product present in the sample, cycle\ntime ( C\nt) was determined. The average Ct value was\ncalculated from triplicate wells for each sample with\neach primer set. Most duplicate samples varied by!0.5\nCt. The relative gene expression for individual cDNA\nsamples was determined by calculating DCt values (DCt)\nby subtraction of the Ct value for GAPDH primers from\nthe Ct value for CD82 primers. The relative fold\nexpression of CD82 was determined compared with the\ncontrol. The experiments were carried out in triplicate.\nCD82 silence in ESCs\nFor short interfering RNA (siRNA) transfection, ESCs\nfrom eutopic endometrium with endometriosis ( nZ6)\nwere seeded in 96-well plates. When cells had reached\nconfluency, medium was changed to OPTIMEM\n(Invitrogen). The siRNA oligonucleotides targeting\nCD82 (set of three oligonucleotides; Stealth Select\nRNAi; Invitrogen) and Lipofectamine 2000 (Invi-\ntrogen) were mixed in OPTIMEM, and then added to\nthe cells at room temperature with non-targeting siRNA\noligonucleotides as negative control, without any\ntreatment group as blank control. After 6 h incubation,\nthe cells were incubated in DMEM for further 72 h in\n5% CO\n2 at 37 8C until the successful gene knockdown\nwas confirmed by in-cell Western and western blot. The\nsequences for three dsRNA oligonucleotides were as\nfollows: (CD82-HSS105652) 5\n0-AUC AGG AGC AGG\nAAA GCA AAG UAC A-3 0 (forward) and 5 0-UGU ACU\nUUG CUU UCC UGC UCC UGA U-30 (reverse); (CD82-\nHSS105653) 5 0-UGC CCA UGU UGA AGU AGA AGA\nGGG C-30 (forward) and 5 0-GCC CUC UUC UAC UUC\nAAC AUG GGC A-30 (reverse); and (CD82-HSS105654)\n50-UCU CGA AUG AGC UCA GUC ACG AUG\nC-30(forward) and 5 0-GCA UCG UGA CUG AGC UCA\nUUC GAG A-3 0 (reverse). The results showed that the\nsilencing efficiency of the first one is best, so in all\nsubsequent experiments, we used this siRNA to silence\nCD82 expression in eutopic ESCs, with non-targeting\nsiRNA as control.\nTreatment in vitro with E 2 and TCDD\nAfter starvation for 12 h, the ESCs (1 !105 cells/well;\nFCS of cultured media was the charcoal stripped FCS)\nfrom women with ( nZ6) or without endometriosis\n(both the sample number of eutopic and ectopic was 6)\nwere treated, respectively, with TCDD (Sigma) or E\n2\nCD82 suppression in the endometriotic milieu . M-QL I and others 197\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\n(Sigma) at concentrations ranging from 10 K12 to\n10K7 M for 48 h, to observe the effect of TCDD or E 2\non CD82 expression in ESCs. In the subsequent\ninvestigation, ESCs were treated with TCDD at\n10\nK9 M, E 2 at 10 K8 M, or the combination of TCDD\nwith E 2 for 48 h, respectively, with vehicle dimethyl\nsulfoxide (DMSO) as control. Each experiment was\ncarried out in triplicate, and repeated three times.\nContact co-culture of two sorts of cells\nThe eutopic ESCs ( nZ6) from endometrium with\nendometriosis were cultured in 24-well plates at a\nconcentration of 1 !10\n5 cells/well until adhering to\nthe plastic. The media was removed, and then the U937\ncells (2!104,1 !105,o r5 !105 cells/well) were applied.\nThe proportion of ESCs and U937 cells was 5:1, 1:1, and\n1:5 respectively. The cells were cultured in a final volume\nof 200 ml fresh DMEM/F-12 with 10% FCS for 48 h. The\nESCs of 1 !10\n5 cells/well cultured alone were used as\ncontrols. Then, we discarded the suspended U937 cells\nand co-cultured supernatant, and used in-cell Western to\nanalysis the CD82 expression. Each experiment was\ncarried out in triplicate, and repeated three times.\nTreatment in vitro with CCR2 antagonist RS102895\nand anti-CCL2 neutralizing antibody\nThe primary ESCs ( nZ6) or CD82-silenced ESCs\n(nZ6) from normal endometrium were treated with\nvarious concentration of RS102895 (a CCR2 antagonist,\n0–500 ng/ml, Sigma) or anti-CCL2 neutralizing\nantibody (0–5 mg/ml, R&D Systems, Abingdon, UK),\n0\n.1% DMSO was used as control. After 24, 48, and 72 h\nof culture, the cells were detected by Matrigel invasion\nassay. In the subsequent investigation, these ESCs and\nCD82-silenced ESCs were treated with RS102895 at\n100 ng/ml or anti-CCL2 neutralizing antibody at\n1 mg/ml for 48 h. Each experiment was carried out in\ntriplicate, and repeated three times.\nMatrigel invasion assay\nThe invasion of the ESCs (nZ6) or CD82-silenced ESCs\n(nZ6) from normal endometrium across Matrigel was\nevaluated objectively in an invasion chamber, based on\nour previous procedure (Yu et al. 2008). Briefly, the cell\ninserts (8 mm pore size, 6\n.5 mm diameter; Corning,\nNew York, NY, USA) coated with 15–25 ml Matrigel\nwere placed in a 24-well plate. The primary ESCs or\nsiRNA-transfected ESCs of 2 !104 were plated in the\nupper chamber (the media contained 1% charcoal\nstripped FCS). TCDD or/and E\n2,M A P Ki n h i b i t o r\nU0126 (30 mM), anti-integrin b1 neutralizing antibody\n(1 mg/106 cells, R&D Systems), RS102895, or anti-CCL2\nneutralizing antibody were added respectively. The\nlower chamber (the media contained 5% charcoal\nstripped FCS) was filled with 800 ml medium. The cells\nwere then incubated at 37 8C for 48 h. The inserts were\nremoved, washed in PBS and the non-invading cells\ntogether with the Matrigel were removed from the\nupper surface of the filter by wiping with a cotton bud.\nThe inserts were then fixed in methanol for 10 min at\nroom temperature and stained with hematoxylin. The\nresult was observed under Olympus BX51 CDP70\nmicroscope (Olympus, Tokyo, Japan). The cells that\nhad migrated to the lower surfaces were counted at a\nmagnification of !200. At same time, we seeded the\nsame cells with Matrigel invasion assay in a 96-well plate,\nand detected the protein concentration by BCA Protein\nAssay (Beyotime Institute of Biotechnology, China).\nThe invasion index of each group was calculated as the\nratio of the number of cells migrated to the lower\nsurfaces to the protein concentration. Each experiment\nwas carried out in triplicate, and repeated three times.\nIn-cell Western\nAccording to the description byEgorina et al. (2006),w e\nused a newly set up assay called in-cell Western to\ndetermine the in-cell protein level of CD82, CCR2,\nMMP2, MMP9, TIMP1, TIMP2, integrinb1, and integri-\nnanb3. Further details are given in the Supplementary\nMaterials and methods, and the precision analysis of the\nin-cell Western has been provided in Supplementary\nFigure 1(see section onsupplementary data given at the\nend of this article). The procedure was as follows:\nnormal ESCs (nZ6) or siRNA-transfected normal ESCs\n(nZ6) in 96-well plate were incubated with or without\nU0126 (30 mmol/l; Cell Signaling Technology, Danvers,\nMA, USA), and anti-integrin b1 neutralizing antibody\n(1 mg/10\n6 cells; R&D Systems) for another 24 h, or\ntreated with TCDD or E2 or combination of TCDD and\nE2 for 48 h, respectively, with vehicle as control. Then\ncells were immediately fixed with 4% formaldehyde in\nPBS for 20 min at room temperature. After washing with\n0\n.1% Triton, the cells were blocked by adding 150 mlo f\nLI-COR Odyssey Blocking Buffer (LI-COR Biosciences,\nLincoln, NE, USA) for 90 min at room temperature.\nThe cells were incubated with mouse anti-human CD82\n(20 mg/ml, SC-15572; Santa Cruz Biotechnology, Santa\nCruz, CA, USA) or goat anti-human CCR2 (1:80; Abcam,\nCambridge, MA, USA) or mouse anti-human MMP2\n(20 mg/ml, R&D Systems), MMP9 (20 mg/ml, R&D\nSystems), TIMP1 (15 mg/ml, R&D Systems) or TIMP2\n(15 mg/ml, R&D Systems) or integrin b1 (10 mg/ml,\nR&D Systems), or intergrin anb3( 1 0 mg/ml, R&D\nSystems) antibody. To assess the housekeeping\nprotein actin, rabbit anti-human actin (Santa Cruz\nBiotechnology) was added to each well at the same\ntime as an internal control. After overnight treatment\nM-QL I and others . CD82 suppression in the endometriotic milieu198\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nat 4 8C, the wells were incubated with corresponding\nsecond IRDye 700DX-conjugated affinity purified (red\nfluorescence) anti-mouse and IRDye 800DX-conjugated\naffinity purified (green flu orescence) anti-rabbit.\nHowever, for the CCR2 detection group, the wells were\nincubated with corresponding second IRDye 700DX-\nconjugated affinity purified (red fluorescence) anti-\nrabbit and IRDye 800DX-conjugated affinity purified\n(green fluorescence) anti-goat, fluorescence antibody\nrecommended by the manufacturer (Rockland, Inc.,\nGilbertsville, PA, USA). This procedure must be carried\nout in the dark. Images of target gene were obtained\nusing the Odyssey Infrared Imaging System (LI-COR\nBiosciences). The expression level of the correspondent\nmolecules was calculated as the ratio of the intensity of\ntarget gene to actin. The experiments were carried out\nin triplicate, and repeated three times.\nWestern blot analysis\nTotal protein extracted from primary cultured ESCs\nfrom the endometriotic ( nZ6) or endometrial tissues\n(both the sample number of eutopic and normal groups\nwas 6) was prepared using RIPA buffer. Then 30 mg\nprotein was loaded onto 10% polyacrylamide-SDS gels.\nThe resolved protein was transferred onto polyvinyl-\nidene difluoride membranes (Bio-Rad), and incubated\nwith a 1:500 dilution of mouse anti-human CD82\nmonoclonal antibody (SC-15572, Santa Cruz Bio-\ntechnology) and a 1:1000 dilution of mouse anti-\nhuman b-actin monoclonal antibody (Santa Cruz\nBiotechnology) in PBS containing 0\n.05% Tween-20\nand 5% FCS respectively. After an extensive washing,\nthe bound primary antibodies were detected by a 1:5000\ndilution of HRP-conjugated goat anti-mouse IgG\n(Southern Biotechnology Associates, Inc., Birmingham,\nAL, USA), respectively, with a chemiluminescent detec-\ntion system. The experiments were repeated three times.\nELISA for determination of CCL2\nESCs or siRNA-transfected ESCs (2!105 cells/well) from\nnormal endometrium in 24-well plates were treated with\nTCDD or/and E 2, or treated with U0126 (30 mmol/l)\nand anti-integrin b1 neutralizing antibody for 48 h,\nrespectively, and then the culture supernatant was\nharvested, centrifuged to remove cellular debris, and\nstore at K80 8C until being assayed by ELISA. The CCL2\nconcentration in the supernatant was quantified by\nELISA kits (R&D Systems) according to the manufac-\nturer’s instruction. At the same time, we detected the\nprotein concentration of each group, and the CCL2 level\nof each group was calculated as the ratio of the CCL2\nconcentration of supernatant to the protein concen-\ntration. Each experiment was carried out in triplicate.\nStatistical analysis\nAll values were shown as the mean GS.E.M. Data were\nanalyzed by one-way ANOVA and least significant\ndifference (equal variances assumed) or Tamhane’s\ntest (equal variances not assumed) was used post hoc for\nmultiple comparisons with Statistical Package for the\nSocial Sciences software version 11.5 (SPSS Inc.,\nChicago, IL, USA). Differences were considered as stati-\nstically significant at P!0\n.05.\nResults\nThe expression of CD82 is decreased in primary ESCs\nfrom patients with endometriosis\nTo clarify the relationship of CD82 expression in\nendometriosis, we collected the endometriotic and\nendometrial tissues from women with or without\nendometriosis, and then detected the mRNA and\nprotein levels of CD82 in ESCs by quantitative real-\ntime PCR, western blot and in-cell Western. As shown,\nthe mRNA level of CD82 in the normal ESCs without\nendometriosis ( nZ6) is 2\n.824-fold ( P!0.05) and\n11.636-fold ( P!0.01) higher than that of the eutopic\n(nZ6) and ectopic ESCs ( nZ6) with endometriosis,\nrespectively ( Fig. 1a ). Consistent with transcription\nlevel, the normal ESCs show a significant higher CD82\nprotein expression than that of eutopic ESCs (P!0.05),\nand the latter is further higher significantly than that of\nthe ectopic ESCs ( P!0.05 or P!0.01) by western blot\nand in-cell Western (Fig. 1b and c). These results above\nsuggest that low expression of CD82 in the eutopic and\nectopic ESCs may be involved in the occurrence and\ndevelopment of endometriosis.\nThe combination of TCDD with E 2 or co-culture with\nU937 downregulates CD82 expression in ESCs\nTCDD alone can significantly inhibit expression of\nCD82 in the ESCs from endometrium with or without\nendometriosis, especially the concentration 10 K9 M\n(P!0.05 or P!0.01; Fig. 2a), but E2 alone increases the\nexpression of CD82 in normal ESCs and eutopic ESCs,\nespecially the concentration 10\nK8 M( P!0.05; Fig. 2b).\nEither TCDD or E2 alone shows no obvious effect on the\nCD82 expression in the ectopic ESCs. Interestingly, the\ncombination of TCDD with E\n2 has a further inhibition\non the CD82 expression in the ESCs from endome-\ntrium with or without endometriosis (P!0.05; Fig. 2c),\nbut the expression of CD82 in the ectopic ESCs has not\nsignificantly changed. The results indicate that the\ncombination of TCDD with estrogen may downregulate\nthe expression of CD82 in the eutopic ESCs with\nendometriosis.\nCD82 suppression in the endometriotic milieu . M-QL I and others 199\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nTo further explore the possible roles of the abnormal\nlower CD82 expression in the ectopic ESCs, we\nestablished the contact co-culture of U937 cells and\neutopic ESCs from endometrium with endometriosis.\nU937 cells are monocytes, which may differentiate into\nmacrophages in tissues or in response to stimuli. First,\nwe observed, respectively, ESCs and U937 cells by an\nOlympus BX51 fluorescence microscope (Olympus)\nbefore and after the co-culture ( Supplementary\nFigure 2a, see section on supplementary data given at\nthe end of this article). Moreover, we used immuno-\ncytochemical staining to identify the cells after super-\nnatants of co-culture were discarded ( Supplementary\nFigure 2b, see section on supplementary data given at\nthe end of this article). As shown in Fig. 2d, U937 can\ninhibit CD82 expression in the ESCs in a dosage-\ndependent manner ( P!0\n.05 or P!0.01), which\nsuggests that CD82 expression in the retrograded\nendometrial cells into the peritoneal cavity is further\ndecreased owing to interaction with the increased active\nmacrophages in peritoneal fluid with endometriosis.\nSilencing of CD82 enhances the invasion of ESCs\nTo test the effects of CD82 on the invasion of ESCs, we\nsilenced the CD82 expression of the primary normal\nESCs ( nZ6) by siRNA transfection ( P!0.01; Fig. 3a\nand b), and then a matrigel-based transwell was carried\nout. The silenced ESCs were added to the upper\nchamber, the number of cells migrating to the lower\nsurface was counted in 72 h of incubation. Meanwhile,\nthe total protein concentration of ESCs in each group\nwas analyzed. The invasion index of each group was\ncalculated as the ratio of the number of cells migrated\nto the lower surfaces to the protein concentration.\nAs shown in Fig. 3c , the CD82 silence in ESCs can\nsignificantly enhance the invasiveness of ESCs\ncompared with the si-negative control ( P!0\n.01).\nThereafter, we further investigated invasiveness of\nthe silenced ESCs without endometriosis after treated\nwith TCDD or/and E\n2 for another 48 h. The results\nshow that TCDD or/and E 2 also increase the invasion\nof ESCs ( P!0.05 or P!0.01; Fig. 3d), which echoes a\nprevious result in eutopic ESCs from women with\nendometriosis (Yu et al. 2008). Therefore, combination\nof TCDD with E\n2 presents a synergistic role with CD82\nsilence in the upregulation of ESC invasiveness\n(P!0.01; Fig. 3d).\nCD82 suppresses ESCs invasion by inhibiting the\nCCL2 secretion and CCR2 expression\nTo clarify whether CD82 regulates CCL2 secretion\nand CCR2 expression in ESCs, ELISA and in-cell\nWestern were used to analyze CCL2 secretion and\nCCR2 expression in ESCs. The results show that CD82\ncan significantly inhibit CCL2 secretion ( P!0\n.05;\nFig. 4a ) and CCR2 expression in the normal ESCs\n(P!0.01; Fig. 4b) from patients without endometriosis\n(nZ6). We further evaluated the invasiveness of ESCs\ntreated with various concentrations of RS102895 or\nanti-CCL2 neutralizing antibody. It is shown in Fig. 4c\nand d that both RS102895 and anti-CCL2 neutralizing\nantibody can decrease the invasiveness of ESCs\n(P!0\n.05 or P!0.01), the optimal concentration is\n100 ng/ml and 1 mg/ml respectively.\nThe results above indicate that CD82 may inhibit\nthe invasion of ESCs through downregulating CCL2\nproduction and CCR2 expression to some extent.\nTherefore, it can be speculated that the decreased\nexpression of CD82 in ectopic ESCs may recruit more\nmacrophages into the peritoneal cavity via upregulating\nCCL2 secretion, which in turn leads to the further\ndecline of CD82 expression in the ESCs, and such a\nvicious circle in the endometriotic milieu.\nTo elucidate the regulatory mechanism of CD82\nexpression on ESCs invasion, we evaluated the invasion\nof the CD82-silenced ESCs after treated with RS102895\nor anti-CCL2 neutralizing antibody. The results show\nthat both RS102895 and anti-CCL2 neutralizing\nantibody can abolish completely the increased invasion\nof the CD82-silenced ESCs ( P!0\n.01; Fig. 4e).\n1·2\n(a)\nCD82 mRNA level\n1\n0·8\n0·6\n0·4\n0·2\n0\nNormal Eutopic\n**\n**\n#\nEctopic (ESCs)\n(b)\nCD82 protein level\n1\n0·8\n0·6\n0·4\n0·2\n0\nNormal\nCD82\n(46 kDa)\nβ-Actin\n(43 kDa)\nEutopic\n*\n**\n#\nEctopic (ESCs)\n1·6\n(c)\nCD82 mRNA level\n1·2\n0·8\n0·4\n0\nNormal Eutopic\n*\n**\n##\nEctopic (ESCs)\nFigure 1 The expression of CD82 is decreased in the primary\nESCs from eutopic endometrium and endometriotic tissues.\nCD82 mRNA and protein levels in human ESCs were analyzed\nby quantitative real-time PCR (a), western blot (b), or in-cell\nWestern (c). The relative fold mRNA and protein levels of CD82 in\nESCs were compared between the eutopic ESCs, ectopic ESCs,\nand normal ESCs. CD82 (red) and actin (green). Results were\nhighly reproducible in three independent experiments. In this\nstudy, normal, ESCs from endometrium without endometriosis;\neutopic and ectopic, ESCs from eutopic endometrium and ectopic\ntissue with endometriosis respectively. Error bars depict the\nS.E.M.\n*P!0.05 and **P!0.01 compared with the control. #P!0.05\nand ##P!0.01 compared with the eutopic.\nM-QL I and others . CD82 suppression in the endometriotic milieu200\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nCD82 inhibits invasion of ESCs by downregulating\nCCL2 secretion and CCR2 expression via MAPK\nand integrinb1 signal pathway\nAlterations of MMPs, TIMPs and integrins in eutopic\nendometrium are important factors in the development\nof endometriosis (Yoshimura 2002, Collette et al. 2006,\nYu et al . 2008 ). Moreover, the expression of integrins\nwas intimately associated with the function of CD82.\nOur previous work has confirmed that DSCs-expressed\nCD82 controls the invasiveness of trophoblasts through\nsuppressing the integrin b1/MAPK/ERK1/2 signal\npathway (Li et al. 2010). Therefore, we next investigated\nthe protein levels of the invasion-relative molecules\nand integrins in the CD82-silenced ESCs from patients\nwithout endometriosis ( nZ6). It has been clearly\ndemonstrated in Fig. 5a that CD82 silence can obviously\ninhibit the expression of TIMP1 ( P!0.01) and TIMP2\n(P!0.01) and enhance the expression of integrin b1\n(P!0.01) and integrin anb3( P!0.05), but has no\n1·6\nNormal\nEutopic\nEctopic\nNormal\nEutopic\nEctopic\n(a)\nCD82 protein level\n1·2\n0·8\n0·4\n0\nCon TCDD\n**\n* *\n## ## ## ##\n* 1·5\n1·8\nNormal\nEutopic\nEctopic\nNormal\nEutopic\nEctopic\n(b)\nCD82 protein level\n1·2\n0·6\n0·9\n0·3\n0\nCon E 2\n**\n*\n*\n# #\n# #\n* *\n1·6\n2\nNormal\nEutopic\nEctopic\nNormal\nEutopic\nEctopic\n(c)\nCD82 protein level\n1·2\n0·8\n0·4\n0\nE2:–\nTCDD: –\n+\n–\n–\n+\n+\n+\n**\n**\n∆\n##\n∆\n* *\n*\n##\n#\n1\n1·2\n(d)\nCD82 protein level\n0·8\n0·4\n0·6\n0·2\n0\nCon U937\n**\n*\nFigure 2 The combination of TCDD with 17 b-estradiol or co-culture with U937\ndownregulates CD82 expression in ESCs. (a–c) ESCs were treated, respectively, with\ndifferent concentrations of TCDD (from 10K12 to 10K8 M), 17b-estradiol (from 10K11 to\n10K7 M) or the combination of 10K9 M TCDD and 10K8 M1 7b-estradiol for 48 h, with\nvehicle as controls. In-cell Western indicates that TCDD alone can significantly decrease,\nand 17b-estradiol alone can increase the expression of CD82 in ESCs, but 17b-estradiol\ncan coordinate with TCDD in downregulating CD82 expression. (d) ESCs (1 !10\n5\ncell/well) from eutopic endometrium were co-cultured with U937 of 2 !104,1 !105,o r\n5!105 cell/well for 48 h, respectively, with ESCs cultured alone as control. Thereafter,\nin-cell Western was used to detect the CD82 expression in ESCs. CD82 (red) and actin\n(green). Results were highly reproducible in three independent experiments. Error bars\ndepict the\nS.E.M.* P!0.05 and **P!0.01 compared with the normal ESCs control or\nESCs without co-culture. #P!0.05 and ##P!0.01 compared with the eutopic ESCs\ncontrol. OP!0.05 compared with TCDD treatment.\nCD82 suppression in the endometriotic milieu . M-QL I and others 201\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\neffect on the expression of MMP2 and MMP9 in the\nESCs. These results indicate that the decreased CD82\nexpression in ESCs may lead to not only the abnormal\nincrease in invasion through suppressing the TIMP1\nand TIMP2 expression but also the abnormal enhanced\nadhesion of ESCs to ECMs through increasing the\nexpression of integrin b1 and integrin anb3, which is\nattributed to the onset and development of\nendometriosis.\nTo explore the molecular mechanisms of CCL2\nand CCR2 expression regulated by CD82 in ESCs,\nwe used U0126 or anti-integrin b1 neutralizing\nantibody to treat the CD82-silenced ESCs for another\n24 h. We have found U0126 and anti-integrin b1\nneutralizing antibody can decrease CCL2 production\nand CCR2 expression in ESCs ( P!0\n.05 or P!0.01;\nFig. 5b). Furthermore, either U0126 or anti-integrin b1\nneutralizing antibody can abolish completely the\nstimulating effects on CCL2 secretion and CCR2\nexpression induced by CD82 silence (Fig. 5b).\nTo understand the relationship of CCL2/CCR2 to\nTIMPs, we treated the CD82-silenced ESCs with U0126\nor anti-integrin b1 neutralizing antibody for another\n24 h, or anti-CCL2 neutralizing antibody or RS102895\nfor another 48 h. As shown in Fig. 5c, all the treatments\ncan significantly promote TIMP1 and TIMP2 pro-\nduction, and completely reverse the inhibition effects\non TIMP1 and TIMP2 induced by CD82 silence.\nTherefore, CCL2/CCR2 interaction downregulates\nthe expression of TIMP1 and TIMP2 in an autocrine\nmanner, which controls the ESC invasion.\nMoreover, we tested whether MAPK and integrin b1\nsignal pathway was involved in the regulation of\nESCs invasion by CD82. As shown, either U0126 or\n1·5\n(a)\nCD82 protein level\n1·2\n0·9\n0·6\n0·3\n0\nBlank Control Silence\n**\n140\n(b)CD82 protein level\n(change from blank %)\n120\n100\n80\n20\n40\n60\n0\nBlank Control Silence\n**\n**250\n(c)Invasion index\n(change from control %)\n50\n100\n150\n200\n0\nControl Silence\n700\n(d)Invasion index\n(change from control %)\n100\n200\n300\n600\n500\n400\n0\nSilence:\nE2:\nTCDD:\n–\n–\n–\n+\n–\n–\n–\n+\n–\n+\n+\n–\n–\n–\n+\n+\n–\n+\n–\n+\n+\n+\n+\n+\n**\n**\n*\n** ##\n# #\nCD82\n(46 kDa)\nβ-Actin\n(43 kDa)\nFigure 3 CD82 silence enhances the invasion of ESCs. The ESCs from endometrium\nwithout endometriosis were silenced for CD82 for 72 h, and the efficiency was\ndemonstrated by in-cell Western (a) and western blot (b) respectively. CD82 (red) and actin\n(green). (c) After CD82 in these ESCs was silenced, the invasiveness of ESCs was\nenhanced apparently. (d) After CD82 had been knocked down, ESCs were treated with\nTCDD, 17b-estradiol or the combination of both for another 48 h, and then the invasiveness\nof ESCs was detected. TCDD, 17b-estradiol or the combination can promote the invasion\nof ESCs. Moreover, TCDD or the combination shows a synergistic effect with the CD82\nsilence in elevating invasiveness (the ratio of the cells migrated to the lower chamber to the\nprotein concentration of total cells) of ESCs. These pictures are representatives of three\nindividual experiments. Blank, no transfection; control, the non-targeting siRNA\noligonucleotides; silence, CD82 is knocked down. Error bars depict the\nS.E.M.* P!0.05 and\n**P!0.01 compared with the negative control.#P!0.05 and ##P!0.01 compared with the\nCD82 silence.\nM-QL I and others . CD82 suppression in the endometriotic milieu202\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nanti-integrinb1 neutralizing antibody can also abolish\ncompletely the pro-invasion effect on ESCs resulted\nfrom CD82 silence ( P!0.01; Fig. 5d).\nThus, our research above supports the idea that\nCD82 inhibits ESCs invasion by the downregulation of\nCCL2 secretion, CCR2 expression, and the upregula-\ntion of TIMP1 and TIMP2 expression via MAPK and\nintegrinb1 signal pathway.\nThe combination of TCDD with E 2 stimulates CCL2\nsecretion, CCR2 expression and invasion of ESCs\nby downregulating CD82 expression\nTo further test the effect of TCDD or/and E2 on CCL2\nsecretion and CCR2 expression via CD82, we treated\nnormal ESCs (nZ6) or the CD82-silenced ESCs (nZ6)\nwith TCDD, E2 or the combination of both. The results\nshow that TCDD alone or combined with E 2 stimulates\nCCL2 secretion and CCR2 expression in ESCs\n(P!0.01), and presents the synergistic action with\nCD82 silence (Fig. 6a and b).\nMoreover, our results show that CCR2 blocker,\nRS102895, or anti-CCL2 neutralizing antibody\nabolishes completely the increased invasiveness\ninduced by the combination of TCDD with E 2\n(P!0.01; Fig. 6c).\nFrom the results above, it may be concluded that the\ncombination of TCDD with E 2 stimulates the ESCs\ninvasion through downregulating CD82 expression and\nupregulating CCL2 secretion and CCR2 expression,\nwhich eventually leads to the occurrence and pro-\ngression of endometriosis.\nDiscussion\nEndometriosis is considered to be a pathological\ndisorder caused by interaction of multiple molecules\nincluding steroid exposure, immunological disturb-\nances, genetic predisposition, and environmental\ntoxin exposure ( Osteen et al . 2003 ). The adherence\nand invasion of the retrograded endometrial cells into\nthe peritoneum is a key step for the early stage of\nendometriosis, and the retrograded ESCs are respon-\nsible for the adherence and implantation of endome-\ntrium to peritoneum in the early stage of endometriosis\n(Witz et al . 2001 ). It has been suggested that several\n4000\n(a) (b)\n(c) (d) (e)\nCCL2 secretion\n(pg/100 ug protein)\nInvasion index\n(change from control %)\nInvasion index\n(change from control %)\n3500\n3000\n2500\n2000\n1500\n1000\n500\n0\n120 250\n200\n150\n100\n50\n0\n100\n80\n60\n40\n20\nCon Conα -CCL2 RS102895\nSilence:\nRS102895:\n–+–+–+\n––++––\nα -CCL2: ––––++\n** ** **\n*\n**\n**\n**\n**\n** ## ##\n0\nInvasion index\n(change from control %)\n120\n100\n80\n60\n40\n20\n0\n72h 96h 120h\n*\nBlank\nBlank\n2\n1·6\n1·2\nCCR2 protein level\n0·8\n0·4\n0\nControl\nControl\nSilence\nSilence\nFigure 4 CD82 suppresses ESCs invasion by inhibiting CCL2 secretion and CCR2\nexpression. (a and b) CCL2 secretion and CCR2 expression in ESCs from endometrium\nwithout endometriosis were increased at 120 h after the CD82 silence. The CCL2 level was\ncalculated as the ratio of the secretion of CCL2 in supernatant by ELISA to the protein\nconcentration of total cells. CCR2 (green) and actin (red). ESCs were treated with anti-CCL2\nneutralizing antibody (0, 0\n.2, 1, and 5 mg/ml) or RS102895 (0, 10, 50, 100, and 500 ng/ml),\nthen the invasion (c and d) of ESCs was detected by Matrigel invasion assay. The results\nindicate that both RS102895 and anti-CCL2 neutralizing antibody can inhibit the\ninvasiveness of ESCs. (e) After CD82 was silenced in ESCs, the ESCs were treated,\nrespectively, with anti-CCL2 neutralizing antibody at 1 mg/ml or RS102895 at 100 ng/ml for\nanother 48 h, and then the invasiveness of ESCs was detected. RS102895 or anti-CCL2\nneutralizing antibody can block the increased invasiveness of ESCs induced by CD82\nsilence. The cell invasiveness was calculated as the ratio of the cells migrated to the lower\nchamber to the protein concentration of total cells. The pictures are representatives of three\nindividual experiments. In this study, control, the non-targeting siRNA oligonucleotides;\nsilence, CD82 is knocked down. Error bars depict the\nS.E.M.* P!0.05 and **P!0.01\ncompared with the control. ##P!0.01 compared with CD82 silence.\nCD82 suppression in the endometriotic milieu . M-QL I and others 203\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nProtein level\n0\n0·2\n0·4\n0·6\n0·8\n1\nProtein levelProtein level\nProtein level\n0\n0·4\n0·8\n1·2\nProtein level\n2\n1·6\n0\n0·2\n0·4\n0·6\n0·8\n1\n1·2\nCCR2 protein level\n1·8\n1·6\n1·4\n0\n0·2\n0·4\n0·6\n0·8\n1\n1·2\n0\n0·5\n1\n1·5\nIntegrinανβ 3\nIntegrinανβ 3\nIntegrinβ1\nIntegrinβ1 2 ***\n1·6\n1·4\nProtein level\nProtein level\nSilence\n**\nControlBlankSilenceControlBlank\nSilenceControlBlankSilenceControlBlank Silence ControlBlank\nSilenceControlBlank 0\n0·2\n0·4\n0·6\n0·8\n1\n0\n0·2\n0·4\n0·6\n0·8\n1·0\n1·2\n1·6\n1·4\n0\n0·2\n0·4\n0·6\n0·8\n1\n1·2\nMMP2 MMP9 TIMP1\n–\n+\n–\n–\n+\n+\n–\n–\n+\n+\n–\n+\n+\n–\n–\n–\n–\n–\nα -Integrinβ1:\nU0126:\nSilence:\n–\n+\n–\n–\n+\n+\n+\n–\n+\n–\n–\n+\n–\n+\n–\n–\n–\n–\n–\n–\n–\n–\n–\n–\n–\n–\n+\n–\n–\n–\n–\n–\n–\n–\n–\n+\n–\n–\n+\n+\n–\n+\n+\n–\n–\n–\n–\n–\n–\n–\nTIMP2\nTIMP1\nRS102895:\nα -CCL2:\nα -Integrinβ1:\nU0126:\nSilence:\n–\n+\n–\n–\n+\n+\n–\n–\n+\n+\n–\n+\n+\n–\n–\n–\n–\n–\nα -Integrinβ1:\nU0126:\nSilence:\n–\n+\n–\n–\n+\n+\n–\n–\n+\n+\n–\n+\n+\n–\n–\n–\n–\n–\nα -Integrinβ1:\nU0126:\nSilence:\n0\n500\n1000\n1500\n2000\n2500\n3000\n3500\nCCL2 secretion\n(pg/100 µg protein)\n##\n####\n##\n**\n**\n**\n**\n****\n**** **\n**\n**\n**\n** ####\n0\n50\n100\n150\nInvasion index\n(change from control %)\n200\n250\n##\n####\n####\n###### ****\n*\n*\n4000\nTIMP2\nTIMP2\nTIMP2\nTIMP1\nTIMP1MMP9MMP2(a)\n(b)\n(c)\n(d)\n1·4\nFigure 5 CD82 inhibits the invasion of ESCs by downregulating CCL2 secretion and CCR2 expression via MAPK and integrinb1 signal\npathway. (a) CD82 upregulates TIMP1 and TIMP2 expression, downregulates integrinb1 and integrinanb3 expression in ESCs, but has\nno influence on MMP2 and MMP9 expression. In this study, MMP2, MMP9, TIMP1, TIMP2, integrinb1, and integrinanb3 are shown in red\nand actin is shown in green. (b) After the silence, ESCs were treated with U0126 or anti-integrinb1 neutralizing antibody for another 24 h,\nand then the CCL2 secretion and CCR2 expression were detected by ELISA and in-cell Western, respectively, and the invasion of ESCs\n(d) by invasion assay. The results show that U0126 or anti-integrinb1 neutralizing antibody can abolish completely the increase of CCL2\nsecretion, CCR2 expression and invasion of ESCs induced by CD82 silence. CCR2 (green) and actin (red). (c) We treated CD82-silenced\nESCs with U0126 or anti-integrinb1 neutralizing antibody for another 24 h, or anti-CCL2 neutralizing antibody or RS102895 for another\n48h, and then TIMP1 and TIMP2 expression were evaluated by in-cell Western. TIMP1 and TIMP2 (red) and actin (green). The CCL2\nlevel was calculated as the ratio of the secretion of CCL2 in supernatant by ELISA to the protein concentration of total cells. The cell\ninvasiveness was calculated as the ratio of the cells migrated to the lower chamber to the protein concentration of total cells. Results\nwere highly reproducible in three independent experiments. In this study, control, the non-targeting siRNA oligonucleotides; silence,\nCD82 is knocked down. Error bars depict the\nS.E.M.* P!0.05 and **P!0.01 compared with the negative control. ##P!0.01 compared\nwith CD82 silence.\nM-QL I and others . CD82 suppression in the endometriotic milieu204\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\ntumor relative genes are involved in susceptibility to\nendometriosis. In view of that, we first evaluated the\nmRNA and protein levels of CD82 in primary ESCs from\nendometrium with or without endometriosis or the\nectopic ESCs. We have found that the expression of\nmetastasis suppressor CD82 in the eutopic and ectopic\nESCs with endometriosis is significantly decreased,\nespecially for the ectopic ESCs, which suggests that\nthe abnormal lower CD82 in the eutopic and ectopic\nESCs may induce abnormal increase of its invasion,\ninitiating the invasion and implantation of the shed\nendometrium to peritoneum.\nTo analyze the potential mechanisms of CD82\nreduction in the eutopic and ectopic tissue, we detected\nthe CD82 expression in the eutopic and ectopic ESCs\nafter treatment with TCDD or/and E\n2. TCDD has\nbeen viewed as a toxic compound with both estrogenic\nand anti-estrogenic activity ( Grochowalski et al . 2001).\nIn this study, we have demonstrated that E\n2 alone\nincreases and TCDD alone decreases CD82 expression,\nrespectively, while CD82 in the eutopic ESCs is reduced\nfurther after the treatment with the combination of\nTCDD with E\n2. Hence, TCDD and estrogen have the\nsynergistic effect on CD82 expression. Moreover,\nthe expression of CD82 is decreased when the eutopic\nESCs were co-cultured with macrophages, which\nsuggests that the abnormal decrease of CD82\nexpression in the ectopic ESCs is a direct response to\nthe specific microenvironment in the ectopic milieu.\nIn agreement with our previous studies ( Shi et al .\n2006, Yu et al . 2008 ), the immuno-inflammatory\nmicroenvironments mediated by numerous cytokines\nand growth factors, especially for chemokine, are now\nbelieved to play an important role in the progression\nof endometriosis. However, previous studies about\nCCL2 focused mainly on its chemotactic effect on\nmonocyte–macrophages. In this study, we investigated\nthe role and regulating mechanism of metastasis\nsuppressor CD82 in the onset and development of\nendometriosis, and analyzed whether TCDD, E\n2, CCL2,\nand receptor CCR2 participated in the CD82-mediated\nprogression. We have demonstrated that the ESC-\nexpressed CD82 inhibits the invasion of ESCs them-\nselves. Interestingly, our observation has shown that\nCD82 in ESCs downregulates the CCL2 secretion and\nCCR2 expression of ESCs, and CCR2 antagonist,\nRS102895, or anti-CCL2 neutralizing antibody\nabolishes the increased invasiveness of ESCs induced\nby CD82 silence. Thus, it can be concluded that\nthe abnormal decreased CD82 expression in the\nectopic ESCs may lead to invasiveness increase of\nESCs through stimulating the CCR2 expression and\nCCL2 secretion.\nThis study has shown that CD82 promotes the\nexpression of TIMP1 and TIMP2 in ESCs via inhibiting\nCCL2 and CCR2 production, but does not change\n6000\n5000\n4000\n*\n** **## ##\n∆\nCCL2 secretion pg/100 ug protein)\n3000\n2000\n1000\n2\nSilence:\nTCDD:\nE\n2:\nSilence: –\n–\n– –\n–\n–\n––\n–\n–\n–\n–+\n+\n++\n+\n+\n++\n+\n+\n+\n+\nTCDD:\nE2:\nRS102895:\nα -CCL2:\nTCDD:\nE2:\n** **\n**\n∆# #\n#\n1·6\n1·2\nCCR2 protein level\n0·8\n0·4\n0\n700\n*\n**\n**\n## ## ##\n600\n500\nInvasion index\n(change from control %)\n400\n300\n200\n100\n0\n0\n–––\n–\n–\n–\n––\n–\n–\n–\n–\n++\n++\n+\n+\n++\n++\n+\n+\n–\n–\n–\n–\n–\n–\n––\n––\n––\n–\n–\n+\n++\n++\n+\n+++\n+\n+\n+\n+\n+\n(a)\n(b)\n(c)\nFigure 6 The combination of TCDD with 17b-estradiol stimulates\nCCL2 secretion, CCR2 expression and invasion of ESCs by\ndownregulating CD82 expression. (a and b) After CD82 was\nsilenced in normal ESCs (nZ6), the ESCs were treated with\nTCDD, 17b-estradiol or the combination of both for another 48 h,\nand then CCL2 secretion and CCR2 expression of ESCs were\ndetected by ELISA and in-cell Western respectively. TCDD not\nonly stimulates CCL2 secretion and CCR2 expression in ESCs,\nbut also recovers the increase of CCL2 secretion and CCR2\nexpression in ESCs induced by CD82 silence. CCR2 (green) and\nactin (red). (c) The ESCs were treated with TCDD, 17b-estradiol,\nor the combination of both with RS102895 or/and anti-CCL2\nneutralizing antibody for 48 h respectively. Thereafter, the\ninvasion of ESCs was detected by Matrigel invasion assay. The\nresults indicate that either RS102895 or anti-CCL2 neutralizing\nantibody abolishes completely the invasion enhancement of ESCs\ninduced by the combination of TCDD and 17b-estradiol. The\nCCL2 level was calculated as the ratio of the secretion of CCL2 in\nsupernatant by ELISA to the protein concentration of total cells.\nMeanwhile, the cell invasiveness was calculated by the ratio of the\ncells migrated to the lower chamber to the protein concentration of\ntotal cells. These pictures are representatives of three individual\nexperiments. Error bars depict the\nS.E.M.* P!0.05 and **P!0.01\ncompared with the negative control. #P!0.05 and ##P!0.01\ncompared with CD82 silence. DP!0.05 compared with E2 treat-\nment group.\nCD82 suppression in the endometriotic milieu . M-QL I and others 205\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nMMP2 and MMP9 expression. Indeed, the invasion-\nrelative molecules, such as MMPs and their inhibitor\nTIMPs, are correlated to onset and progression of\nendometriosis, which suggests that the CD82-regulated\nTIMP1 and TIMP2 expression decrease may upregulate\nthe ESCs invasion, which plays roles in pathogenesis\nof endometriosis.\nMeanwhile, we have found that CD82 can inhibit\nintegrinb1 and integrin anb3 expression in ESCs. Cell\nadhesion molecules, such as integrins and cadherins,\nhave been shown to be involved in the shedding of\nendometrium during menstruation and in the\nadhesion of endometrial cells to the peritoneum\n(Lessey et al . 1994 , Lessey & Young 1997 ). A recent\nstudy has shown that interaction between galectin-3 and\nintegrinb3 promotes endometrial cell proliferation and\nadhesion (Lei et al. 2009). The adhesion of endometrial\nstromal cells to various ECM proteins induces an\nupregulation in CCL2 gene expression and protein\nsecretion by integrin b1( Garcia-Velasco et al . 1999 ).\nIn our study, abnormal decrease of CD82 expression in\nthe eutopic and ectopic ESCs from endometrium with\nendometriosis may promote the adhesion and invasion\nof ESCs, stimulates CCL2 secretion, and results in the\ndevelopment of endometriosis through upregulating\nintegrinb1 and integrinanb3 expression via MAPK and\nintegrinb1 signal pathway, which is consistent with\nother studies about CCL2 ( Garcia-Velasco et al . 1999 ,\nHe et al. 2007).\nInterestingly, in our Matrigel invasion test, the\nincreased invasiveness of ESCs induced by TCDD or\ncombination with E 2 can be reversed completely by\nRS102895 or anti-CCL2 neutralizing antibody. On the\nother hand, TCDD alone or combined with E 2 can\ndownregulate CD82 expression, and increase CCL2\nsecretion and CCR2 expression in ESCs.\nIn conclusion, based on our results, a hypothetical\nmodel may be proposed to illustrate the complicated\npathogenesis of endometriosis ( Fig. 7 ). The endome-\ntrium with the abnormal repressed CD82 expression\nowing to inherent defects or the combined effect\nof TCDD and estrogen regurgitates into the peritoneal\ncavity, which upregulates the CCL2 secretion and CCR2\nexpression, invasion, and adhesion of ESCs through\nMAPK and integrin b1 signal pathway. In this pro-\ngression, downregulation of TIMP1 and TIMP2 as\neffective molecules is involved in the invasiveness\nincrease of ESCs. On the other hand, after more\nmacrophages are recruited into the ectopic milieu by\nthe increased CCL2 secretion derived of ESCs, the\nCD82 levels are further downregulated, which leads to a\nvicious circle, and eventually the onset and develop-\nment of endometriosis. Meanwhile, TCDD and estro-\ngen can coordinate to evoke and aggravate the\ninflammatory progression by stimulating other pro-\ninflammatory cytokine secretion, such as RANTES and\nMIP-1a in the endometriotic milieu ( Yu et al. 2008).\nSupplementary data\nThis is linked to the online version of the paper at http://dx.doi.org/\n10.1530/JME-10-0165.\nDeclaration of interest\nThe authors declare that there is no conflict of interest that could be\nperceived as prejudicing the impartiality of the research reported.\nFunding\nThis study was supported by National Basic Research Program of\nChina (2006CB944007) to D-J L; Major International Joint Research\nProject of NSFC (30910103909) to D-J L; National and Shanghai\nLeading Academic Discipline Project (211XK22) to D-J L; Program for\nOutstanding Medical Academic Leader of Shanghai to D-J L; and\nCreative Research Fund for Outstanding Graduate in Key Discipline of\nFudan University (EHF157201) to M-Q L.\nReferences\nAkoum A, Lemay A, McColl SR, Paradis I & Maheux R 1996a Increased\nmonocyte chemotactic protein-1 level and activity in the peripheral\nblood of women with endometriosis. American Journal of Obstetrics\nand Gynecology 175 1620–1625. (doi:10.1016/S0002-9378(96)\n70115-1)\nAkoum A, Turcot-Lemay L, Lemay A, Maheux R & McColl S 1996 b\nElevated concentration and biologic activity of monocyte chemo-\ntactic protein-1 in the peritoneal fluid of patients with endome-\ntriosis. Fertility and Sterility 66 17–23.\nAkoum A, Jolicoeur C & Boucher A 2000 Estradiol amplifies\ninterleukin-1-induced monocyte chemotactic protein-1 expression\nTCDD and estrogen\nCD82\nRecruit\nParacrine CCR2\nMϕ\nMAPK ESC\nERK1/2\nCCL2\nTIMPs\nInvasion\nCCR2\nAutocrine\nIntegrinβ1 Adhesion\nFigure 7 Schematic roles of CD82 regulation in the origin\nand development of endometriosis. The ESCs with the abnormal\nrepressed CD82 expression owing to inherent defects or the\ncombined effect of TCDD and estrogen regurgitates into the\nperitoneal cavity, which upregulates CCL2 secretion and CCR2\nexpression through MAPK and integrinb1 signal pathway.\nDownregulation of TIMP1 and TIMP2 as effective molecules is\ninvolved in the invasiveness increase of ESCs. The increased\nCCL2 secretion recruits more macrophages into the peritoneal\ncavity in a paracrine manner, and these cells further downregulate\nCD82 expression in ESCs, which leads to a vicious circle, and\neventually the onset and development of endometriosis.\nM-QL I and others . CD82 suppression in the endometriotic milieu206\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nby ectopic endometrial cells of women with endometriosis.\nJournal of Clinical Endocrinology and Metabolism 85 896–904. (doi:10.\n1210/jc.85.2.896)\nArici A, Tazuke SI, Oral E, Olive DL & Attar E 1997 Monocyte\nchemotactic protein-1 concentration in peritoneal fluid of women\nwith endometriosis and its modulation of expression in mesothelial\ncells. Fertility and Sterility 67 1065–1072. (doi:10.1016/S0015-\n0282(97)81440-9)\nBandyopadhyay S, Zhan R, Chaudhuri A, Watabe M, Pai SK, Hirota S,\nHosobe S, Tsukada T, Miura K, Takano Y et al. 2006 Interaction of\nCD82 on tumor cells with DARC on vascular endothelium leads to\nmetastasis suppression. Nature Medicine 12 933–938. (doi:10.1038/\nnm1444)\nBirnbaum LS & Cummings AM 2002 Dioxins and endometriosis: a\nplausible hypothesis. Environmental Health Perspectives 110 15–21.\n(doi:10.1289/ehp.0211015)\nBruner-Tran KL, Eisenberg E, Yeaman GR, Anderson TA, McBean J &\nOsteen KG 2002 Steroid and cytokine regulation of matrix\nmetalloproteinase expression in endometriosis and the establish-\nment of experimental endometriosis in nude mice. Journal of\nClinical Endocrinology and Metabolism 87 4782–4791. (doi:10.1210/jc.\n2002-020418)\nCollette T, Maheux R, Mailloux J & Akoum A 2006 Increased\nexpression of matrix metalloproteinase-9 in the eutopic endo-\nmetrial tissue of women with endometriosis. Human Reproduction 21\n3059–3067. (doi:10.1093/humrep/del297)\nEgorina EM, Sovershaev MA & Osterud B 2006 In-cell Western assay: a\nnew approach to visualize tissue factor in human monocytes.\nJournal of Thrombosis and Haemostasis 4 614–620. (doi:10.1111/j.\n1538-7836.2005.01781.x)\nGarcia-Velasco JA, Seli E & Arici A 1999 Regulation of monocyte\nchemotactic protein-1 expression in human endometrial stromal\ncells by integrin-dependent cell adhesion. Biology of Reproduction 61\n548–552. (doi:10.1095/biolreprod61.2.548)\nGellersen B, Briese J, Obernsororfer M, Redlin K, Samalecos A, Richter\nDU, Loning T, Schulte HM & Bamberger AM 2007 Expression of\nthe metastasis suppressor CD82 in decidual cells at the human\nmaternal–fetal interface: regulation and functional implications.\nAmerican Journal of Pathology 170 126–139. (doi:10.2353/ajpath.\n2007.060175)\nGonzalez M, Neufeld J, Reimann K, Wittmann S, Samalecos A, Wolf\nA, Bamberger A-M & Gellersen B 2011 Expansion of human\ntrophoblastic spheroids is promoted by decidualized\nendometrial stromal cells and enhanced by heparin-binding\nepidermal growth factor-like growth factor and interleukin-1\nbeta. Molecular Human Reproduction 17 421–433. ( doi:10.1093/\nmolehr/gar015)\nGrochowalski A, Chrzaszcz R, Pieklo R & Gregoraszczuk EL 2001\nEstrogenic and antiestrogenic effect of in vitro treatment of\nfollicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Chemosphere\n43 823–827. (doi:10.1016/S0045-6535(00)00440-9)\nHe YY, Du MR, Gou PF, He XJ, Zhou WH, Zhu XY & Li DJ 2007\nRegulation of C-C motif chemokine ligand2 and its receptor in\nhuman decidual stromal cells by pregnancy-associated hormones in\nearly gestation. Human Reproduction 22 2733–2742. (doi:10.1093/\nhumrep/dem208)\nHynes RO 1992 Integrins: versatility, modulation, and signaling\nin cell adhesion. Cell 69 11–25. ( doi:10.1016/0092-8674(92)\n90115-S)\nKang S, Zhao XW, Wang N, Chen SC, Zhou RM & Li Y 2008\nAssociation of polymorphisms of the MMP-2 and TIMP-2\ngenes with the risk of endometriosis in North Chinese women.\nFertility and Sterility 90 2023–2029. ( doi:10.1016/j.fertnstert.2007.\n09.068)\nKim JY, Kim H, Suh CS, Kim SH, Choi YM & Kim JG 2008 The\nG(K2518)A polymorphism of monocyte chemotactic protein-1\n(MCP-1) and its serum and peritoneal fluid levels in Korean women\nwith endometriosis. European Journal of Obstetrics, Gynecology, and\nReproductive Biology 139 106–110. (doi:10.1016/j.ejogrb.2007.10.\n009)\nKlemmt PAB, Carver JG, Koninckx P, McVeigh EJ & Mardon HJ 2007\nEndometrial cells from women with endometriosis have increased\nadhesion and proliferative capacity in response to extracellular\nmatrix components: towards a mechanistic model for endome-\ntriosis progression. Human Reproduction 22 3139–3147. (doi:10.\n1093/humrep/dem262)\nKress S & Greenlee WF 1997 Cell-specific regulation of human CYP1A1\nand CYP1B1 genes. Cancer Research 57 1264–1269.\nLei CX, Zhang W, Zhou JP & Liu YK 2009 Interaction between\ngalectin-3 and integrin beta3 in regulating endometrial cell\nproliferation and adhesion. Human Reproduction 24 2879–2889.\n(doi:10.1093/humrep/dep250)\nLessey BA & Young SL 1997 Integrins and other cell adhesion\nmolecules in endometrium and endometriosis. Seminars in\nReproductive Endocrinology 15 291–299. (doi:10.1055/s-2008-\n1068759)\nLessey BA, Castelbaum AJ, Sawin SW, Buch CA, Schinnar R, Bilker W &\nStrom BL 1994 Aberrant integrin expression in the endometrium\nof women with endometriosis. Journal of Clinical Endocrinology and\nMetabolism 79 643–649. (doi:10.1210/jc.79.2.643)\nLi MQ, Hou XF, Shao J, Tang CL & Li DJ 2010 DSCs-expressed CD82\npromoted the expression of TIMP1 and inhibited the invasion of\ntrophoblast cells by integrin b1/MAPK/Erk1/2 signaling pathway\nat materno-fetal interface of human first-trimester pregnancy.\nBiology of Reproduction 82 968–979. (doi:10.1095/biolreprod.109.\n080739)\nMannion BA, Berditchevski F, Kraeft SK, Chen LB & Hemler ME 1996\nTransmembrane-4 superfamily proteins CD81 (TAPA-1), CD82,\nCD63, and CD53 specifically associated with integrin alpha 4 beta 1\n(CD49d/CD29). Journal of Immunology 157 2039–2047.\nMimura J & Fujii-Kuriyama Y 2003 Functional role of AhR in the\nexpression of toxic effects by TCDD. Biochimica et Biophysica Acta\n1619 263–268. (doi:10.1016/S0304-4165(02)00485-3)\nOdintsova E, Sugiura T & Berditchevski F 2000 Attenuation of EGF\nreceptor signaling by a metastasis suppressor, the tetraspanin\nKAI1/CD82. Current Biology 10 1009–1012. (doi:10.1016/S0960-\n9822(00)00652-7)\nOsteen KG, Yeaman GR & Bruner-Tran KL 2003 Matrix metallopro-\nteinases and endometriosis. Seminars in Reproductive Medicine 21\n155–164. (doi:10.1055/s-2003-41322)\nRier S & Foster WG 2002 Environmental dioxins and endometriosis.\nT oxicological Sciences 70 161–170. (doi:10.1093/toxsci/70.2.161)\nRier S & Foster WG 2003 Environmental dioxins and endometriosis.\nSeminars in Reproductive Medicine 21 145–154. (doi:10.1055/s-2003-\n41321)\nRizner TL 2009 Estrogen metabolism and action in endometriosis.\nMolecular and Cellular Endocrinology 307 8–18. (doi:10.1016/j.mce.\n2009.03.022)\nSampson JA 1925 Heterotopic or misplaced endometrial tissue.\nAmerican Journal of Obstetrics and Gynecology 10 649–664.\nShi YL, Lou XZ, Zhu XY, Hua KQ, Zhu Y & Li DJ 2006 Effects of\ncombined 17b-estradiol with TCDD on secretion of chemokine IL-8\nand expression of its receptor CXCR1 in endometriotic focus-\nassociated cells in co-culture. Human Reproduction 21 870–879.\n(doi:10.1093/humrep/dei414)\nSridhar SC & Miranti CK 2006 Tetraspanin KAI1/CD82 suppresses\ninvasion by inhibiting integrin-dependent crosstalk with c-Met\nreceptor and Src kinases. Oncogene 25 2367–2378. (doi:10.1038/sj.\nonc.1209269)\nSugiura T & Berditchevski F 1999 Function of alpha3beta1-tetraspanin\nprotein complexes in tumor cell invasion. Evidence for the role of\nthe complexes in production of matrix metalloproteinase 2 (MMP-\n2). Journal of Cell Biology 146 1375–1389. (doi:10.1083/jcb.146.6.\n1375)\nCD82 suppression in the endometriotic milieu . M-QL I and others 207\nwww.endocrinology-journals.org Journal of Molecular Endocrinology (2011) 47, 195–208\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access\n\n\nTakaoka A, Hinoda Y, Satoh S, Adachi Y, Itoh F, Adachi M & Imai K\n1998 Suppression of invasive properties of colon cancer cells by a\nmetastasis suppressor CD82 gene. Oncogene 16 1443–1453. (doi:10.\n1038/sj.onc.1201648)\nUlukus M, Cakmak H & Arici A 2006 The role of endometrium in\nendometriosis. Journal of the Society for Gynecologic Investigation 13\n467–476.\nWitz CA, Thomas MR, Montoya-Rodriguez IA, Nair AS, Centonze VE &\nSchenken RS 2001 Short-term culture of peritoneum explants\nconfirms attachment of endometrium to intact peritoneal\nmesothelium. Fertility and Sterility 75 385–390. (doi:10.1016/S0015-\n0282(00)01699-X)\nWu MH, Shoji Y, Wu MC, Chuang PC, Lin CC, Huang MF & Tsai SJ\n2005 Suppression of matrix metalloproteinase-9 by prostaglandin\nE\n2 in peritoneal macrophage is associated with severity of\nendometriosis. American Journal of Pathology 167 1061–1069. (doi:10.\n1016/S0002-9440(10)61195-9)\nYang X, Wei LL, Tang C, Slack R, Mueller S & Lippman ME 2001\nOverexpression of CD82 suppresses in vitro invasiveness\nand in vivo metastasis in breast cancer cells. Cancer Research 61\n5284–5288.\nYoshimura Y 2002 The role of integrins in the human reproductive\nprocess. Journal of Reproduction and Development 48 215–232.\n(doi:10.1262/jrd.48.215)\nYu J, Wang Y, Zhou WH, Wang L, He YY & Li DJ 2008 Combination\nof estrogen and dioxin is involved in the pathogenesis of\nendometriosis by promoting chemokine secretion and invasion\nof endometrial stromal cells. Human Reproduction 23 1614–1626.\n(doi:10.1093/humrep/den125)\nReceived in final form 12 June 2011\nAccepted 17 June 2011\nMade available online as an Accepted Preprint 17 June 2011\nM-QL I and others . CD82 suppression in the endometriotic milieu208\nJournal of Molecular Endocrinology (2011) 47, 195–208 www.endocrinology-journals.org\nDownloaded from Bioscientifica.com at 06/07/2026 10:10:00PM\nvia free access","source_license":"public-domain-us","license_restricted":false}