Molecular Human Reproduction, Vol. 8, No. 12, 1071-1078,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Molecular endocrinology |
Effects of GnRH analogues, `add-back' steroid therapy, antiestrogen and antiprogestins on leiomyoma and myometrial smooth muscle cell growth and transforming growth factor-ß expression*
Department of Obstetrics and Gynecology, University of Florida College of Medicine, Box 100294, Gainesville, FL 32610, USA
Abstract
The objective of this study was to elucidate the biological significance of GnRH and antiprogestins and antiestrogen in leiomyoma and their interactions with ovarian steroid `add-back' therapy. Leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) were isolated and exposed to GnRH agonist (leuprolide acetate, LA), 17ß-estradiol (E2), medroxyprogesterone acetate (MPA), GnRH antagonist (Antide), estrogen antagonist, ICI182780 (Fulvestrant) and progesterone antagonists RU486 (Mifepristone) and ZK98299 (Onapristone) and combinations thereof. The rate of DNA synthesis, cell proliferation and transforming growth factor-ß (TGF-ß) expression were then determined. In both cell types, we found that in a dose-dependent manner, LA inhibited, whereas E2, MPA and the combination of E2 + MPA stimulated, the rate of DNA synthesis in these cells. Antide reversed the inhibitory effect of LA, while LA partly inhibited the stimulatory effect of the steroids. In addition, RU486, ICI182780 and ZK98299 at 0.1µmol/l or higher doses inhibited the rate of DNA synthesis and partly reversed the effects of E2 and/or MPA. We also found that LSMC expressed elevated levels of TGF-ß1 compared with MSMC. In both cell types, the effects of LA, E2, MPA, RU, ZK and ICI and combinations thereof on TGF-ß1 production were reflective of their effects on DNA synthesis. In line with this, TGF-ß1 was found to stimulate DNA synthesis and the E2-, TGF-ß1- or E2 + TGF-ß1-induced DNA synthesis was found to be inhibited by TGF-ß1 neutralizing antibodies and/or LA. In conclusion, the results provide further evidence that GnRH agonist- and RU486-induced leiomyoma regression is mediated in part through an interactive mechanism that results in altered cell growth and suppression of TGF-ß production.
antiestrogen/antiprogestins/GnRH/leiomyoma/ovarian steroids
Introduction
Leiomyomata are benign uterine tumours that originate from the transformation of myometrial smooth muscle cells during the reproductive years. The molecular environment that initiates such transformation is unknown; however, ovarian steroids are critical to leiomyoma growth and GnRH analogue therapy is often used for their medical management (Friedman, 1993
; Kettel et al., 1993; Takeuchi et al., 2000). Daily administration of anti-progestin RU486 (Mifepristone) has also been shown to cause leiomyoma regression (Reinsch et al., 1994; Murphy et al., 1995). Because uterine tissue, including myometrium and leiomyoma, expresses GnRH and GnRH receptors, an autocrine/paracrine role for GnRH and an additional site of action for GnRH agonist on various uterine cell types has been proposed (Chegini et al., 1996; Raga et al., 1998, 1999). Several in-vitro studies have provided support for the direct action of GnRH by demonstrating changes in cell growth, cell cycle progression, apoptosis, and expression of several growth factors, proteases and protease inhibitors in endometrial, myometrial and leiomyoma cells and other steroid-sensitive cell types. These changes are mediated through GnRH receptor-activated signalling transduction (Chegini et al., 1996; Dou et al., 1996, 1997a; Raga et al., 1998, 1999; Chegini, 2000; Cheng and Leung 2000; Everest et al., 2001; Grundker et al., 2001a,b; Kraus et al., 2001). GnRH agonist therapy is often associated with adverse side-effects that prevent prolonged use; however, combination therapy with low doses of continuous estrogen and/or progesterone, i.e. `add-back' therapy, appear to reduce some of the adverse side-effects, allowing prolonged therapy without stimulating leiomyoma growth (Friedman 1993; Takeuchi et al., 2000).
Compared with myometrium, leiomyomata overexpress estrogen and progesterone receptors, as well as several growth factors and cytokines, whose local expression is regulated in part by ovarian steroids in steroid-sensitive tissues such as the uterus (Brandon et al., 1993, 1995; Regidor et al., 1995; Fujimoto et al., 1998; Nisolle et al., 1999; Chegini, 2000; Zasawski et al., 2001). In particular, leiomyomata overexpress transforming growth factor-ß (TGF-ß) which is widely accepted as a key factor in the pathogenesis of tissue undergoing fibrosis, such as leiomyoma (Branton and Kopp, 1999). The action of TGF-ß on tissue fibrosis is believed to occur through excess cell migration, cell growth, extracellular matrix (ECM) production and deposition as well as alteration of proteases expression, all of which are among the common characteristics of tissue undergoing fibrosis (Branton and Kopp, 1999). We have previously demonstrated that leiomyomata overproduce TGF-ß and TGF-ß receptors compared to myometrium (Dou et al., 1996) and TGF-ß is reported to stimulate myometrial and leiomyoma smooth muscle cell (MSMC and LSMC) growth, and to alter the expression of MMP, TIMP and ECM molecules (Dou et al., 1996, 1997a; Tang et al., 1997; Chegini et al., 1999; Arici and Sozen, 2000; Lee and Nowak, 2001). GnRH agonist therapy causes down-regulation of TGF-ß, TGF-ß receptors and TIMP, whilst increasing expression of MMP (Chegini et al., 1996; Dou et al., 1996, 1997a; Raga et al., 1999).
To further elucidate the molecular mechanisms of GnRH and RU486 action on leiomyoma regression and their interactions with ovarian steroids in `add-back' therapy, the present study examined their direct action on the rate of DNA synthesis, cell proliferation and TGF-ß1 expression in leiomyoma and myometrial smooth muscle cells. The effects of leuprolide acetate (LA, a GnRH agonist), and RU486 (a type II progesterone antagonist), either alone or in combinations with 17ß-estradiol (E2) and medroxyprogesterone acetate (MPA) were comparatively analysed with the effects of ICI182780 (Fulvestrant, a pure estrogen antagonist) and ZK98299 (Onapristone, a type I progesterone antagonist) as well as Antide (a GnRH antagonist). We also determined whether E2-induced TGF-ß expression is altered following treatment with TGF-ß and TGF-ß-type receptor neutralizing antibodies and antisense oligomers.
Materials and methods
Reagents
All the materials for isolation of LSMC and MSMC, culture media, cell proliferation and RT–PCR were purchased from commercial sources as previously described (Tang et al., 1994; Chegini et al., 1999). E2, MPA, LA, RU486 and Antide as well as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit were purchased from Sigma Chemical Co. (St. Louis, MO, USA). [3H]thymidine (83 Ci/mmol) was purchased from Amersham–Pharmacia Biotech (Piscataway, NJ, USA). Recombinant human TGF-ß1 neutralizing antibodies to TGF-ß1 and TGF-ß type II receptors were purchased from R&D (Minneapolis, MN, USA) and TGF-ß1 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Promega (Madison, WI, USA). ICI182780 (Fulvestrant) was purchased from Tocris Cookson, Inc. (Ballwin, MO, USA). ZK98299 (Onapristone) was a gift from Schering (Berlin, Germany).
Tissue collection, cell isolation, culture, treatments and assays
Portions of leiomyoma and unaffected myometrium were collected from pre-menopausal women scheduled to undergo hysterectomy for symptomatic leiomyomas. These patients were not receiving any hormonal treatments at the time of surgery. The tissues were collected at the University of Florida-affiliated Shands Hospital, with the approval of the Institutional Review Board. Immediately after collection, leiomyoma and myometrial smooth muscle cells were isolated and cultured as previously described (Rossi et al., 1992). The isolated cells were cultured in Dulbecco's modified Eagle's medium containing antimycotic, antibiotics and 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified 5% CO2 incubator until reaching confluence. Prior to their use in these experiments, the cell cultures were characterized using immunofluoresence microscopy and antibodies to a smooth muscle actin, desmin and vimentin as previously described (Rossi et al., 1992).
To determine the effect of GnRH, progesterone antagonists, estrogen antagonist and ovarian steroids on DNA synthesis and cell proliferation, LSMC and MSMC were subcultured in 48- or 96-well dishes at an approximate density of 2.5x104 and 2.5x103 cells/well respectively, and incubated with phenol red-free media containing 10% charcoal-stripped FBS for 48 h. The cells were made quiescent under serum-free conditions for 48 h, and then treated with several concentrations of LA, E2, MPA or E2 + MPA, Antide, RU486, ZK98299 or ICI182780 either alone or in appropriate combinations, added to phenol red-free medium containing 2% charcoal-stripped FBS (cFBS) (Hyclone, Logan, UT, USA). [3H]thymidine (2 µCi/ml) was added to each well and the rate of incorporation into DNA was determined after 24 h of incubation (Rossi et al., 1992). The rate of cell proliferation was determined after similar treatments, following 48 h of incubation, using a cell proliferation assay (MTT) as previously described (Tang et al., 1994).
To determine whether LA and RU486 alter TGF-ß expression, LSMC and MSMC were cultured, made quiescent as above, and then treated with LA (2.5 µmol/l), E2 (0.1 µmol/l), MPA (0.1 µmol/l), Antide (5 µmol/l), ICI182780, RU486 or ZK98299 (10 µmol/l) either alone or in appropriate combinations, added to phenol red-free medium containing 2% cFBS. After 24 h of incubation, the culture condition media were collected and assayed for TGF-ß1 as previously described (Chegini et al., 1999). The level of TGF-ß1 was determined by ELISA and compared to known concentrations of standard.
To determine whether LA, E2 and TGF-ß interactions alter the rate of DNA synthesis, LSMC and MSMC were cultured and made quiescent as above and then treated with LA (2.5 µmol/l), E2 (0.1 µmol/l), TGF-ß (1 ng/ml) or combinations thereof, with and without co-treatment with TGF-ß1 neutralizing antibody (5 µg/ml) added to phenol red-free medium containing 2% cFBS. The rate of DNA synthesis was determined after 24 h of treatment as described above. To determine whether TGF-ß1 regulates its own expression, LSMC and MSMC were cultured in 24-well dishes as above in the presence of 10% FBS for 48 h. The cells were washed, incubated in phenol red-free medium containing 2% cFBS and treated with either TGF-ß1 (1 ng/ml), TGF-ß1 antisense and/or sense oligomers (5 µmol/l) (Dou et al., 1997b), added 24 h prior to addition of TGF-ß1, or TGF-ß type II receptor antibody (5 µg/ml) added 2 h prior to addition of TGF-ß1. After incubation for 24 h, the culture-conditioned media were collected and assayed for TGF-ß1.
Results
We have previously shown that LSMC and MSMC cultured under serum-free conditioned media become quiescent, and are then fully stimulated to re-enter the cell cycle after incubation with 10% FBS, or show half-maximal stimulation to re-enter the cell cycle when incubated with 2% FBS (Rossi et al., 1992; Tang et al., 1994). Using LSMC and MSMC cultured with charcoal-stripped serum and phenol red-free media and subsequently made quiescent under serum-free conditions, we evaluated the effect of LA, E2, MPA, Antide, RU486, ZK98299 and ICI182780, either alone or in appropriate combinations on the rate of DNA synthesis, cell proliferation and TGF-ß1 expression. Exposure of LSMC (Figure 1) and MSMC (data not shown) to LA in a dose dependent manner inhibited the rate of [3H]thymidine incorporation by ~25% (P < 0.05), without affecting their proliferation (data not shown). In contrast, the GnRH antagonist Antide did not cause any significant effect, although it moderately stimulated the rate of [3H]thymidine incorporation into LSMC (Figure 1). E2, in a dose-dependent manner, increased the rate of [3H]thymidine incorporation, but did not affect cell proliferation. The effects of E2 were markedly higher with LSMC than with MSMC (P < 0.05; Figure 2, data shown only for LSMC). MPA had a smaller but significant effect on [3H]thymidine incorporation but no effect on the proliferation of both cell types (Figure 2, data shown only for LSMC). RU486, ICI182780, and ZK98299 all showed limited effects on the rate of [3H]thymidine incorporation and cell proliferation of LSMC and MSMC at doses <0.1 µmol/l, while at higher doses they inhibited these parameters from 10 to 55% (P < 0.05; Figure 2 shown only for LSMC). While E2 + MPA at 0.1 µmol/l concentrations increased DNA synthesis in MSMC and LSMC, their actions either alone or in combination were partly inhibited by LA (P < 0.05, Figure 3). Co-treatment of the LSMC and MSMC with ICI182780, RU486 and ZK98299 partly reversed the stimulatory actions of E2, MPA and E2 + MPA on the rate of [3H]thymidine incorporation (Figure 3, data not shown for ZK982299).
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0.05) difference compared with the control (**, CTRL, 2% charcoal-stripped fetal bovine serum in phenol red-free media).
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) and cell proliferation (MTT: •) of leiomyoma smooth muscle cells (LSMC). The cells were cultured for 48 h, washed and incubated in serum-free media for 24 h, then treated with E2 and MPA for 24 h with addition of 2 µCi of [3H]thymidine or for 48 h for assaying cell proliferation. The data are expressed as mean ± SEM of percentage change from three separate experiments assayed in duplicates. * indicates a statistically significant (P
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We also found that LSMC express more TGF-ß1 compared with MSMC (P < 0.05). Treatments with E2, MPA and E2 + MPA significantly increased total TGF-ß1 expression, with a higher production of both total and active forms in MPA and MPA + E2-treated cells (P < 0.05; Figure 4
). In contrast, LA inhibited TGF-ß1 expression in both cell types, and this was reversed following co-treatment with Antide (Figure 4). LA also reduced the stimulatory action of E2, MPA and E2 + MPA in LSMC and MSMC (P < 0.05; Figure 4). Treatment of the cells with ICI182780, RU486 or ZK98299 has a limited inhibitory effect on TGF-ß1 expression; however, they partly reversed the stimulatory actions of E2 and MPA (Figure 4). These treatments altered the total TGF-ß1 production with limited effects on the production of active TGF-ß1, with the exception of MPA and/or MPA + E2 (Figure 4; P < 0.05).
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We next analysed the effect of TGF-ß on LSMC and MSMC and found that TGF-ß1 increased the rate of [3H]thymidine incorporation into both cell types (Figure 5
). Co-treatment of LSMC with E2 and TGF-ß1 enhanced the rate of [3H]thymidine incorporation, while addition of LA or neutralizing antibody to TGF-ß1 (TGF-Ant) prior to TGF-ß1 treatment partly reversed E2 + TGF-ß1 action (Figure 5; P < 0.05). A neutralizing antibody to TGF-ß1 also reduced E2 + TGF-ß1 action, suggesting that E2 mediates its action in part through the induction of TGF-ß1 (Figure 5; P < 0.05). TGF-ß1 was also found to up-regulate its own expression in both cell types, and co-treatment with TGF-ß1 antisense, but not with sense oligomers reduced this production of TGF-ß1 (Figure 6; P < 0.05). Pretreatment with TGF-ß type II receptor antibody prior to exposure to TGF-ß1 also resulted in down-regulation of TGF-ß1 expression (Figure 6; P < 0.05), suggesting a TGF-ß receptor-mediated action.
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Discussion
In the present study, we provide further evidence that GnRH directly acts on leiomyoma and myometrium as demonstrated by alteration of the rate of DNA synthesis and TGF-ß expression in LSMC and MSMC following treatment with LA. LA is an agonist of GnRH which exhibits antagonistic properties after prolonged exposure due to a depletion of GnRH receptors. The LA actions occurred in a dose-dependent manner and were competitively reversed by a GnRH antagonist, Antide, which alone had limited action on these cells. Because GnRH agonist therapy is associated with adverse side-effects, prolonged use is not advised; however, low doses of continuous estrogen and/or progesterone, or `add-back', are often used to overcome some of the side-effects such as bone loss and hot flushes, allowing prolonged therapy (Friedman, 1993; Kettel et al., 1993; Takeuchi et al., 2000). Despite overexpression of estrogen and progesterone receptors and growth dependency of leiomyoma on ovarian steroids, add-back intervention appears not to alter their growth (Friedman, 1993; Kettel et al., 1993; Takeuchi et al., 2000). In addition, a few limited clinical observations have provided evidence that daily administration of RU486 causes leiomyoma regression, implying the importance of progesterone in leiomyoma growth (Reinsch et al., 1994; Murphy et al., 1995). These clinical observations also indicate that GnRH agonist and RU486 therapy have different effects on leiomyoma and myometrial regression, with GnRH agonist therapy affecting non-myoma tissue more than leiomyoma (Kettel et al., 1993; Reinsch et al., 1994; Takeuchi et al., 2000). Using a defined culture condition with respect to steroid content, we demonstrated that both LSMC and MSMC respond to ovarian steroids, as well as GnRH agonist (LA), RU486 (a type II anti-progesterone), ICI182780 (an estrogen antagonist) and ZK98299 (Onapristone, a type I anti-progesterone), with actions that seem to follow their actions seen in vivo. As expected, we found that E2 acts as a mitogen for these cells and that co-treatments with LA altered the ovarian steroid's action, providing experimental support for the add-back concept. We also demonstrated that RU486, as well as ZK98299, inhibited the rate of DNA synthesis in LSMC and MSMC in a dose-dependent manner, and competitively reversed the MPA actions, providing experimental support for RU486-induced leiomyoma regression.
The inhibitory action of GnRH agonist in endometrial, ovarian, breast, prostate and hepatic carcinoma cell lines has been suggested to occur through alteration of cell cycle progression, programmed cell death and the expression of growth factors and cytokines and their receptors (Mullen et al., 1991; Thompson et al., 1991; Connor et al., 1994; Borri et al., 1998; Mizutani et al., 1998; Takeuchi et al., 1998; Chegini, 2000; Imai and Tamaya, 2000). In ovarian and breast tumour cell lines, GnRH agonist is reported to arrest the cells in G0/G1 of the cell cycle, and is reported to enhance programmed cell death in endometrial carcinoma cells (Mullen et al., 1991; Thompson et al., 1991; Mizutani et al., 1998; Takeuchi et al., 1998; Grundker et al., 2001b). Our finding that LA inhibits DNA synthesis without affecting cell proliferation of LSMC and MSMC, indicates a G0/G1 cell cycle arrest, as has been reported for LSMC (Mizutani et al., 1998). RU486 at comparable, or higher, doses to that used in our study, has also been shown to inhibit the growth of endometrial cell lines and primary cultures of endometrial and endometriosis cells, and at low concentrations has limited or no antiprogesterone activity, but retains its antiproliferative actions (Schneider et al., 1998; Murphy et al., 2000; Prange-Kiel et al., 2000). The inhibitory action of RU486 on cell growth has been reported to occur through G0/G1 and G2-M cell cycle arrest (Mizutani et al., 1998; Schneider et al., 1998; Chwalisz et al., 2000; Murphy et al., 2000; Prange-Kiel et al., 2000; Slayden et al., 2001). Because RU486 induces androgen receptor expression in endometrial glandular epithelial and stromal cells and androgen antagonizes estrogen actions, the antiproliferative action of RU486 is suggested to occur in part due to antiestrogenic effects (Chwalisz et al., 2000; for review). Under various clinical settings, RU486 acts as an antiprogestin and antiglucocorticoid agent, but it is reported to have a very weak estrogenic action in cells stably transfected with the estrogen receptor (Chwalisz et al., 2000). Other progestin antagonists, ZK137316 and ZK230211 have been shown to have neither antiestrogenic nor estrogenic activity (Thomas et al., 1994; Dowsett et al., 1995; Schneider et al., 1998; Chwalisz et al., 2000; Elger et al., 2000; Prange-Kiel et al., 2000; Slayden et al., 2001), whereas ZK112993 acts as a potent antagonist, without effecting endometrial cell growth (Elger et al., 2000; Murphy et al., 2000). Our results suggest that RU486 and ZK98299 act as progesterone antagonists and/or have antiestrogenic effects on LSMC and MSMC. We found that ICI182780 also inhibits the rate of DNA synthesis and reduces E2-induced stimulation of LSMC and MSMC. ICI182780 is a type II antiestrogen that binds to uterine estrogen receptor proteins with high affinity and has limited or no uterotrophic activity (Thomas et al., 1994; Austin et al., 1999; Robertson et al., 2001).
We also found that LA and ovarian steroids differentially regulate the expression of TGF-ß1 in LSMC and MSMC. LA reduced E2- or MPA-stimulated TGF-ß1 production, but had less of an effect on TGF-ß1 production induced by E2 + MPA in both LSMC and MSMC. The limited action of LA inhibiting E2 + MPA-induced TGF-ß1 production in vitro appears to parallel the in-vivo condition in which excess leiomyoma growth and TGF-ß production occurs during the secretory phase of the menstrual cycle with elevated circulating levels of ovarian steroids. ICI182780, RU486 and ZK98299 also reversed the E2- and MPA-induced TGF-ß expression, although these antagonists had limited inhibitory actions alone. Interestingly, both RU486 and ZK98299 partially inhibited the E2-induced TGF-ß expression by LSMC and MSMC, possibly due to the weak antiestrogenic activity of RU486. These treatments altered the total (latent + active) TGF-ß1 production with limited effect on the production of active TGF-ß1 alone. Such limited effects on active TGF-ß1 could be due to immediate binding of active TGF-ß1 to cell surface receptors, making it unavailable to measure. The LA-induced TGF-ß1 inhibition in LSMC and MSMC adds more weight to previous reports that leiomyomata express elevated levels of TGF-ß but show reduced expression in women who received GnRH agonist therapy (Dou et al., 1996; Arici and Sozen 2000; Lee and Nowak 2001). GnRH agonist therapy has been shown to inhibit the expression of several other growth factors and cytokines including insulin-like growth factor, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and interleukin-2 as well as TGF-ß1–3 and TGF-ß type I–III receptors in leiomyoma and myometrial cells (Dou et al., 1996; Chegini 2000).
RU486 and ZK98299 have also been reported to inhibit progestin-induced prolactin, insulin-like growth factor binding protein-1, and calcitonin expression in endometrial cells as well as in LSMC and MSMC (Stewart et al., 1996; Gao and Tseng, 1997; Kumar et al., 1998; Austin et al., 1999; Brosens et al., 1999). Under the conditions of our study, both RU486 and ZK98299 appear to have had antagonistic effects on MPA-induced TGF-ß1 production, whereas inhibition of E2-induced TGF-ß1 expression may be mediated in part through antiestrogenic effects, particularly that of RU486. ICI182780 has been reported to inhibit E2-induced PDGF production, a key mitogen and autocrine mediator of cell growth in leiomyoma (Rossi et al., 1992; Barbarisi et al., 2001) and to reverse E2-induced down-regulation of VEGF expression in an endometrial carcinoma cell line, HEC-1A (Stoner et al., 2000). ICI182780 is reported to act both as an E2 agonist by increasing the endometrial weight, and as an E2 antagonist by inhibiting E2-induced progesterone receptor, cyclophilin, actin and c-fos expression (Robertson et al., 2001) as well as VEGF expression in human endometrial stromal cells (Huang et al., 1998). In human endometrial glandular epithelial and stromal cells, ICI182364, ICI182780 and RU486 have been reported to inhibit E2- and P4-induced EGF receptor, VEGF, TGF-ß and granulocyte macrophage colony-stimulating factor expression (Watson et al., 1998; Classen-Linke et al., 2000; Riply et al., 2001). Several analogues of ICI182780 have also been shown to inhibit E2-induced proliferation of breast cancer (MCF-7) and endometrial (Ishikawa) cell lines (Nuttall et al., 2001).
We have also shown that E2 and TGF-ß interactively regulate LSMC DNA synthesis and that a TGF-ß1 neutralizing antibody reduces E2 action. In addition, blocking TGF-ß receptor mediated action resulted in reduction in E2-induced DNA synthesis and TGF-ß production. These results suggest that the effects of E2 are at least in part mediated by TGF-ß activity. Crosstalk between TGF-ß and estrogen receptor signalling pathways may also regulate these and other cellular events in leiomyoma. Since mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) are activated by various growth factor receptors (Massague and Wotton, 2000; Pearson et al., 2001), ovarian steroids independent of their nuclear receptors (Lange et al., 1999; Hall et al., 2001), GnRH receptor (Cheng and Leung, 2000; Barbarisi et al., 2001) and RU486 through activation of cell surface receptor signaling (Beck et al., 1996), their differential activation by these receptors may serve to implement their growth-promoting or inhibitory actions on leiomyoma. Our preliminary results indicate that TGF-ß and GnRH agonist activate mitogen-activated protein kinase (MAPK) and Smad in LSMC and MSMC and that their crosstalk alters the expression, induction and activation of these pathways (Chegini and Kornberg, 2002; Xu et al., 2002).
In summary, the present study provides further evidence in support of GnRH autocrine/paracrine action in myometrium and leiomyoma, and demonstrates that GnRH agonist as well as estrogen receptor and progesterone receptor antagonists alter the rate of DNA synthesis and TGF-ß1 expression induced by GnRH, E2 and MPA in these cells. Since TGF-ß is a key regulator of tissue fibrosis (Branton and Kopp, 1999), a characteristic of leiomyoma, lowering of its expression by GnRH agonist and RU486 may further serve to alter TGF-ß, regulated genes that are involved in ECM deposition and degradation, resulting in leiomyoma regression.
Acknowledgements
This work was supported by grant HD42372 from the NIH.
Notes
* Presented in part at the 53rd Annual Meeting of the American Society for Reproductive Medicine, San Diego, CA, USA, 2000. 
1 To whom correspondence should be addressed. E-mail: cheginin{at}obgyn.ufl.edu
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Submitted on May 6, 2002; resubmitted on July 2, 2002; accepted on September 9, 2002.
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