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Department of Physiology (L.-Y.C.W., H.-M.C., S.-J.T.), Institute of Basic Medical Sciences (P.-C.C., S.-J.T.), and Institute of Clinical Medicine and Department of Obstetrics/Gynecology (M.-H.W.), National Cheng Kung University Medical College, Tainan 701, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Shaw-Jenq Tsai, Ph.D., Department of Physiology, National Cheng Kung University Medical College, 1 University Road, Tainan 701, Taiwan, Republic of China. E-mail: seantsai{at}mail.ncku.edu.tw.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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FGFs regulate numerous important biological processes such as embryogenesis, angiogenesis, neuroprotection, cell proliferation and migration, implantation, and tumorigenesis (14). These heparin-binding polypeptides comprise a family of at least 23 structurally related proteins that are expressed in specific spatial and temporal patterns (14, 15). FGF-9, originally isolated from human glioma cells (16) has been found to be a potent mitogen and survival factor for numerous nerve cells (17, 18), prostatic cells (19), and lung mesenchymal cells (20) and is necessary for fetal testicular development (21). In the uterus, FGF-9 was found to be an autocrined estromedin that induced endometrial stromal but not epithelial cell proliferation during menstrual cycle (22).
Receptors for FGFs (FGFR1–4) are tyrosine kinase receptors consisting of an intracellular tyrosine kinase domain, a single transmembrane domain, and an extracellular portion that contains three Ig-like domains. FGFR1–3 undergo alternative mRNA splicing that generates three different isoforms (designated as IIIa, IIIb, and IIIc), which possess distinct FGF binding properties and tissue-specific expression patterns (23, 24, 25, 26). The splicing variant IIIa of FGFR is a secreted protein, whereas IIIb and IIIc are both membrane-bound receptors containing mutually exclusive Ig-like domains. It is generally believed that the IIIb isoform of FGFRs is expressed in epithelial lineages, whereas the IIIc variant is restricted to mesenchymal origin (27, 28, 29) except in the uterine endometrium where FGFR3IIIb is expressed in both stroma and epithelium (22).
Previously, we reported that FGF-9 is expressed in normal eutopic endometrial stroma and is regulated by 17ß-estradiol but not progesterone (22). Because ectopic endometriotic lesion aberrantly produces significant amounts of 17ß-estradiol, we sought to determine the effect of this autonomously produced steroid on the expression of FGF-9 and its high affinity receptors because it may control an important step in the development of endometriosis. The functional roles of FGF-9 in the development and/or maintenance of endometriosis were also investigated.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Tissues from patients with ovarian endometrioma (n = 20; 11 proliferative and nine secretory phase samples) and pelvic endometriotic implant (n = 10; six proliferative and four secretory phase samples) were collected for this study. Endometriosis was graded according to the revised classification of the American Society of Reproductive Medicine (30) and was histologically confirmed. Samples graded as stages I and II were classified as early endometriosis, whereas stages III and IV samples were classified as severe endometriosis. All ovarian endometrioma samples were derived from severe endometriosis, whereas pelvic endometriotic implants were obtained from early endometriosis in this study. Ectopic endometriotic samples were collected at the time of laparoscopy or laparotomy at the Department of Obstetrics and Gynecology, National Cheng Kung University Hospital. Endometriomas were removed by stripping the cyst wall from the ovarian endometriosis, whereas peritoneal implants were carefully dissected from surrounding tissues to avoid contaminations. Patients were of reproductive age with normal menstrual cycles and were not receiving any endocrine therapy, such as GnRH analog, danazol, or pseudopregnancy therapy. This study was approved by the Clinical Research Ethics Committee at the National Cheng Kung University Medical Center, and informed consent was obtained from each patient.
Tissue collection and cell culture
Tissues were immersed in Hank’s solution supplemented with HEPES and antibiotics and transported to the laboratory for further processing. Part of the tissues was snap frozen in liquid nitrogen and stored at -80 C for mRNA and protein analysis. Another part of tissues was fixed in 4% paraformaldehyde and paraffin embedded for histochemical study. The rest were minced and subjected for isolation of stromal cells as previously described (7, 31). Stromal cells were cultured in medium consisting of DMEM/F12, 10% fetal bovine serum (FBS), penicillin (100 µg/ml), streptomycin (100 U/ml), and fungizone (50 µg/ml) in a humidified atmosphere with 5% CO2 at 37 C. The medium was changed every other day. When the cells reached confluence, they were subcultured in phenol red-free DMEM/F12 medium supplemented with 10% FBS, penicillin (100 µg/ml), streptomycin (100 U/ml), and fungizone (50 µg/ml). Purity of the cell was immunostained with vimentin (stromal cell specific) and cytokeratin (epithelial cell specific) specific antibodies as previously described (7). The stromal cell population was free of epithelial cell contamination, whereas greater than 95% of the epithelial cells were vimentin negative. To confirm that the stromal cell collected for this study was endometrium origin, decidualization was induced by treatment with 8-bromo-cAMP for 3, 6, and 9 d, and culture media were collected for measurement of prolactin (PRL). Cell morphology was observed under a phase contrast microscope, and concentrations of PRL in conditioned media were measured using the electrochemiluminescence immunoassay kit and Modular Analytics E170 immunoassay analyzer (Roche Diagnostics GmbH, Mannheim, Germany). The sensitivity was 0.47 ng/ml, and the intraassay and interassay coefficients of variation were less than 10%. In addition, conditioned media of stromal cells and fibroblast cells were collected and concentrations of 17ß-estradiol were determined as previously described (7). The sensitivity (80% bound) was 14.4 pg/ml, and the intraassay and interassay coefficients of variation were less than 10%.
Cell cultures
Stromal cells were subcultured in 24-well culture plates (2 x 104 cells/well) in phenol red-free DMEM/F12 in the presence of 10% FBS until 70% confluence was reached. After serum starvation for 36 h, the cells were then stimulated with vehicle, 17ß-estradiol (10-11 to 10-7 M) for 24 h in phenol red-free DMEM/F12 without FBS. Cells were directly lysed in the well using lysis buffer (4 M guanidinium isothiocyanate; 10 mM Tris-HCl, pH 8.0; 0.5% sodium dodecyl sulfate; and 1% dithiothreitol) and subjected to mRNA isolation as described (7, 32). For protein analysis, stromal cells were cultured in a 10-cm petri dish (5 x 105 cells/dish), serum starved, and then treated with vehicle or 17ß-estradiol (10-9 M) in the presence or absence of ICI for 24 h. Culture medium was then collected for FGF-9 determination.
Effect of FGF-9 and 17ß-estradiol (E2) on endometriotic stromal cell proliferation
Subcultured stromal cells were deprived of serum for 36 h and then treated with different doses of recombinant human FGF-9 (0.1–200 ng/ml, from Sf21 cell, R&D Research, San Diego, CA) for 48 h in the presence or absence of 1% charcoal-stripped FBS and subjected to 3H-thymidine incorporation assay as previously described (22, 33). In brief, cells were incubated with 3H-thymidine (1 µCi/ml) for 24 h and then washed twice with PBS. Cells were incubated with 10% ice-cold trichloroacetic acid for 20 min and then washed with PBS. The acid-insoluble fractions were dissolved by the addition of 1 N NaOH and then neutralized with an equal volume of 1 N HCl to a final concentration of 0.5 N. Five-hundred-microliter aliquots were transferred to a scintillation vial containing 3.5 ml counting fluid (Ready safe, Beckman Coulter, Fullerton, CA). The radioactivity was measured by a liquid scintillation counter. To address the effect of spontaneous E2 biosynthesis on endometriotic stromal cell proliferation, cells were treated with ICI 182,870 (0.1, 1, and 10 µM) and 4-hydroxyandrostenedione (4-HA, 50 µM) in the presence or absence of E2 (10-9 M) or FGF-9 (30 ng/ml), and 3H-thymidine incorporation was determined.
Quantification of mRNA concentrations using standard curve quantitative, competitive RT-PCR (QC-RT-PCR) methodology
Procedures for preparation of native and competitive plasmids for in vitro transcription of native and competitive RNA has been described previously (7, 32, 34). Specific primer pairs for FGF-9, FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, and FGFR3IIIc were designed according to sequences deposited in GenBank as described before (22). Specific RNA was in vitro transcribed by procedures routinely used in our laboratory (7, 34, 35). Each RNA aliquot was used only one time to reduce variation due to potential degradation of RNA after repeated freezing and thawing. The detailed procedure of standard curve QC-RT-PCR was described previously (22, 36).
Detection of FGF-9 protein by Western blotting
Expression of FGF-9 in endometriotic tissues was detected by Western blot using standard procedure (7). Goat anti-FGF-9 polyclonal antibody (200 µg/ml, R&D) and horseradish peroxidase-conjugated rabbit antigoat Ig (1:2000 dilution, Sigma, St. Louis, MO) were used as primary and secondary antibodies, respectively. Effect of estrogen and ICI on expression of FGF-9 was determined by loading equal volume of culture media onto 12.5% SDS-PAGE, separated by electrophoresis, transferred onto polyvinyl difluoride membrane (Millipore Co., Bedford, MA), and subjected to immunoblotting. The remaining cells were lysed for determination of DNA contents using Hoechst 33258 fluorescent dye and a fluorometer (DyNA Quant 200, Pharmacia, Piscataway, NJ). Amount of FGF-9 was quantified by AlphaImager software (Alpha Innotech Corp., San Leandro CA) and normalized with contents of DNA.
Immunohistochemical staining
Paraffin-embedded tissues were sectioned at 5-µm thickness and mounted onto polylysine-coated slides, deparaffinized, and rehydrated. Tissue sections were incubated with 0.1% trypsin at room temperature for 10 min followed by incubating with 0.1 µg/ml trypsin inhibitor (Sigma) for 5 min. The sections were then rinsed with PBS before blocking with 10% normal goat serum for 15 min at room temperature. The sections were again rinsed in PBS solution and incubated with rabbit antihuman FGFR2 (amino acid 362–374, Sigma) or rabbit-antihuman FGFR3 (amino acid 359–372, Sigma) at a 1:2000 dilution. The tissue sections were then rinsed three times (5 min each) in PBS and incubated with a biotinylated sheep antirabbit Ig (1:500, Sigma) for 60 min at room temperature. The sections were then quenched with endogenous peroxidase activity (3% H2O2 in PBS at room temperature for 10 min) and rinsed briefly in PBS. Amplification of the antigen-antibody complex was achieved by using avidin-biotin-peroxidase (ABC kit, Vector Laboratories, Burlingame, CA) for 60 min at room temperature. The color reaction was precipitated using 3-amino-9-ethylcarbazole (Vector Laboratories) for 10 min at room temperature. The tissue sections were counterstained with hematoxylin, and coverslips were mounted using an aqueous mounting medium (Dako Corp., Carpinteria, CA). Nonspecific staining was assessed by replacing primary antibody with nonimmunized rabbit serum.
Statistical analysis
The data were expressed as mean ± SEM. Differences in a given mRNA among groups were analyzed with the one-way ANOVA through use of general linear model of the Statistical Analysis System (37). Dunnett’s procedure was used to test difference between individual treatment group and control in 3H-thymidine incorporation, and FGF-9 mRNA expression levels once significance was found by F-test.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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To determine ectopic endometriotic tissues used in this study were originated from endometrium, stromal cells derived from these tissues were placed in culture for 3, 6, and 9 d with or without 0.5 mM 8-bromo-cAMP. In the presence of 8-bromo-cAMP, PRL production was measurable on d 6 and further elevated on d 9 after treatment (Fig. 1A
). In contrast, unstimulated stromal cells and fibroblasts (NIH3T3, stimulated or unstimulated) did not produce any detectable concentrations of PRL (Fig. 1A and data not shown). Morphologically, the untreated cells showed a fibroblast-like spindle-shaped appearance, whereas the cells treated with 8-bromo-cAMP were transformed into large polygonal cells, resembling decidual cells (data not shown). When cultured in serum-free, phenol red-free medium, ectopic endometriotic stromal cells produced significant amounts of E2 (Fig. 1B), whereas eutopic endometrial stromal cells or fibroblasts did not (data not shown).
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Concentrations of mRNA encoding for FGF-9 were greater in endometriotic tissues obtained from pelvic implants, compared with that in ovarian endometrioma (Fig. 2A). The expression of FGF-9 transcripts in endometriotic implants, though fluctuating, was not different during the menstrual cycle (Table 1). Western blot analysis further confirmed that FGF-9 protein expressed in pelvic implants was greater than that in ovarian endometrioma (Fig. 2B). Results from RT-PCR demonstrated that FGF-9 is predominantly expressed by endometriotic implant, whereas a minute amount was detected in uterine myometrium (Fig. 2C). In contrast, fibroblast cells and peritoneum had no detectable amount of FGF-9 mRNA (Fig. 2C). Immunohistostaining demonstrated that FGFR2 and FGFR3 were expressed in ectopic endometriotic lesion (Fig. 3). Positive stain was found in both epithelium and stroma (Fig. 3). Further analysis of subtype of FGFRs in purified stromal cell using standard curve QC-RT-PCR indicated that the "c" variants were the major isotype of FGFR2 (Fig. 4A). In contrast, the "b" and "c" variants of FGFR3 were expressed at roughly equal amount (Fig. 4B).
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In purified stromal cells, expression of FGF-9 was induced by administration of E2 in a dose-dependent manner with greatest effect found at 10-9 M (Fig. 5A). In contrast, the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was not altered by treatment with E2 (Fig. 5B). Time-dependent experiments found that E2 stimulated FGF-9 expression at 24 h after treatment (data not shown). Effect of estrogen on the expression of "c" variant was further addressed because FGF-9 binds to this particular splicing variant with high affinity. Administration of E2 induced both FGFR2IIIc and FGFR3IIIc expression in endometriotic stromal cells (Fig. 6). The maximal induction was seen at 10-9 and 10-10 M for FGFR2IIIc and FGFR3IIIc, respectively.
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FGF-9 stimulated proliferation of ectopic endometriotic stromal cells in a dose-dependent manner (Fig. 7). The lowest effective concentration was found at 10 ng/ml. Maximal induction (6-fold over control) was found at concentration of 100 ng/ml. This mitogenic effect of FGF-9 was seen in the presence (Fig. 7) or absence (data not shown) of 1% charcoal-stripped serum.
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To characterize the biological significance of endogenously produced E2 by ectopic endometriotic implants, estrogen receptor (ER) antagonist as well as aromatase inhibitor was administered to the cultured cell, and the expression of FGF-9 and concomitantly cell proliferation were determined. Treatment of endometriotic stromal cells with ICI inhibited both FGF-9 mRNA and protein expression in a dose-dependent manner (Fig. 8). The inhibitory effect of ICI was reversed by adding E2 (1 nM) back to the culture system, indicating it is an ER-mediated event. Administration of 4-HA also inhibited FGF-9 expression, but the effect was not as evident as that of ICI 182,870 (data not shown). Proliferation of endometriotic stromal cell was inhibited by treatment with ICI (Fig. 9A). The inhibitory effect of ICI was dose dependent (Fig. 9A). Treatment of endometriotic stromal cells with 4-HA also inhibited 3H-thymidine incorporation (Fig. 9A). Inhibition of endometriotic stromal cell proliferation by ICI was reversed by adding exogenous E2 or FGF-9 back to the culture medium (Fig. 9B). In contrast, addition of FGF-7 and FGF-10 failed to release the inhibitory effect caused by ICI (Fig. 9B), indicating this is not a random effect.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Studies using ER
knockout mice have demonstrated that estrogen-stimulated uterine responses and cell proliferation are paracrine/autocrine events mediated by ER-positive stroma (39, 40, 41). EGF, IGF-I, and FGFs are believed to be the major paracrine/autocrine factors carrying out the estrogen-stimulated growth effect (10, 11, 12, 13). However, attempts to characterize the expression patterns of these growth factors in association with severity of endometriosis have not found strong correlations. The expression of EGF and EGF receptor are not different between ectopic endometriotic implants and eutopic endometrial tissues (42, 43). Concentration of IGF-I in peritoneal fluid of women with endometriosis was higher (44), no different (45), or lower (46) as compared with that without endometriosis. Increased FGF-2 and FGFR1 expression in glandular epithelium, and stroma was found in adenomyosis, compared with autologous endometrium (47). However, concentrations of FGF-2 in peritoneal fluid and immunoreactive FGF-2 in pelvic endometriotic lesions were not different from those of normal or eutopic counterparts (43, 48, 49). Moreover, using competitive RT-PCR methodology, Di Blasio et al. (50) demonstrated that FGF-2 transcripts in ectopic endometriotic stromal cells were lower, compared with those in eutopic endometrial stroma. Nevertheless, the lack of positive correlation does not exclude the importance of these peptide growth factors in the development of endometriosis. It is well established that cyclic involution of uterine endometrium is due to the lack of support from peptide growth factors regulated by ovarian hormones. The ectopic endometriotic tissue can escape this destiny if it evolves a self-supporting system that enables autonomous production of growth factors when they are needed the most. Thus, the critical role of peptide growth factor in contributing to the development of endometriosis may not be determined by the amount but the time of expression.
In the current study, we demonstrated that the expression of FGF-9 was regulated by estrogen in ectopic endometriotic tissue just like that in eutopic endometrium (22). The slight difference that distinguishes estrogen effect in these two counterparts was that expression of FGF-9 in ectopic endometriotic lesions is not changed throughout the menstrual cycle. In contrast, FGF-9 expression in eutopic endometrium was greatest in late follicular phase when serum estrogen reaches the maximal concentration (22). This may be due to the fact that ectopic endometriotic tissue is able to synthesize estrogen so as to reduce the dependency on estrogen of ovarian origin. During late follicular phase, ovarian estrogen represents the dominant source for FGF-9 induction in both eutopic and ectopic endometrial stromal cells. When serum estrogen of ovarian origin falls, especially during menstruation or early follicular phase, expression of FGF-9 in eutopic endometrial stroma decreases due to lack of estrogen stimulation. On the contrary, estrogen synthesized by ectopic endometriotic stroma (51) serves as primary source for induction of FGF-9 via an autocrine mechanism when systemic estrogen is low. As a result, the expression of FGF-9 in ectopic endometriotic stroma persisted. The effect of self-producing estrogen on FGF-9 expression was more evident when stromal cells were placed in culture with no exogenous estrogen administrated. Under phenol-red free, serum-free condition, stromal cells obtained from ectopic endometriotic lesion effectively produced E2 and FGF-9. The production of FGF-9 was inhibited by administration of ER antagonist or aromatase inhibitor, whereas administration of estrogen reversed inhibition of FGF-9 expression by ICI provides further evidence to support this notion.
We have previously shown that expression of StAR by endometriotic stromal cells leads to concomitant elevation of estrogen in the peritoneal fluid, which was greatest in patients with early endometriosis (7). Amounts of FGF-9 and its receptors in ectopic endometriotic tissues were highly associated with concentrations of estrogen in the peritoneal fluid. In accordance with these results, our in vitro study also demonstrated that FGF-9 and FGFRs were indeed controlled by treatment with E2. The biological significance of elevation of FGF-9 system in early endometriosis may be that it is important for the growth and/or survival of endometriotic implants. Because FGF-9 is one of the major growth factors for stromal cell proliferation (19, 22), it is reasonable to hypothesize that early endometriotic lesions can survive the hostile microenvironment of the peritoneal cavity may, in some part, owing to the proliferative, and possibly antiapoptotic, effect exerted by expressing FGF-9 in the ectopic endometriotic stroma. It is known that FGF-9 binds to FGFR2IIIc and FGFR3IIIc with high affinity and that binding of FGF-9 to both receptors elicits potent mitogenic effect (52). Our in vitro study clearly demonstrated that FGF-9 is a potent mitogen for endometriotic stromal cells and is in agreement with results of previous reports using other cell types (19, 52). Therefore, the elevation of both FGF-9 and its high affinity receptors in the early stage may result in rapid cell proliferation and increase the chance for ectopic endometriotic lesions to survive in battling against the immune system. Nevertheless, further studies are necessary to resolve this hypothesis.
The development and maintenance of endometriosis is clearly estrogen dependent. Deprivation of estrogen by surgical or pharmacological approaches leads to immediate growth retardation or even regression of endometriotic implants. However, pharmacological induction of hypoestrogenism seldom results in complete involution of endometriotic lesions (53, 54, 55). The 5-yr recurrence rate of patients receiving GnRH analog treatment is 53.4% for all stages of endometriosis and can be as high as 74.4% for those with severe endometriosis (54). The underlining mechanisms are not known, but aberrant production of estrogen by endometriotic lesion per se may account for one of the factors. It may also explain, at least in part, the failure of danazol or GnRH agonists, used for the systemic depletion of LH-stimulated ovarian estrogen, to eliminate ectopic endometriotic lesions (56). In contrast, the use of aromatase inhibitors for the systemic and local inhibition of estrogen biosynthesis has shown promising results in the treatment of endometriosis (55). Obviously, there are many other factors involve in the pathogenesis of endometriosis, and the results of our current study are primarily based on in vitro experiments, which may not represent well what actually happens in vivo. The value of our current report is to provide a new thinking direction of reevaluating effects of estrogen and peptide growth factors on the etiology of endometriosis by focusing on the temporal instead of quantitative expression of these growth promoting/antiapoptotic agents.
In conclusion, our current results demonstrated that expression of FGF-9 and its receptors in ectopic endometriotic lesions was regulated by estrogen and controlled endometriotic stromal cell proliferation. More importantly, the expression of FGF-9 system in ectopic endometriotic tissues was induced by estrogen of endometriotic stromal cell origin in an autocrine fashion. This may play a critical role in promoting ectopic retrograded endometrial survival and/or propagation, especially during menstruation or early follicular phase when ovarian estrogen output is at the nadir or limited. Our present report may provide new insights into the pathophysiological effect of abnormal local production of estrogen in the etiology of endometriosis.
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Footnotes |
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Abbreviations: E2, 17ß-Estradiol; EGF, epidermal growth factor; ER, estrogen receptor; FBS, fetal bovine serum; FGF, fibroblast growth factor; FGFR, FGF receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 4-HA, 4-hydroxyandrostenedione; PRL, prolactin; QC-RT-PCR, quantitative, competitive RT-PCR; StAR, steroidogenic acute regulatory protein.
Received April 14, 2003.
Accepted July 30, 2003.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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knockout mouse. Biol Reprod 59:470–475 on in vitro luteinized bovine granulosa cells. Reproduction 122:245–253[Abstract]
(PGF2) is associated with PGF2 resistance or susceptibility in human granulosa-luteal cells. Mol Hum Reprod 7:415–423This article has been cited by other articles:
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