Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells

Takashi Yamada, Satoshi Nakago, Osamu Kurachi, Jiayin Wang, Shigeki Takekida, Hiroya Matsuo and Takeshi Maruo1

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan

1 To whom correspondence should be addressed. e-mail: maruo{at}kobe-u.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Insulin-like growth factor-I (IGF-I) plays crucial roles in uterine leiomyoma cell growth through stimulating proliferation and inhibiting apoptosis. The present study was conducted to elucidate whether progesterone affects IGF-I and its receptor expression in cultured leiomyoma cells. METHODS: Isolated leiomyoma cells were subcultured in Phenol Red-free Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 48 h in the presence or absence of 17{beta}-estradiol (E2) (10 ng/ml) or progesterone (100 ng/ml). IGF-I and its receptor mRNA and immunoreactive IGF-I in the cultured cells were assessed by quantitative RT–PCR with Southern blot analysis and by radioimmunoassay with Seppak C18 chromatography, respectively. The presence of estrogen receptor (ER) and progesterone receptor (PR) in cultured leiomyoma cells was immunocytochemically examined. RESULTS: Both treatment with progesterone alone and treatment with E2 and progesterone combined significantly decreased IGF-I mRNA and protein expression in cultured leiomyoma cells compared with that in untreated cultures, but treatment with E2 alone did not. IGF-I receptor mRNA expression in those cells was not affected by treatment with either E2 or progesterone. Immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells maintained in a serum-free condition for 48 h whereas ER protein expression in the cells remarkably decreased after 24 h culture under the serum-free condition. CONCLUSIONS: The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.

Key words: cultured leiomyoma cells/estrogen/IGF-I/progesterone/uterine leiomyoma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Uterine leiomyoma is the most common benign tumour in women of reproductive years, occurring in as many as 30% of women aged >35 years (Vollenhoven et al., 1997Go). In most countries, uterine leiomyomas are the most frequent cause of indication for hysterectomy in pre-menopausal women and therefore present a major health issue (Vollenhoven, 1998Go). The growth of leiomyomas is dependent on the biological activity of ovarian steroid hormones (Rein and Nowak, 1992Go) mediated by locally derived growth factors. We have demonstrated that progesterone up-regulates the expression of epidermal growth factor (EGF) and proliferating cell nuclear antigen (PCNA) in cultured human leiomyoma cells, whereas 17{beta}-estradiol (E2) up-regulates the expression of EGF receptor and PCNA in those cells (Shimomura et al., 1998Go). As EGF is known to play a vital role in the autocrine/paracrine regulation of leiomyoma growth (Hofmann et al., 1984Go; Yeh et al., 1991Go), it is conceivable that progesterone and E2 act in combination to stimulate leiomyoma cell proliferation through the induction of EGF and EGF receptor in those cells. We have also shown that progesterone up-regulates the expression of Bcl-2 protein, an apoptosis-inhibiting gene product, and down-regulates the expression of tumour necrosis factor-{alpha} (TNF-{alpha}), a cytokine which induces apoptosis, in the cultured leiomyoma cells (Matsuo et al., 1997Go; Kurachi et al., 2001Go). This suggests that progesterone may also participate in leiomyoma cell survival through the inhibition of apoptosis of those cells. Furthermore, our recent study has indicated that E2 down-regulates p53 protein content, a tumour suppressor gene product, in the cultured leiomyoma cells, but that progesterone does not (Gao et al., 2002Go).

Insulin-like growth factor-I (IGF-I), a major anabolic agent responsible for growth and differentiation as the mediator of growth hormone (Schoenle et al., 1982Go), is a single-chain peptide that has a high degree of amino acid homology with pro-insulin. In the human uterus, IGF-I mRNA expression in leiomyomas was demonstrated to be greater than that in myometrium (Boehm et al., 1990Go). Recently we have demonstrated that IGF-I plays a crucial role in uterine leiomyoma growth, not only through promoting cell proliferation but also through inhibiting apoptosis in cultured leiomyoma cells (Gao et al., 2001Go). Several investigators also reported that IGF-I promotes leiomyoma cell growth (Van Der Ven et al., 1994Go, 1996; Strawn et al., 1995Go; Howe et al., 1996Go). IGF-I expression in the rat uterus is induced by estrogen (Murphy et al., 1987Go). Giudice et al. (1993Go) demonstrated that IGF-I mRNA expression in human uterine leiomyoma is significantly higher in the proliferative phase of the menstrual cycle relative to that in the secretory, progesterone-dominated, phase of the menstrual cycle. Furthermore, Englund et al. (2000Go) reported that IGF-I mRNA expression in uterine leiomyoma from GnRH agonist-treated women is lower compared with that in untreated women and that there is a significant positive correlation between serum E2 concentration and IGF-I mRNA expression in both myometrium and uterine leiomyoma except in pregnant women. Rein et al. (1990Go) found that tissue explants of leiomyoma could secrete IGF-I in culture, and leiomyoma tissues obtained from GnRH agonist-treated hypo-estrogenic women secreted significantly less IGF-I. Little evidence is, however, available regarding the direct effects of sex steroid hormones on IGF-I and its receptor expression in uterine leiomyoma cells. Thus, we conducted the present study to investigate the effects of sex steroid hormones on IGF-I and its receptor expressions in human uterine leiomyoma cells cultured in vitro in serum-free conditions.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue collection
Uterine leiomyoma tissues were obtained from women with regular menstrual cycles who underwent abdominal hysterectomy for medical indicated reasons at Kobe University Hospital. A total of 10 uterine leiomyomas were collected, of which five were from the proliferative phase and five from the secretory phase of the menstrual cycle. Each uterine specimen was examined histologically by a pathologist and the endometrium dated. Endometrial tissues were obtained from extirpated uteri, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (1950Go). Informed consent for the present study was obtained from each patient before surgery. The institutional review board approved the use of uterine tissues for culture experiments. The patients ranged in age from 31 to 45 years with a mean age of 38.0 years and no patients received any hormonal therapy for at least six menstrual cycles before surgery. Samples were excluded from the present study if accurate menstrual cycle dates could not be assigned or if unexpected pathology (e.g. adenomyosis or leiomyosarcoma) was found.

Cell culture
Cell cultures of uterine leiomyoma cells were carried out as previously described (Matsuo et al., 1997Go). In brief, uterine leiomyoma tissues, dissected from endometrial cell layers, were cut into small pieces and digested in 2.0% collagenase (wt/vol) at 37°C for 3–5 h. The leiomyoma cells were collected by centrifugation at 460 g for 5 min and washed several times with Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Inc., USA) containing 1% antibiotic solution (1x107 IU/l penicillin and 0.5 mg/l streptomycin). The cell viability was determined by Trypan Blue exclusion test. The isolated leiomyoma cells were plated at a density of ~106 cells/dish in 10 cm2 culture dishes (Iwaki Glass Corp., Japan). The leiomyoma cells were subcultured for 120 h at 37°C in a humidified atmosphere of 5% CO2/95% air in Phenol Red-free DMEM (Berthois et al., 1986Go; Welshons et al., 1988Go) supplemented with 10% fetal bovine serum (FBS) (vol/vol; Life Technologies). Characterization of the cultured cells was confirmed immunocytochemically as previously described (Matsuo et al., 1977Go). The cultured cells were immunostained for the muscle-specific protein desmin and {alpha}-smooth muscle actin, but not immunostained for either cytokeratin 19, a cytoskeletal protein specific for epithelial cells, or vimentin, a class of intermediate filament protein present in fibroblast. The monolayer cultures at ~60% confluence were treated with E2 (10 ng/ml), progesterone (100 ng/ml) or E2 (10 ng/ml) plus progesterone (100 ng/ml) in serum-free, Phenol Red-free DMEM for an additional 48 h. The cultured cells were immediately stored at –80°C for subsequent analysis.

Quantitative RT–PCR analysis with Southern blot analysis
Total RNA was isolated from cultured leiomyoma cells using RNeasy Mini Kit (Qiagen, GmbH, Germany) according to the manufacturer’s protocol. Expression of IGF-I mRNA and IGF-I receptor mRNA was assessed by RT–PCR following first-strand synthesis and precipitation of complementary DNA (cDNA). First strand cDNA was synthesized from 4 µg total RNA using a cDNA synthesis kit (Qiagen) according to the manufacturer’s protocol. PCR was performed using 0.1 µg cDNA as template in a 25 µl reaction buffer (10 mmol/l Tris–HCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l of MgCl2 and 0.01% gelatin) containing 6.25 pmol/l of each primer (IGF-I sense primer: 5'-ATGCACACCATGTCCTCCTC-3'; IGF-I antisense primer: 5'-GCACGGACAGAGCGAGCTGA-3'; IGF-I receptor sense primer: 5'-AGCCGATGTGTGAGAAGACC-3'; IGF-I receptor antisense primer: 5'-TTCTTCCTCACAGACCTTCG-3') synthesized by Gibco Life Technologies (USA), 2.5 mmol/l dNTP and 0.125 IU taq DNA polymerase (all from Qiagen). Reactions were amplified by a Gene Amp PCR System 9600-R (Perkin Elmer Corp., USA) using the following thermal profile: initial denaturation step at 95°C for 5 min, followed by 20 or 21 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and a final elongation of 72°C for 5 min. To test for the possibility of RNA degradation or RNA transcription default, separate reactions were performed using primers specific for the housekeeping gene, {beta}-actin (sense primer: 5'-CTTCTACAATGAGCTGCGTG-3'; antisense primer: 5'-TCATGAGGTAGTCAGTCAGG-3'). The PCR product specific for {beta}-actin was visualized under UV light following gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

The PCR products specific for IGF-I and IGF-I receptor were electrophoresed on 3% agarose gel and transferred to a nylon membrane filter for Southern blot analysis after denaturation with alkaline solution. The DNA on the membrane was fixed by UV irradiation. Subsequently, a 5'-32P end-labelled oligonucleotide probe specific for IGF-I (5'-GCTGGTGGATGCTCTTCAGTTCGT GTGTGG-3') or IGF-I receptor (5'-CAACTACTACTATGC CGGTGTCTGTGTGCC-3') was allowed to hybridize to membranes at 65°C for 18 h after prehybridization at 65°C for 2 h. The probe specific for {beta}-actin was the full-length cDNA of {beta}-actin labelled with 32P. Membrane washing was allowed to proceed twice by washing buffer (300 mmol/l NaCl and 30 mmol/l trisodium citrate, pH 7.0) with 0.1% sodium dodecyl sulphate (SDS) at room temperature for 20 min, followed by incubation in a buffer (30 mmol/l NaCl and 3 mmol/l trisodium citrate, pH 7.0) with 0.1% SDS at 60°C for 5 min. Membranes were visualized by exposure to X-Omat film (Eastman Kodak Co., USA). The radioautograms were then scanned with Scanjet 3 C/ADF (Hewlett–Packard, USA) and quantified with NIH Image version 1.58. The amount of mRNA was expressed relative to the abundance of {beta}-actin mRNA. Data were presented as fold increase over the control value and the mean ± SD. The PCR products were cloned and sequence analysis revealed their specificity.

Determination of immunoreactive IGF-I in cultured leiomyoma cells
Protein extraction from cultured leiomyoma cells was performed as described previously (Shimomura et al., 1998Go). At the termination of culture, cultured cells were lysed at 4°C for 20 min in the presence of a lysis buffer (150 mmol/l NaCl, 2 mmol/l phenylmethylsulphonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/l aprotinin, 0.1% SDS and 50 mmol/l Tris–HCl, PH 7.5). The lysates were subsequently centrifuged at 13 000 g for 30 min at 4°C and the supernatants were collected. Protein content in the supernatants was estimated by the Bradford (1976Go) assay, with bovine serum albumin as a standard. Immunoreactive IGF-I concentrations in the cell extracts were measured by radioimmunoassay as previously described (Nakago et al., 1999Go), after removal of IGF binding proteins (IGFBP) from samples by Seppak C18 cartridge chromatography (Daughaday et al., 1986Go).

Immunocytochemical analysis of ER and PR protein expression
Leiomyoma cells cultured for 0, 24 and 48 h under a serum-free, Phenol Red-free condition following the subculture for 120 h in DMEM with 10% FBS were fixed in 90% ethanol at 4°C for 20 min, and washed with PBS for three times. The fixed cells were subjected to immunostaining by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitags, USA). An affinity-purified rabbit polyclonal antibody (ER{alpha} HC-20: sc-543; Santa Cruz Biotechnology, Inc., USA) raised against a peptide mapping at the carboxy terminus of human ER{alpha} and an affinity-purified rabbit polyclonal antibody (PR C-19: sc-538; Santa Cruz Biotechnology) raised against a peptide mapping at the carboxy terminus of human PR which reacts with PR-A and PR-B were used at a dilution of 1:200 as the primary antibodies. To assure the specificity of the immunological reaction, control cells were subjected to the same immunoperoxidase method, except that the primary antibody was replaced by non-immune rabbit IgG at the same dilution as the specific antibody. The replacement of the specific antibody with non-immune rabbit IgG resulted in a lack of positive immunostaining.

The ER-positive rate and PR-positive rate of cultured leiomyoma cells were determined by observing >1000 nuclei for each experimental sample. Experiments were repeated four times. Data are presented as mean ± SD.

Statistical analysis
Statistical analysis was performed using one-way analysis of variance with Stat View 4.1 software (SAS Institute, Inc., USA) for Macintosh, followed by post hoc testing using Fisher’s protected least-significant-difference test. P < 0.05 was considered statistically significant.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Effect of E2 and progesterone on IGF-I mRNA expression
Quantitative RT–PCR analysis with Southern blot analysis specific for IGF-I of total RNA extracts from cultured leiomyoma cells obtained in the proliferative phase of the menstrual cycle showed that IGF-I mRNA expression in leiomyoma cells treated with progesterone (100 ng/ml) was less abundant than that in untreated control cultures. IGF-I mRNA expression in leiomyoma cells concomitantly treated with E2 (10 ng/ml) and progesterone (100 ng/ml) was also less abundant relative to that in untreated control cultures. By contrast, treatment with E2 (10 ng/ml) showed no effect on the abundance of IGF-I mRNA expression in cultured leiomyoma cells compared to that in untreated control cultures (Figure 1). The densitometric analysis of IGF-I mRNA expression revealed that either the treatment with progesterone alone (P < 0.01) or the combined treatment with E2 and progesterone (P < 0.01) significantly decreased IGF-I mRNA expression in cultured leiomyoma cells compared to that in untreated control cultures, whereas there was no significant difference in IGF-I mRNA expression in cultured leiomyoma cells between E2-treated cultures and untreated control cultures. Similar results were obtained with the use of total RNA extracts of cultured leiomyoma cells obtained in the secretory phase of the menstrual cycle.



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Figure 1. Effects of 17{beta}-estradiol (E2) and progesterone on insulin-like growth factor-I (IGF-I) mRNA expression in cultured leiomyoma cells, as assessed by quantitative RT–PCR analysis with Southern blot analysis. Experiments were repeated five times with similar results for each. Lane 1, untreated control cultures; lane 2, E2 (10 ng/ml)-treated; lane 3, progesterone (100 ng/ml)-treated; lane 4, combined treatment with E2 (10 ng/ml) and progesterone (100 ng/ml); lane 5, negative control. After densitometric analysis the amount of IGF-I mRNA was expressed relative to the abundance of {beta}-actin mRNA. Data are presented as the fold increases over the untreated control value and as the mean ± SD. *P < 0.01.

 
Effect of E2 and progesterone on IGF-I receptor mRNA expression
Quantitative RT–PCR analysis with Southern blot analysis specific for IGF-I receptor of total RNA extracts from cultured leiomyoma cells obtained in the proliferative phase of the menstrual cycle showed that neither the treatment with E2 (10 ng/ml) or progesterone (100 ng/ml) nor the combined treatment with E2 and progesterone affected IGF-I receptor mRNA expression in cultured leiomyoma cells relative to that in untreated control cultures (Figure 2). The densitometric analysis of IGF-I receptor mRNA expression revealed that there were no significant differences in IGF-I receptor mRNA expression in cultured leiomyoma cells between either E2- or progesterone-treated cultures and untreated control cultures. Similar results were obtained with the use of total RNA extracts of cultured leiomyoma cells obtained in the secretory phase of the menstrual cycle.



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Figure 2. Effects of 17{beta}-estradiol (E2) and progesterone on insulin-like growth factor-I (IGF-I) receptor mRNA expression in cultured leiomyoma cells, as assessed by quantitative RT–PCR analysis with Southern blot analysis. Experiments were repeated five times with similar results for each. Lane 1, untreated control cultures; lane 2, E2 (10 ng/ml)-treated; lane 3, progesterone (100 ng/ml)-treated; lane 4, combined treatment with E2 (10 ng/ml) and progesterone (100 ng/ml); lane 5, negative control. After densitometric analysis the amount of IGF-I receptor mRNA was expressed relative to the abundance of {beta}-actin mRNA. Data were presented as the fold increases over the untreated control value and as the mean ± SD.

 
Effect of E2 and progesterone on intracellular immunoreactive of IGF-I
Table I shows the immunoreactive IGF concentrations in leiomyoma cells cultured in serum-free medium in the absence or presence of E2 and progesterone. Measurement of immunoreactive IGF-I in the protein extracts from cultured leiomyoma cells obtained in the proliferative phase of the menstrual cycle revealed that either the treatment with progesterone alone or the combined treatment with E2 and progesterone significantly (P < 0.05) decreased the immunoreactive IGF-I concentrations in cultured leiomyoma cells compared to those in untreated control cultures. On the other hand, there was no significant difference in the immunoreactive IGF-I concentrations in cultured leiomyoma cells between E2-treated cultures and untreated control cultures. Similar results were obtained with the use of protein extracts from cultured leiomyoma cells obtained in the secretory phase of the menstrual cycle.

Immunocytochemical analysis of ER and PR protein expression
Immunocytochemical staining demonstrated that ER and PR protein were immunolocalized to the nuclei of cultured leiomyoma cells. ER{alpha} protein expression in the cultured cells appeared less abundant after culture for 24 and 48 h relative to that before culture under the serum-free condition (Figure 3A, B, C). By contrast, there were no apparent changes in the abundance of PR protein expression among 0, 24 and 48 h cultures under serum-free, Phenol Red-free conditions (Figure 3E, G, F).



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Figure 3. Immunocytochemical staining of estrogen receptor (ER) and progesterone receptor (PR) protein in cultured leiomyoma cells. ER and PR protein were immunolocalized to the nuclei of cultured leiomyoma cells. ER protein expression in the cultured leiomyoma cells appeared less abundant after culture for 24 h (B) and 48 h (C) relative to that before culture (A) in a serum-free condition, whereas there were no apparent changes in the abundance of PR protein expression among 0 h (E), 24 h (F), and 48 h (G) cultures. Replacement of the primary antibody with non-immune rabbit IgG showed a lack of positive immunostaining in the cultured leiomyoma cells (D). Bars = 5 µm; original magnification: x400.

 
The ER{alpha}-positive rate of leiomyoma cells cultured for 0, 24 and 48 h was estimated to be 37.6 ± 3.6, 22.5 ± 2.8 and 3.4 ± 0.6% respectively. There was a significant decrease in the ER{alpha}-positive rate in leiomyoma cells after 24 h culture (P < 0.05) compared to that before culture under serum-free conditions. On the other hand, the PR-positive rate of leiomyoma cells cultured for 0, 24 and 48 h was estimated to be 72.4 ± 3.8, 76.0 ± 4.1 and 69.1 ± 4.0% respectively. No significant difference in the PR-positive rate was noted in leiomyoma cells in the course of culture for 48 h under serum-free conditions.


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The circulating concentrations of E2 and progesterone are considered to be the major determinants of uterine leiomyoma growth, as these tumours stop growing and decrease in size after menopause. Actually, Brandon et al. (1993, 1995) have demonstrated increased expression of estrogen receptor (ER) and PR receptor in uterine leiomyomas compared to those in normal myometrium. Most clinicians, thus, have believed until recently that uterine leiomyomas increase in size naturally during pregnancy in response to the increased circulating concentrations of E2 and progesterone. This is, however, not uniformly accepted. In the management of pregnant women complicated by large uterine leiomyomas, the authors have noted that it is very rare to find an increase in the size of uterine myomas despite the remarkable increase in the total size of the uterus over the course of pregnancy. Actually, an ultrasonographic study by Lev-toaff et al. (1987Go) revealed that uterine myomas only occasionally increased in size during the first trimester of pregnancy, and very few continued to grow during the course of pregnancy. Phelan (1995Go) also described that most uterine myomas identified early in pregnancy remained the same size or even shrank over the course of pregnancy. The reason for the lack of growth response of uterine myomas during pregnancy despite an overwhelming increase in circulating concentrations of sex steroid hormones remains unknown.

On the other hand, it is now evident that the use of the levonorgestrel (a progestin)-releasing intrauterine system (LNG-IUS) is effective for long-term management of menorrhagic women with uterine leiomyomas because of a striking reduction in menstrual bleeding volume (Maruo et al., 2001aGo). Although the LNG-IUS insertion uniformly caused the atrophic change of the endometrium associated with decreased proliferation and increased apoptosis, the effect of LNG-IUS on the size of uterine leiomyomas varied remarkably: the size of uterine myomas during the use of LNG-IUS was noted to increase, remain the same or decrease in each one-third of the cases examined (Maruo et al., 2001bGo). The reason why uterine myoma during the use of LNG-IUS decreased in size in some cases but increased in size in other cases remains unknown.

In this context, it has been shown that local growth factors such as EGF (Hofmann et al., 1984Go; Yeh et al., 1991Go) and IGF-I (Gao et al., 2001Go) play crucial roles in promoting uterine leiomyoma growth by up-regulating the cell proliferation and down-regulating apoptosis in those cells and that a complex network, in which sex steroid hormones cooperate with the local modulators, may exist and regulate the net growth of leiomyoma. The results obtained in our previous studies (Matsuo et al., 1997Go; Shimomura et al., 1998Go; Kurachi et al., 2001Go) have suggested that progesterone contributes only to the promotion of uterine leiomyoma growth and survival through up-regulating EGF and Bcl-2 expression in cultured leiomyoma cells as well as through down-regulating TNF-{alpha} expression in those cells. Furthermore, the present study has provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured human uterine leiomyoma cells, whereas E2 does not affect IGF-I mRNA and protein expression in those cells. Consistent with this observation, the immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells was maintained without significant changes in the PR-positive rate of the leiomyoma cells in the course of culture for 48 h under serum-free conditions. This suggests the maintenance of PR protein expression in cultured leiomyoma cells used in the present study. Scatchard analysis of progestin binding to the cytosol preparation would be needed for understanding the functional nature of the PR in the cultured leiomyoma cells. On the other hand, Severino et al. (1996Go) reported a rapid reduction in ER and PR protein content in human leiomyoma minced explants within 8 h of incubation. The discrepancy between the two studies might partly be due to the difference of culture methods used: Severino et al. examined PR maintenance using minced explants of leiomyoma whereas we used leiomyoma cells in monolayer culture. It is possible that PR expression is maintained in leiomyoma cells in monolayer culture for ≥48 h even under serum-free conditions, but not in minced leiomyoma explants in culture.

Unlike the PR maintenance in cultured leiomyoma cells, we noted that ER{alpha} protein expression in the cultured cells fell to very low levels after stepping down to serum-free conditions for 24 h and further decreased after 48 h in culture under serum-free conditions. This study dealt with the change in classical ER, at present ER{alpha}. While this may be an explanation for the lack of effect of E2 in the present study, we have noted that under the same conditions, treatment with E2 significantly decreased p53 protein content in cultured leiomyoma cells compared with the control cultures, whereas treatment with progesterone did not affect p53 protein content in those cells (Gao et al., 2002Go). Nevertheless, the rapid reduction in ER protein expression in primary culture conditions has been reported by Severino et al. (1996Go) with minced leiomyoma explants and by Freyschuss et al. (1993Go) with cultured rat hepatocytes. Thus, in vitro studies on the effects of sex steroids should examine this phenomenon.

That progesterone down-regulates IGF-I expression in cultured leiomyoma cells is supported by the observation of Giudice et al. (1993Go) who demonstrated lower expression of IGF-I mRNA in leiomyoma tissues in the secretory, progesterone-dominated, phase of the menstrual cycle relative to that in the proliferative phase. Nevertheless, Englund et al. (2000Go) showed higher expression of IGF-I mRNA in uterine leiomyoma than in the corresponding myometrium, with no differences related to phase of the menstrual cycle. These discrepancies might partly be due to the fact that uterine leiomyomas are individual tumours in which the expression of IGF-I mRNA varies considerably between uterine myomas even from the same patient (Englund et al., 2000Go). The present study has also showed that IGF-I receptor mRNA expression in cultured leiomyoma cells are not affected by the treatment with either E2 or progesterone. That neither E2 nor progesterone modulates the IGF-I receptor expression in human uterine leiomyoma is consistent with the report by Giudice et al. (1993Go), who demonstrated that IGF-I receptor gene expression in leiomyomas was not dependent on phase of the menstrual cycle or in vivo estrogen status.

Since IGF-I evidently plays a pivotal role in leiomyoma growth, not only in stimulating the proliferative potential but also in inhibiting apoptosis in leiomyoma cells, the present study suggests for the first time that progesterone may also contribute to the inhibition of uterine leiomyoma growth and survival through down-regulating IGF-I expression in those cells. Taking these notions into account, it seems likely that progesterone may have dual actions on uterine leiomyoma growth: one is to stimulate leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 expression and through down-regulating TNF-{alpha} expression in those cells, and the other is to inhibit leiomyomas cell growth and survival through down-regulating IGF-I expression in those cells. This may explain, at least in part, why the size of uterine leiomyomas during the use of LNG-IUS decreases in some but increases in other instances. Whether uterine leiomyomas either decrease or increase in size during LNG-IUS use may be dependent on the local autocrine/paracrine growth factor conditions around each leiomyoma. Furthermore, this may also explain in part why it is very seldom to find the increase in the size of uterine leiomyomas over the course of pregnancy despite the overwhelming increase in circulating concentrations of sex steroid hormones. Further studies will be needed to evaluate the sex steroidal regulation of the synthesis of IGF binding proteins in uterine leiomyoma cells in order to determine the molecular mechanism by which IGF-I and sex steroid hormones interact in regulating human uterine leiomyoma growth.


    Acknowledgements
 
This work was supported in part by Grants-in-Aid for Scientific Research no. 12877263 from the Japanese Ministry of Education, Science and Culture and by the Ogyaa-Donation Foundation of the Japan Association of Maternal Welfare.


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Table I. Immunoreactive IGF-I concentrations in human uterine leiomyoma cells cultured in serum-free conditions for 48 h in the absence or presence of E2 (10 ng/ml) or P4 (100 ng/ml)
 

    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 24, 2003; resubmitted on September 29, 2003; accepted on November 26, 2003.





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