Gonadotropin-releasing hormone and TGF-{beta} activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells

Xiaoping Luo, Li Ding, and Nasser Chegini

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

Submitted 6 May 2004 ; accepted in final form 12 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gonadotropin-releasing hormone analog (GnRHa) is used for medical management of endometriosis and premature luteinizing hormone surge during controlled ovarian stimulation. Human endometrium expresses GnRH receptors, and GnRHa alters the expression of transforming growth factor-{beta} (TGF-{beta}) and receptors in endometrial cells. Because the diverse biological actions of GnRHa and TGF-{beta} are mediated in part through the MAPK pathway, we determined whether utilization of MAPK/ERK and transcriptional activation of immediate early genes c-fos and c-jun result in differential regulation of fibronectin, known as key regulator of embryo implantation and endometriosis progression. Using endometrial stromal cells (ESC) and the endometrial epithelial cell line HES, we demonstrated that GnRHa and TGF-{beta}, in a dose-, time-, and cell-dependent manner, increased the level of phosphorylated ERK1/2 (pERK1/2). GnRH antagonist Antide also increased pERK1/2 induction in ESC and HES, whereas pretreatment reduced GnRHa-induced pERK2 in ESC but not in HES. Cotreatments with GnRHa plus TGF-{beta}1 did not have an additive or an inhibitory effect on pERK1/2 induction compared with GnRHa or TGF-{beta}1 action alone. TGF-{beta}1 and GnRHa increased ERK1/2 nuclear accumulation and inversely regulated the expression of c-fos and c-jun and that of fibronectin in a cell-specific manner. Pretreatment with U-0126, a MEK1/2 inhibitor, blocked basal, as well as GnRHa- and TGF-{beta}1-induced pERK1/2; however, it differentially affected c-fos, c-jun, and fibronectin expression. In conclusion, the results indicate that GnRHa and TGF-{beta} signaling through MAPK/ERK results in differential regulation of fibronectin expression in endometrial cells, a molecular mechanism where short- and long-term GnRHa therapy and locally expressed TGF-{beta} could influence embryo implantation and endometriosis implants, respectively.

endometrial cells; endometriosis; extracellular signal-regulated kinase; fibronectin; gonadotropin-releasing hormone analog; transforming growth factor-{beta}; regulation


GONADOTROPIN-RELEASING HORMONE ANALOG (GnRHa) therapy is often used for medical management of several uterine abnormalities, including endometriosis (47, 49). Short-term administration of GnRHa is used to prevent premature luteinizing hormone (LH) surge in women undergoing controlled ovarian stimulation (2, 3, 25, 29, 40). GnRHa therapy is traditionally believed to act at the level of the pituitary-gonadal axis, regulating the ovarian steroid production and steroid-dependent activities of the target tissues such as endometrium and endometriosis implants. Clinical observations indicate that GnRHa administration to prevent premature LH surge is associated with a reduced rate of embryo implantation (2, 3, 25, 29, 40). Although changes in hormonal milieu affecting endometrial preparation could account for lower implantation rate, accumulating evidence suggests that GnRHa acts directly on endometrium and other peripheral tissues expressing GnRH receptors (12, 21, 28). GnRHa administration is reported to alter the endometrial expression of ovarian steroid receptors and to induce antimitotic effects compared with endometrium of the natural cycle (3). Other in vivo and in vitro studies also reported GnRHa-induced alteration in cell growth and expression of cell cycle proteins, growth factors, cytokines, proteases, and protease inhibitors in several peripheral tissues/cells, including endometrial carcinoma cell lines, stromal cells, ectopic endometrial cells, and leiomyoma and myometrial cells (511, 14, 15, 18, 19, 2124, 26, 28, 33, 36, 43, 52, 54). These observations and identification of GnRH and GnRH receptor expression in these tissues and cells have led to the proposal of an autocrine/paracrine role for GnRH and a direct action for GnRHa in peripheral tissues (1113, 17, 22, 28, 33, 42).

Ovarian steroids are key regulators of many uterine growth factor, cytokine, and chemokine expressions, including transforming growth factor-{beta} (TGF-{beta}), a multifunctional cytokine expressed in human endometrium throughout the menstrual cycle. TGF-{beta} is known to regulate various cellular activities, including cell growth and differentiation, apoptosis, inflammatory and immune responses, and extracellular matrix (ECM) turnover (1, 38, 48, 56). Altered expression of TGF-{beta} has been associated with several abnormalities, including endometriosis and endometrial cancer (1, 11, 34, 38, 44, 45, 48, 56). In the endometrium, TGF-{beta} regulates its own expression and that of ECM, adhesion molecules, proteases, and proteases inhibitors, thus regulating trophoblast invasion, angiogenesis, and tumor metastasis, which occur during embryo implantation, endometriosis, and endometrial cancer, respectively (4, 9, 33, 34, 44, 45). GnRHa suppresses the expression of TGF-{beta} and TGF-{beta} receptors (11), ovarian steroid-induced TGF-{beta} expression in leiomyoma and myometrial smooth muscle cells, and matrix metalloproteinases and their inhibitors in endometrial stromal and decidual cells (10, 1417, 43).

Signaling pathways activated by GnRH receptors include protein kinase A (PKA), PKC, G protein-coupled receptor kinases, calcium-calmodulin (Ca2+-CaM), and the mitogen-activated protein kinase (MAPK) cascade and may also involve epidermal growth factor receptor tyrosine and c-Src kinases (12, 28, 30). In contrast, TGF-{beta} receptor signaling is mediated mainly through the Smad pathway, although activation of other pathways, including MAPKs, and their functional interactions with Smads have been documented in several cell types (38, 48, 56). Functional interactions between GnRH and TGF-{beta} receptors involving MAPK and Smad pathways have been reported to result in differential regulation of LH, GnRH, GnRH receptor, and Smad expression in pituitary gonadotropes, leiomyoma and myometrial smooth muscle cells, and endometrial stromal cells, respectively (20, 33, 39, 54, 55).

Fibronectin is a major component of ECM whose expression is regulated by ovarian steroids and TGF-{beta} (4, 41, 50, 53). Fibronectin is a key regulator of various cellular activities including cell-cell and cell-ECM communications that are central to endometrial preparation for embryo implantation, trophoblast invasion, angiogenesis, and tissue turnover (4, 50, 53). Attachment of endometriosis implants to the peritoneal surface also involves cellular adhesion, invasion, angiogenesis, and ECM turnover (45). Because of the diverse actions of GnRHa and TGF-{beta} in the endometrium, we hypothesized that their receptors signaling through MAPK/ERK and transcriptional activation of immediate early response genes c-fos and c-jun result in differential regulation of fibronectin expression. To test our hypothesis, we used primary culture of endometrial stromal cells and an endometrial epithelial cell line (HES).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All the materials used for isolation and culturing of endometrial stromal cells, GnRH analogs, leuprolide acetate and Antide, {alpha}-smooth muscle actin, and vimentine antibodies were purchased from Sigma Chemical (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-{beta}1 was purchased from R&D Systems (Minneapolis, MN), and affinity-purified monoclonal anti-phosphospecific ERK1/2 and rabbit anti-ERK1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). MEK1/2 synthetic inhibitors U-0126 and PD-98059, and the p38 MAPK inhibitor SB-203580 were purchased from Calbiochem (San Diego, CA).

Tissue collection and cell culture. Portions of endometrial tissues were collected from premenopausal women (n = 3) who were undergoing hysterectomy for medically indicated reasons, excluding endometrial cancer. The patients had not received any medications (including hormonal therapy) during the previous 3 mo before surgery. The tissues were collected at the University of Florida-affiliated Shands Hospital with the approval of the Institutional Review Board. Immediately after collection, small portions of endometrial tissues were prepared for isolation of endometrial stromal cells (ESC) and cultured in DMEM-F-12 until reaching visual confluence, as previously described (33). Before use in these experiments, the isolated cells were seeded in eight-well culture slides (Nalge Nunc, Naperville, IL) and after 24 h of culturing were characterized by immunofluroscence microscopy using antibodies to vimentin and {alpha}-smooth muscle actin, as previously described (34). In addition, a human endometrial epithelial cell line (HES), derived from spontaneous transformation of isolated endometrial surface epithelial cells from benign proliferative endometrium [(16) kindly provided by Dr. D. Kniss at Ohio State University, Columbus, Ohio] was used in parallel with ESC. HES cells were cultured in M-199 containing 10% FBS.

GnRH receptor I and II mRNA expression. Total cellular RNA isolated from ESC and HES was subjected to RT-PCR to determine GnRH I and II receptor mRNA by use of the following primers, respectively: sense 5'-CATCAACAACAGCATCCCAC-3' and antisense 5'-ATCCAGTGGCATGACAATCA-3', product size 247 bp, and sense 5'-GCAAGAGACCACCTATAACCT-3' and antisense 5'-GGTGTCCAGCAGAGGATGAAGGTCAG-3', product size 660 bp (37, 51).

MAPK activity. HES and ESC were seeded in six-well plates at an approximate density of 106 cells/well in supplemented medium containing 10% FBS. After reaching visual confluence, the cells were washed with serum-free medium and incubated under serum-free, phenol-free condition for 24 h. ERK1/2 activation was determined following treatment with TGF-{beta}1 (2.5 ng/ml) and/or GnRHa (0.1 µM) for 5, 15, and 30 min (time dependence) or with TGF-{beta}1 (1, 2.5, 5, and 10 ng/ml) and/or GnRHa (0.01, 0.1, 1, and 10 µM) for 15 min (dose dependence). The specificity of GnRHa on ERK1/2 activation was determined after pretreatment of HES and ESC with 10 µM of the GnRH antagonist Antide for 2 h followed by GnRHa (0.1 µM) for 15 min. To determine possible autocrine/paracrine action of TGF-{beta}1 on ERK activation, HES and ESC were treated with TGF-{beta} type II receptor antisense or sense oligonucleotides (1 µM) for 24 h, and then the cells were washed and treated with TGF-{beta}1 (2.5 ng/ml) for 15 min. The specificity of TGF-{beta}1- and GnRHa-induced pERK1/2 was assessed using MEK1- and MEK1/2-specific inhibitors PD-98059 and U-0126. A parallel experiment was also performed using the p38 MAPK inhibitor SB-203580. HES and ESC were treated with 20 µM PD-98059, U-0126, or SB-203580 for 2 h before treatment with TGF-{beta}1 (2.5 ng/ml) or GnRHa (0.1 µM) for 15 min. The cell lysate was prepared and subjected to immunoblotting.

Western blot analysis. Cell lysates were centrifuged, the supernatants were collected, and their total protein content was determined using a conventional method (Pierce, Rockford, IL), as previously described (33, 54). Equal amounts of sample proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane, as previously described (33, 54). Immunostained proteins were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ), and the band intensity corresponding to pERK1/2 (44/42 kDa) and ERK1/2 was determined as previously described (33, 54).

Immunostaining. TGF-{beta}1- and GnRHa-induced pERK1/2 translocation into the nucleus was assessed in HES and ESC cultured in eight-well culture slides for 24 h. The cells were washed and further incubated under serum-free, phenol red-free condition for 24 h and then treated with TGF-{beta}1 (2.5 ng/ml) or GnRHa (0.1 µM) for 5, 15, and 30 min. The cells were immunostained with anti-ERK antibodies by FITC-labeled indirect method and Vectashield with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) as the mounting medium (33).

c-fos, c-jun, and fibronectin expression. HES and ESC were cultured as above and treated with TGF-{beta}1 (2.5 ng/ml) and GnRHa (0.1 µM) for 0.5, 1, 2, 4, 6, and 12 h. Additional experiments were performed by pretreating the cells with PD-98059, U-0126, and SB-203580 at the aforementioned doses for 2 h followed by treatment with TGF-{beta}1 (2.5 ng/ml) and GnRHa (0.1 µM) for 1 or 6 h selected from the time course study. Total RNA was isolated from treated and untreated control and subjected to real-time PCR.

Real-time PCR. cDNA was generated from 2 µg of total RNA by use of Taqman reverse transcription (RT) reagent. The RNA was incubated in 100 µl of RT reaction mixture (1x RT buffer, 5.5 mM MgCl2, 2 mM dNTP, 2.5 µM random hexamers, 0.4 U RNasin, and 1.25 U MultiScribe reverse transcriptase) for 10 min at 25°C and for 30 min at 48°C. The reverse transcriptase was inactivated by heating at 95°C for 5 min. PCR was performed in 96-well optical reaction plates on cDNA equivalent to 100 ng of RNA in a volume of 50 µl, containing 25 µl of TaqMan Universal Master Mix and optimized concentrations of FAM-labeled probe, forward and reverse primers selected from Assay on Demand (Applied Biosystems). Real-time PCR was performed for c-fos, c-jun, fibronectin, and 18S ribosomal RNA gene by means of ABI-Prism 7700 Sequence System at the following conditions: 2 min at 50°C and 10 min at 95°C for 1 cycle, and 15 s at 95°C and 1 min at 60°C for 40 cycles. 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 the 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.

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


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
GnRHa- and TGF-{beta}-induced MAPK/ERK activation in ESC and HES. Human endometrial tissues and their isolated epithelial and stromal cells, as well as HES, an endometrial surface epithelial cell line, express GnRH I and II as well as GnRH I and II receptor mRNA (Refs. 17, 22; Fig. 1). Because these cells also express TGF-{beta} isoforms and TGF-{beta} receptors, and MAPK/ERK pathway is activated by both GnRH and TGF-{beta} receptors as part of their intracellular signaling in other cell types, we determined the involvement of this pathway in mediating GnRHa and TGF-{beta} actions in HES and ESC. The results show that HES and ESC contain varying levels of constitutively activated ERK1/2, and treatments of serum-starved cells with GnRHa and TGF-{beta}1 in a dose- (Fig. 2, A and B) and time- (Fig. 3, A and B) dependent manner increased the level of pERK1/2 in both cell types compared with untreated controls (P < 0.05). Cotreatment of HES and ESC with GnRHa plus TGF-{beta}1 did not have an additive and/or inhibitory effect on pERK1/2 induction compared with levels induced by TGF-{beta}1 or GnRHa (not shown). Treatment of HES and ESC with Antide also increased pERK1/2 induction (P < 0.05); however, pretreatment with Antide had a minimal effect on GnRHa-induced pERK1/2 in ESC as opposed to HES (Fig. 4). Pretreatment of HES and ESC with TGF-{beta} receptor type II antisense oligonucleotide also reduced TGF-{beta}1-induced pERK1/2 (P < 0.05), although a partial inhibition also occurred with TGF-{beta} receptor type II sense oligomer, particularly in ESC (Fig. 5).



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Fig. 1. RT-PCR of total RNA isolated from endometrial epithelial cell line HES and endometrial stromal cells (ESC) cultured for 48 h in supplemented medium containing 10% FBS. The predicted 247- and 660-bp fragments of gonadotropin-releasing hormone (GnRH) I and GnRH II receptors (arrows) are shown for both cell types. M, DNA marker.

 


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Fig. 2. Dose-dependent action of transforming growth factor (TGF)-{beta}1 (1, 2.5, 5, and 10 ng/ml) and GnRH analog (GnRHa; 0.01 to 10 µM) on the rate of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and total ERK (ERK) induction in HES and ESC. Top: serum-starved cells were treated with TGF-{beta}1 (A) and GnRHa (B) for 15 min, and cell lysates were prepared from treated and untreated control (Ctrl) cells and subjected to immunoblotting using pERK and ERK antibodies. Bottom: bar graphs show means ± SE of fold change in pERK1/2 induction in HES and ESC from 3 different experiments. * and **Significantly different from untreated controls; * differs from ** and ** from *** (P < 0.05).

 


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Fig. 3. Time-dependent action of TGF-{beta}1 (2.5 ng/ml) and GnRHa (0.1 µM) on the rate of pERK1/2 and total ERK1/2 induction in HES and ESC. Top: serum-starved cells were treated with TGF-{beta}1 (A) and GnRHa (B) for 5, 15, and 30 min, and cell lysates from treated and untreated control cells (time 0) were prepared and subjected to immunoblotting using pERK1/2 and ERK antibodies. Bottom: bar graphs show means ± SE of fold change in pERK1/2 induction in HES and ESC from 3 different experiments. *Significantly different from controls (P < 0.05).

 


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Fig. 4. Effect of GnRH antagonist (Antide, An), GnRHa, and GnRHa + Antide (An+GnRH) on the rate of pERK1/2 induction in HES (left) and ESC (right). Top: serum-starved cells were pretreated with 10 µM Antide for 2 h and then treated with 0.1 µM GnRHa for an additional 15 min. Cell lysates from treated and untreated control cells were prepared and subjected to immunoblot analysis using pERK1/2 and ERK antibodies. Bottom: bar graphs show means ± SE of fold change in pERK1/2 induction from 3 different experiments. *Significantly different from controls (P < 0.05).

 


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Fig. 5. Effect of TGF-{beta}1 on the rate of pERK1/2 induction in HES and ESC following pretreatment with TGF-{beta} type II receptor antisense (1 µM) and sense (1 µM) oligoneucleotides. Top: cells were treated with antisense (A) or sense (S) oligomers for 24 h (medium contained 2% FBS), washed, and then treated with TGF-{beta}1 (2.5 ng/ml) for 15 min. Cell lysates were prepared from TGF-{beta} (TGF), TGF-{beta}1 + TGF-{beta} type II receptor antisense (T+A) and TGF-{beta}1 + TGF-{beta} type II receptor sense (T+S) treated and untreated (Ctrl) cells and subjected to immunoblotting using pERK1/2 and ERK antibodies. Bottom: bar graphs show means ± SE of fold change in pERK1/2 induction in HES and ESC from 3 different experiments. *Significantly different from untreated controls; **P < 0.05.

 
Pretreatment with U-0126 (MEK1/2 inhibitor) inhibited basal as well as TGF-{beta}1- and GnRHa-induced pERK1/2 in HES and ESC (P < 0.05; Fig. 6). However, pretreatment with PD-98059 (MEK1 inhibitor) was less effective in inhibiting basal and did not affect TGF-{beta}1-induced pERK1/2 in HES but prevented GnRHa-induced pERK1/2 in both cells and TGF-{beta}1 action in ESC (P < 0.05; Fig. 6). Pretreatment of HES with SB-203580, a p38 MAPK inhibitor, had no significant effect on TGF-{beta}1 but increased GnRHa-induced pERK1/2 (P < 0.05; Fig. 6). In contrast, pretreatment of ESC with SB-203580 increased TGF-{beta}-induced pERK1/2, while reducing that in GnRHa-treated cells (P < 0.05, Fig. 6).



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Fig. 6. Effect of TGF-{beta}1 and GnRHa on pERK1/2 induction following pretreatment of HES and ESC with MEK1/2 synthetic inhibitors PD-98059 (PD) and U-0126 (U) and p38 MAPK inhibitor SB-203580 (SB). Top: cells were treated with PD, U, or SB at 20 µM for 2 h, washed, and then treated without (–) or with TGF-{beta}1 (T, 2.5 ng/ml) or GnRHa (G, 0.1 µM) for 15 min. Cell lysates were subjected to immunoblotting using pERK1/2 and ERK antibodies. Bottom: bar graphs show means ± SE of fold change in pERK1/2 induction in HES and ESC from 3 different experiments. *Significantly different from untreated controls; **P < 0.05.

 
GnRHa and TGF-{beta} differently regulate transcriptional activation of c-fos and c-jun genes and fibronectin expression. By use of indirect fluorescent immunocytochemistry, ERK1/2 was localized in the cytoplasmic and nuclear regions of HES and ESC. Treatments with TGF-{beta}1 (2.5 ng/ml) or GnRHa (0.1 µM) for 5, 15, and 30 min resulted in an increase in ERK1/2 nuclear labeling (Fig. 7, shown for 15 min). Nuclear translocation of activated ERK1/2 is accompanied by transcriptional activation of immediate early response genes, including c-fos and c-jun. GnRHa and TGF-{beta}1 induced a rapid and time-dependent expression of c-fos mRNA in ESC, which was inhibited in HES after prolonged exposure (P < 0.05; Fig. 8). GnRHa did not significantly alter c-fos expression during the early time points; however, prolonged exposure resulted in gradual inhibition in both ESC and HES (P < 0.05; Fig. 8). Unlike c-fos, TGF-{beta}1 significantly increased c-jun expression in HES, whereas in ESC TGF-{beta}1 action was limited, with only a slight, but significant, increase in c-jun expression after 2–6 h (P < 0.05; Fig. 8). GnRHa treatment resulted in a rapid induction of c-jun expression in HES while inhibiting that in ESC (P < 0.05; Fig. 8).



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Fig. 7. Immunofluorescence localization of ERK in HES and ESC. Cells were incubated under serum-free condition for 24 h and then treated with TGF-{beta}1 (2.5 ng/ml) or GnRHa (0.1 µM) for 5, 15, and 30 min. Note cytoplasmic/nuclear localization of ERK1/2 in untreated control (A and G) and increased nuclear localization in TGF-{beta}1- (B and H) and GnRHa- (C and I) treated cells shown after 15 min of treatments. FITC staining was used to localize ERK and 4,6-diamidino-2-phenylindole (DAPI) staining for the nuclei (D, E, F, J, K and L).

 


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Fig. 8. Real-time PCR analysis of c-fos, c-jun, and fibronectin mRNA expression in HES and ESC following treatments with TGF-{beta}1 (2.5 ng/ml) or GnRHa (0.1 µM) for 0.5 to 12 h. Cells were also pretreated with U-0126 (U, 20 µM), for 2 h followed by treatment with TGF-{beta}1 (T) for 1 h or GnRHa (G) for 6 h. Bar graphs show means ± SE of relative expression of c-fos, c-jun, and fibronectin mRNA from 3 different experiments. *Significantly different from untreated controls (Ctrl); ** differs from * and *** from ** and *, with arrows pointing out differences in c-fos, c-jun, and fibronectin mRNA expression in each cell type and treatment (P < 0.05).

 
Transcriptional activation of c-fos and c-jun leads to regulation of various genes possessing activating protein (AP)-1 binding sites on their promoters such as fibronectin. As expected, TGF-{beta} increased the expression of fibronectin mRNA in HES and ESC, with a progressive inhibition by GnRHa compared with untreated controls (P < 0.05; Fig. 8). Pretreatment with U-0126 and PD-98059 resulted in differential expression of basal and TGF-{beta}1 and GnRHa actions on c-fos, c-jun, and fibronectin mRNA expression (P < 0.05; Fig. 8). PD-98095 had only a slight, but significant, effect on TGF-{beta}1-induced c-fos, c-jun, and fibronectin expression in HES and ESC but reversed the inhibitory action of GnRHa on fibronectin expression (P < 0.05; Fig. 9). Furthermore, pretreatment with SB-203580 was equally effective in altering TGF-{beta}1 and GnRHa actions on c-fos, c-jun and fibronectin expression, suggesting a potential cross talk between ERK1/2 and p38 MAPK in mediating their actions (Fig. 9). Overall the influence of TGF-{beta}1 on c-fos, c-jun, and fibronectin expression in HES and ESC differed by 2- to 20-fold compared with GnRHa action (Figs. 8 and 9).



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Fig. 9. Real-time PCR analysis of c-fos, c-jun, and fibronectin mRNA expression in HES and ESC following treatment with TGF-{beta}1 (T, 2.5 ng/ml) and GnRHa (G, 0.1 µM) for 6 h or pretreatment with PD-98059 (P) or SB-203580 (SB) at 20 µM for 2 h before treatment with T and G. Bar graphs show means ± SE of relative expression of c-fos, c-jun, and fibronectin mRNA from 3 different experiments. *, **, and ***Significantly different from their respective untreated controls. * differs from ** and ***, with arrows pointing out differences between c-fos and c-jun expression in each cell type and treatment (P < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the present study, we demonstrated that human endometrial epithelial and stromal cells, as well as HES, an endometrial surface epithelial cell line, express GnRH I and II receptors, and we showed that GnRHa and TGF-{beta}1 activates the MAPK/ERK1/2 pathway, which translocated into the nucleus and differentially regulated the expression of c-fos, c-jun, and fibronectin in these cells. Because of the sensitivity of GnRHa- and TGF-{beta}1-induced pERK1/2 as well as c-fos, c-jun, and fibronectin expression to U-0126, the results suggest the involvement of MAPK/ERK signaling downstream from MEK1/2 in mediating their actions in endometrial cells. In support of our observations with ESC and HES are previous reports demonstrating the expression of GnRH and GnRH receptors in endometrium and endometrial cell lines (17, 22, 42) and direct action of GnRHa in several cell types, including endometrial cell lines, endometrial stromal cells, ectopic endometrial cells, and leiomyoma and myometrial smooth muscle cells. Treatment of these cells with GnRHa resulted in alteration of their rate of cell growth and apoptosis and the expression of cell cycle proteins, growth factors, cytokines, proteases, and protease inhibitors (511, 14, 15, 18, 19, 2124, 26, 33, 36, 52, 54). Furthermore, human endometrium expresses all the components of the TGF-{beta} family and TGF-{beta} receptors throughout the menstrual cycle, and TGF-{beta}1, through an autocrine/paracrine action, influences various endometrial biological activities, including cell growth and differentiation, apoptosis, inflammatory and immune responses, and ECM turnover (9, 34, 44, 45).

Several specific signaling pathways, including MAPK/ERK, are utilized by GnRH receptors to regulate these processes in several cell types, including endometrial cancer cell lines; however, the signaling pathway activated by GnRH receptor in the endometrium has not been investigated (12, 23, 24, 28, 33, 35, 39, 46, 54). In contrast, TGF-{beta} receptor signaling occurs mainly through the activation of the Smad pathway; it may also involve MAPKs in certain cell types (38, 48, 56). We (33) have recently reported the endometrial expression of Smads and their regulation by GnRHa and demonstrated the activation of Smad3 by TGF-{beta}1 in ESC and HES. To our knowledge, our observation is the first to demonstrate the activation of MAPK/ERK by GnRHa and TGF-{beta}1 in ESC and HES, suggesting that both Smad and MAPK pathways are involved in mediating GnRH and TGF-{beta} receptor signaling in the endometrium (Fig. 10). We found that, under the culture condition of our study, ESC and HES contain constitutively active ERK, possibly induced by many autocrine/paracrine growth factors and cytokines expressed by these cells, thus contributing toward moderate activation of ERK1/2 after GnRH and TGF-{beta}1 treatments. Because GnRHa therapy results in inhibition of TGF-{beta} isoform, TGF-{beta} receptor, and Smad expression (19, 33), as such we expected cotreatment with GnRHa to inhibit/reduce TGF-{beta}-induced pERK in HES and ESC. Lack of inhibitory and/or additive effect of GnRHa on TGF-{beta}-induced pERK1/2 suggests that GnRHa inhibitory action on TGF-{beta} and TGF-{beta} receptor expression may occur through pathways independent of MAPK/ERK. Because GnRH and TGF-{beta} receptors utilize signaling pathways including PKC, Ca2+-CaM, and other members of the MAPK pathway, such as p38 MAPK and c-Jun NH2-terminal protein kinase (JNK), their activation and/or cross talk could mediate GnRH inhibitory action on TGF-{beta} and TGF-{beta} receptor expression. Additionally, we have recently reported that, in HES and ESC, GnRHa alters the expression of Smads, specifically antagonist Smad, Smad7, which interacts with TGF-{beta} type I receptor kinase and receptor-activated Smad3 and, through a feedback regulatory mechanism, controls TGF-{beta} receptor signaling (Refs. 38, 48, and 56 and Fig. 10). GnRHa-induced Smad7 expression could alternatively influence TGF-{beta} self-regulatory action, including ERK activation. GnRHa is also reported to regulate Smad expression and activation in leiomyoma and myometrial smooth muscle cells (54) and by interacting with TGF-{beta} and activin, through a mechanism involving Smad and MAPK pathways, regulate the expression of LH, GnRH, and GnRH receptor in pituitary gonadotropes (20, 39, 55). Using U-0126, PD-98059, and SB-203580 to identify the specificity of MAPK signaling in mediating GnRHa and TGF-{beta} actions implicated MEK1/2 with possible cross talk with p38 MAPK in HES and ESC.



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Fig. 10. Diagram shows GnRH and TGF-{beta} receptor signaling through MAPK and Smad pathways leading to transcription activation of their target genes in endometrial cells. We showed that activation of MEK/ERK1/2 by GnRH and TGF-{beta} following interaction with their specific receptors (GnRH I and possibly II receptors, GnRHR I and GnRHR II) and TGF-{beta} receptor type II (TGF-{beta}R II) results in transcriptional activation of c-fos and c-jun, whose activation/binding to activating protein (AP)-1 sites of promoters of their target gene results in differential transcription of target genes, i.e., fibronectin. Binding of TGF-{beta} to TGF-{beta}R II leads to activation of serine-threonine kinase activity of the type I receptor (TGF-{beta}R I) resulting in phosphorylation of receptor-activated Smad (Smad2/3). pSmad2/3 associates with the common Smad (Smad4) and translocates into the nucleus, where they regulate transcription through association with DNA-binding proteins and recruitment of transcriptional coactivators or corepressors to the promoters of target genes. Inhibitory Smad (Smad7) negatively regulates the pathway by preventing phosphorylation of TGF-{beta}R I and Smad2/3 and association with Smad4. Smad pathway controls gene expression through Smad2/3-Smad4 heterodimer direct interaction with Smad-binding elements (SBEs) in regulatory regions of TGF-{beta} target genes. TGF-{beta} and GnRH receptor signaling through p38 MAPK, or upregulation of Smad7 expression, may also serve as a molecular mechanism in regulating TGF-{beta} action in the endometrial environment (33, 34).

 
Despite the similarity between GnRHa- and TGF-{beta}1-induced pERK1/2 in ESC and HES, their effects on transcriptional regulation of c-fos, c-jun, and fibronectin differed significantly and occurred in cell-specific manners. Although TGF-{beta} resulted in a rapid induction of c-fos expression in ESC, GnRHa action was a delayed response. This contrasted with c-jun expression, as GnRHa, and specifically TGF-{beta}, caused a rapid induction of c-jun in HES, whereas TGF-{beta} action in ESC was a delayed response, with GnRHa inhibiting c-jun expression. MAPK signaling involving MEK1/2 with possible interaction with p38 MAPK appears to mediate GnRHa and TGF-{beta} actions on c-fos and c-jun transcriptional activation in HES and ESC. We have recently reported a similar interaction between GnRHa and TGF-{beta} actions in leiomyoma and myometrial smooth muscle cells (16a). In pituitary-derived {alpha}T3-1 cells, treatment with PD-98059 has been reported to block GnRH-activated ERK, whereas pretreatment resulted in an increase in GnRH-induced mitogen-activated protein kinase phosphatase 2 (MKP-2) (31, 32, 35, 46, 57, 58). In contrast, SB-203580 is reported to inhibit p38-dependent activation of c-fos promoter in these cells without affecting GnRH-induced MKP-2 (46, 57). GnRH-induced ERK, but not JNK, has also been reported to be sensitive to U-0126, with potential dependence on Raf-1 activation by PKC, although PKC is reported not to mediate GnRH receptor signaling in cell types derived from peripheral tissues (12, 23, 24, 31, 32, 46, 57, 58). Additionally, Smad interacts with various components of the MAPK pathway, leading to inhibition and/or enhancement of ERK activity (38, 48, 56). For instance, activation of ERK2 results in increased Smad2 activation and nuclear localization, with Smad3 serving as substrate for ERK2 (38, 48, 56). Interestingly, overexpression of Smad2/3 increases GnRH receptor activation in {alpha}T3-1 cells (39).

We found that the GnRH antagonist Antide increased pERK1/2 induction in HES and ESC; however, cotreatment with GnRHa plus Antide did not have an inhibitory and/or an additive effect on GnRHa-induced pERK1 in HES; however, it partly increased pERK in ESC. Although a detailed investigation of native GnRH and GnRH antagonist signaling in these cells is needed before any conclusion is reached, the results provide support for the presence of GnRH receptor subtypes with different ligand selectivity in HES and ESC cells. Interestingly, Antide acting through the well-characterized GnRH I receptors is reported to convert an antagonistic action into an agonist (12, 35). Because GnRH I and II receptors are expressed in the endometrium with surface and glandular epithelial cells as the major sites of GnRH I receptor (Refs. 8, 13, 17, 22 and our unpublished data), both receptors may participate in mediating GnRHa actions in ESC and HES. With respect to TGF-{beta} action, blocking/reducing TGF-{beta} type II receptor expression in part reduced TGF-{beta}-induced pERK1/2 in HES, reaching near control levels in ESC. The results support that TGF-{beta}-induced pERK1/2 is receptor mediated; however, inadequate inhibition, or overexpression, of TGF-{beta} receptor type II in HES may have been a limiting factor in preventing the action of exogenously added TGF-{beta}1 and endogenously expressed TGF-{beta} isoforms. TGF-{beta} receptor content, MAPK activation by other autocrine/paracrine factors, and the extent of ERK1/2 activation in response to TGF-{beta} could also account for the differences between HES and ESC.

TGF-{beta} is known to regulate the expression of a wide range of genes in a variety of cell types (1, 38, 48, 56). However, only a limited number of genes are known to be the target of GnRHa action in gonadotropes and tumor cells (31, 32, 35, 46, 57, 58). Recent reports indicate that GnRH can also target the expression of a large number of genes, including several components of ECM and proteases (10, 14, 15, 18, 43). Fibronectin is a major component of ECM whose expression is regulated by ovarian steroids and TGF-{beta} (41, 50, 53). Fibronectin is involved in various cellular activities, including cell-cell and cell-ECM communications that are central to endometrial preparation for embryo implantation, trophoblast invasion, angiogenesis, and tissue turnover (33, 41, 48, 50, 53). Endometriosis implant adherence to the peritoneal surface also involves cellular invasion, angiogenesis, and ECM turnover (26, 45). Thus the interaction between GnRH and TGF-{beta} receptor signaling may serve to direct their diverse actions in their target tissues including endometrium, reflected in their gene expression profile. As expected, we found that TGF-{beta} increases the expression of fibronectin and discovered an inhibitory action for GnRHa both occurring through a signaling pathway downstream from MEK1/2 and potential cross talk with p38 MAPK. Fibronectin and its receptor components, integrins, are important elements in endometrial preparation for embryo implantation and endometriosis implant attachment (50, 53), and its inhibition may represent a molecular mechanism by which short- and long-term GnRHa therapies influence the outcome of these events. GnRHa is reported to induce an imbalance in endometrial ovarian steroid receptor expression with an antimitotic effect, altering the endometrial preparation for embryo implantation in patients who received GnRHa therapy to prevent premature LH surge (2, 3, 25, 29, 40).

In conclusion, we demonstrated that GnRHa and TGF-{beta} signaling through MAPK/ERK downstream from MEK1/2 with potential interaction with p38 MAPK results in differential regulation of c-fos and c-jun transcriptional activation, altering the endometrial expression of fibronectin in a cell-specific manner. Because fibronectin plays a key role in endometrial preparation for embryo implantation and endometriosis implant attachment into the peritoneal cavity, alteration of fibronectin expression may represent a molecular mechanism whereby short- and long-term GnRHa therapies and locally expressed TGF-{beta} could influence embryo implantation and endometriosis implants, respectively.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported in part by Grant HD-37432 from the National Institute of Child Health and Human Development.


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


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Chegini, Dept. of OB/GYN, Univ. of Florida, Box 100294, Gainesville, FL 32610 (E-mail: cheginin{at}obgyn.ufl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Blobe GC, Schiemann WP, and Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 342: 1350–1358, 2000.[Free Full Text]
  2. Bourgain C and Devroey P. The endometrium in stimulated cycles for IVF. Hum Reprod Update 9: 515–22, 2003.[Abstract/Free Full Text]
  3. Bourgain C, Ubaldi F, Tavaniotou A, Smitz J, Van Steirteghem AC, and Devroey P. Endometrial hormone receptors and proliferation index in the periovulatory phase of stimulated embryo transfer cycles in comparison with natural cycles and relation to clinical pregnancy outcome. Fertil Steril 78: 237–244, 2002.[ISI][Medline]
  4. Chakraborty C, Gleeson LM, McKinnon T, and Lala PK. Regulation of human trophoblast migration and invasiveness. Can J Physiol Pharmacol 80: 116–124, 2002.[CrossRef][ISI][Medline]
  5. Chegini N and Kornberg L. Gonadotropin releasing hormone analogue therapy alters signal transduction pathways involving mitogen-activated protein and focal adhesion kinases in leiomyoma. J Soc Gynecol Investig 10: 21–26, 2003.[CrossRef][ISI][Medline]
  6. Chegini N, Luo X, Ding L, and Ripley D. The expression of Smads and transforming growth factor beta receptors in leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. Mol Cell Endocrinol 80: 9–16, 2003.
  7. Chegini N, Rong H, Dou Q, Kipersztok S, and Williams RS. Gonadotropin releasing hormone (GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells interaction with ovarian steroids in vitro. J Clin Endocrinol Metab 81: 3215–3221, 1996.[Abstract]
  8. Chegini N, Tang XM, and Dou Q. The expression, activity and regulation of granulocyte macrophage-colony stimulating factor in human endometrial epithelial and stromal cells. Mol Hum Reprod 5: 459–466, 1999.[Abstract/Free Full Text]
  9. Chegini N, Tang XM, Ma C, and Williams RS. The effects of gonadotropin releasing hormone analogues, add-back, antiestrogen and antiprogestins on leiomyoma and myometrial smooth muscle cells growth and transforming growth factor beta expression. Mol Hum Reprod 8: 1071–1078, 2002.[Abstract/Free Full Text]
  10. Chegini N, Verala J, Luo X, Xu J, and Williams RS. Gene expression profile of leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. J Soc Gynecol Investig 10: 161–171, 2003.[CrossRef][ISI][Medline]
  11. Chegini N and Williams RS. Implication of growth factor and cytokine networks in leiomyomas. In: Cytokines in Human Reproduction, Edited by Hill J. New York: Wiley, 2000, p. 133–162.
  12. Cheng KW and Leung PC. The expression, regulation and signal transduction pathways of the mammalian gonadotropin-releasing hormone receptor. Can J Physiol Pharmacol 78: 1029–1052, 2000.[CrossRef][ISI][Medline]
  13. Cheon KW, Lee HS, Parhar IS, and Kang IS. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod 7: 447–452, 2001.[Abstract/Free Full Text]
  14. Chou CS, MacCalman CD, and Leung PC. Differential effects of gonadotropin-releasing hormone I and II on the urokinase-type plasminogen activator/plasminogen activator inhibitor system in human decidual stromal cells in vitro. J Clin Endocrinol Metab 88: 3806–3815, 2003.[Abstract/Free Full Text]
  15. Chou CS, Zhu H, MacCalman CD, and Leung PC. Regulatory effects of gonadotropin-releasing hormone (GnRH) I and GnRH II on the levels of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinases-1 in primary cultures of human extravillous cytotrophoblasts. J Clin Endocrinol Metab 88: 4781–4790, 2003.[Abstract/Free Full Text]
  16. Desai NN, Kennard EA, Kniss DA, and Friedman CI. Novel human endometrial cell line promotes blastocyst development. Fertil Steril 61: 760–766, 1994.[ISI][Medline]
  17. Ding L, Xu J, Luo X, and Chegini N. Gonadotropin releasing hormone and transforming growth factor beta activate MAPK/ERK and differentially regulate fibronectin, type I collagen, and PAI-1 expression in leiomyoma and myometrial smooth muscle cells. J Clin Endocrinol Metab. In press.
  18. Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, and Roberts JL. Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. Mol Hum Reprod 4: 893–898, 1998.[Abstract]
  19. Dou Q, Tarnuzzer RW, Williams RS, Schultz GS, and Chegini N. Differential expression of matrix metalloproteinases and their tissue inhibitors in leiomyomata: a mechanism for gonadotrophin releasing hormone agonist-induced tumour regression. Mol Hum Reprod 3: 1005–1014, 1997.[Abstract]
  20. Dou Q, Zhao Y, Tarnuzzer RW, Rong H, Williams RS, Schultz GS, and Chegini N. Suppression of TGF-{beta}s and TGF-{beta} receptors mRNA and protein expression in leiomyomata in women receiving gonadotropin releasing hormone agonist therapy. J Clin Endocrinol Metab 81: 3222–3230, 1996.[Abstract]
  21. Ellsworth BS, Burns AT, Escudero KW, Duval DL, Nelson SE, and Clay CM. The gonadotropin releasing hormone (GnRH) receptor activating sequence (GRAS) is a composite regulatory element that interacts with multiple classes of transcription factors including Smads, AP-1 and a forkhead DNA binding protein. Mol Cell Endocrinol 206: 93–111, 2003.[CrossRef][ISI][Medline]
  22. Emons G, Grundker C, Gunthert AR, Westphalen S, Kavanagh J, and Verschraegen C. GnRH antagonists in the treatment of gynecological and breast cancers. Endocr Relat Cancer 10: 291–299, 2003.[Abstract/Free Full Text]
  23. Grundker C, Gunthert AR, Millar RP, and Emons G. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87: 1427–1430, 2002.[Abstract/Free Full Text]
  24. Grundker C, Schlotawa L, Viereck V, and Emons G. Protein kinase C-independent stimulation of activator protein-1 and c-Jun N-terminal kinase activity in human endometrial cancer cells by the LHRH agonist triptorelin. Eur J Endocrinol 145: 651–658, 2001.[ISI][Medline]
  25. Grundker C, Volker P, and Emons G. Antiproliferative signaling of luteinizing hormone-releasing hormone in human endometrial and ovarian cancer cells through G protein {alpha}(i)-mediated activation of phosphotyrosine phosphatase. Endocrinology 142: 2369–2380, 2001.[Abstract/Free Full Text]
  26. Hernandez ER. Embryo implantation and GnRH antagonists: embryo implantation: the rubicon for GnRH antagonists. Hum Reprod 15: 1211–1216, 2000.[Abstract/Free Full Text]
  27. Imai A, Takagi A, and Tamaya T. Gonadotropin-releasing hormone analog repairs reduced endometrial cell apoptosis in endometriosis in vitro. Am J Obstet Gynecol 182: 1142–1146, 2000.[CrossRef][ISI][Medline]
  28. Kakar SS, Winters SJ, Zacharias W, Miller DM, and Flynn S. Identification of distinct gene expression profiles associated with treatment of L{beta}T2 cells with gonadotropin-releasing hormone agonist using microarray analysis. Gene 308: 67–77, 2003.[CrossRef][ISI][Medline]
  29. Klausen C, Chang JP, and Habibi HR. Multiplicity of gonadotropin-releasing hormone signaling: a comparative perspective. Prog Brain Res 141: 111–128, 2002.[Medline]
  30. Kolibianakis EM, Albano C, Camus M, Tournaye H, Van Steirteghem AC, and Devroey P. Initiation of gonadotropin-releasing hormone antagonist on day 1 compared with day 6 of stimulation: effect on hormonal levels and follicular development in in vitro fertilization cycles. J Clin Endocrinol Metab 88: 5632–5637, 2003.[Abstract/Free Full Text]
  31. Kraus S, Benard O, Naor Z, and Seger R. c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells. J Biol Chem 278: 32618–32630, 2003.[Abstract/Free Full Text]
  32. Liu F, Austin DA, Mellon PL, Olefsky JM, and Webster NJ. GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol Endocrinol 16: 419–434, 2002.[Abstract/Free Full Text]
  33. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, et al. Involvement of both G(q/11) and G(s) proteins in gonadotropin-releasing hormone receptor-mediated signaling in Lbeta T2 cells. J Biol Chem 277: 32099–32108, 2002.[Abstract/Free Full Text]
  34. Luo X, Xu J, and Chegini N. Gonadotropin releasing hormone analogue (GnRHa) differentially regulates the expression and activation of Smads in human endometrial epithelial and stromal cells. Reprod Biol Endocrinol 1: 125–138, 2003.[CrossRef][Medline]
  35. Luo X, Xu J, and Chegini N. The expression of Smads in human endometrium and regulation and induction in endometrial epithelial and stromal cells by TGF-{beta}. J Clin Endocrinol Metab 88: 4967–4976, 2003.[Abstract/Free Full Text]
  36. McArdle CA, Franklin J, Green L, and Hislop JN. Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. J Endocrinol 173: 1–11, 2002.[Abstract/Free Full Text]
  37. Meresman GF, Bilotas MA, Lombardi E, Tesone M, Sueldo C, and Baranao RI. Effect of GnRH analogues on apoptosis and release of interleukin-1beta and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Hum Reprod 18: 1767–1771, 2003.[Abstract/Free Full Text]
  38. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, et al. A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98: 9636–9641, 2001.[Abstract/Free Full Text]
  39. Moustakas A, Pardali K, Gaal A, and Heldin CH. Mechanisms of TGF-{beta} signaling in regulation of cell growth and differentiation. Immunol Lett 82: 85–91, 2002.[CrossRef][ISI][Medline]
  40. Norwitz ER, Xu S, Xu J, Spiryda LB, Park JS, Jeong KH, McGee EA, and Kaiser UB. Direct binding of AP-1 (Fos/Jun) proteins to a SMAD binding element facilitates both gonadotropin-releasing hormone (GnRH)- and activin-mediated transcriptional activation of the mouse GnRH receptor gene. J Biol Chem 277: 37469–37478, 2002.[Abstract/Free Full Text]
  41. Olivennes F, Cunha-Filho JS, Fanchin R, Bouchard P, and Frydman R. The use of GnRH antagonists in ovarian stimulation. Hum Reprod Update 8: 279–290, 2002.[Abstract/Free Full Text]
  42. Pankov R and Yamada KM. Fibronectin at a glance. J Cell Sci 115: 3861–3863, 2002.[Free Full Text]
  43. Raga F, Casan EM, Kruessel JS, Wen Y, Huang HY, Nezhat C, et al. Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod 59: 661–669, 1998.[Abstract/Free Full Text]
  44. Raga F, Casan EM, Wen Y, Huang HY, Bonilla-Musoles F, and Polan ML. Independent regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-3 in human endometrial stromal cells by gonadotropin-releasing hormone: implications in early human implantation. J Clin Endocrinol Metab 84: 636–642, 1999.[Abstract/Free Full Text]
  45. Ripley D, Tang XM, Ma C, and Chegini N. The expression and action of granulocyte macrophage-colony stimulating factor and its interaction with TGF-{beta} in endometrial carcinoma. Gynecol Oncol 81: 301–309, 2001.[CrossRef][ISI][Medline]
  46. Seli E and Arici A. Endometriosis: interaction of immune and endocrine systems. Semin Reprod Med 21: 135–144, 2003.[CrossRef][ISI][Medline]
  47. Shah BH, Soh JW, and Catt KJ. Dependence of gonadotropin-releasing hormone-induced neuronal MAPK signaling on epidermal growth factor receptor transactivation. J Biol Chem 278: 2866–2875, 2003.[Abstract/Free Full Text]
  48. Shalev E and Leung PC. Gonadotropin-releasing hormone and reproductive medicine. J Obstet Gynaecol Can 25: 98–113, 2003.[Medline]
  49. Shi Y and Massague J. Mechanisms of TGF-{beta} signaling from cell membrane to the nucleus. Cell 113: 685–700, 2003.[ISI][Medline]
  50. Takeuchi H, Kobori H, Kikuchi I, Sato Y, and Mitsuhashi N. A prospective randomized study comparing endocrinological and clinical effects of two types of GnRH agonists in cases of uterine leiomyomas or endometriosis. J Obstet Gynaecol Res 26: 325–331, 2000.[Medline]
  51. Tseng L, Tang M, Wang Z, and Mazella J. Progesterone receptor (hPR) upregulates the fibronectin promoter activity in human decidual fibroblasts. DNA Cell Biol 22: 633–40, 2003.[CrossRef][ISI][Medline]
  52. Van Biljon W, Wykes S, Scherer S, Krawetz SA, and Hapgood J. Type II gonadotropin-releasing hormone receptor transcripts in human sperm. Biol Reprod 67: 1741–1749, 2002.[Abstract/Free Full Text]
  53. Vignali M. Molecular action of GnRH analogues on ectopic endometrial cells. Gynecol Obstet Invest 45, Suppl 1: 2–5, 1998.[CrossRef][ISI][Medline]
  54. Wang J and Armant DR. Integrin-mediated adhesion and signaling during blastocyst implantation. Cells Tissues Organs 172: 190–201, 2002.[CrossRef][ISI][Medline]
  55. Xu J, Luo X, and Chegini N. Differential expression, regulation, and induction of Smads, transforming growth factor-beta signal transduction pathway in leiomyoma, and myometrial smooth muscle cells and alteration by gonadotropin-releasing hormone analog. J Clin Endocrinol Metab 88: 1350–1361, 2003.[Abstract/Free Full Text]
  56. Yamada Y, Yamamoto H, Yonehara T, Kanasaki H, Nakanishi H, Miyamoto E, and Miyazaki K. Differential activation of the luteinizing hormone beta-subunit promoter by activin and gonadotropin-releasing hormone: a role for the mitogen-activated protein kinase signaling pathway in LbetaT2 gonadotrophs. Biol Reprod 70: 236–243, 2004.[Abstract/Free Full Text]
  57. Yue J and Mulder KM. Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway. J Biol Chem 275: 30765–30773, 2000.[Abstract/Free Full Text]
  58. Zhang T, Mulvaney JM, and Roberson MS. Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone. Mol Cell Endocrinol 172: 79–89, 2001.[CrossRef][ISI][Medline]
  59. Zhang T, Wolfe MW, and Roberson MS. An early growth response protein (Egr) 1 cis-element is required for gonadotropin-releasing hormone-induced mitogen-activated protein kinase phosphatase 2 gene expression. J Biol Chem 276: 45604–45613, 2001.[Abstract/Free Full Text]




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