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Departments of Gynecology and Obstetrics (K.F., M.Ka., M.Ki., H.S., T.K., C.K., A.O., S.F.) and Clinical Molecular Biology (K.F., J.F.), Faculty of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Shingo Fujii, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507, Japan. E-mail: . sfu{at}kuhp kyoto-u.ac.jp
Abstract
Secreted frizzled related protein 1 (sFRP1) is a modulator of Wnt signaling. Recently, aberrations of Wnt signaling were reported to be involved in the pathology of various human neoplasms. We investigated the expression and function of sFRP1 in uterine leiomyomas. Secreted FRP1 expression was increased in leiomyomas, compared with normal myometrium using Northern and Western blot analyses. Expression was strongest in the late follicular phase (high estrogenic milieu) of the menstrual cycle. Interestingly, expression was negligible in leiomyomas treated with GnRH agonist. Expression was also prominent in cells during E2 treatment, serum deprivation, and hypoxia. Moreover, induction of apoptosis by serum deprivation in a leiomyosarcoma cell line was enhanced by antisense inhibition of sFRP1. These results suggest that sFRP1 expression was associated with uterine leiomyomas, particularly under high estrogenic conditions. Secreted FRP1 expression was not associated with cell proliferation but rather occurred during cell protection against apoptosis in vitro. Strong sFRP1 expression under high estrogenic conditions seems to contribute to the development of uterine leiomyomas through the antiapoptotic effect of sFRP1, which appear to be independent of cell proliferation.
UTERINE LEIOMYOMAS ARE the most common tumors found in the female genital tract (1). They cause symptoms such as abnormal genital bleeding, pelvic pain, and/or infertility. Leiomyomas have been reported to grow under the influence of ovarian steroids (estrogen and progesterone) and express the receptors thereof (2, 3). In addition, growth factors, such as epidermal growth factor, IGF, TGF-ßs, and/or their receptors have been reported to be associated with the growth of leiomyomas (4, 5, 6, 7). However, the factors that promote the initial development of leiomyomas and regulate their growth in vivo remain poorly understood.
Recently secreted frizzled related protein 1 (sFRP1) was identified as a modulator of Wnt signaling. Wnt proteins are secreted signaling molecules that are involved in the development of many organs (8, 9). The Wnt signal acts through the cytoplasmic protein Disheveled to inhibit the activity of the serine-threonine kinase, glycogen synthase kinase (GSK) 3ß. GSK3ß appears to bind through a bridging molecule, axin, to the adenomatous polyposis coli and ß-catenin complex and phosphorylates ß-catenin, causing its rapid degradation. Inhibition of GSK3ß by Wnt leads to ß-catenin stabilization and accumulates the uncomplexed soluble form of ß-catenin (10, 11, 12). The latter form can interact with T-cell factor/lymphoid enhancer factor transcription factor, move to the nucleus, and activate target genes. Secreted FRP1 has a binding site for Wnt and modulates its signaling (13, 14, 15).
There is evidence that aberrations of Wnt signaling, as well as sFRPs, can contribute to the neoplastic process (16, 17). Inappropriate expression of these ligands because of promoter insertion of the mouse mammary tumor virus (8) or targeted expression in transgenic mice causes mammary tumor formation (18). Genetic alterations affecting adenomatous polyposis coli or ß-catenin, which are associated with increased uncomplexed ß-catenin levels, have been observed in human colon cancers (19), melanomas (20), and hepatocellular carcinomas (21). Additionally, sFRP1 is down-regulated in breast tumors (22), and sFRP4 is up-regulated in endometrial neoplasms (23), indicating that aberrations of Wnt signaling pathways are critical to the development of these tumors.
Therefore, to determine whether sFRP1 is associated with the pathogenesis of uterine leiomyomas, we analyzed mRNA and protein expression of sFRP1 in leiomyomas and matched normal myometrium. In addition, the regulation of sFRP1 expression and its effects were examined in smooth muscle cells cultured from the myometrium and leiomyoma and cells of the leiomyosarcoma cell line. Moreover, sFRP1 was reported to increase under some stresses like starvation (22). We tried to observe sFRP1 expression under hypoxic condition because we had a hypothesis that hypoxia may play an important role in the pathogenesis of uterine leiomyomas (24).
Materials and Methods
Tissue collection
Matched myometrial and leiomyoma tissues were obtained from 25 patients with regular menstrual cycles who underwent surgery for uterine leiomyomas at Kyoto University Hospital. The patients ranged in age from 37 to 49 yr (mean age, 43 yr). Twenty-one patients did not receive any hormonal treatment for at least 3 months before surgery. On the other hand, four patients underwent presurgical GnRH agonist (GnRHa) treatment. Informed consent was obtained from each patient before surgery by means of consent forms, and protocols were approved by the Human Investigation Committee of the hospital. Myometrial tissues were obtained mostly from the uterine fundus and leiomyoma tissues from midway between the center and the periphery of each tumor. In the cases of multiple leiomyoma, the sample was obtained from each nodule. Endometrial dating of the patient was carried out by the method of Noyes et al. (25). Totally, there were seven patients in early follicular phase, seven in late follicular phase, seven in luteal phase, and four in GnRHa treatment. In the cases of multiple leiomyoma, the tissues from the largest nodule were regarded as a representative data during the following statistical analyses to avoid bias.
Northern blotting
Total RNA was prepared from surgical specimens by homogenization in Trizol reagent (Life Technologies, Frederick, MD). Total RNA (20 µg per lane) was separated by 1% agarose gel electrophoresis in the presence of formaldehyde. The separated RNA was then transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech Ltd., Little Chalfont, UK) by capillary action and cross-linked by UV light. A small EcoRI-XhoI-restricted fragment of sFRP1 was radiolabeled with 
-32P-dCTP using the random priming technique. The small fragment of S26 ribosomal protein was also radiolabeled with -32P-dCTP for normalization. Membranes were hybridized and washed according to established methods. All autoradiography was performed using intensifying screens at -70 C. The autoradiographic bands were quantified with the use of a densitometry (ATTO Densitograph version 4.0 software, ATTO Corp., Bunkyoku, Japan). Each sFRP1 band was normalized, using the value for the corresponding S26 ribosomal protein mRNA and was corrected for any variation in the amount of RNA applied to each lane.
Western blotting
Normal myometrium and leiomyoma tissue were homogenized with a Dounce homogenizer at 4 C in the lysis buffer containing 150 mM NaCl, 50 mM Tris-Cl, 5 mM EDTA, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Homogenates were subsequently centrifuged at 15,000 g for 30 min, and the supernatant was collected for protein analysis. The protein concentration of each sample was determined using a commercial protein assay kit (DC protein assay, Bio-Rad Laboratories, Inc., Richmond, CA). Proteins resolved by SDS-PAGE were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). All subsequent steps were performed at room temperature. After brief washing in TBST [25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1% Tween 20], membranes were blocked with 5% skim milk in TBST for 1 h and then washed three times with TBST. Membranes were incubated for 2 h with goat-polyclonal anti-sFRP1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1/1000 in 5% skim milk/TBST. After three washes with TBST, membranes were incubated for 2 h with horseradish peroxidase conjugated to antigoat antibody (DAKO Corp., Glostrup, Denmark). After three washes with TBST, bound antibodies were visualized by chemiluminescence (ECLplus, Amersham Pharmacia Biotech Ltd., Little Chalfont, UK).
Immunostaining
Frozen tissue sections were treated with 0.3% hydrogen peroxide to quench endogenous peroxidase activity. Sections were rinsed three times in PBS (5 min each rinse) and then immersed in normal blocking serum in PBS for 1 h at room temperature. Goat-polyclonal anti-sFRP1 antibody was then added to the sections that were incubated overnight at 4 C. After washing in PBS, avidin-biotin complex (Vector Laboratories, Inc., Burlingame, CA) conjugated to rabbit antigoat antibody was added for 1 h at room temperature. Sections were again washed in PBS before development with diaminobenzidine as chromagen. Slides were finally washed in distilled water and counterstained with hematoxylin. The specific staining of positive control was observed in small vessels (26), and slides treated with nonimmune control antibodies yielded negative results.
Cell cultures
Matched myometrial and leiomyoma tissues were minced and digested in PBS (-) containing 0.08% collagenase for 4 h at 37 C with agitation. The dispersed myometrial and leiomyoma cells were plated in DMEM (Nikken Biomedicals, Kuze, Kyoto, Japan) containing penicillin-streptomycin (2%, vol/vol, ICN Biomedicals, Inc., Aurora, OH) and FBS (IWAKI, 10%, vol/vol, Asahi Technoglass, Funabashi, Japan) and maintained in a 37 C incubator ventilated with 5% CO2 in room air. The medium was changed every 2–3 d and cells were subcultured after 5–7 d. The human leiomyosarcoma cell line, SK-LMS-1 (27), was maintained in DMEM with 10% FBS in a 37 C incubator ventilated with 5% CO2 in room air. The medium was changed every 2–3 d and cells were subcultured after 4–5 d.
Analysis of sFRP1 expression
For analysis of sFRP1 expression, the primary culture cells and SK-LMS-1 cells were plated at a density of 5 x 105 cells in a 100-mm diameter dish (IWAKI, Asahi Technoglass). After plating in DMEM with 10% FBS for 12 h, the medium was changed to fresh DMEM in the absence or presence of 10% FBS and cultured for an additional 96 h. Total RNA was isolated as described above for Northern blot analysis at 0, 1, 3, 6, 9, and 12 h or 0, 24, 48, 72, and 96 h after the medium exchange. After reaching confluence, the myometrial and leiomyoma cells were cultured in 1%, 5%, and 20% O2 in a 37 C incubator ventilated with 5% CO2 in room air for an additional 48 h and then harvested for analysis. To determine the hormonal regulation of sFRP1 expression, the culture medium was changed to Phenol Red-free DMEM (Life Technologies, Grand Island, NY) containing 10% charcoal-stripped FBS for the secondary culture to avoid the effect of steroids. After reaching confluence, cyclodextrin-encapsulated 17ß-estradiol (ß-E2-water soluble, Sigma, St. Louis, MO) was added to the culture medium at concentrations of 0.01, 0.1, and 1 µM. In addition, cyclodextrin-encapsulated progesterone (progesterone-water soluble, Sigma) was added to the culture medium at concentrations of 0.1, 1, and 10 µM in the absence or presence of E2 (0.01 µM). 2-Hydroxypropyl-ß-cyclodextrin (Sigma) was added at the same concentrations to act as control for this study. The cell cultures were harvested for analysis after 24 h of treatment.
Inhibition of sFRP1 gene expression
Antisense and sense phosphorothioate oligodeoxynucleotides (S-ODNs) for sFRP1 were custom synthesized by Japan Bioservice (Asagaya, Japan) (antisense: 5'-GCTGCGCCCGATGCCCATGCC-3'; sense: 5'-GGCATGGGCATCGGGCGCAGC-3'). SK-LMS-1 cells were plated at a density of 1.3 x 104 cells in 24-well flat-bottomed tissue-culture plates (IWAKI, Asahi Technoglass) or a density of 2.5 x 103 cells in 96-well flat-bottomed tissue-culture plates (IWAKI, Asahi Technoglass). After plating for 12 h, the medium was changed to fresh DMEM without FBS, and S-ODNs were added directly to the culture medium at concentrations of 0.1, 0.5, and 1 µM. The cultured cells that had been treated with S-ODNs were harvested for Northern blot analysis after 6 h, and the conditioned medium was collected for Western blot analysis after 12 h. Cell viability was determined using the Trypan Blue dye exclusion assay or MTT colorimetric method (Premix, WST-1; cell proliferation assay system: TaKaRa Biomedicals, Otsu, Japan) at the designated intervals.
Apoptosis detection
Using an ELISA kit for apoptosis detection (Cell Death Detection ELISA Plus, Roche Molecular Biochemicals, Mannheim, Germany), apoptosis induced by S-ODNs was evaluated. Briefly, SK-LMS-1 cells were plated at a density of 7.0 x 104 cells in 6-well flat-bottomed tissue-culture plates (IWAKI, Asahi Technoglass). After plating for 12 h, the medium was changed to fresh DMEM in the absence or presence of 10% FBS, and S-ODNs were added directly to the culture medium at concentrations of 0.1 and 1 µM. Twelve hours after treatment, the mixture of cell lysate and culture supernatant was placed in a streptavidin-coated microtiter plate. Based on quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones, mono- and oligonucleosomes in the cytoplasmic fraction of the cell lysate were determined.
Statistical methods
The experiments were repeated on at least three different occasions with SK-LMS-1 cells, and the cells prepared from different myometrial and leiomyoma tissues. Data are presented as means plus SEM. The significance of differences was assessed using paired t test at the comparison of the data between leiomyoma and matched myometrium. Comparisons between groups were made using ANOVA with Scheffé’s correction for multiple comparisons. Spearman’s test was used to evaluate correlation between sFRP1 expression and hormonal treatments or hypoxia. Differences were considered to be significant when P was less than 0.05.
Results
SFRP1 mRNA levels in normal myometrium and leiomyoma
Of the 25 patients, 23 showed higher expression of sFRP1 mRNA in leiomyoma than the matched normal myometrium; however, two pairs in the luteal phase did not show this tendency (Fig. 1A
, representative data shown). In five uteri with multiple leiomyoma, all leiomyoma nodules were examined. Each nodule showed similar high expression of sFRP1 mRNA, compared with the matched myometrium (Fig. 1A, groups 1 and 4). Overall, the sFRP1 mRNA level in the leiomyoma tissues was 3.1-fold higher than that in the normal myometrium samples (P < 0.01, Fig. 1B).
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). Western blot analysis and immunostaining of sFRP1
Having found higher sFRP1 mRNA levels in leiomyomas than in normal myometrium, the expression pattern of the sFRP1 protein was examined in these tissues of three representative cases by Western blot analysis to confirm the protein secretion also reflecting RNA expression. Using goat polyclonal anti-sFRP1 antibody, we could detect the band that indicates 36-kDa protein as previously reported (13) (Fig. 2A). The Western blot data clearly revealed an abundance of sFRP1 protein in leiomyoma, compared with much lower level in the myometrium, which parallels the findings for the mRNA transcripts.
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). There was no immunoreactivity in fibroblasts surrounding the vessels (Fig. 2B). In leiomyoma tissue, the cytoplasms of the tumor cells were more strongly stained than those of the myometrial cells (Fig. 2, C and D). Effect of E2 and/or progesterone treatment on sFRP1 gene expression in vitro
Because results during the menstrual cycle suggest that sFRP1 gene expression is under the influence of ovarian steroids, we evaluated the effect of E2 and/or progesterone on the expression of sFRP1 mRNA in smooth muscle cells cultured from the myometrium and leiomyoma. In the control pairs without steroid hormonal treatment, cells from the leiomyoma showed a 1.7-fold higher level of sFRP1 mRNA expression than cells from the myometrium on the average. Smooth muscle cells cultured from the myometrium exhibited no significant induction of sFRP1 mRNA in response to treatment with E2 and/or progesterone (Fig. 3, A through D). On the other hand, cells cultured from leiomyomas showed significant dose-dependent induction of sFRP1 mRNA in response to treatment with E2 (Fig. 3, A and B, r = 0.797, P < 0.05). However, progesterone had no effect on sFRP1 even when coapplied with E2 (Fig. 3, A, C, and D).
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In culture with 10% FBS, low expression of sFRP1 mRNA was observed during smooth muscle cell proliferation (in both leiomyoma and myometrium), but expression increased as the culture became quiescent at confluence. In serum-deprived culture (without 10% FBS), high expression of sFRP1 mRNA was observed in smooth muscle cells from both leiomyoma and myometrium even at low confluence (Fig. 4A).
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). In the presence of 10% FBS, sFRP1 mRNA was induced when cells became subconfluent and was increased as the culture became quiescent. On the other hand, serum deprivation caused rapid induction of sFRP1 mRNA after only 1 h of starvation, which reached a plateau by 6 h (Fig. 4C).
Furthermore, when the smooth muscle cells cultured from leiomyomas were placed under hypoxic conditions, there was a significant negative correlation between sFRP1 expression level and O2 concentration in leiomyoma cells (r = -0.725, P < 0.05). However, the smooth muscle cells cultured from the myometrium showed no significant correlation between sFRP1 expression and O2 concentration (Fig. 5, A and B).
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To determine the effect of the antisense S-ODN for sFRP1 on the expression of sFRP1, total RNA and secreted protein were evaluated in SK-LMS-1 cells of nontreatment control and the cells treated with sense and antisense S-ODN. As shown in Fig. 4C, we confirmed that the RNA expression of sFRP1 becomes maximum in by 6 h if we may cultured the cells without FBS. Therefore, we decided on the 6 h time point for collection of cells for the purpose of RNA analysis after treatment with the S-ODNs. Then Northern blot analysis demonstrated slight reduction of sFRP1 mRNA expression in the cells treated with antisense S-ODN, compared with sense S-ODN (Fig. 6A), and Western blot analysis demonstrated apparent reduction of sFRP1 protein secretion in the antisense treatment cells, compared with sense treatment (Fig. 6B). The densitometric quantification showed about 28% decrease of sFRP1 mRNA and about 70% decrease in the protein level on the average of three independent experiments in the cells treated with antisense S-ODN, compared with sense S-ODN (data not shown). This was probably caused by the different working mechanism of antisense S-ODN because the binding between antisense oligo and target RNA sometimes leads to the early degeneration of target RNA. Furthermore, the antisense oligo could also inhibit RNA translation to protein, so there existed additional decrease in protein level.
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, C and D). In addition, using the cell death detection ELISA kit, cells treated with S-ODNs for 12 h were evaluated for apoptosis. In the presence of FBS, apoptosis in the cells treated with both sense and antisense S-ODNs were as low as control. In the absence of FBS, apoptosis in nontreated cells was increased, and the level of apoptosis was significantly high especially in the group treated with antisense-ODN, compared with the group treated with sense S-ODN and controls (Fig. 7).
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This is the first report showing that sFRP1 expression is increased in human uterine leiomyomal tissue, compared with normal myometrial tissue. The increased expression was observed at the transcriptional level by Northern blotting. In addition, the expression of the protein at the posttranscriptional level was confirmed by Western blotting. Immunohistochemically, sFRP1 was stained in the cytoplasm of leiomyomal cells, myometrial cells, and vascular smooth muscle cells but not in fibroblasts surrounding blood vessels. This implies that sFRP1 is produced from the smooth muscle cells in leiomyoma, myometrium, and blood vessels.
Interestingly, particularly in leiomyoma, the expression of sFRP1 varied during the menstrual cycle with the highest level in the late follicular phase. In addition, downexpression of sFRP1 was observed during GnRHa treatment, which decreases estrogen secretion from the ovary. These observations suggest that the expression of sFRP1 could be under the control of estrogen. This was confirmed by in vitro experiments using smooth muscle cells cultured from leiomyoma that showed induction of sFRP1 mRNA by an addition of E2. It has been reported that the gene expression of estrogen receptors in leiomyoma is stronger than that in the myometrium (3). This implies that leiomyoma possesses increased sensitivity to E2. Therefore, the estrogen-dependent expression of sFRP1 in leiomyoma could be associated with the growth and the pathogenesis of leiomyoma.
However, as reported previously, leiomyomas show proliferative activity in the luteal phase of the menstrual cycle, which is under the influence of both estrogen and progesterone (28, 29). In our study, the expression of sFRP1 was shown to be low in proliferating cultured leiomyomal cells and increase as the cells became confluent. A high level of expression was maintained after confluence. Therefore, sFRP1 expression does not seem directly related to the proliferation of leiomyomal cells but rather appears associated with cells whose growth is inhibited by contact in tissue culture. In addition, both hypoxic conditions and serum deprivation induced increased expression of sFRP1 in leiomyoma cells. This suggests that sFRP1 may protect the cells from the damage caused by these stresses. In this respect, the effect of sFRP1 was examined using sense or antisense S-ODNs for sFRP1. In experiments using cells of the leiomyosarcoma cell line, the incidence of apoptosis under conditions of serum deprivation condition was compared when sense or antisense S-ODNs for sFRP1 were applied. The incidence of apoptosis was high in the condition of serum deprivation, and it was further increased by the application of the antisense S-ODN for sFRP1. This implies that sFRP1 has antiapoptotic effects in this experiment. This kind of apoptosis-modulating function has previously been reported for sFRP1 and sFRP2 (22, 30, 31).
Secreted FRP1 is reported to act as a modulator of the Wnt signaling pathway. The Wnt and Frizzled protein families have been reported in adult mouse and human tissues such as heart, brain, eye, lung, liver, kidney, and testis (8, 32). Consequently, sFRP1 probably exists in the myometrium and leiomyoma as a modulator of the Wnt signaling pathway if Wnt and its receptor can be detected in the myometrium and leiomyoma. Recently Wnt7a was reported to be decreased in leiomyoma tissue, compared with myometrium (33), and Wnt 4 was reported to be necessary in the development of mullerian duct (34). Getting together, Wnt-Frizzled signal pathway seems to play an important role in differentiation of uterine smooth muscle cell, and the aberration of this pathway caused by increased sFRP1 or decrease of Wnt 7a may cause unregulated growth of smooth muscle cells.
We hypothesize that leiomyomas result from proliferation of smooth muscle cells in the myometrial tissue that survives the repeated ischemic-reperfusion stress experienced during the menstrual cycle (24). The blood supply to the myometrium in vivo is known to decrease during uterine contraction, particularly during menstruation, and this ischemic-reperfusion state is emulated in the present in vitro studies by the serum deprivation and hypoxic conditions imposed on the cell cultures. In each luteal phase of the menstrual cycle, myometrial smooth muscles exhibit proliferative activity in preparation for pregnancy. However, if pregnancy does not occur, the proliferative activity of the myometrial smooth muscle cells may be interrupted at the time of menstruation. Myometrial contraction, which results in the cessation of menstrual bleeding, probably induces an ischemic/hypoxic state in the myometrial smooth muscle cells. Ischemic injury could occur in these cells that are in the proliferative phase. It is suggested that these injured cells could become candidates for progenitor cells of leiomyomas. Somatic mutation could well be induced in these cells after surviving many repeats of the menstrual cycle (24).
In this respect, the fact that the overexpression of sFRP1 in leiomyoma under high estrogenic conditions was unrelated to the proliferative activity of the cells is intriguing. Moreover, an association of sFRP1 with antiapoptotic function is interesting if we are to speculate on the pathogenesis of leiomyoma because impairment of appropriate apoptosis in damaged cells is thought to be one of the mechanisms of tumor genesis. This kind of trigger can occur in uterine smooth muscle cells after menstruation at the time of the late follicular phase, i.e. high estrogenic conditions.
Strong sFRP1 expression under high estrogenic conditions seems to contribute to the development of uterine leiomyomas through the antiapoptotic effect of sFRP1, which appear to be independent of cell proliferation.
Acknowledgments
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (no. 13307047 and 13877272) from the Ministry of Education and Sports, Japan.
Abbreviations: GnRHa, GnRH agonist; GSK, glycogen synthase kinase; sFRP1, secreted frizzled related protein 1; S-ODN, sense phosphorothioate oligodeoxynucleotides.
Received July 26, 2001.
Accepted December 22, 2001.
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