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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 11 5549-5557
Copyright © 2004 by The Endocrine Society

Gonadotropin Releasing Hormone and Transforming Growth Factor ß Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells

Li Ding, Jingxia Xu, Xiaoping Luo and Nasser Chegini

Department of Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Dr. Nasser Chegini, Department of OB/GYN, University of Florida, Box 100294, Gainesville Florida 32610. E-mail: cheginin{at}obgyn.ufl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH analog (GnRHa) and TGF-ß act directly on leiomyoma/myometrial smooth muscle cells (LSMCs and MSMCs) regulating diverse activities resulting in leiomyoma growth and regression. Because GnRH and TGF-ß receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of c-fos and c-jun, result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression, whose products are known to influence extracellular matrix turnover, which is critical in leiomyoma growth and GnRHa-induced regression. We found that GnRHa and TGF-ß in a dose- and time-dependent manner increased the level of phosphorylated ERK1/2 (pERK1/2) in LSMCs and MSMCs. GnRHa and TGF-ß increased ERK1/2 nuclear accumulation resulting in differential regulation of c-fos and c-jun mRNA expression via downstream signaling from MAPK kinase (MEK)1/2, because pretreatment with U0126, a synthetic inhibitor of MEK1/2, abolished basal and GnRHa- and TGF-ß-induced pERK1/2 and the expression of c-fos and c-jun. LSMCs and MSMCs also express fibronectin, type I collagen, and PAI-1 mRNA, and GnRHa and TGF-ß altered their expression in a cell-specific manner through MEK1/2. We concluded that GnRHa and TGF-ß acting through a MAPK/ERK pathway and transcriptional activation of c-fos/c-jun results in differential regulation of specific genes whose products may in part influence the outcome of leiomyoma growth and regression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEIOMYOMAS ARE OVARIAN steroid-dependent benign uterine tumors, which develop during the reproductive years and are suppressed with menopause (1). Because of the growth dependency of leiomyomas on ovarian steroids, long lasting GnRH analog (GnRHa) therapy is often used for their medical management (2, 3). GnRHa is traditionally believed to act primarily at the level of the pituitary-gonadal axis and by suppressing the ovarian steroid production, resulting in leiomyoma regression. However, the expression of the classical form of GnRH (GnRH I) and the second mammalian form of GnRH (GnRH II) as well as GnRH I and II receptors in several peripheral tissues, including the uterus, has provided evidence for an autocrine/paracrine role for GnRH and additional sites of action for GnRHa therapy (4, 5, 6, 7, 8, 9). With respect to GnRH binding sites in peripheral tissues and cell lines, they are of low affinity/high capacity and/or high affinity/low capacity, implying the presence of two distinct GnRH receptors in these tissues and cells (1, 6). In vitro studies have provided strong support for the direct action of GnRHa as well as GnRH I and II on various cell types derived from these tissues, which include changes in the rate of cell growth, apoptosis, and the expression of cell cycle proteins, growth factors, cytokines, proteases, and protease inhibitors (1, 4, 6, 10, 11, 12, 13, 14, 15, 16).

TGF-ß is among the cytokines whose expression is altered because of GnRHa therapy or because of in vitro treatment with GnRHa (11, 17, 18). TGF-ß isoforms (TGF-ß1, -ß2, and -ß3) and TGF-ß receptors (type I, II, and III) are expressed in various tissues and cell types where they act as key regulators of cell growth and differentiation and the expression of extracellular matrix, adhesion molecules, proteases, and protease inhibitors (19, 20, 21). Altered expression of TGF-ß has been correlated with several disorders, including tissue fibrosis, a characteristic of leiomyoma, in which extracellular matrix turnover plays a central role (19, 20, 21). In reproductive tissue disorders, elevated expression of TGF-ß isoforms and TGF-ß receptors has been demonstrated in endometriosis implants, endometrial cancer, and leiomyoma (17, 18, 22, 23). GnRHa therapy suppresses TGF-ß isoforms and TGF-ß receptor expression in leiomyoma and myometrium, ovarian steroid-induced TGF-ß1 expression in leiomyoma and myometrial smooth muscle cells (LSMCs and MSMCs), and matrix metalloproteinases and their inhibitors in endometrial stromal cells (10, 11, 17, 18, 24).

GnRH receptors mediate their actions through recruitment of a diverse group of intracellular signaling pathways, including protein kinase (PK)A, PKC, G protein-coupled receptor kinases, calcium-calmodulin (Ca2+/CaM), and the MAPK cascade, in primary cultures and cell lines derived from the pituitary (25, 26, 27, 28). The GnRH receptor signaling cascade in leiomyoma and myometrial cells is poorly characterized, although many of the above signaling pathways, specifically MAPK, are linked to GnRH receptor signaling in cell types derived from other peripheral tissues, including endometrial carcinoma cell lines (13, 25, 28, 29, 30). In contrast, binding of TGF-ß isoforms to TGF-ß type II receptor and activation of type I receptor mainly results in recruitment and activation of the Smad signaling pathway (19, 20). TGF-ß receptors also use components of other pathways, including MAPK, as part of their intracellular signaling, with functional interactions with PKC, Ca2+/CaM, and Smads (19, 20, 31, 32). Recent reports have documented the functional interactions between MAPK and Smad pathways in mediating the biological actions of GnRH, TGF-ß, and activin, a member of the TGF-ß family, in regulating the expression of gonadotropin, GnRH, and GnRH receptors in pituitary gonadotropes (33, 34, 35). We have reported that GnRHa therapy altered the expression of MAPK/ERK and Smads in leiomyoma and myometrium (18, 36). We have further demonstrated that GnRHa alters the expression of Smads and the TGF-ß-activated Smad pathway in LSMCs and MSMCs (12).

Because GnRH and TGF-ß receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of immediate early-response genes c-fos and c-jun result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression. The products of these genes are known to influence extracellular matrix turnover, a critical process in leiomyoma growth and GnRHa-induced regression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All the materials used for isolation and culturing of LSMCs and MSMCs, GnRH analogs (leuprolide acetate), and {alpha}-smooth muscle actin, desmin, and vimentine antibodies were purchased from Sigma Chemical Co. (St. Louis, MO). The materials for immunoblotting, real-time PCR and immunocytochemistry were purchased from Bio-Rad (Hercules, CA), Applied Biosystems (Foster City, CA), and Vector Laboratories (Burlingame, CA), respectively. Recombinant human TGF-ß1 was purchased from R&D Systems (Minneapolis, MN), and affinity-purified monoclonal anti-phospho-specific ERK1/2 and rabbit anti-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Synthetic inhibitors PD98059, U0126, and SB203580 were purchased from CalBiochem (San Diego, CA).

Portions of leiomyoma and matched myometrium were collected from premenopausal patients (n = 3) scheduled to undergo hysterectomy for symptomatic uterine leiomyomas at the University of Florida affiliated Shands Hospital. The patients did not receive any medications (including hormonal therapy) during the previous 3 months before surgery. The tissue specimens were obtained under a study protocol approved by the institutional review board at the University of Florida without requiring written informed consent. After collection, the tissues were processed for isolation of smooth muscle cells as previously described (11, 12). To maintain a standard, leiomyomas used in this study were 2–3 cm in diameter. LSMCs and MSMCs were isolated and cultured in DMEM until reaching visual confluence (11, 12). Before use in these experiments, the primary cultures were seeded in eight-well culture slides (Nalge Nunc, Naperville, IL), and after 24 h they were characterized by immunofluorescence microscopy using antibodies to {alpha}-smooth muscle action, desmin, and vimentin (11).

The effect of GnRHa and TGF-ß on ERK1/2 activation was determined using LSMCs and MSMCs cultured in six-well plates at an approximate density of 106 cells per well in DMEM-supplemented media containing 10% fetal bovine serum. After the cells reached visual confluence, they were washed in serum-free media and incubated under serum-free/phenol red-free conditions for 24 h (11). Time- and dose-dependent effects of GnRHa and TGF-ß1 were determined by treating the cells with GnRHa (0.1 µM) and TGF-ß1 (2.5 ng/ml) for 5, 15, and 30 min or with TGF-ß1 (1, 2.5, and 5 ng/ml) and GnRHa (0.001, 0.01, 0.1, 1, and 10 µM) for 15 min. Total protein was isolated and subjected to immunoblotting to determine the level of phosphorylated ERK1/2 (pERK1/2) and total ERK. Briefly, the cells were lysed in a lysis buffer and centrifuged, and the supernatants were collected and their total protein content was determined using a conventional method (Pierce, Rockford, IL) as previously described (11, 12). Equal amounts of sample proteins were subjected to PAGE, transferred to polyvinylidene difluoride membrane, and after further processing, the blots were incubated with pERK1/2 and ERK1/2 antibodies for 1 h at room temperature. The blots were washed with washing buffer and exposed to corresponding horseradish peroxidase-conjugated IgG, and immunostained proteins were visualized using enhanced chemiluminescence reagents (Amersham-Pharmacia Biotech, Piscataway, NJ). The band intensity corresponding to ERK1/2 (44/42 kDa) and pERK1/2 was analyzed as previously described (12). The specificity of GnRHa and TGF-ß1 actions on pERK1/2 induction was determined by pretreatment of LSMCs and MSMCs with 20 µM PD98059 or U0126 (MEK1/2 inhibitors) for 2 h followed by treatment with TGF-ß1 (2.5 ng/ml) or GnRHa (0.1 µM) for 15 min. Parallel experiments were also performed using the p38 MAPK inhibitor SB203580 at 20 µM instead of MEK1/2 inhibitors to determine their potential interactions. The cell lysates were prepared and subjected to immunoblot analysis.

To determine the effects of GnRHa and TGF-ß1 on c-fos, c-jun, fibronectin, type I collagen, and PAI-1 mRNA expression, the cells were cultured as above and treated with GnRHa (0.1 µM) or TGF-ß1 (2.5 ng/ml) for 1, 2, 4, and 6 h. In additional experiments, the cells were pretreated with U0126 for 2 h and then treated with GnRHa (0.1 µM) or TGF-ß1 (2.5 ng/ml) for 1 and 2 h. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), and the levels of c-fos, c-jun, fibronectin, type I collagen, and PAI-1 mRNA were determined using real-time PCR performed on ABI-Prism 7700 sequence detection system (Applied Biosystems). Briefly, complimentary DNA was generated from 2 µg of total RNA using TaqMan RT reagent, and newly synthesized cDNA was used for PCR. PCR was performed in 96-well optical reaction plates with cDNA equivalent to 100 ng RNA in a volume of 50 µl system, containing 1x TaqMan Universal Master Mix, optimized concentrations of 6-carboxyfluorescein (FAM)-labeled probe and specific forward and reverse primers for c-fos, c-jun, type I collagen, fibronectin, or PAI-1 selected from Assay on Demand (Applied Biosystems). Controls included RNA subjected to RT-PCR without reverse transcriptase and PCR with water replacing cDNA. All the controls gave a threshold cycle (Ct) value of 40, indicating no detectable PCR product under these cycle conditions. The cycle number at which fluorescence emission crossed the automatically determined threshold level (Ct) was determined using Applied Biosystems software. The results were analyzed using a comparative method, and the values were normalized to the 18S rRNA expression by subtracting mean Ct of 18S rRNA from mean target Ct for each sample, to obtain the mean {Delta}Ct. The mean {Delta}Ct values were then converted into fold change based on a doubling of PCR product in each PCR cycle, according to the manufacturer’s guidelines.

All the experiments were performed at least three times in duplicate using independent cell cultures. Where appropriate, the results are expressed as mean ± SEM and statistically analyzed using unpaired Student’s t test and Tukey test (ANOVA). A probability level of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRHas and TGF-ß-induced MAPK/ERK activation in LSMCs and MSMCs

Because GnRH and TGF-ß receptors activate the MAPK/ERK pathway as part of their intracellular signaling in several of their target cells, we determined the effect of GnRHa and TGF-ß1 on ERK1/2 activation in LSMCs and MSMCs. GnRHa (Fig. 1Go) and TGF-ß1 (Fig. 2Go) in a dose- and time-dependent manner increased the level of pERK1/2 in quiescent LSMCs and MSMCs compared with untreated controls, which contain varying levels of constitutively activated ERK1/2 (Figs. 1Go and 2Go). Cotreatments with GnRHa (0.1 µM) plus TGF-ß1 (2.5 ng/ml) did not have an additive effect on the level of pERK1/2 induction in LSMCs and MSMCs compared with GnRHa- or TGF-ß1-treated cells (data not shown). GnRHa- and TGF-ß1-induced pERK1/2 in LSMCs and MSMCs was sensitive to pretreatment with U0126, a MEK1/2 inhibitor, resulting in a near total inhibition of basal as well as GnRHa- and TGF-ß1-induced pERK1/2 in both cells (Fig. 3Go). In contrast, pretreatments of LSMCs and MSMCs with PD98059, a MEK1 inhibitor, increased pERK1/2 induction compared with untreated control, with partial inhibition of GnRHa- and TGF-ß1-induced pERK1/2 in LSMCs and MSMCs, respectively (Fig. 3Go). Pretreatment with SB203580, a p38 MAPK inhibitor, reduced basal pERK1/2 in LSMCs, but not in MSMCs, and had no significant inhibitory effect on GnRHa- and TGF-ß1-induced pERK1/2 in both LSMCs and MSMCs (Fig. 3Go). The total ERK1/2 levels were not affected in any of the experiments. The results indicate that GnRHa and TGF-ß receptor signaling in LSMCs and MSMCs at least in part is mediated through MAPK/ERK downstream from MEK1/2.



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  		FIG. 1. Dose and time response of GnRHa on pERK1/2 induction in LSMCs and MSMCs. Serum-starved MSMCs and LSMCs were treated with GnRHa at 0.001–10 µM for 15 min (A) or with 0.1 µM of GnRHa for 5, 15, and 30 min (B). Cell lysates were prepared from treated and untreated control (Ctrl) and subjected to immunoblotting using phosphospecific (pERK1/2) and ERK antibodies. Immunoblots show pERK1/2 and total ERK1/2 from a representative experiment with bar graphs displaying the mean ± SEM of band densities from three experiments performed using independent cell cultures from different tissues. * and **, Statistically different from control with arrows pointing to differences between pERK1 and pERK2 (P < 0.05).

 


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  		FIG. 2. Dose and time response of TGF-ß1 on pERK1/2 induction in LSMCs and MSMCs. Serum-starved MSMCs and LSMCs were treated with TGF-ß1 at 1, 2.5, and 5 ng/ml for 15 min (A) or with 2.5 ng/ml of TGF-ß1 for 5, 15, and 30 min (B). Cell lysates were prepared from treated and untreated control (Ctrl) and subjected to immunoblotting using phosphospecific ERK1/2 (pERK1/2) and ERK antibodies. Immunoblots show pERK1/2 and total ERK1/2 from a representative experiment, with bar graphs displaying the mean ± SEM of band densities from three experiments performed using independent cell cultures from different tissues. * and **, Statistically different from control with arrow pointing to difference between pERK1 and pERK2 (P < 0.05).

 


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  		FIG. 3. The effect of TGF-ß1 and GnRHa on pERK1/2 induction after pretreatment of LSMCs and MSMCs with PD98059 (PD), U0126 (U), and SB203580 (SB). Serum-starved cells were treated with the inhibitors at 20 µM for 2 h, washed, and then treated with 2.5 ng/ml TGF-ß1 (T), 0.1 µM GnRHa (G), or without treatment (–) for 15 min. The cell lysates were prepared from treated and untreated cells and subjected to immunoblotting using phosphospecific ERK1/2 (pERK1/2) and ERK antibodies. Immunoblots show pERK1/2 and total ERK1/2 from a representative experiment, with bar graphs displaying the mean ± SEM of band densities from three experiments performed using independent cell cultures from different tissues. * and **, Statistically different from control and *** with arrows pointing to differences between pERK1 and pERK2 (P < 0.05).

 
GnRHa- and TGF-ß-induced transcriptional activation of c-fos and c-jun genes

Indirect fluorescent immunocytochemistry showed that ERK1/2 is present in cytoplasmic and nuclear regions of LSMCs and MSMCs, and treatments with TGF-ß1 or GnRHa resulted in an increase in ERK1/2 nuclear labeling (data not shown). Because activated ERK nuclear translocation is accompanied by transcriptional activation of immediate early response genes, including c-fos and c-jun, we determined the effect of GnRHa and TGF-ß1 on their expression in LSMCs and MSMCs. GnRHa (0.1 µM) and TGF-ß1 (2.5 ng/ml) in a time-dependent manner differentially regulated the expression of c-fos and c-jun mRNA, lasting for several hours in LSMCs compared with MSMCs (P < 0.05; Fig. 4Go). Although c-fos expression was rapidly induced by TGF-ß1 and remained elevated for several hours in LSMCs and MSMCs, GnRHa action was observed after 2 h and was limited to LSMCs with a lower rate of induction compared with TGF-ß1 (Fig. 4Go). In contrast, GnRHa and TGF-ß1 increased c-jun expression in LSMCs and MSMCs after 2 h of treatment and remained elevated during the 6 h, with a lower rate of induction by TGF-ß1 compared with GnRHa (Fig. 4Go). Pretreatment with U0126 inhibited the basal (c-fos) as well as GnRHa- and TGF-ß1-regulated c-fos and c-jun mRNA expression in LSMCs and MSMCs (P < 0.05; Fig. 4Go). The results further suggest that GnRH and TGF-ß receptors signaling downstream from MEK1/2 are involved in transcriptional activation of c-fos and c-jun in LSMCs and MSMCs in a cell- dependent manner.



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  		FIG. 4. Real-time PCR analysis of c-fos and c-jun mRNA expression in LSMCs and MSMCs after treatment with TGF-ß1 (2.5 ng/ml) or GnRHa (0.1 µM) for 1–6 h. In parallel experiments, the cells were pretreated with U0126 (U) for 2 h and then treated with or without TGF-ß (T) and GnRH (G) for an additional 1 or 2 h. Total RNA was isolated from treated and untreated control (Ctrl) cells and subjected to real-time PCR. Bar graphs show the mean ± SEM of relative c-fos and c-jun mRNA expression performed using three independent cell cultures prepared from different tissues. * and **, Differences in c-fos and c-jun expression in TGF-ß- and GnRHa-treated cells compared with control, or in pretreated groups with U compared with TGF-ß- and GnRHa-treated groups and untreated control, respectively, with arrows pointing to differences between LSMCs and MSMCs (P < 0.05).

 
GnRHa and TGF-ß alter the expression of type I collagen and PAI-1in LSMCs and MSMCs

MSMCs and LSMCs express fibronectin, type I collagen, and PAI-1 mRNA, and their expression was differentially regulated by GnRHa and TGF-ß1 (Fig. 5Go). As expected, TGF-ß1 increased the expression of type I collagen and PAI-1 mRNA in LSMCs and MSMCs, with a significantly higher rate of induction in LSMCs and MSMCs, respectively (P < 0.05; Fig. 5Go). TGF-ß1 moderately increased fibronectin expression in MSMCs after 4 h of treatment (P < 0.05; Fig. 5Go). In contrast, GnRHa inhibited fibronectin mRNA expression in MSMCs with a moderate increase in its expression in LSMCs, while inhibiting type I collagen expression in both MSMCs and LSMCs after 4–6 h of treatments (P < 0.05; Fig. 5Go). GnRHa increased PAI-1 mRNA expression in both cell types, but at significantly lower levels compared with TGF-ß1 (Fig. 5Go). Pretreatment with U0126 had a variable effect on basal and GnRHa- and TGF-ß1-regulated expression of fibronectin, type I collagen, and PAI-1 in MSMCs and LSMCs in a cell- and gene-specific manner (Fig. 5Go).



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  		FIG. 5. Real-time PCR analysis of fibronectin (FN), type I collagen (Coll-1), and PAI-1 mRNA expression in LSMCs and MSMCs after treatments with TGF-ß1 (2.5 ng/ml) or GnRHa (0.1 µM) for 2, 4, and 6 h, or in cells pretreated with MEK1/2 synthetic inhibitor U0126 (U) for 2 h and then treated with or without TGF-ß (T) and GnRHa (G) for an additional 2 h. Total RNA was isolated from treated and untreated control (Ctrl) cells and subjected to real-time PCR. Bar graphs show the mean ± SEM of relative mRNA expression of FN, Coll-1, and PAI-1 performed using three independent cell cultures prepared from different tissues. * and **, Differences in FN, Coll-1, and PAI-1 expression in TGF-ß- and GnRHa-treated cells compared with control, or in pretreated groups with U (***) compared with TGF-ß- and GnRHa-treated groups (* and **) and untreated control, respectively, with arrows pointing to differences between LSMCs and MSMCs (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that in LSMCs and MSMCs GnRHa and TGF-ß1 also use MAPK/ERK as part of their signaling pathway. The effect of GnRHa and TGF-ß on pERK1/2 induction in LSMCs and MSMCs was relatively similar and moderate, possibly because of the presence of constitutively activated ERK, induced by many autocrine/paracrine growth factors and cytokines expressed by these cells. However, GnRH and TGF-ß receptors can use other pathways, including PKC, Ca2+/CaM, Smad, and other members of MAPK pathway, such as p38 MAPK and c-Jun N-terminal kinase (JNK), respectively (19, 20, 25, 27, 29, 31, 32), that could serve as their main signaling pathways in LSMCs and MSMCs. We have recently reported that TGF-ß1 activates the Smad pathway in LSMCs and MSMCs, and GnRHa either directly or through activation of other pathways alters the expression and activation of Smad in these cells (12). However, the involvement of these signaling pathways and their crosstalk with MAPK/ERK in mediating GnRH and TGF-ß receptor actions in LSMCs and MSMCs remains to be investigated.

GnRHa and TGF-ß1 treatment resulted in an increased nuclear translocation of ERK1/2 in LSMCs and MSMCs, which was accompanied by alteration in immediate early response genes c-fos and c-jun mRNA expression. Unlike pERK1/2 induction, there were significant differences between GnRHa and TGF-ß actions and between LSMCs and MSMCs on c-fos and c-jun expression, suggesting divergence at post-receptor signaling, which occurred downstream from MEK1/2. Furthermore, GnRHa and TGF-ß1 through this signaling pathway alter the expression of fibronectin, type I collagen and PAI-1 mRNA in LSMCs and MSMCs in a cell- and gene-specific manner. Because the products of these genes regulate diverse biological activities, specifically extracellular matrix turnover, their differential expression may play a critical role in leiomyoma growth and regression.

However, GnRH and GnRH receptors are expressed at low levels and GnRH binding sites detected in several peripheral tissues and cells, including normal endometrial and myometrial cells, and endometrial cancer cell lines are of low affinity/high capacity and/or high affinity/low capacity (1, 4, 25, 26). Thus, GnRH expressed in these tissues and cells must act in an autocrine/paracrine manner, although exposure of these two classes of GnRH receptors to pharmacological doses during GnRHa therapy could result in their activation and generation of the necessary signaling. Several in vitro cell culture systems derived from peripheral tissues, including normal and cancer cells from endometrium, ovary, prostate, and breast have provided support for the direct action of GnRHa (1, 10, 11, 12, 13, 14, 15, 16, 25, 26, 29, 30, 37, 38). These results indicate that GnRHa acting through GnRH receptor-mediated signaling alters the rate of cell growth, apoptosis, and the expression profile of many genes, which include cell cycle regulatory factors, growth factors, cytokines, proteases, and protease inhibitors (10, 11, 12, 13, 14, 15, 16, 17, 18, 30, 39).

GnRHa therapy is reported to inhibit the expression of TGF-ß isoforms, TGF-ß receptors, ERK1/2, and Smads in leiomyoma and myometrium and that of TGF-ß1 in LSMCs and MSMCs in vitro (11, 17, 18, 36, 39). However, GnRHa did not interfere with TGF-ß1-induced pERK1/2 in LSMCs and MSMCs, possibly because of inadequate TGF-ß receptor inhibition by GnRHa (17), and/or GnRHa-induced signaling through MAPK/ERK may not alter TGF-ß receptors signaling involving MAPK/ERK. We have recently reported that GnRHa alters the expression of TGF-ß-induced Smad3 and antagonistic Smad7 expression in these cells (12). GnRHa and TGF-ß1 signaling through the MAPK pathway is known to regulate the transcriptional activation of immediate early response genes and their target genes (25, 26, 27, 28, 29, 30, 31). GnRHa- and TGF-ß1 through this pathway regulated the expression of c-fos and c-jun as well as fibronectin, type I collagen and PAI-1 mRNA in LSMCs and MSMCs. GnRHa- and TGF-ß1-induced pERK1/2 was highly sensitive to U0126, an effective MEK1/2 inhibitor; however, pretreatment with PD98059, a MEK1 inhibitor, either was without effect or increased pERK1/2, with partial blocking of GnRHa action in LSMCs. Because GnRH and TGF-ß receptor signaling is also mediated through p38 MAPK and their crosstalk interacts with MAPK/ERK, we pretreated LSMCs and MSMCs with SB203580, a p38 MAPK inhibitor, and found that the treatment altered the basal pERK1/2 in LSMCs but had no effect on GnRHa- and TGF-ß1-induced pERK1/2 in both LSMCs and MSMCs. In addition, GnRHa and TGF-ß1 actions on c-fos, c-jun, fibronectin, type I collagen, and PAI-1 mRNA expression also displayed different sensitivity to U0126 pretreatment. Although the results further support that MAPK/ERK signaling downstream from MEK1/2 is involved in mediating GnRH and TGF-ß receptor signaling in LSMCs and MSMCs, their cell-specific activation of this pathway and their target genes with the possibility of crosstalk with other pathways requires detailed investigation.

In the gonadotrope-derived {alpha}T3–1 cell line, GnRHa- induced pERK is reported to be sensitive to both U0126 and PD98059 (28, 40) without affecting activin A-induced pERK (35). In addition, mechanical stretch-induced ERK1/2 in rat myometrial smooth muscle cells is attenuated by PD98059, whereas it was completely abolished by U0126 at 5-fold lower concentration (41). U0126 has been reported to have approximately 100-fold higher affinity for MEK compared with PD98059 (42), and that may account for differences in their actions in our study. In LßT2 gonadotropes, GnRHa-induced nuclear accumulation of ERK and activation of c-fos resulted in transcriptional regulation of LH genes (35), whereas in GT1–7 cells, GnRHa caused a rapid induction of c-fos without nuclear translocation of ERK (27). GnRHa-induced p38 MAPK has also been reported to activate c-fos promoter in {alpha}T3–1 cells, which was inhibited by SB203580, but failed to inhibit GnRHa-induced MAPK phosphatase-2 expression (28). In addition, c-jun expression is reported to occur after 1–6 h of treatments with TGF-ß in a Smad- dependent manner, which is rapidly activated by TGF-ß (19, 20, 43, 44). Consistent with these observations, c-fos and c-jun expression in LSMCs and MSMCs was induced within 1–4 h of treatments with GnRHa and TGF-ß1 in a cell-specific manner. We have also reported a rapid activation of Smad3 in LSMCs and MSMCs by TGF-ß1 and induction of an antagonistic Smad, Smad7, after GnRHa treatment, a molecular mechanism in which GnRHa can block TGF-ß action in these cells or their expression at tissue level (12). Although our observations are the first to demonstrate the expression of c-fos and c-jun and their differential regulation by GnRHa and TGF-ß1 in LSMCs and MSMCs, previous studies have demonstrated that leiomyoma and myometrium express c-fos and c-jun, with significantly lower expression in leiomyoma (45, 46). The reports also indicated no relationship between the level of c-fos and c-jun expression in leiomyoma and myometrium and the phases of the menstrual cycle or GnRHa treatment (45, 46).

Increased transcriptional activation and phosphorylation of c-fos and c-jun through MAPK and/or several other signaling pathways leads to regulation of specific genes whose promoters contain activator protein (AP)-1 sites. Heterodimerization of c-fos with members of the Jun family of transcription factors form the AP-1 complex, which is critical in a wide range of cellular activities. We demonstrated that the expression of fibronectin, type I collagen and PAI-1, whose promoters contain several AP-1 sites, are the target of GnRHa and TGF-ß1 actions in LSMCs and MSMCs (47, 48). As expected, TGF-ß1 increased the expression of type I collagen and PAI-1 mRNA in LSMCs and MSMCs, whereas fibronectin expression was only moderately increased in MSMCs and by GnRHa treatment in LSMCs. In contrast, GnRHa inhibited fibronectin expression in MSMCs and type I collagen expression in both MSMCs and LSMCs. GnRHa increased PAI-1 mRNA expression in both cell types but at significantly lower levels compared with TGF-ß. Unlike c-fos and c-jun expression, the expression of these genes displayed different sensitivities to U0126 pretreatments in LSMCs and MSMCs, with GnRHa action on PAI-1 and TGF-ß1 action on type I collagen and PAI-1 expression showing sensitivity to U0126. Although the results support the involvement of signaling downstream from MEK1/2 in mediating GnRHa and TGF-ß1actions on transcriptional regulation of these genes in LSMCs and MSMCs, their expression and influence of GnRHa and TGF-ß1 occurred in a cell-specific manner. TGF-ß-induced type I collagen and PAI-1 expression has been reported to involve both ERK and Smad activation and to occur in an AP-1- and Smad-dependent manner, although activation of ERK is reported to inhibit collagen expression and act as a negative regulator of TGF-ß-induced collagen expression (19, 20, 45, 46, 47, 48, 49, 50).

Considerable data are available regarding the regulation of type I collagen, fibronectin, and PAI-1 by TGF-ß; however, the direct action of GnRHa on the expression of type I collagen and fibronectin has not been previously reported. A previous study has reported that leiomyoma and myometrium express type I and type III collagens and fibronectin mRNA and demonstrated that collagens, but not fibronectin, are overexpressed in leiomyoma (51). However, the levels of collagens and fibronectin expression in leiomyoma and myometrium from women who received GnRHa therapy were similar to that seen in untreated tissues from the proliferative phase of the menstrual cycle (51). A recent report also indicated that GnRH regulates the expression of urokinase-type plasminogen activator and PAI-1 mRNA and protein in isolated first-trimester decidual cells (15). Similar to our observation in LSMCs and MSMCs, GnRH I is reported to increase, whereas GnRH II to decrease the expression of PAI-1 in endometrial stromal cells (15). The biological significance of GnRHa action in regulating collagens, fibronectin, and PAI-1 expression in uterine/leiomyoma is not possible from our study, although in decidual cells GnRH regulation of urokinase-type plasminogen activator and PAI-1 is considered to play a key regulatory role in the extracellular matrix remodeling events that occur at the maternal-fetal interface during pregnancy (15). TGF-ß fibrinogenic activity is mediated in part through modulation of extracellular matrix and proteolytic enzymes expression (21, 48, 49).

In conclusion, we demonstrated that GnRHa and TGF-ß1 mediate their actions in LSMCs and MSMCs in part through the MAPK/ERK pathway downstream from MEK1/2 and transcriptional activation of c-fos and c-jun, leading to differential expression of fibronectin, type I collagen and PAI-1. The results suggest that GnRHa and TGF-ß signaling through this pathway, with possible interactions with other pathways activated by TGF-ß and GnRH receptors, serve to mediate their diverse biological activities leading to leiomyoma growth and regression, respectively.


   Footnotes
 
This work was supported by Grant RO1 HD37432 from the National Institutes of Health.

This work was presented in part at the 50th Annual Meeting of the Society for Gynecological Investigation, Washington, DC, March 2003.

Abbreviations: AP, Activator protein; CaM, calmodulin; Ct, threshold cycle; GnRHa, GnRH analog; LSMC, leiomyoma smooth muscle cell; MSMC, myometrial smooth muscle cell; PAI, plasminogen activator inhibitor; pERK, phosphorylated ERK; PK, protein kinase.

Received January 30, 2004.

Accepted August 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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