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Increased expression of c-fos protein associated with increased matrix metalloproteinase-9 protein expression in the endometrium of endometriotic patients
Increased expression of c-fos protein associated with increased matrix metalloproteinase-9 protein expression in the endometrium of endometriotic patients Hong Pan, M.D.,a Jian-Zhong Sheng, Ph.D.,b Li Tang, M.D.,a Rong Zhu, M.D.,a Tian-Hua Zhou, Ph.D.,c and He-Feng Huang, M.D.a a Department of Reproductive Endocrinology, Women’s Hospital; c Department of Medical Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China; and b Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
Objective: To investigate c-fos and matrix metalloproteinase-9 (MMP-9) expression in the endometrium from women with or without endometriosis throughout the menstrual cycle, and to explore the correlation of c-fos expression with MMP-9 expression and 17b-E2 levels in serum. Design: Molecular studies in human tissue. Setting: A women’s hospital in China. Patient(s): Fifty-five premenopausal women (25 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery or hysterectomy. Intervention(s): Eutopic and ectopic endometrium tissue were obtained at the time of surgery. Peripheral sera were also collected on the same day. Main Outcome Measure(s): Immunohistochemical localization of c-fos in the endometrium, c-fos and MMP-9 protein levels in the endometrium, and 17b-E2 levels in the serum. Result(s): c-fos protein was predominantly located in the nuclei of glandular epithelial cells and stromal cells. c-fos and MMP-9 protein levels in paired eutopic and ectopic endometria from women with endometriosis were significantly higher than those in the endometrium from women without endometriosis. No significant difference in c-fos or MMP-9 protein levels was observed between paired eutopic and ectopic endometria. c-fos protein levels in endometrium positively correlated with endometrial MMP-9 levels and serum 17b-E2 levels. Conclusion(s): Expression of c-fos in the human endometrium may be regulated by 17b-E2, and c-fos may be involved in development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants. (Fertil Steril 2008;90:1000–7. 2008 by American Society for Reproductive Medicine.) Key Words: c-fos, MMP-9, endometriosis, pathogenesis
Endometriosis is defined as the presence of endometrial tissue that lines the inside of the uterus outside of its normal location, and affects about 10% to 15% of women of reproductive age (1). To date, the most widely accepted etiologic theory of endometriosis is that retrograde flow of menstrual fluid through the fallopian tubes deposits viable endometrial tissues, which implant outside of the uterine cavity, such as peritoneal surfaces (2). After implantation, the ectopic endometrium degrades the extracellular matrix and subsequently invades the surrounding tissue with corresponding cell proliferation and neoangiogenesis. Endometriotic lesions, although derived from normal endometrium, usually present decreased expression of adhesion Received May 21, 2007; revised July 19, 2007; accepted July 30, 2007. Supported by National Basic Research Program of China (No. 2006CB944006) and Basic Research Program of China (No. 973: 2006CB504004). The authors have no conflict of interest to disclose. Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, P.R. China (FAX: 86-571-87061878; E-mail: [email protected]).
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molecules and increased expression of proteolytic enzymes, which may contribute to establishment of endometrial glands and stroma at ectopic sites. Endometriotic cells with this behavior are more similar to cancer cells than normal endometrial cells (3, 4). Thus, endometriosis is also treated as an invasive disease (3, 5), and it is not surprising that endometriosis may share the same characteristic of invasion behavior and biochemical mediators with malignant tumors. It has been shown that matrix metalloproteinases (MMPs) and c-fos play very important roles in the invasion of malignant tumors (6–9). MMPs are a broad family of zinc-binding endopeptidases that are responsible for degradation of the extracellular matrix (10). In the normal human endometrium, numerous MMPs are cyclically expressed, suggesting that these proteases are probably related to ovarian steroid hormones, and may be involved in endometrial remodeling and establishment of menstruation (11). Although endometrial expression of the MMP family is normally tightly regulated during the menstrual cycle, altered expression patterns of MMPs have been reported in endometrial tissues obtained from patients with endometriosis, which suggests that MMPs
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.07.1386
may play critical roles in the occurrence and development of endometriosis (12, 13). It is well established that endometriosis is an estrogendependent disease (14–16). The proto-oncogene, c-fos, is one of the estrogen-target genes, and a functional estrogenresponsive element has been identified on the human and mouse c-fos genes (17, 18). Reis et al. (19) demonstrated that c-fos expression was higher in the proliferative phase of the menstrual cycle, correlating with E2 levels in serum, and could be suppressed by medroxyprogesterone acetate. C-fos represents a prototypical ‘‘immediate early’’ gene, because its expression is rapidly and dramatically induced by different extracellular stimuli including estrogen (20). Once activated, c-fos protein dimerizes with the Jun family proteins and forms the activator protein 1 (AP-1) complex, which binds to sites identified in the regulatory region of target genes modulating the late-response expression of critical factors for a wide range of biologic processes, such as cell proliferation, differentiation, apoptosis, oncogenesis, and invasion (21). Studies on malignant tumor invasion have demonstrated that c-fos could promote cancer cell invasion, which was mediated by MMPs (8, 22). Inhibition of c-fos expression was associated with reduced MMPs expression (9). MMP-9, an important enzyme for degradation of the basement membrane, is reported to be involved not only in tumor invasion and metastasis (22–25), but also in progression of endometriosis (26–28). Elucidation of the mechanism underlying regulation of MMP-9 and other MMP gene expression in the endometrium will be helpful for the understanding and treatment of endometriosis. The MMP-9 gene is encoded on chromosome 20, and its expression is under the control of a 2.2-kb upstream regulatory sequence harboring binding sites for AP-1 (29), implying the critical role played by AP-1 subunit c-fos in regulating MMP-9 expression. Recent studies have shown that there was a tight relationship between MMP-9 and c-fos expression in malignant tumor invasion (22, 30). Because of the roles of c-fos and MMP-9 in tumor invasion and the invasive characteristic of endometriosis, it is important to investigate c-fos expression and possible correlation between c-fos and MMP-9 expression levels in eutopic and ectopic endometrium of women with endometriosis. In the present study, we examined [1] the immunohistochemical localization of c-fos protein in normal endometria as well as in paired eutopic and ectopic endometria from the endometriotic patients; [2] c-fos and MMP-9 protein expression in paired eutopic and ectopic endometria of the endometriotic patients and in eutopic endometria from the control patients throughout the menstrual cycle; [3] the correlation between c-fos protein and MMP-9 protein levels in the endometrium; and [4] the correlation between endometrial c-fos protein levels and serum 17b-E2 levels to explore whether c-fos was involved in the pathogenesis of endometriosis. Fertility and Sterility
MATERIALS AND METHODS Patients and Tissue Samples The subjects in this study were the women of reproductive age who were undergoing laparoscopic surgery or hysterectomy in the Women’s Hospital, Zhejiang University School of Medicine between 2005 and 2006. Ethical approval for this project was granted by the institutional review board and the ethics committee of the School of Medicine, Zhejiang University, and a written informed consent was obtained from each subject before tissue collection. All patients exhibited normal menstrual cycles and no history of hormonal treatment, pregnancy, breast feeding, or using an intrauterine device within the last 6 months. Patients with pelvic inflammatory disease, adenomyosis, and dysfunctional uterine bleeding were excluded. Stages of the disease were classified according to the revised American Fertility Society classification (31). Endometrial samples for Western blotting analysis were obtained from 55 women aged 25–47 years. Paired eutopic and ectopic endometrial tissues (at the proliferative phase, n ¼ 14; at the secretory phase, n ¼ 11) were collected from 25 patients with endometriosis at stages II–IV. Control eutopic endometria (at the proliferative phase, n ¼ 17; at the secretory phase, n ¼ 13) were collected from 30 patients without endometriosis confirmed by laparoscopic surgery. The menstrual cycle phase was confirmed by histologic dating. Shortly after collection, each tissue was stored at 70 C for protein extraction. Meanwhile, peripheral sera were also collected for measurement of serum 17b-E2 levels on the same day of endometrium collection. Paired eutopic and ectopic endometrial tissues (at the proliferative phase, n ¼ 3) for immunohistochemistry analysis were obtained from three patients with endometriosis. Normal eutopic endometria (at the proliferative phase, n ¼ 3) for immunohistochemistry analysis were collected from three patients without endometriosis. Shortly after collection, the tissue was fixed in 10% formalin and embedded in paraffin.
Immunohistochemistry Analysis Tissue samples embedded in paraffin were sectioned at 4 mm thickness and then deparaffinized in xylene and rehydrated through graded alcohol. Antigen retrieval was performed according to the method reported by Gresz et al. (32). After endogenous peroxidase was blocked with 3% hydrogen peroxide, the sections were incubated for 2 hours at room temperature with the primary antibody against c-fos (Santa Cruz sc-52, 1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Tissue sections were then washed with phosphate-buffered saline (PBS) and incubated with the horseradish peroxidaseconjugated secondary antibody for 1 hour at room temperature. After washing again, the sections were reacted with diaminobenzidine (DAKO Cytomation, Carpinteria, CA) to visualize the signal. The sections were counterstained with hematoxylin to enhance color contrast. The negative control was performed with substituting PBS for the primary antibody. 1001
Staining intensity was assigned with a semiquantitative H-SCORE, which was calculated with the following equation: H-SCORE ¼ S Pi (i þ 1), where i is the intensity of the stained endometrial cell (1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong), and Pi is the percentage of stained endometrial cells for each intensity varying from 0% to 100%. The pathologists who read the slides were blind to the grouping information. Protein Extraction After washing with ice-cold PBS, samples of each tissue were homogenized in lysis buffer (10 mM Tris–HCl, pH 7.4, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, and 1 mg/mL Leupeptine) on ice, incubated in lysis buffer for 30 minutes, and then centrifuged at 12,000 g for 15 minutes at 4 C. The supernatants were stored at 70 C until required. Western Blotting Analysis The procedure was performed as previously described (33). Protein concentrations were determined by the Braford assay. Equal amounts of protein from endometrial tissue lysates (50 mg) were solubilized in 2 SDS sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 5%–10% 2-mercaptoethanol, and 0.004% bromphenol blue), denatured in 95 C for 5 minutes, and separated on SDS-10% polyacrylamide gels. The separated proteins were transferred onto nitrocellulose membrane using a Bio-Rad transfer apparatus (Bio-Rad, Hercules, CA) for 1.5 hours at a constant voltage of 100 volts. Nonspecific binding sites were blocked by immersing the membrane in blocking solution (TBST, 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20 [v/v]) containing 5% skim milk powder) at 37 C for 1 hour. After blocking, blots were incubated with polyclonal antibodies raised against human MMP-9 (Santa Cruz sc-6840, 1: 200 dilution; Santa Cruz Biotechnology), c-fos (Santa Cruz sc-52, 1: 400 dilution; Santa Cruz Biotechnology), and actin (Santa Cruz sc-1616-R, 1: 5000 dilution; Santa Cruz Biotechnology), respectively, at room temperature for 2 hours. The blots were washed with TBST buffer and then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. After washing again, the blots were transferred to freshly made ECL solution (Amersham Biosciences, Arlington Heights, IL) for 2 minutes and exposed to roentgengram film. Protein bands were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer (Bio-Rad). Normalized densities were determined with the ratio of a band density of each target protein to a band density of actin that was used as an internal reference. Measurements of 17b-E2 Levels in Serum 17b-Estradiol levels in serum were measured by an electrochemiluminescene immunoassay. The reagents were purchased from Roche (Roche Diagnostics, Indianapolis, IL) and assays were conducted according to the manufacturer’s instruction. 1002
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Statistics Statistical analysis was performed by Statistical Package for the Social Sciences software version 13.0 (SPSS, Chicago, IL). All data were expressed as mean SEM. One-way analysis of variance (ANOVA) was used to analyze the difference among groups. Multiple comparisons were performed using the LSD t procedure. An independent sample t test was used to analyze c-fos or MMP-9 expression levels between two groups of samples. Correlation between c-fos protein and MMP-9 protein levels in the endometrium and correlation between endometrial c-fos protein levels and serum 17b-E2 levels were examined with Pearson’s correlation coefficient. A P-value of < .05 was considered statistically significant. RESULTS Distribution of c- fos Protein in Different Groups of Endometria Figure 1 shows distribution of c-fos protein in normal eutopic endometrium (Fig. 1A), as well as in paired eutopic (Fig. 1B) and ectopic (Fig. 1C) endometrium from endometriotic patients. In the three groups of endometria, positive c-fos immunostaining was predominantly located in the nuclei of glandular epithelial cells and stromal cells. No staining was detected in the negative control (Fig. 1D). H-SCORE analysis shows that, compared with the normal eutopic endometrium, the intensities of c-fos protein staining in both eutopic and ectopic endometrium from the endometriotic patients were significantly higher (P<.05; Fig. 1B). Expression of c-fos Protein c-fos protein was detected in 25 paired eutopic and ectopic endometria from patients with endometriosis and in 30 normal eutopic endometria (Fig. 2A, B). Compared with the normal eutopic endometrium, both eutopic and ectopic endometrium from the endometriotic patients showed significantly higher c-fos protein expression levels throughout the menstrual cycle (P<.05; Fig. 2B). However, no significant difference in c-fos protein expression levels was observed between eutopic and ectopic endometria from the patients with endometriosis throughout the menstrual cycle (P>.05; Fig; 2B). All three groups of endometria showed higher c-fos protein levels at the proliferative phase compared with those at the secretory phase (P<.05; Fig. 2B). Expression of MMP-9 Protein MMP-9 protein was also detected in 25 paired eutopic and ectopic endometria from the patients with endometriosis and in 30 normal eutopic endometria (Fig. 2C, D). Compared with normal eutopic endometrium, both eutopic and ectopic endometrium from the patients with endometriosis expressed significantly higher MMP-9 protein levels throughout the menstrual cycle (P<.05; Fig. 2D). However, no significant difference in MMP-9 protein expression levels was observed between eutopic and ectopic endometria from the patients with endometriosis throughout the menstrual cycle (P>.05; Fig. 2D). Normal eutopic endometrium showed higher Vol. 90, No. 4, October 2008
FIGURE 1 (A) Immunostaining of c-fos protein in different groups of endometria. A, Normal eutopic endometrium; B, eutopic endometrium from endometriotic patients; C, ectopic endometrium from endometriotic patients; and D, negative control. Original magnification: 400. (B) H-SCORE of c-fos protein staining in different groups of endometria. *P< .05, compared with normal eutopic endometria.
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
MMP-9 protein levels at the proliferative phase compared with those at the secretory phase (P<.01; Fig. 2D). Correlation of c-fos Protein Levels in Human Endometrium with Endometrial MMP-9 Protein Levels and Serum 17b-E2 Levels As shown in Figure 3A, Pearson correlation analysis showed a significantly positive correlation between c-fos and MMP-9 protein expression levels in all the 80 endometrial tissues Fertility and Sterility
(P<.01, r ¼ .511). In contrast, c-fos protein levels in eutopic endometria from the women with or without endometriosis (Fig. 3B, C) at the proliferative phase were also shown to have positive correlation with 17b-E2 levels in sera (P<. 01, r ¼ .855; P<.05, r ¼ .614). DISCUSSION The c-fos is a kind of proto-oncogene involved in wide range of biologic processes including cell proliferation, differentiation, apoptosis, oncogenesis, and invasion (21). It has been 1003
FIGURE 2 Western blotting analysis of c-fos and MMP-9 protein expression in different groups of endometria throughout the menstrual cycle. (A, C) Expression of c-fos and MMP-9 proteins in endometria from the patients with or without endometriosis, respectively. A, Eutopic endometrium at the proliferative phase from the endometriotic patients; B, ectopic endometrium at the proliferative phase; C, eutopic endometrium at the proliferative phase from the controls; D, eutopic endometrium at the secretory phase from the endometriotic patients; E, ectopic endometrium at the secretory phase; F, eutopic endometrium at the secretory phase from the controls. (B, D) Summary data of c-fos and MMP-9 protein expression, respectively. Euto, eutopic endometria from the endometriotic patients; Ecto, ectopic endometria; Endo, eutopic endometria from the controls. *P< .05, compared with normal eutopic endometria at the same menstrual phase. #P< .05, ##P< .01, compared with the corresponding secretory endometria, respectively.
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
shown that overexpression of c-fos is closely associated with invasive phenotype of various cancers. A recent study demonstrated that c-fos mRNA and protein expression levels in the eutopic endometrium of the induced baboon model of endometriosis were upregulated, suggesting a potential role of c-fos in endometriosis development (34). In the present study we observed that c-fos protein was expressed in human endometrium and mainly located in the nuclei of the endometrial stromal cells and glandular cells. Both the immunohistochemistry and the Western blotting analyses showed that, compared with normal eutopic endometrium, c-fos protein levels in paired eutopic and ectopic endometrium from endometriotic patients were significantly higher, suggesting that c-fos protein might be abnormally expressed in the endometrium of patients with endometriosis, and c-fos might be involved in the progression of endometriosis. 1004
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It is well known that the c-fos gene is an estrogen-target gene, and a functional estrogen-responsive element has been identified in human and mouse c-fos genes (17, 18). 17b-Estradiol could induce rapid, marked, and transient expression of c-fos mRNA in the uterus of ovariectomized rats (35). The results from the present and other studies (19) demonstrated that c-fos expression in endometrium was higher at the proliferative phase and had a positive correlation with E2 levels in sera. Our results support the idea that endometrial c-fos may be regulated by sex steroid hormones during the menstrual cycle. Although it is well known that endometriosis is an estrogen-dependent disorder, the mechanisms underlying occurrence and development of endometriosis are still unclear. The results of the present study suggest that c-fos protein may play a key role in estrogen-dependent etiology of endometriosis. Vol. 90, No. 4, October 2008
FIGURE 3 (A) Correlation between c-fos protein levels and MMP-9 protein levels in all the 80 endometrial tissues examined (P< .01, r ¼ .511); (B) Correlation between c-fos protein levels in eutopic endometria and 17b-E2 concentrations in sera of the endometriotic patients at the proliferative phase (P< .01, r ¼ .855); (C) Correlation between c-fos protein levels in eutopic endometria and 17b-E2 concentrations in sera of the controls at the proliferative phase (P< .05, r ¼ .614).
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
Several recent studies showed increased expression of various proteases including MMP-1 (36), MMP-2 (11), MMP-3 (37), and some apoptosis-related proteins (38) in the ectopic endometrium of the women with endometriosis. In contrast, the tissue inhibitors of metalloproteinases, endogenous inhibitors of MMPs, also play significant roles in normal endometrial remodeling and development of endometriosis (39). However, only a few studies addressed possible changes in Fertility and Sterility
MMPs, including MMP-9 expression in the eutopic endometrium of endometriotic patients (12, 26). In the present study, we analyzed MMP-9 protein expression in paired eutopic and ectopic endometria from endometriotic patients and in normal eutopic endometrium from the women without endometriosis. Consistent with the results from other laboratories (26, 27), our data showed that MMP-9 protein expression in both eutopic and ectopic endometria from the endometriotic patients was significantly higher than that in normal eutopic endometrium. The overexpression of MMP-9 in both eutopic and ectopic endometria of the patients with endometriosis suggests that the endometrium of the patients with endometriosis may be abnormal and have the potential to be implanted in a place outside of the uterus. Our results also provide further evidence that MMPs, including MMP-9, play important roles in the development of endometriosis. The roles of c-fos in the invasion of tumors have been studied extensively. Transient transfection with c-fos expression vector significantly enhanced the invasion of the carcinoma cell line MCF7 (30). In contrast, the roles of MMP-9 in tumor invasion and angiogenesis were also investigated (6, 7). The MMP-9 gene has been found to be regulated by AP-1 (29). An accumulating body of evidence shows that induction of invasion by c-fos may be mediated by MMPs including MMP-9. Expression of c-fos positively correlated with MMP-9 protein levels in carcinoma cells (30). Fibronectin, which stimulates human lung carcinoma cell invasion and proliferation, increases MMP-9 expression in human lung carcinoma cells through increased AP-1/DNA binding and c-fos protein expression (22). Treatment of human lung carcinoma cells with c-fos siRNA not only eliminated the stimulatory effect of fibronectin on MMP-9 protein expression, but also attenuated the effects of fibronectin on MMP-9 promoter activity in human lung carcinoma cells (22). Like malignant tumors, endometriosis also exhibits the characteristics of invasion and migration. Because expression of MMP-9 was shown to have a significant and tight correlation with c-fos in the invasion of malignant tumor cells, we focused on a possible correlation between c-fos and MMP-9 in eutopic and ectopic endometrium of the women with endometriosis in the present study. We found that both c-fos and MMP-9 proteins were elevated in the endometrium from the patients with endometriosis, and there was a positive correlation between endometrial c-fos and MMP-9 protein levels. These results provide evidence that c-fos might play an important role in the development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants. Additional studies are needed to clarify the specific molecular interaction between c-fos and MMP-9 in the development of endometriosis. In contrast, the correlation between c-fos and other types of MMPs in endometrium of women with endometriosis needs to be investigated in future studies. It is noticed that, in the present study, both eutopic and ectopic endometria of the patients with endometriosis expressed higher c-fos and MMP-9 proteins than normal 1005
endometrium, and there was no difference between eutopic and ectopic endometria of the patients with endometriosis. These results suggest that the endometrium of the patients with endometriosis may be more prone to implantation and invasion. We suppose that the changes of the internal environment around the endometrium or other unknown factors might induce the abnormal expression of some genes such as c-fos and MMPs, which control the invasion and proliferation of cells, resulting in implantation and invasion of endometrial cells in a place outside of the uterus cavity and the development of endometriosis. Further study is needed to clarify this hypothesis. In conclusion, the present study demonstrates that c-fos expression is increased in the endometrium from the patients with endometriosis. A positive correlation between endometrial c-fos and MMP-9 protein levels suggests that roles of c-fos in the development of endometriosis may be mediated by promoting MMP-9 gene expression and subsequently creating the invasive potential of endometrial explants. The positive correlation between endometrial c-fos protein levels and estrogen levels in serum indicates that the estrogendependent etiology of endometriosis might be at least partly mediated by c-fos. To the best of our knowledge, this is the first report to demonstrate c-fos protein expression in the endometrium from endometriotic patients, and the association of endometrial c-fos protein expression with endometrial MMP-9 protein expression and serum 17b-E2 levels. These observations are potentially important in understanding the pathogenesis of endometriosis, an aggressive tumor-like and estrogen-dependent benign disease, and might provide a novel therapeutic target for this disease. Acknowledgments: We are very grateful to Dr. Qiong Luo (Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine) for the review and revision of the manuscript.
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28. Collette T, Bellehumeur C, Kats R, Maheux R, Mailloux J, Villeneuve M, et al. Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9. Hum Reprod 2004;19:1257–64. 29. Nair RR, Boyd DD. Expression cloning of novel regulators of 92 kDa type IV collagenase expression. Biochem Soc Trans 2005;33:1135–6. 30. Milde-Langosch K, Roder H, Andritzky B, Aslan B, Hemminger G, Brinkmann A, et al. The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas. Breast Cancer Res Treat 2004;86:139–52. 31. American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997;67:817–21. 32. Gresz V, Kwon TH, Hurley PT, Varga G, Zelles T, Nielsen S, et al. Identification and localization of aquaporin water channels in human salivary glands. Am J Physiol Gastrointest Liver Physiol 2001;281: G247–54. 33. Cai J, Lou HY, Dong MY, Lu XE, Zhu YM, Huang HF, et al. Poor ovarian response to gonadotropin stimulation is associated with low expression of follicle-stimulating hormone receptor in granulosa cells. Fertil Steril 2007;87:1350–6.
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34. Hastings JM, Jackson KS, Mavrogianis PA, Fazleabas AT. The estrogen early response gene FOS is altered in a baboon model of endometriosis. Biol Reprod 2006;75:176–82. 35. Mendoza-Rodriguez CA, Camacho-Arroyo I, Garcia GA, Cerbon MA. Variations of progesterone receptor and c-fos gene expression in the rat uterus after treatment with norethisterone and its A-ring reduced metabolites. Contraception 1999;59:339–43. 36. Hudelist G, Keckstein J, Czerwenka K, Lass H, Walter I, Auer M, et al. Estrogen receptor beta and matrix metalloproteinase 1 are coexpressed in uterine endometrium and endometriotic lesions of patients with endometriosis. Fertil Steril 2005;84(Suppl 2):1249–56. 37. Uzan C, Cortez A, Dufournet C, Fauvet R, Siffroi JP, Dara€ı E. Eutopic endometrium and peritoneal, ovarian and bowel endometriotic tissues express a different profile of matrix metalloproteinases-2, -3 and -11, and of tissue inhibitor metalloproteinases-1 and -2. Virchows Arch 2004;445:603–9. 38. Dufournet C, Uzan C, Fauvet R, Cortez A, Siffroi JP, Darai E. Expression of apoptosis-related proteins in peritoneal, ovarian and colorectal endometriosis. J Reprod Immunol 2006;70:151–62. 39. Sharpe-Timms KL, Cox KE. Paracrine regulation of matrix metalloproteinase expression in endometriosis. Ann N Y Acad Sci 2002;955: 147–56. discussion 157–8, 396–406.
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Objective: To investigate c-fos and matrix metalloproteinase-9 (MMP-9) expression in the endometrium from women with or without endometriosis throughout the menstrual cycle, and to explore the correlation of c-fos expression with MMP-9 expression and 17b-E2 levels in serum. Design: Molecular studies in human tissue. Setting: A women’s hospital in China. Patient(s): Fifty-five premenopausal women (25 with endometriosis and 30 without endometriosis) undergoing laparoscopic surgery or hysterectomy. Intervention(s): Eutopic and ectopic endometrium tissue were obtained at the time of surgery. Peripheral sera were also collected on the same day. Main Outcome Measure(s): Immunohistochemical localization of c-fos in the endometrium, c-fos and MMP-9 protein levels in the endometrium, and 17b-E2 levels in the serum. Result(s): c-fos protein was predominantly located in the nuclei of glandular epithelial cells and stromal cells. c-fos and MMP-9 protein levels in paired eutopic and ectopic endometria from women with endometriosis were significantly higher than those in the endometrium from women without endometriosis. No significant difference in c-fos or MMP-9 protein levels was observed between paired eutopic and ectopic endometria. c-fos protein levels in endometrium positively correlated with endometrial MMP-9 levels and serum 17b-E2 levels. Conclusion(s): Expression of c-fos in the human endometrium may be regulated by 17b-E2, and c-fos may be involved in development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants. (Fertil Steril 2008;90:1000–7. 2008 by American Society for Reproductive Medicine.) Key Words: c-fos, MMP-9, endometriosis, pathogenesis
Endometriosis is defined as the presence of endometrial tissue that lines the inside of the uterus outside of its normal location, and affects about 10% to 15% of women of reproductive age (1). To date, the most widely accepted etiologic theory of endometriosis is that retrograde flow of menstrual fluid through the fallopian tubes deposits viable endometrial tissues, which implant outside of the uterine cavity, such as peritoneal surfaces (2). After implantation, the ectopic endometrium degrades the extracellular matrix and subsequently invades the surrounding tissue with corresponding cell proliferation and neoangiogenesis. Endometriotic lesions, although derived from normal endometrium, usually present decreased expression of adhesion Received May 21, 2007; revised July 19, 2007; accepted July 30, 2007. Supported by National Basic Research Program of China (No. 2006CB944006) and Basic Research Program of China (No. 973: 2006CB504004). The authors have no conflict of interest to disclose. Reprint requests: He-Feng Huang, M.D., Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, P.R. China (FAX: 86-571-87061878; E-mail: [email protected]).
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molecules and increased expression of proteolytic enzymes, which may contribute to establishment of endometrial glands and stroma at ectopic sites. Endometriotic cells with this behavior are more similar to cancer cells than normal endometrial cells (3, 4). Thus, endometriosis is also treated as an invasive disease (3, 5), and it is not surprising that endometriosis may share the same characteristic of invasion behavior and biochemical mediators with malignant tumors. It has been shown that matrix metalloproteinases (MMPs) and c-fos play very important roles in the invasion of malignant tumors (6–9). MMPs are a broad family of zinc-binding endopeptidases that are responsible for degradation of the extracellular matrix (10). In the normal human endometrium, numerous MMPs are cyclically expressed, suggesting that these proteases are probably related to ovarian steroid hormones, and may be involved in endometrial remodeling and establishment of menstruation (11). Although endometrial expression of the MMP family is normally tightly regulated during the menstrual cycle, altered expression patterns of MMPs have been reported in endometrial tissues obtained from patients with endometriosis, which suggests that MMPs
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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may play critical roles in the occurrence and development of endometriosis (12, 13). It is well established that endometriosis is an estrogendependent disease (14–16). The proto-oncogene, c-fos, is one of the estrogen-target genes, and a functional estrogenresponsive element has been identified on the human and mouse c-fos genes (17, 18). Reis et al. (19) demonstrated that c-fos expression was higher in the proliferative phase of the menstrual cycle, correlating with E2 levels in serum, and could be suppressed by medroxyprogesterone acetate. C-fos represents a prototypical ‘‘immediate early’’ gene, because its expression is rapidly and dramatically induced by different extracellular stimuli including estrogen (20). Once activated, c-fos protein dimerizes with the Jun family proteins and forms the activator protein 1 (AP-1) complex, which binds to sites identified in the regulatory region of target genes modulating the late-response expression of critical factors for a wide range of biologic processes, such as cell proliferation, differentiation, apoptosis, oncogenesis, and invasion (21). Studies on malignant tumor invasion have demonstrated that c-fos could promote cancer cell invasion, which was mediated by MMPs (8, 22). Inhibition of c-fos expression was associated with reduced MMPs expression (9). MMP-9, an important enzyme for degradation of the basement membrane, is reported to be involved not only in tumor invasion and metastasis (22–25), but also in progression of endometriosis (26–28). Elucidation of the mechanism underlying regulation of MMP-9 and other MMP gene expression in the endometrium will be helpful for the understanding and treatment of endometriosis. The MMP-9 gene is encoded on chromosome 20, and its expression is under the control of a 2.2-kb upstream regulatory sequence harboring binding sites for AP-1 (29), implying the critical role played by AP-1 subunit c-fos in regulating MMP-9 expression. Recent studies have shown that there was a tight relationship between MMP-9 and c-fos expression in malignant tumor invasion (22, 30). Because of the roles of c-fos and MMP-9 in tumor invasion and the invasive characteristic of endometriosis, it is important to investigate c-fos expression and possible correlation between c-fos and MMP-9 expression levels in eutopic and ectopic endometrium of women with endometriosis. In the present study, we examined [1] the immunohistochemical localization of c-fos protein in normal endometria as well as in paired eutopic and ectopic endometria from the endometriotic patients; [2] c-fos and MMP-9 protein expression in paired eutopic and ectopic endometria of the endometriotic patients and in eutopic endometria from the control patients throughout the menstrual cycle; [3] the correlation between c-fos protein and MMP-9 protein levels in the endometrium; and [4] the correlation between endometrial c-fos protein levels and serum 17b-E2 levels to explore whether c-fos was involved in the pathogenesis of endometriosis. Fertility and Sterility
MATERIALS AND METHODS Patients and Tissue Samples The subjects in this study were the women of reproductive age who were undergoing laparoscopic surgery or hysterectomy in the Women’s Hospital, Zhejiang University School of Medicine between 2005 and 2006. Ethical approval for this project was granted by the institutional review board and the ethics committee of the School of Medicine, Zhejiang University, and a written informed consent was obtained from each subject before tissue collection. All patients exhibited normal menstrual cycles and no history of hormonal treatment, pregnancy, breast feeding, or using an intrauterine device within the last 6 months. Patients with pelvic inflammatory disease, adenomyosis, and dysfunctional uterine bleeding were excluded. Stages of the disease were classified according to the revised American Fertility Society classification (31). Endometrial samples for Western blotting analysis were obtained from 55 women aged 25–47 years. Paired eutopic and ectopic endometrial tissues (at the proliferative phase, n ¼ 14; at the secretory phase, n ¼ 11) were collected from 25 patients with endometriosis at stages II–IV. Control eutopic endometria (at the proliferative phase, n ¼ 17; at the secretory phase, n ¼ 13) were collected from 30 patients without endometriosis confirmed by laparoscopic surgery. The menstrual cycle phase was confirmed by histologic dating. Shortly after collection, each tissue was stored at 70 C for protein extraction. Meanwhile, peripheral sera were also collected for measurement of serum 17b-E2 levels on the same day of endometrium collection. Paired eutopic and ectopic endometrial tissues (at the proliferative phase, n ¼ 3) for immunohistochemistry analysis were obtained from three patients with endometriosis. Normal eutopic endometria (at the proliferative phase, n ¼ 3) for immunohistochemistry analysis were collected from three patients without endometriosis. Shortly after collection, the tissue was fixed in 10% formalin and embedded in paraffin.
Immunohistochemistry Analysis Tissue samples embedded in paraffin were sectioned at 4 mm thickness and then deparaffinized in xylene and rehydrated through graded alcohol. Antigen retrieval was performed according to the method reported by Gresz et al. (32). After endogenous peroxidase was blocked with 3% hydrogen peroxide, the sections were incubated for 2 hours at room temperature with the primary antibody against c-fos (Santa Cruz sc-52, 1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Tissue sections were then washed with phosphate-buffered saline (PBS) and incubated with the horseradish peroxidaseconjugated secondary antibody for 1 hour at room temperature. After washing again, the sections were reacted with diaminobenzidine (DAKO Cytomation, Carpinteria, CA) to visualize the signal. The sections were counterstained with hematoxylin to enhance color contrast. The negative control was performed with substituting PBS for the primary antibody. 1001
Staining intensity was assigned with a semiquantitative H-SCORE, which was calculated with the following equation: H-SCORE ¼ S Pi (i þ 1), where i is the intensity of the stained endometrial cell (1 ¼ weak, 2 ¼ moderate, and 3 ¼ strong), and Pi is the percentage of stained endometrial cells for each intensity varying from 0% to 100%. The pathologists who read the slides were blind to the grouping information. Protein Extraction After washing with ice-cold PBS, samples of each tissue were homogenized in lysis buffer (10 mM Tris–HCl, pH 7.4, 0.5% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, and 1 mg/mL Leupeptine) on ice, incubated in lysis buffer for 30 minutes, and then centrifuged at 12,000 g for 15 minutes at 4 C. The supernatants were stored at 70 C until required. Western Blotting Analysis The procedure was performed as previously described (33). Protein concentrations were determined by the Braford assay. Equal amounts of protein from endometrial tissue lysates (50 mg) were solubilized in 2 SDS sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 5%–10% 2-mercaptoethanol, and 0.004% bromphenol blue), denatured in 95 C for 5 minutes, and separated on SDS-10% polyacrylamide gels. The separated proteins were transferred onto nitrocellulose membrane using a Bio-Rad transfer apparatus (Bio-Rad, Hercules, CA) for 1.5 hours at a constant voltage of 100 volts. Nonspecific binding sites were blocked by immersing the membrane in blocking solution (TBST, 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20 [v/v]) containing 5% skim milk powder) at 37 C for 1 hour. After blocking, blots were incubated with polyclonal antibodies raised against human MMP-9 (Santa Cruz sc-6840, 1: 200 dilution; Santa Cruz Biotechnology), c-fos (Santa Cruz sc-52, 1: 400 dilution; Santa Cruz Biotechnology), and actin (Santa Cruz sc-1616-R, 1: 5000 dilution; Santa Cruz Biotechnology), respectively, at room temperature for 2 hours. The blots were washed with TBST buffer and then incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. After washing again, the blots were transferred to freshly made ECL solution (Amersham Biosciences, Arlington Heights, IL) for 2 minutes and exposed to roentgengram film. Protein bands were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer (Bio-Rad). Normalized densities were determined with the ratio of a band density of each target protein to a band density of actin that was used as an internal reference. Measurements of 17b-E2 Levels in Serum 17b-Estradiol levels in serum were measured by an electrochemiluminescene immunoassay. The reagents were purchased from Roche (Roche Diagnostics, Indianapolis, IL) and assays were conducted according to the manufacturer’s instruction. 1002
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Statistics Statistical analysis was performed by Statistical Package for the Social Sciences software version 13.0 (SPSS, Chicago, IL). All data were expressed as mean SEM. One-way analysis of variance (ANOVA) was used to analyze the difference among groups. Multiple comparisons were performed using the LSD t procedure. An independent sample t test was used to analyze c-fos or MMP-9 expression levels between two groups of samples. Correlation between c-fos protein and MMP-9 protein levels in the endometrium and correlation between endometrial c-fos protein levels and serum 17b-E2 levels were examined with Pearson’s correlation coefficient. A P-value of < .05 was considered statistically significant. RESULTS Distribution of c- fos Protein in Different Groups of Endometria Figure 1 shows distribution of c-fos protein in normal eutopic endometrium (Fig. 1A), as well as in paired eutopic (Fig. 1B) and ectopic (Fig. 1C) endometrium from endometriotic patients. In the three groups of endometria, positive c-fos immunostaining was predominantly located in the nuclei of glandular epithelial cells and stromal cells. No staining was detected in the negative control (Fig. 1D). H-SCORE analysis shows that, compared with the normal eutopic endometrium, the intensities of c-fos protein staining in both eutopic and ectopic endometrium from the endometriotic patients were significantly higher (P<.05; Fig. 1B). Expression of c-fos Protein c-fos protein was detected in 25 paired eutopic and ectopic endometria from patients with endometriosis and in 30 normal eutopic endometria (Fig. 2A, B). Compared with the normal eutopic endometrium, both eutopic and ectopic endometrium from the endometriotic patients showed significantly higher c-fos protein expression levels throughout the menstrual cycle (P<.05; Fig. 2B). However, no significant difference in c-fos protein expression levels was observed between eutopic and ectopic endometria from the patients with endometriosis throughout the menstrual cycle (P>.05; Fig; 2B). All three groups of endometria showed higher c-fos protein levels at the proliferative phase compared with those at the secretory phase (P<.05; Fig. 2B). Expression of MMP-9 Protein MMP-9 protein was also detected in 25 paired eutopic and ectopic endometria from the patients with endometriosis and in 30 normal eutopic endometria (Fig. 2C, D). Compared with normal eutopic endometrium, both eutopic and ectopic endometrium from the patients with endometriosis expressed significantly higher MMP-9 protein levels throughout the menstrual cycle (P<.05; Fig. 2D). However, no significant difference in MMP-9 protein expression levels was observed between eutopic and ectopic endometria from the patients with endometriosis throughout the menstrual cycle (P>.05; Fig. 2D). Normal eutopic endometrium showed higher Vol. 90, No. 4, October 2008
FIGURE 1 (A) Immunostaining of c-fos protein in different groups of endometria. A, Normal eutopic endometrium; B, eutopic endometrium from endometriotic patients; C, ectopic endometrium from endometriotic patients; and D, negative control. Original magnification: 400. (B) H-SCORE of c-fos protein staining in different groups of endometria. *P< .05, compared with normal eutopic endometria.
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
MMP-9 protein levels at the proliferative phase compared with those at the secretory phase (P<.01; Fig. 2D). Correlation of c-fos Protein Levels in Human Endometrium with Endometrial MMP-9 Protein Levels and Serum 17b-E2 Levels As shown in Figure 3A, Pearson correlation analysis showed a significantly positive correlation between c-fos and MMP-9 protein expression levels in all the 80 endometrial tissues Fertility and Sterility
(P<.01, r ¼ .511). In contrast, c-fos protein levels in eutopic endometria from the women with or without endometriosis (Fig. 3B, C) at the proliferative phase were also shown to have positive correlation with 17b-E2 levels in sera (P<. 01, r ¼ .855; P<.05, r ¼ .614). DISCUSSION The c-fos is a kind of proto-oncogene involved in wide range of biologic processes including cell proliferation, differentiation, apoptosis, oncogenesis, and invasion (21). It has been 1003
FIGURE 2 Western blotting analysis of c-fos and MMP-9 protein expression in different groups of endometria throughout the menstrual cycle. (A, C) Expression of c-fos and MMP-9 proteins in endometria from the patients with or without endometriosis, respectively. A, Eutopic endometrium at the proliferative phase from the endometriotic patients; B, ectopic endometrium at the proliferative phase; C, eutopic endometrium at the proliferative phase from the controls; D, eutopic endometrium at the secretory phase from the endometriotic patients; E, ectopic endometrium at the secretory phase; F, eutopic endometrium at the secretory phase from the controls. (B, D) Summary data of c-fos and MMP-9 protein expression, respectively. Euto, eutopic endometria from the endometriotic patients; Ecto, ectopic endometria; Endo, eutopic endometria from the controls. *P< .05, compared with normal eutopic endometria at the same menstrual phase. #P< .05, ##P< .01, compared with the corresponding secretory endometria, respectively.
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
shown that overexpression of c-fos is closely associated with invasive phenotype of various cancers. A recent study demonstrated that c-fos mRNA and protein expression levels in the eutopic endometrium of the induced baboon model of endometriosis were upregulated, suggesting a potential role of c-fos in endometriosis development (34). In the present study we observed that c-fos protein was expressed in human endometrium and mainly located in the nuclei of the endometrial stromal cells and glandular cells. Both the immunohistochemistry and the Western blotting analyses showed that, compared with normal eutopic endometrium, c-fos protein levels in paired eutopic and ectopic endometrium from endometriotic patients were significantly higher, suggesting that c-fos protein might be abnormally expressed in the endometrium of patients with endometriosis, and c-fos might be involved in the progression of endometriosis. 1004
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It is well known that the c-fos gene is an estrogen-target gene, and a functional estrogen-responsive element has been identified in human and mouse c-fos genes (17, 18). 17b-Estradiol could induce rapid, marked, and transient expression of c-fos mRNA in the uterus of ovariectomized rats (35). The results from the present and other studies (19) demonstrated that c-fos expression in endometrium was higher at the proliferative phase and had a positive correlation with E2 levels in sera. Our results support the idea that endometrial c-fos may be regulated by sex steroid hormones during the menstrual cycle. Although it is well known that endometriosis is an estrogen-dependent disorder, the mechanisms underlying occurrence and development of endometriosis are still unclear. The results of the present study suggest that c-fos protein may play a key role in estrogen-dependent etiology of endometriosis. Vol. 90, No. 4, October 2008
FIGURE 3 (A) Correlation between c-fos protein levels and MMP-9 protein levels in all the 80 endometrial tissues examined (P< .01, r ¼ .511); (B) Correlation between c-fos protein levels in eutopic endometria and 17b-E2 concentrations in sera of the endometriotic patients at the proliferative phase (P< .01, r ¼ .855); (C) Correlation between c-fos protein levels in eutopic endometria and 17b-E2 concentrations in sera of the controls at the proliferative phase (P< .05, r ¼ .614).
Pan. C-fos and MMP-9 in endometriosis. Fertil Steril 2008.
Several recent studies showed increased expression of various proteases including MMP-1 (36), MMP-2 (11), MMP-3 (37), and some apoptosis-related proteins (38) in the ectopic endometrium of the women with endometriosis. In contrast, the tissue inhibitors of metalloproteinases, endogenous inhibitors of MMPs, also play significant roles in normal endometrial remodeling and development of endometriosis (39). However, only a few studies addressed possible changes in Fertility and Sterility
MMPs, including MMP-9 expression in the eutopic endometrium of endometriotic patients (12, 26). In the present study, we analyzed MMP-9 protein expression in paired eutopic and ectopic endometria from endometriotic patients and in normal eutopic endometrium from the women without endometriosis. Consistent with the results from other laboratories (26, 27), our data showed that MMP-9 protein expression in both eutopic and ectopic endometria from the endometriotic patients was significantly higher than that in normal eutopic endometrium. The overexpression of MMP-9 in both eutopic and ectopic endometria of the patients with endometriosis suggests that the endometrium of the patients with endometriosis may be abnormal and have the potential to be implanted in a place outside of the uterus. Our results also provide further evidence that MMPs, including MMP-9, play important roles in the development of endometriosis. The roles of c-fos in the invasion of tumors have been studied extensively. Transient transfection with c-fos expression vector significantly enhanced the invasion of the carcinoma cell line MCF7 (30). In contrast, the roles of MMP-9 in tumor invasion and angiogenesis were also investigated (6, 7). The MMP-9 gene has been found to be regulated by AP-1 (29). An accumulating body of evidence shows that induction of invasion by c-fos may be mediated by MMPs including MMP-9. Expression of c-fos positively correlated with MMP-9 protein levels in carcinoma cells (30). Fibronectin, which stimulates human lung carcinoma cell invasion and proliferation, increases MMP-9 expression in human lung carcinoma cells through increased AP-1/DNA binding and c-fos protein expression (22). Treatment of human lung carcinoma cells with c-fos siRNA not only eliminated the stimulatory effect of fibronectin on MMP-9 protein expression, but also attenuated the effects of fibronectin on MMP-9 promoter activity in human lung carcinoma cells (22). Like malignant tumors, endometriosis also exhibits the characteristics of invasion and migration. Because expression of MMP-9 was shown to have a significant and tight correlation with c-fos in the invasion of malignant tumor cells, we focused on a possible correlation between c-fos and MMP-9 in eutopic and ectopic endometrium of the women with endometriosis in the present study. We found that both c-fos and MMP-9 proteins were elevated in the endometrium from the patients with endometriosis, and there was a positive correlation between endometrial c-fos and MMP-9 protein levels. These results provide evidence that c-fos might play an important role in the development of endometriosis by promoting MMP-9 gene expression and subsequently the invasive potential of endometrial explants. Additional studies are needed to clarify the specific molecular interaction between c-fos and MMP-9 in the development of endometriosis. In contrast, the correlation between c-fos and other types of MMPs in endometrium of women with endometriosis needs to be investigated in future studies. It is noticed that, in the present study, both eutopic and ectopic endometria of the patients with endometriosis expressed higher c-fos and MMP-9 proteins than normal 1005
endometrium, and there was no difference between eutopic and ectopic endometria of the patients with endometriosis. These results suggest that the endometrium of the patients with endometriosis may be more prone to implantation and invasion. We suppose that the changes of the internal environment around the endometrium or other unknown factors might induce the abnormal expression of some genes such as c-fos and MMPs, which control the invasion and proliferation of cells, resulting in implantation and invasion of endometrial cells in a place outside of the uterus cavity and the development of endometriosis. Further study is needed to clarify this hypothesis. In conclusion, the present study demonstrates that c-fos expression is increased in the endometrium from the patients with endometriosis. A positive correlation between endometrial c-fos and MMP-9 protein levels suggests that roles of c-fos in the development of endometriosis may be mediated by promoting MMP-9 gene expression and subsequently creating the invasive potential of endometrial explants. The positive correlation between endometrial c-fos protein levels and estrogen levels in serum indicates that the estrogendependent etiology of endometriosis might be at least partly mediated by c-fos. To the best of our knowledge, this is the first report to demonstrate c-fos protein expression in the endometrium from endometriotic patients, and the association of endometrial c-fos protein expression with endometrial MMP-9 protein expression and serum 17b-E2 levels. These observations are potentially important in understanding the pathogenesis of endometriosis, an aggressive tumor-like and estrogen-dependent benign disease, and might provide a novel therapeutic target for this disease. Acknowledgments: We are very grateful to Dr. Qiong Luo (Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine) for the review and revision of the manuscript.
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C-fos and MMP-9 in endometriosis
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