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GROWTH FACTORS-CYTOKINES-ONCOGENES |
Departments of Physiology (S.-J.T., H.-M.C., P.-C.C., L.-Y.W.) and Obstetrics & Gynecology (M.-H.W.), National Cheng Kung University Medical College, Tainan 70101, Taiwan, Republic of China
Address all correspondence and requests for reprints to: Lih-Yuh C. Wing, Ph.D., Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China. E-mail: . wing{at}mail.ncku.edu.tw
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Abstract |
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Introduction |
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FGF-9, originally isolated from human glioma cells (10), is widely expressed in rat central nervous system (11). The gene encoding for human FGF-9 is mapped to chromosome 11q11–13 (12). FGF-9 is highly conserved across species with greater than 93% identity among Xenopus, mouse, rat, and human, suggesting that FGF-9 is of vital importance (10, 13). In adult tissue, FGF-9 has been found to be a potent mitogen and survival factor for numerous nerve cells (14, 15). Recently, it was shown that mice lacking FGF-9 exhibit lung hypoplasia with reduced amount of mesenchyme and die shortly after birth (16). In addition, mice lacking FGF-9 result in male-to-female sex reversal during embryogenesis, indicating the critical role of FGF-9 in fetal testicular development (17).
FGF-9 binds to FGFR2 and FGFR3, but not FGFR1 and FGFR4, with high affinity and activates efficiently the "c" splice forms (18, 19, 20). Recently, FGF-9 was found to be expressed in the stroma of the prostate, an androgen-responsive organ, whose growth also requires epithelial-mesenchymal interaction. It has been shown that FGF-9 acts as an autocrine and paracrine factor for prostatic cell proliferation (21). In the uterus, endometrium undergoes cyclic changes in cell proliferation, differentiation, and death under the influences of ovarian steroids. The growth of endometrium includes glands and stroma. Several growth factors have been shown to involve in ovarian steroid regulation of uterine functions (22, 23). For example, FGF-2, FGF-7, and their receptors, FGFR1 and FGFR2IIIb, have been detected in and regulate the function of epithelial cells (24, 25). In contrast, the expression pattern and functional role of FGF-9 and its receptor in female reproductive tract has not been investigated. This study was designed to test the hypothesis that FGF-9 is expressed in human uterus and is a mitogen of uterine endometrial cells. The regulation of FGF-9 expression by ovarian steroid hormones was also investigated.
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Materials and Methods |
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Cell cultures
Eutopic endometrial stromal and epithelial cells (predominantly glandular epithelium) were dissociated and purified as previously described (26). Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and antibiotics 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 24-well culture plates using 1 ml phenol red-free DMEM/F12. Purity of the cell was immunostained with vimentin (stromal cell-specific) and cytokeratin (epithelial cell-specific) specific antibodies as previously described (26). The stromal cell population was free of epithelial cell contamination, whereas greater than 95% of epithelial cells were vimentin negative.
Regulation of FGF-9 gene expression in stromal cells
Subcultured stromal cells were maintained in 24 well plates (2 x 104 cells/well) until 70% confluence was reached. After serum starvation for 12 h, the cells were then incubated in phenol red-free DMEM/F12 supplemented with 1% charcoal-stripped FBS and were stimulated with vehicle, 17ß-estradiol (10-11–10-7 M), progesterone (10-11–10-7 M), or estradiol (10-9 M) plus progesterone (10-9 M) for 24 h. 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 (26, 27). For protein analysis, stromal cells (n = 5 batches of cells) were cultured in 10-cm Petri dish (5 x 105 cells/dish), serum starved and then treated with vehicle or 17ß-estradiol (10-9 M) for 4 or 24 h.
Effect of FGF-9 on stromal and epithelial cell proliferation
Subcultured stromal cells or epithelial cells were deprived of serum for 12 h and then treated with different doses of recombinant human FGF-9 (0.1 ng/ml to 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. In a separated experiment, 50 ng/ml of recombinant FGF-9 or FGF-2 (from Escherichia coli, R&D Research) were added to serum-starved stromal cells in the presence or absence of anti-FGF-9 monoclonal antibody (mouse against recombinant FGF-9, R&D Research) for 48 h and subjected to 3H-thymidine incorporation assay as previously described (28). In brief, cells were incubated with 3H-thymidine (1 µCi/ml) for 24 h and then washed twice with PBS. After the addition of 10% ice-cold trichloroacetic acid for 20 min, trichloroacetic acid was removed and cells were washed with PBS. The acid-insoluble fractions were dissolved by the addition of 1 N NaOH. The contents were 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, Inc., Fullterton, CA). The radioactivity was measured by a liquid scintillation counter.
Quantification of mRNA concentrations using standard curve quantitative competitive (QC)-RT-PCR methodology
Procedures for preparation of native and competitive plasmids for in vitro transcription of native and competitive RNA had been described previously (26, 27, 29). Specific primer pairs for FGF-9, FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, and FGFR3IIIc were designed according to sequences deposited in GenBank (Table 1
). All the plasmids containing native or competitors were sequenced by automated sequencing for verification of the sequences (ABI model 377, Perkin-Elmer Corp., Foster City, CA). Specific RNA was in vitro transcribed by procedures routinely used in our laboratory (26, 27, 29). 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 (30, 31). In brief, 1 attomole/reaction of competitor RNA was added into RT master mix [50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2 (pH 8.3), 10 mM dithiothreitol, 100 pmol random primer, 4 mM deoxy-NTPs and 50 U Moloney murine leukemia virus reverse transcriptase]. Fifteen microliters of this mix was then dispensed into PCR tubes and serial diluted native RNA (12.8–0.1 attomole/reaction) in 5 µl of diethylpyrocarbonate-treated water or 5 µl of RNA samples were added individually to each tube. RT was carried out at 42 C (90 min) followed by heating to 95 C for 10 min and quick chilled to 4 C in a thermocycler (PTC-100, MJ Research, Inc., Watertown, MA). Two microliters of RT products were added to 18 µl of PCR mix [final concentration: 20 mM Tris-HCl (pH 8.4 at 25 C), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxy-NTPs, 0.5 U Taq polymerase, and 0.4 µM of primers]. This was subjected to 30 cycles of amplification (30 sec denaturation at 95 C, 30 sec annealing at 57 C, and 30 sec elongation at 72 C) followed by final elongation at 72 C for 5 min. The PCR products were directly separated on a 5% acrylamide gel and then stained with ethidium bromide and placed on UV illuminator equipped with a camera connected to a computer. The gel image was analyzed using AlphaImager software (Alpha Innotech Corp., San Leandro, CA). Figure 1 demonstrates production of standard curve and calculation of amounts of RNA transcripts in the sample using FGF-9 as an example.
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Immunohistochemical staining
Paraffin-embedded tissues were sectioned at 5-µm thickness and mounted onto poly-lysine-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 three times (5 min each) in PBS before blocking with 10% normal goat serum (15 min at room temperature). The sections were again rinsed in PBS solution and incubated with primary antibody, rabbit-antihuman FGFR2 (amino acid 362–374, Sigma) or rabbit-antihuman FGFR3 (amino acid 359–372, Sigma) at 1:2000 dilution (overnight at 4 C). Following incubation with primary antibody, the tissue sections were rinsed three times (5 min each) in PBS and then incubated with a biotinylated sheep antirabbit immunoglobulin (1:500, Sigma) for 60 min at room temperature. The sections were then quenched of 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, Inc., Burlingame, CA) for 60 min at room temperature. The color reaction was precipitated using 3-amino-9-ethylcarbazole (Vector Laboratories, Inc.) 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 omission of the primary antibody as well as replacing primary antibody with nonimmunized rabbit serum and was undetectable in all instances.
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 (32). Differences in FGF-9 expression in endometria of different phases of menstrual cycle were performed using Tukey’s multiple comparison procedure once significance was found by F test. Dunnett’s procedure was used to test difference between individual treatment group and control in 3H-thymidine incorporation and FGF-9 mRNA expression levels.
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Results |
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). Using standard curve QC-RT-PCR, expression of FGF-9 transcript was the greatest at late proliferative phase (d 8–14 of menstrual cycle). There was no difference in FGF-9 mRNA concentration between early proliferative phase and entire secretory phase. Western blot analysis also showed similar pattern of FGF-9 expression in cell extracts obtained from uterine endometrium (Fig. 2B). Using purified stromal and epithelial cells, we have identified that FGF-9 was expressed primarily in the stromal cells (Fig. 2C).
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). Both of them have been stained positive in both glandular epithelial cells and stromal cells. Control experiments conducted on serial sections of endometrium using normal rabbit serum instead of specific primary antibody showed no immunostaining confirmed the specificity of the immunoreaction (Fig. 3, B and D). Because the antibodies used in this study were not able to distinguish the alternatively spicing variants, we have designed primers to specifically amplify FGFR2IIIb, FGFR2IIIc, FGFR3IIIb, and FGFR3IIIc, respectively. Figure 4 shows that FGFR2IIIc and FGFR3IIIc are primarily expressed in stromal cells, whereas FGFR2IIIb is present mainly in the epithelial cells. FGFR3IIIb is expressed in both epithelial and stromal cells. The amplified FGFR3IIIb PCR product was further subjected to sequence analysis because this is the first report demonstrating the presence of FGFR3IIIb in uterine endometrium. Alignment result showed the 3' end of amplified product is 100% identical with the first 118 bases of exon 8, indicating that it is indeed the IIIb splicing variant of FGFR3 (data not shown).
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). The effective dose of FGF-9 is less than 10 ng/ml. At the dose of 100 ng/ml, FGF-9 stimulated more stromal cells proliferation than 10% serum indicating the strong mitogenic effect of FGF-9 on endometrial stromal cells (Fig. 5A). The mitogenic effect of FGF-9, but not FGF-2, can be blocked by addition of anti-FGF-9 antibody provided the evidence for the specificity of growth effect (Fig. 5B). In contrast to its potent growth effect on stromal cells, FGF-9 exerts no effect on stimulation of epithelial cell proliferation even at the concentration of 100 ng/ml (Fig. 6).
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). On the other hand, progesterone failed to stimulate FGF-9 expression at any concentration tested (10-11–10-7 M). Coadministration of 17ß-estradiol (10-9 M) and progesterone (10-9 M) did not induce more FGF-9 compared with 17ß-estradiol treated alone (Fig. 7A). Treatment of endometrial stromal cells with 17ß-estradiol (10-9 M) induced time-dependent increase in FGF-9 protein as indicated by immunoprecipitation of cell lysate with monoclonal antibody specific to FGF-9 (Fig. 7B).
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shows that administration of 1 nM 17ß-estradiol induces endometrial stromal cell proliferation. The mitogenic effect of estradiol was blocked by cotreatment with anti FGF-9 antibody. Addition of anti FGF-9 antibody to cultured stromal cell, on the other hand, did not cause significant adverse effect (Fig. 8).
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Discussion |
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FGF-9 was first identified as a neuro-protecting and survival growth factor and was widely expressed in the brain (11). It has been shown that FGF-9 is associated with lung and testis development in the fetus and is a prostatic stromal-derived autocrine/paracrine growth factor in adult prostate gland (16, 17, 21). In this study, we demonstrated that FGF-9 was expressed in normal uterine endometrium, especially in stromal cells. Due to its affinity for cell surface and extracellular matrix heparan sulfate, diffusion of FGF-9 from the site of synthesis is limited. Thus, it is unlikely that FGF-9 of uterine origin will exert systematic effect via circulation. In contrast, the most possible target for uterine FGF-9 will be the uterus per se via an autocrine/paracrine mechanism. In addition, expression of FGF-9 was the greatest in late proliferative phase, which is coincident with rapid proliferation of uterine endometrium, suggesting that FGF-9 may play roles in regulation of endometrial cell growth. Indeed, FGF-9 stimulated endometrial stromal cell proliferation in a dose-dependent manner and its effect was blocked by addition of anti-FGF-9 antibody. Our result is in concordance with report by Giri et al. (21) showing that FGF-9 promotes prostatic stromal cell growth and extended the action of FGF-9 to the uterus.
The result that FGF-9 failed to stimulate uterine epithelial cell proliferation is in contrast to the report that FGF-9 is also a mitogen for prostatic epithelial cells (21). The underlying mechanism responsible for this discrepancy is not clear but may be due to tissue-specific expression of different isoforms of FGF receptors. The prostate study did not evaluate the expression of particular splicing variants in epithelial cells, whereas we found that high affinity receptors of FGF-9, namely FGFR2IIIc and FGFR3IIIc, were absence or expressed in very low level in uterine epithelial cells. Although our immunohistochemistry study demonstrated that FGFR2 and FGFR3 were both expressed in endometrial epithelium and stroma, the antibodies used in this study were not able to distinguish the "b" from "c" splicing variants. Using specific primers to identify splicing variants of "b" or "c", we found that FGFR2IIIc and FGFR3IIIc are mainly expressed in stromal cells, whereas FGFR2IIIb is primarily expressed in epithelial cells. The detection of FGFR3IIIb transcript in epithelial and stroma cells is interesting. To the best of our knowledge, there was no information regarding the expression and function of FGFR3IIIb in uterine endometrium in the literature. Our current result represents the first report to document the presence of FGFR3IIIb in both epithelial and stromal cells. It is known that FGF-9 binds to FGFR2 and FGFR3 with high affinity, whereas FGFR1 is not receptors for FGF-9 (18). Activation of FGFR2IIIc or FGFR3IIIc by FGF-9 results in great mitogenic effect, whereas activation of FGFR2IIIb and FGFR3IIIb only leads to weak response (19, 20). Our studies show that both epithelial and stromal cells proliferate well in the presence of serum, however, only stromal cells respond to FGF-9. It seems that some growth factors other than FGF-9 may be involved in epithelial cell proliferation. Epidermal growth factor, IGF-I, and IGF-II were shown to have mitogenic effect on cultured uterine epithelial cells (33, 34). Animal study also showed that the injection of FGF-7 into mice elicited an increased cell proliferation in uterine epithelial cells but not in the stroma. This is probably due to the presence of FGFR2IIIb, the high affinity receptor for FGF-7 in the epithelium but not in the stroma (35). Thus, the presence of FGFR2IIIb and FGFR3IIIb in epithelial cells may respond to other members of FGFs rather than to FGF-9.
Estrogen is known for its mitogenic effect in the uterus. However, estrogen normally does not directly stimulate cell proliferation. The mitogenic effect of estrogen on uterine epithelial cells is elicited via paracrine growth promoting influence from stromal cells (23, 36). It has been shown that the injection of FGF-7, a stroma-derived growth factor, into neonatal mouse stimulated uterine epithelial growth (35). Furthermore, FGF-7, FGF-10 and their receptors FGFR2IIIb are identified in the neonatal ovine uterus (25, 37). Consistent with their findings, we also demonstrate the existence of FGFR2IIIb in human endometrial epithelial cells. In the present study, we further demonstrate the specific expression of FGF-9 and its receptors FGFR2IIIc and FGF3IIIc in endometrial stroma. In association with systemic hormonal profile, the elevated expression of FGF-9 during late proliferative phase suggests that expression of FGF-9 may be regulated by estradiol. Indeed, administration of various doses of estradiol induced marked FGF-9 production by endometrial stromal cells. The results that FGF-9 is induced by estrogen and is able to stimulate uterine stromal cell proliferation make it an excellent candidate for endometrial estromedin growth factor. This hypothesis was substantiated by the finding that anti-FGF-9 antibody, when added to cultured stromal cells along with estrogen, completely inhibited estrogen mediated stromal cell proliferation. This result demonstrates that FGF-9 is, at least one of, the major peptidic growth factors downstream of estrogen-mediated uterine stromal cell proliferation.
To the contrary of estrogen stimulation of FGF-9, progesterone failed to stimulate FGF-9 expression either alone or in combination with estradiol, which echoes the in vivo data showing that FGF-9 concentration was not different between early proliferative phase and entire secretory phase. During the menstrual cycle, both epithelial and stromal cells proliferate during mid to late proliferative phase; however, stromal cells also proliferate during secretory phase (38, 39). Progesterone, which is secreted during secretory phase, stimulates stromal cell proliferation and differentiation. FGF-2 has been reported to control the growth of rat uterine stromal cells, and its effect is dependent on progesterone, not estrogen (40, 41). Taken together, different growth factors may be involved in estrogen and progesterone stimulated uterine stromal cell proliferation.
In conclusion, we have demonstrated that FGF-9 is a potent uterine stromal cells growth factor that was markedly expressed in stromal cell during late proliferative phase. Expression of FGF-9 is regulated by estrogen but not progesterone. Cell-type specific expression of high affinity FGF receptors may determine the target of FGF-9-mediated growth effect in human uterus.
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Footnotes |
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Abbreviations: FBS, Fetal bovine serum; FGF, fibroblast growth factor; FGFR1–4, high affinity tyrosine kinase receptors; QC, quantitative competitive.
Received December 31, 2001.
Accepted for publication March 25, 2002.
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on in vitro luteinized bovine granulosa cells. Reproduction 122:245–253[Abstract]
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