Effect of Follicle-Stimulating Hormone and Estrogen on the Expression of Betaglycan Messenger Ribonucleic Acid Levels in Cultured Rat Granul
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内分泌学杂志 2005年第8期
Department of Obstetrics and Gynecology (Y.O., K.N., S.Y., H.M., T.M.), School of Medicine, Gunma University, Gunma 371-8511; Department of Biochemistry (T.M., K.M.), Fukui Medical University, Fukui 910-1193; and CREST (K.N., H.M., T.M., K.M., T.M.), Japan Science and Technology, Japan
Address all correspondence and requests for reprints to: Kazuto Nakamura, Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Gunma 371-8511, Japan. E-mail: nkazuto@med.gunma-u.ac.jp.
Abstract
Betaglycan (TGF? type III receptor) was recently identified as a coreceptor to enhance the binding of inhibin A to activin type II receptor. This inhibin/betaglycan/activin type II receptor complex prevents activins from binding to their own receptors. The present study was undertaken to identify the expression and the regulation of the betaglycan gene in cultured rat granulosa cells. Northern blot analysis indicated betaglycan mRNA transcript of approximately 6.4 kbp. The treatment of the cells with FSH increased the betaglycan mRNA level, and a concurrent treatment with estradiol brought a significant increase in betaglycan mRNA. The protein kinase A activator, 8-bromoadenosine-cAMP, also increased the expression of its mRNA. Furthermore, betaglycan mRNA was induced additively by estradiol, which was blocked by estrogen receptor antagonists [ICI 182780, (R, R)-cis-diethyltetrahydro-2,8-chrysenediol]. In the luciferase assay, FSH altered the promoter activity of betaglycan. Moreover, when FSH plus estradiol was added to the granulosa cells, a significant increase in the half-life of betaglycan mRNA transcript was seen. In summary, FSH and estradiol increased betaglycan mRNA expression, most possibly through the protein kinase A pathway and the estrogen receptor-?. The increase of betaglycan mRNA was due to an increase in transcription and altered mRNA stability. In ovarian regulatory function, the expression of betaglycan may involve the functional antagonism of inhibin A in activin signal transduction.
Introduction
TGF? IS A MEMBER OF a superfamily of growth factors that elicit a variety of responses, including growth, differentiation, and morphogenesis (1). Several reports have shown that ovarian cells express both TGF? mRNA and proteins (2, 3), involved in ovarian functions (4). Like other growth factors, the signals of TGF? are mediated by the interaction with various cell surface receptors. So far, three distinct receptors, termed types I, II, and III, have been cloned. The types I and II receptors have intracellular kinase domains responsible for receptor activation and signal transduction (5, 6). In contrast, the type III receptor, also called betaglycan, lacks any clearly identifiable signaling motif. Although the type III receptor does not appear to be a signaling receptor, it has been shown to be important for high affinity binding of TGF?s to the type II receptor (7, 8, 9).
Activins isolated from rat follicular fluid (10, 11) also belong to the TGF? superfamily, modulate cell differentiation, cell proliferation, and specific functions (12, 13, 14). Activin receptor has been found in follicular granulosa cells (15) and is examined for its expression pattern in rat granulosa and thecal cells in developing follicles during all stages of the ovarian cycle (16). Previously we have shown activin up-regulates FSH and LH receptor expression in rat granulosa cell culture (17, 18). Recently it has been demonstrated that betaglycan can bind inhibin A with high affinity, interfering with activin A to bind to its type II receptor (2, 19). In the developing ovarian follicles, betaglycan can function in an environment in which the -subunit of inhibin is produced in a 10- to 20-fold excess to ?-subunit of inhibin, favoring the assembly of inhibin dimers over activin dimers (3). Betaglycan protein was also localized in rat thecal cells and granulosa cells and oocytes (20). Moreover, the expression of betaglycan, which was identified in granulosa and thecal cells of normal human ovaries with immunohistochemistry, was induced by FSH in cultured human granulosa-luteal cells (21). These accumulated data lead us to the idea that betaglycan plays an important role in regulating follicular growth.
In this study, we examined the regulation of betaglycan mRNA expression by FSH and estrogen in rat primary granulosa cell culture. We then performed Northern blot analysis and luciferase assay to show that estrogen increased the level of betaglycan mRNA in the presence of FSH. Through these experiments, we found that the induction of betaglycan mRNA transcripts and the prevention of betaglycan mRNA decay was the mechanism of increase in betaglycan mRNA by estrogen.
Materials and Methods
Hormones and reagents
Rat FSH (I-8) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), ?-estradiol, gentamicin sulfate, and (R, R)-cis-diethyltetrahydro-2,8-chrysenediol (R, R-THC) were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182780 was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). DMEM, Ham’s F-12 medium, and Fungizone were purchased from Life Technologies (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Roche Molecular Biochemicals (Mannheim, Germany).
Cell culture
The granulosa cells were obtained from immature female Wister rats injected with 2 mg diethylstilbestrol in 0.1 ml sesame oil daily for 4 d. The ovaries were then excised, and the granulosa cells were released by puncturing follicles with 25-gauge needles. The animals were treated humanly as possible, following the National Institutes of Health guideline at all times. Granulosa cells were washed and collected by brief centrifugation, and the cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham’s F-1/DMEM (1:1, vol/vol) medium supplemented with 20 mg/liter gentamicin sulfate, 500 μl /liter Fungizone, and 1 g/liter BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2-95% air at 37 C.
Preparation of cRNA probe
The rat betaglycan cDNA was subcloned into the EcoRI site of the pcDNA3.1 (+) vector and linearized with XbaI. Digoxigenin-labeled betaglycan cRNA probes corresponding to 335-2893 bases were produced by in vitro transcription with T7 RNA polymerase and the RNA labeling kit (Boehringer, Mannheim, Germany). A digoxin-labeled glycelaldehyde-3-phosphate dehydrogenase (GAPDH) cRNA probe was obtained by the same method.
RNA isolation and analysis
The granulosa cells were cultured in 60-mm dishes containing 5 x 106 viable cells in 5 ml of medium, and the reagents were added to the medium after 24 h of cell culture. The granulosa cells were further incubated, and the cultures were stopped at the selected times as indicated by using Isogen (Nippon Gene, Toyama, Japan). The final RNA pellet was dissolved in diethylpyrocarbonate-treated H2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 10 μg RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove, NY). In accordance with the standard protocol for the nucleic acid detection kit (Roche Molecular Biochemicals), the membranes were then exposed on X-Omat film (Eastman Kodak Co., Rochester, NY). Luminescence detection was quantified using the LKB 2202 UnitroScan laser densitometer (LKB Produkter AB, Bromma, Sweden), normalized against the corresponding amount of GAPDH mRNA for each sample and expressed in the relative densitometric units.
Vector preparation and transfection
Plasmid pGL3-basic is a luciferase vector lacking the eukaryotic promoter and the enhancer sequences (Promega Corp., Madison, WI). The PGL3-control contains a simian virus 40 promoter and a simian virus 40 enhancer inserted into the structure of pGL3-Basic (Promega). The fragment of the 5'-flanking region of –2013 to –253 bp relative to the translational initiation site was generated from the genomic DNA via a PCR using primers specific to the rat betaglycan sequences. The cDNA was amplified 30 cycles by PCR containing Taq DNA polymerase, and 1 μM each of the two primers at 94 C for 15 sec for betaglycan were 5'-CAGGACTCCAACGAGAAACACACC and 3'-ATCGCGCAGCGAAAGTGGCCTGGA. The isolated PCR-synthesized cDNA fragments were subcloned into pT7 blue-2 vector (Novagen, San Diego, CA) and characterized by nucleotide sequencing analysis. The sequence of the cDNA fragment was identical with the published sequence of rat betaglycan promoter cDNA (22). To evaluate promoter activity, these fragments were ligated to a luciferase reporter vector (pGL3-basic) and named BG-Luc. Using FuGENE (Roch Molecular Biochemicals, Indianapolis, IN), a total of 1 μg of plasmid DNA was transfected into the primary granulosa cell culture plates (2.5 x 105 cells per 0.5 ml of medium in a 20-mm dish). To assay the regulatory elements, the granulosa cells were cultured for the indicated time as described in figure legend in a hormone-free condition medium before transfection. Thirty hours after transfection, the cells were treated with hormones for 6 h. The cells were then harvested, and the luciferase activity was measured. The luciferase assay was performed using the dual-luciferase reporter system (Promega), in which the transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of renilla luciferase.
Transcription stability analysis
Cells were preincubated with FSH alone, estradiol alone, FSH and estradiol, or FSH, estradiol, and R, R-THC for 12 h before 5 μM actinomycin D addition to arrest a new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after the addition of the inhibitor for RNA extraction and Northern blot analysis.
Data analysis
The data represent the mean ± SE from at least three independent experiments. Comparisons between groups were performed by one-way ANOVA. The significance of the differences between the mean values of the control group and each treated group was determined by Duncan’s multiple-comparison test. A value of P < 0.05 was considered significant.
Results
The effect of FSH and estradiol on betaglycan mRNA transcription over time
To investigate the effect of FSH and estradiol on betaglycan mRNA in relation to time, we analyzed the betaglycan mRNA in the primary rat granulosa cell culture. Granulosa cells were cultured for 24 h after plating on a cell culture dish, and this point was considered time 0. Then the cells were cultured another 24–96 h in the presence of FSH (30 ng/ml) and with or without estradiol (5 μM). Figure 1, A and B, represents the autoradiograph of Northern blot, and Fig. 1C shows the relative amount of betaglycan mRNA to GAPDH mRNA. Northern blot analysis indicated betaglycan mRNA transcription of approximately 6.4 kb. In the control cells (with no treatment), the betaglycan mRNA levels were highest at time 0 and decreased gradually over time. On the other hand, after the treatment with FSH, the decline of betaglycan mRNA levels was suppressed. Concurrent treatment with FSH and estradiol increased betaglycan mRNA, approximately twice, from 24 to 96 h. The level remained the same (as the control) with only estradiol.
FIG. 1. Time course of FSH and estradiol (E2) effect on betaglycan mRNA. A and B, Granulosa cells from DES-primed immature rats were cultured alone for 24 h. These cells were further incubated without FSH and estradiol (no treatment), with FSH (30 ng/ml) (A), with estradiol (5 μM), or with a combination of FSH (30 ng/ml) plus estradiol (5 μM) (B). After various incubation times, total RNA was extracted and betaglycan mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The difference between A and B is due to the exposure time. C, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA in time 0, with no treatment, was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to time 0. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
We then investigated the dose-dependent effect of FSH and estradiol at 72 h. An increase in FSH concentration brought about a dose-dependent increase in betaglycan mRNA as shown in Fig. 2. Granulosa cells were cultured in the absence or presence of FSH with or without increasing concentrations of estradiol (1–100 μM) as shown in Fig. 3. Betaglycan mRNA, on the other hand, increased minimally with estradiol alone, and a marked increase of betaglycan mRNA was observed with the concurrent treatment with both FSH and estradiol. Betaglycan mRNA levels were approximately 12 times the control with FSH (30 ng/ml) and estradiol (100 μM).
FIG. 2. Dose-related effect of FSH on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with increasing concentrations of FSH for 72 h. Levels of betaglycan mRNA were measured using Northern blot analysis as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of FSH dose 0 at P < 0.01.
FIG. 3. Dose-related effect of estradiol on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and were then cultured with increasing concentration of estradiol (E2) for 72 h with or without FSH (30 ng/ml). Levels of betaglycan mRNA were measured using Northern blot analysis, as described in Materials and Methods. The Northern blot analysis is representative of three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of FSH (–) at each time at P < 0.01.
Regulation of betaglycan expression by protein kinase A modulator
The protein kinase A pathway is the main signal transduction system of the FSH receptor. We investigated whether the effect of FSH on betaglycan mRNA accumulation involves protein kinase A activation. As shown in Fig. 4, an increase in 8-bromoadenosine-cAMP (8-Br-cAMP) concentration brought a dose-dependent increase of betaglycan mRNA production, suggesting that this pathway is mediated by protein kinase A.
FIG. 4. Effect of 8-Br-cAMP on betaglycan mRNA expression. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and were then cultured with FSH (30 ng/ml) or increasing concentration of 8-Br-cAMP for 48 h. Northern blot analysis was performed as described in Materials and Methods. The Northern blot analysis is representative of three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
The effect of estrogen receptor (ER) antagonist on betaglycan mRNA
The granulosa cells were cultured in the presence of ICI 182780 or R, R-THC with FSH (30 ng/ml) and estradiol (10 μM). ICI 182780 is a high-affinity ER antagonist and R, R-THC is a pure antagonist through ER? and a partial agonist through ER (23). As shown in Fig. 5, A and B, betaglycan mRNA was decreased by ICI. Furthermore, as shown in Fig. 5, C and D, estradiol and FSH-induced betaglycan mRNA and was blocked by the addition of R, R-THC. This suggests that betaglycan mRNA was stimulated by estradiol through estrogen receptor, especially ER?.
FIG. 5. Dose-related effect of ER antagonists on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with FSH, estradiol (E2), and increasing concentrations of ICI 182780 (ICI) for 72 h. Levels of betaglycan mRNA were measured using Northern blot analysis as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured with estradiol and FSH treatment was taken as100%. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control (FSH 30 ng/ml plus E2 10 μM). The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01. C, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with FSH, estradiol, and increasing concentration of R, R-THC for 72 h. The Northern blot analysis is representative of the three experiments. D, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured with estradiol and FSH treatment was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control (FSH 30 ng/ml plus E2 10 μM). The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and are represented as mean ± SEM of the three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
The effect of FSH and estradiol on betaglycan mRNA transcription
We then examined whether the regulation of betaglycan mRNA by FSH and estradiol was dependent on gene transcription and/or receptor mRNA stability. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the 1760 bp of the betaglycan promoter in rat granulosa cells. We tested the basal promoter activity of betaglycan at different times of transfection because they were harvested from DES-treated rat ovaries, suggesting the effect of estrogen perhaps remaining in the cells just after the beginning of culture. As shown in Fig. 6A, the basal promoter activity was induced most in cells transfected 24 h after plating, compared with the cells transfected at 48 or 72 h. Therefore, we used the cells at 72 h for the luciferase assay. The result (Fig. 6B) suggested that the treatment with either FSH (30 ng/ml) alone, cAMP (0.5 mM and 1.0 mM) alone, or FSH plus estradiol amplified the activity of the 1760 bp of the betaglycan 5'-flanking region.
FIG. 6. Effect of FSH and estradiol on betaglycan-luciferase (BG-Luc) expression in rat granulosa cells A, Granulosa cells from DES-primed immature rats were cultured for 24–72 h in hormone-free conditions and then cotransfected with BG (1760 bp)-Luc and pRL-CMV-Rluc at the respective times. Thirty-six hours after transfection, the luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. The luciferase activity at 24 h was taken as 100%. Each bar represents the mean ± SEM of the three independent experiments. *, Difference from the value of control (24 h) at P < 0.01. B, Granulosa cells from DES-primed immature rats were cultured for 72 h in hormone-free conditions and then cotransfected with BG (1760 bp)-Luc and pRL. Thirty hours after transfection, cells were treated with FSH (3 ng/ml, 30 ng/ml), 8-Br cAMP (0.5 μM, 1.0 μM), estradiol (E2, 10 μM), or a combination of FSH (30 ng/ml) plus estradiol (10 μM) for 6 h and then processed. Luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. Each bar represents the mean ± SEM of three independent experiments. *, Difference from the value of control at P < 0.01.
Transcription stability analysis
To asses the rates of degeneration of betaglycan mRNA transcripts, we preincubated the granulosa cells with FSH, or estradiol, FSH and estradiol, or estradiol, FSH, and R, R-THC treatment for 12 h. After this preincubation, 5 μM actinomycin-D was added to arrest a new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after the transcription inhibitor addition, and the betaglycan mRNA levels were quantitated by Northern blot analysis. The amount of betaglycan mRNA at time 0 (the time of addition of actinomycin-D) in each group was assigned a value of 100%, and other values in each group at different times were expressed as a percentage, respective to this value. As shown in Fig. 7, the decay curves for the 6.4-kb betaglycan mRNA transcript in the primary granulosa cells show the presence of FSH, and estradiol significantly increases the stability of betaglycan mRNA. A slight increase in mRNA stability was seen in FSH treatment, not just due to estradiol. In contrast, the effect of estradiol on betaglycan mRNA stability was completely abolished by R, R-THC, demonstrating that estradiol increased mRNA stability through the ER? receptor. There were no statistical differences among treatment with FSH and estradiol, FSH, estradiol, and R, R-THC.
FIG. 7. Effect of FSH and estradiol (E2) on betaglycan mRNA transcript. A, Granulosa cells were preincubated with FSH, estradiol, a combination of FSH and estradiol, or FSH, estradiol, and R. R-THC for 12 h. After this preincubation period, 5 μM actinomycin-D were added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after addition of the transcription inhibitor, and betaglycan mRNA levels were quantitated by Northern blot analysis. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The mRNA level at time 0 was assigned a relative value of 100%, and mRNA levels at all other times are expressed as a percentage to this value. The ordinate is plotted on a logarithmic scale. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and are represented as mean ± SEM of the three independent experiments in the bar graph. Cont, Control. **, Difference from the value of E2 at P < 0.01. *, Difference from the value of control at P < 0.01.
Discussion
TGF? type III receptor, betaglycan, is distributed in many tissues, in which betaglycan attracts and enhances TGF? binding to TGF? type II receptor and form a stable complex between receptor and ligand (23). Findlay et al. (3) indicated that betaglycan might play an important role in the growth and differentiation of follicles, supported by the result that betaglycan mRNA was detected in the postnatal rat ovary. Moreover, recent reports also show that betaglycan, with the activin type II receptor, becomes an inhibin coreceptor and antagonize activin action (2). In this study, we investigated the regulation of betaglycan mRNA expression in the granulosa cell culture system. Our results show that estradiol, in the presence of FSH, induce both the promoter activity and the level of betaglycan mRNA through increasing mRNA stability. These accumulated data indicate a possibility that betaglycan regulates follicular development inside the ovary.
Previously we reported activin induces FSH as well as LH receptor expression in the primary granulosa cell culture from DES-treated rats (17, 18), consistent with other reports (24, 25). In the study, we showed that FSH receptor levels reached maximum at 24 h of culture. However, the effect of activin was terminated for a short time due to the reduction in activin level and the increase of follistatin, binding to activin and antagonizing its effect (26). As shown in Fig. 1, without estrogen, betaglycan mRNA peaked at time 0, followed by a decline as incubation continued. A similar result was seen with estradiol treatment alone, whereas a concomitant treatment with FSH and estradiol increased betaglycan mRNA more than double, compared with the control level. This result suggests FSH and estradiol synergistically up-regulate betaglycan mRNA level in rat granulosa cell culture. Additionally, FSH induced the secretion of both inhibin A and B, especially inhibin A, in immature granulosa cell culture (27). Collectively Lewis et al. (2) found betaglycan protein in follicular granulosa cells, thecal cells, and oocytes in rat ovary through immunohistochemistry. Thus, we speculate that betaglycan plays an important role for both follicular growth and oocyte maturation in the rat ovary.
Luciferase assay was performed to elucidate the mechanism of betaglycan mRNA regulation by FSH and estradiol. To investigate the hormonal regulation of the 5'-flanking region of betaglycan, we examined the clone –2013 to –253 bp of the 5'-flanking region. The sequencing of betaglycan promoter region showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding motif such as GC box, activator protein-2, and specificity protein 1 (Sp 1) existed in this region (22). The cAMP-responsive element (28) and activator protein-2 (29) are well known as most characterized cAMP-responsive elements. This is consistent with the data that the granulosa cells, transfected with a luciferase reporter construct containing this region of the 5'-flanking region of the betaglycan, were responsive to FSH and 8-Br-cAMP in Fig. 6B. In a few cases, estrogen can bind to the half-site of estrogen-responsive elements (EREs) (ggtca) when assisted by the binding of Sp 1 to nearby GC-rich motifs (30). In addition, within the 5'-flanking region of betaglycan, there is no ERE, but there are two half-sites of ERE, which is considered to function when located near Sp1 and GC-rich motif. This indicates that estradiol may not affect the betaglycan promoter.
The basal promoter activity was induced most in cells transfected 24 h after plating, followed by a gradual decline toward 72 h, as shown in Fig. 6A. Combined with the data in Fig. 1, the effect of DES treatment for rats remains in the granulosa cells, especially at the beginning of the culture, supporting that estradiol is an important factor for betaglycan induction. Despite the persistent effect by DES treatment, the addition of estradiol into the culture medium did not induce an additive increase of the promoter activity by FSH. This raises the possibility that the ER pathway does not play a role in the transcription of betaglycan. However, because the promoter region we cloned might not include ERE, we cannot exclude the involvement of the ER pathway in the transcription of betaglycan. Further experiments are required to complete our understanding of the transcriptional mechanisms activating betaglycan in rat granulosa cells. It is well accepted that the signaling cascade of FSH receptor is mainly intermediated by cAMP after subsequent activation of adenylate cyclase. As shown from results in Fig. 4, the protein kinase A pathway consistently plays an important role in the induction of betaglycan mRNA. In the primary cultures of human granulosa-luteal cells from in vitro fertilization patients, Liu et al. (21) suggested that the protein kinase C (PKC) signal transduction pathway is involved in betaglycan induction because the PKC inhibitor, staurosporine, increased the betaglycan mRNA level. However, they failed to demonstrate that the PKC activator, tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate, increases betaglycan mRNA. The study using a stable cell line permanently overexpressing FSH receptor showed that FSH receptor coupled to the PKC pathway with a very high FSH dosage (100 ng/ml or more) (31). The capability of inducing inositol phosphate release remains unclear in both Sertoli and granulosa cells. Thus, we believe that there is no interaction between the PKC signaling pathway and the induction of betaglycan mRNA. Recently several emerging reports in the literature (32) have reported that cAMP intermediates the signaling pathway by FSH not only for PKA, but also for exchange protein activated by cAMP (Epac). In rat granulosa cell culture, Gonzalez-Robayna et al. (33) reported that FSH and 8-Br-cAMP stimulate phosphorylation of protein kinase B through Epacs. To date, there is no clear evidence of Epacs intermediating the regulation of betaglycan expression, and we need to examine this further.
To ascertain the effect of estradiol on betaglycan mRNA through ER, we first used ICI, which can antagonize both isoforms of ER (ER and ER?). In Fig. 5, A and B, ICI 182780 reduced the mRNA level of betaglycan in a dose-dependent manner. ER is known to be expressed in universal tissues (i.e. ovary, uterus, lung, neural tissue) (34). Inside the ovary, ER is expressed in thecal cells and interstitial cells, whereas ER? is thought to be mainly located in granulosa cells (35). Thus, R. R-THC, a weak agonist for ER and an antagonist for ER?, consistently diminished the mRNA level of betaglycan, as in the case of ICI. This suggests that ER? mainly intermediates estradiol signal transduction to increase betaglycan mRNA in the granulosa cells.
The data observed in Fig. 7 suggest FSH and estrogen, when administered together, alters betaglycan mRNA stability in rat granulosa cells. According to the promoter assay, the concomitant treatment with both FSH and estradiol just increased the promoter activity by 1.5-fold. This indicates that the mRNA stability dominantly controls the level of betaglycan mRNA rather than its transcription. The results above provide us one of the mechanisms underlying the regulation of betaglycan expression. However, questions concerning the limitations of this method, the measurement betaglycan mRNA decay, still remain. This assay may not truly reflect the physiological transcript stability, and important elements, modulating the function of gonadotropin receptors, ER, and signal transduction, which can be blocked by the actinomycin-D treatment because there are still limitations to this method.
Previously we have shown that activin, IGF-I, and TGF? increased the level of LH receptor mRNA by changing its stability (18, 36, 37). In the LH receptor, an mRNA binding protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH receptor mRNA stability in the rat ovary (38, 39). To date, there is no evidence supporting the same mechanism, as in the case of LH receptor mRNA. However, this might be one of pivotal roles in controlling mRNA expression of betaglycan.
In this report, we demonstrated that betaglycan is markedly induced by estradiol, assisting FSH synergistically in rat granulosa cell culture although the possibility of other hormones and growth factors is not eliminated (i.e. activin, IGF-I, etc.). Ginther et al. (40) reported that future dominant follicle showed a transient elevation of activin A and estradiol in follicular fluid. Given that betaglycan opposes activin action in the presence of inhibin A, we are inspired to speculate that estradiol is abundant in the dominant follicle, inducing betaglycan, and consequently antagonizing activin in the granulosa cells of the preovulatory follicle.
Here we have first demonstrated the regulatory mechanism of betaglycan mRNA in the granulosa cells. Although the physiological role of betaglycan in the ovary has not been cleared to date, it is likely that betaglycan plays a major role in ovarian functions. The autocrine/paracrine actions of activin inside ovary have been well accepted. Similarly, betaglycan could be an important factor in controlling the ovarian function to modulate the effects of activin. Further investigations are obviously necessary to address this issue.
Acknowledgments
We thank Dr. Yumiko Abe for her excellent technical assistance. We also thank the National Hormone and Pituitary Distribution Program (National Institute of Diabetes and Digestive and Kidney Disease) for supplying the rat FSH assay kit.
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Address all correspondence and requests for reprints to: Kazuto Nakamura, Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Gunma 371-8511, Japan. E-mail: nkazuto@med.gunma-u.ac.jp.
Abstract
Betaglycan (TGF? type III receptor) was recently identified as a coreceptor to enhance the binding of inhibin A to activin type II receptor. This inhibin/betaglycan/activin type II receptor complex prevents activins from binding to their own receptors. The present study was undertaken to identify the expression and the regulation of the betaglycan gene in cultured rat granulosa cells. Northern blot analysis indicated betaglycan mRNA transcript of approximately 6.4 kbp. The treatment of the cells with FSH increased the betaglycan mRNA level, and a concurrent treatment with estradiol brought a significant increase in betaglycan mRNA. The protein kinase A activator, 8-bromoadenosine-cAMP, also increased the expression of its mRNA. Furthermore, betaglycan mRNA was induced additively by estradiol, which was blocked by estrogen receptor antagonists [ICI 182780, (R, R)-cis-diethyltetrahydro-2,8-chrysenediol]. In the luciferase assay, FSH altered the promoter activity of betaglycan. Moreover, when FSH plus estradiol was added to the granulosa cells, a significant increase in the half-life of betaglycan mRNA transcript was seen. In summary, FSH and estradiol increased betaglycan mRNA expression, most possibly through the protein kinase A pathway and the estrogen receptor-?. The increase of betaglycan mRNA was due to an increase in transcription and altered mRNA stability. In ovarian regulatory function, the expression of betaglycan may involve the functional antagonism of inhibin A in activin signal transduction.
Introduction
TGF? IS A MEMBER OF a superfamily of growth factors that elicit a variety of responses, including growth, differentiation, and morphogenesis (1). Several reports have shown that ovarian cells express both TGF? mRNA and proteins (2, 3), involved in ovarian functions (4). Like other growth factors, the signals of TGF? are mediated by the interaction with various cell surface receptors. So far, three distinct receptors, termed types I, II, and III, have been cloned. The types I and II receptors have intracellular kinase domains responsible for receptor activation and signal transduction (5, 6). In contrast, the type III receptor, also called betaglycan, lacks any clearly identifiable signaling motif. Although the type III receptor does not appear to be a signaling receptor, it has been shown to be important for high affinity binding of TGF?s to the type II receptor (7, 8, 9).
Activins isolated from rat follicular fluid (10, 11) also belong to the TGF? superfamily, modulate cell differentiation, cell proliferation, and specific functions (12, 13, 14). Activin receptor has been found in follicular granulosa cells (15) and is examined for its expression pattern in rat granulosa and thecal cells in developing follicles during all stages of the ovarian cycle (16). Previously we have shown activin up-regulates FSH and LH receptor expression in rat granulosa cell culture (17, 18). Recently it has been demonstrated that betaglycan can bind inhibin A with high affinity, interfering with activin A to bind to its type II receptor (2, 19). In the developing ovarian follicles, betaglycan can function in an environment in which the -subunit of inhibin is produced in a 10- to 20-fold excess to ?-subunit of inhibin, favoring the assembly of inhibin dimers over activin dimers (3). Betaglycan protein was also localized in rat thecal cells and granulosa cells and oocytes (20). Moreover, the expression of betaglycan, which was identified in granulosa and thecal cells of normal human ovaries with immunohistochemistry, was induced by FSH in cultured human granulosa-luteal cells (21). These accumulated data lead us to the idea that betaglycan plays an important role in regulating follicular growth.
In this study, we examined the regulation of betaglycan mRNA expression by FSH and estrogen in rat primary granulosa cell culture. We then performed Northern blot analysis and luciferase assay to show that estrogen increased the level of betaglycan mRNA in the presence of FSH. Through these experiments, we found that the induction of betaglycan mRNA transcripts and the prevention of betaglycan mRNA decay was the mechanism of increase in betaglycan mRNA by estrogen.
Materials and Methods
Hormones and reagents
Rat FSH (I-8) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Diethylstilbestrol (DES), ?-estradiol, gentamicin sulfate, and (R, R)-cis-diethyltetrahydro-2,8-chrysenediol (R, R-THC) were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182780 was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). DMEM, Ham’s F-12 medium, and Fungizone were purchased from Life Technologies (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Roche Molecular Biochemicals (Mannheim, Germany).
Cell culture
The granulosa cells were obtained from immature female Wister rats injected with 2 mg diethylstilbestrol in 0.1 ml sesame oil daily for 4 d. The ovaries were then excised, and the granulosa cells were released by puncturing follicles with 25-gauge needles. The animals were treated humanly as possible, following the National Institutes of Health guideline at all times. Granulosa cells were washed and collected by brief centrifugation, and the cell viability was determined by trypan blue exclusion. The granulosa cells were then cultured in Ham’s F-1/DMEM (1:1, vol/vol) medium supplemented with 20 mg/liter gentamicin sulfate, 500 μl /liter Fungizone, and 1 g/liter BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2-95% air at 37 C.
Preparation of cRNA probe
The rat betaglycan cDNA was subcloned into the EcoRI site of the pcDNA3.1 (+) vector and linearized with XbaI. Digoxigenin-labeled betaglycan cRNA probes corresponding to 335-2893 bases were produced by in vitro transcription with T7 RNA polymerase and the RNA labeling kit (Boehringer, Mannheim, Germany). A digoxin-labeled glycelaldehyde-3-phosphate dehydrogenase (GAPDH) cRNA probe was obtained by the same method.
RNA isolation and analysis
The granulosa cells were cultured in 60-mm dishes containing 5 x 106 viable cells in 5 ml of medium, and the reagents were added to the medium after 24 h of cell culture. The granulosa cells were further incubated, and the cultures were stopped at the selected times as indicated by using Isogen (Nippon Gene, Toyama, Japan). The final RNA pellet was dissolved in diethylpyrocarbonate-treated H2O. Total RNA was quantified by measuring the absorbance of samples at 260 nm. For Northern blot analysis, 10 μg RNA from each dish were separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove, NY). In accordance with the standard protocol for the nucleic acid detection kit (Roche Molecular Biochemicals), the membranes were then exposed on X-Omat film (Eastman Kodak Co., Rochester, NY). Luminescence detection was quantified using the LKB 2202 UnitroScan laser densitometer (LKB Produkter AB, Bromma, Sweden), normalized against the corresponding amount of GAPDH mRNA for each sample and expressed in the relative densitometric units.
Vector preparation and transfection
Plasmid pGL3-basic is a luciferase vector lacking the eukaryotic promoter and the enhancer sequences (Promega Corp., Madison, WI). The PGL3-control contains a simian virus 40 promoter and a simian virus 40 enhancer inserted into the structure of pGL3-Basic (Promega). The fragment of the 5'-flanking region of –2013 to –253 bp relative to the translational initiation site was generated from the genomic DNA via a PCR using primers specific to the rat betaglycan sequences. The cDNA was amplified 30 cycles by PCR containing Taq DNA polymerase, and 1 μM each of the two primers at 94 C for 15 sec for betaglycan were 5'-CAGGACTCCAACGAGAAACACACC and 3'-ATCGCGCAGCGAAAGTGGCCTGGA. The isolated PCR-synthesized cDNA fragments were subcloned into pT7 blue-2 vector (Novagen, San Diego, CA) and characterized by nucleotide sequencing analysis. The sequence of the cDNA fragment was identical with the published sequence of rat betaglycan promoter cDNA (22). To evaluate promoter activity, these fragments were ligated to a luciferase reporter vector (pGL3-basic) and named BG-Luc. Using FuGENE (Roch Molecular Biochemicals, Indianapolis, IN), a total of 1 μg of plasmid DNA was transfected into the primary granulosa cell culture plates (2.5 x 105 cells per 0.5 ml of medium in a 20-mm dish). To assay the regulatory elements, the granulosa cells were cultured for the indicated time as described in figure legend in a hormone-free condition medium before transfection. Thirty hours after transfection, the cells were treated with hormones for 6 h. The cells were then harvested, and the luciferase activity was measured. The luciferase assay was performed using the dual-luciferase reporter system (Promega), in which the transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of renilla luciferase.
Transcription stability analysis
Cells were preincubated with FSH alone, estradiol alone, FSH and estradiol, or FSH, estradiol, and R, R-THC for 12 h before 5 μM actinomycin D addition to arrest a new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after the addition of the inhibitor for RNA extraction and Northern blot analysis.
Data analysis
The data represent the mean ± SE from at least three independent experiments. Comparisons between groups were performed by one-way ANOVA. The significance of the differences between the mean values of the control group and each treated group was determined by Duncan’s multiple-comparison test. A value of P < 0.05 was considered significant.
Results
The effect of FSH and estradiol on betaglycan mRNA transcription over time
To investigate the effect of FSH and estradiol on betaglycan mRNA in relation to time, we analyzed the betaglycan mRNA in the primary rat granulosa cell culture. Granulosa cells were cultured for 24 h after plating on a cell culture dish, and this point was considered time 0. Then the cells were cultured another 24–96 h in the presence of FSH (30 ng/ml) and with or without estradiol (5 μM). Figure 1, A and B, represents the autoradiograph of Northern blot, and Fig. 1C shows the relative amount of betaglycan mRNA to GAPDH mRNA. Northern blot analysis indicated betaglycan mRNA transcription of approximately 6.4 kb. In the control cells (with no treatment), the betaglycan mRNA levels were highest at time 0 and decreased gradually over time. On the other hand, after the treatment with FSH, the decline of betaglycan mRNA levels was suppressed. Concurrent treatment with FSH and estradiol increased betaglycan mRNA, approximately twice, from 24 to 96 h. The level remained the same (as the control) with only estradiol.
FIG. 1. Time course of FSH and estradiol (E2) effect on betaglycan mRNA. A and B, Granulosa cells from DES-primed immature rats were cultured alone for 24 h. These cells were further incubated without FSH and estradiol (no treatment), with FSH (30 ng/ml) (A), with estradiol (5 μM), or with a combination of FSH (30 ng/ml) plus estradiol (5 μM) (B). After various incubation times, total RNA was extracted and betaglycan mRNA levels were measured using Northern blot analysis as described in Materials and Methods. The difference between A and B is due to the exposure time. C, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA in time 0, with no treatment, was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to time 0. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
We then investigated the dose-dependent effect of FSH and estradiol at 72 h. An increase in FSH concentration brought about a dose-dependent increase in betaglycan mRNA as shown in Fig. 2. Granulosa cells were cultured in the absence or presence of FSH with or without increasing concentrations of estradiol (1–100 μM) as shown in Fig. 3. Betaglycan mRNA, on the other hand, increased minimally with estradiol alone, and a marked increase of betaglycan mRNA was observed with the concurrent treatment with both FSH and estradiol. Betaglycan mRNA levels were approximately 12 times the control with FSH (30 ng/ml) and estradiol (100 μM).
FIG. 2. Dose-related effect of FSH on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with increasing concentrations of FSH for 72 h. Levels of betaglycan mRNA were measured using Northern blot analysis as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of FSH dose 0 at P < 0.01.
FIG. 3. Dose-related effect of estradiol on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and were then cultured with increasing concentration of estradiol (E2) for 72 h with or without FSH (30 ng/ml). Levels of betaglycan mRNA were measured using Northern blot analysis, as described in Materials and Methods. The Northern blot analysis is representative of three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of FSH (–) at each time at P < 0.01.
Regulation of betaglycan expression by protein kinase A modulator
The protein kinase A pathway is the main signal transduction system of the FSH receptor. We investigated whether the effect of FSH on betaglycan mRNA accumulation involves protein kinase A activation. As shown in Fig. 4, an increase in 8-bromoadenosine-cAMP (8-Br-cAMP) concentration brought a dose-dependent increase of betaglycan mRNA production, suggesting that this pathway is mediated by protein kinase A.
FIG. 4. Effect of 8-Br-cAMP on betaglycan mRNA expression. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and were then cultured with FSH (30 ng/ml) or increasing concentration of 8-Br-cAMP for 48 h. Northern blot analysis was performed as described in Materials and Methods. The Northern blot analysis is representative of three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured without treatment was taken as ratio 1. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
The effect of estrogen receptor (ER) antagonist on betaglycan mRNA
The granulosa cells were cultured in the presence of ICI 182780 or R, R-THC with FSH (30 ng/ml) and estradiol (10 μM). ICI 182780 is a high-affinity ER antagonist and R, R-THC is a pure antagonist through ER? and a partial agonist through ER (23). As shown in Fig. 5, A and B, betaglycan mRNA was decreased by ICI. Furthermore, as shown in Fig. 5, C and D, estradiol and FSH-induced betaglycan mRNA and was blocked by the addition of R, R-THC. This suggests that betaglycan mRNA was stimulated by estradiol through estrogen receptor, especially ER?.
FIG. 5. Dose-related effect of ER antagonists on betaglycan mRNA. A, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with FSH, estradiol (E2), and increasing concentrations of ICI 182780 (ICI) for 72 h. Levels of betaglycan mRNA were measured using Northern blot analysis as described in Materials and Methods. The Northern blot analysis is representative of the three experiments. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured with estradiol and FSH treatment was taken as100%. Data were normalized for GAPDH mRNA levels in each samples and expressed as a value relative to the control (FSH 30 ng/ml plus E2 10 μM). The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and represents of the mean ± SEM of three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01. C, Granulosa cells from DES-primed immature rats were cultured alone for 24 h and then cultured with FSH, estradiol, and increasing concentration of R, R-THC for 72 h. The Northern blot analysis is representative of the three experiments. D, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The amount of betaglycan mRNA cultured with estradiol and FSH treatment was taken as 100%. Data were normalized for GAPDH mRNA levels in each sample and expressed as a value relative to the control (FSH 30 ng/ml plus E2 10 μM). The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and are represented as mean ± SEM of the three independent experiments in the bar graph. *, Difference from the value of control at P < 0.01.
The effect of FSH and estradiol on betaglycan mRNA transcription
We then examined whether the regulation of betaglycan mRNA by FSH and estradiol was dependent on gene transcription and/or receptor mRNA stability. To investigate the hormonal regulation of the 5'-flanking region, we analyzed the 1760 bp of the betaglycan promoter in rat granulosa cells. We tested the basal promoter activity of betaglycan at different times of transfection because they were harvested from DES-treated rat ovaries, suggesting the effect of estrogen perhaps remaining in the cells just after the beginning of culture. As shown in Fig. 6A, the basal promoter activity was induced most in cells transfected 24 h after plating, compared with the cells transfected at 48 or 72 h. Therefore, we used the cells at 72 h for the luciferase assay. The result (Fig. 6B) suggested that the treatment with either FSH (30 ng/ml) alone, cAMP (0.5 mM and 1.0 mM) alone, or FSH plus estradiol amplified the activity of the 1760 bp of the betaglycan 5'-flanking region.
FIG. 6. Effect of FSH and estradiol on betaglycan-luciferase (BG-Luc) expression in rat granulosa cells A, Granulosa cells from DES-primed immature rats were cultured for 24–72 h in hormone-free conditions and then cotransfected with BG (1760 bp)-Luc and pRL-CMV-Rluc at the respective times. Thirty-six hours after transfection, the luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. The luciferase activity at 24 h was taken as 100%. Each bar represents the mean ± SEM of the three independent experiments. *, Difference from the value of control (24 h) at P < 0.01. B, Granulosa cells from DES-primed immature rats were cultured for 72 h in hormone-free conditions and then cotransfected with BG (1760 bp)-Luc and pRL. Thirty hours after transfection, cells were treated with FSH (3 ng/ml, 30 ng/ml), 8-Br cAMP (0.5 μM, 1.0 μM), estradiol (E2, 10 μM), or a combination of FSH (30 ng/ml) plus estradiol (10 μM) for 6 h and then processed. Luciferase activity was corrected for the amount of renilla luciferase activity detected in each lysate. Each bar represents the mean ± SEM of three independent experiments. *, Difference from the value of control at P < 0.01.
Transcription stability analysis
To asses the rates of degeneration of betaglycan mRNA transcripts, we preincubated the granulosa cells with FSH, or estradiol, FSH and estradiol, or estradiol, FSH, and R, R-THC treatment for 12 h. After this preincubation, 5 μM actinomycin-D was added to arrest a new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after the transcription inhibitor addition, and the betaglycan mRNA levels were quantitated by Northern blot analysis. The amount of betaglycan mRNA at time 0 (the time of addition of actinomycin-D) in each group was assigned a value of 100%, and other values in each group at different times were expressed as a percentage, respective to this value. As shown in Fig. 7, the decay curves for the 6.4-kb betaglycan mRNA transcript in the primary granulosa cells show the presence of FSH, and estradiol significantly increases the stability of betaglycan mRNA. A slight increase in mRNA stability was seen in FSH treatment, not just due to estradiol. In contrast, the effect of estradiol on betaglycan mRNA stability was completely abolished by R, R-THC, demonstrating that estradiol increased mRNA stability through the ER? receptor. There were no statistical differences among treatment with FSH and estradiol, FSH, estradiol, and R, R-THC.
FIG. 7. Effect of FSH and estradiol (E2) on betaglycan mRNA transcript. A, Granulosa cells were preincubated with FSH, estradiol, a combination of FSH and estradiol, or FSH, estradiol, and R. R-THC for 12 h. After this preincubation period, 5 μM actinomycin-D were added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, 9, and 12 h after addition of the transcription inhibitor, and betaglycan mRNA levels were quantitated by Northern blot analysis. B, Levels of betaglycan mRNA (6.4 kb) was quantified by densitometric scanning. The mRNA level at time 0 was assigned a relative value of 100%, and mRNA levels at all other times are expressed as a percentage to this value. The ordinate is plotted on a logarithmic scale. The absorbance values obtained from this experiment as well as two other experiments were standardized in relation to the control and are represented as mean ± SEM of the three independent experiments in the bar graph. Cont, Control. **, Difference from the value of E2 at P < 0.01. *, Difference from the value of control at P < 0.01.
Discussion
TGF? type III receptor, betaglycan, is distributed in many tissues, in which betaglycan attracts and enhances TGF? binding to TGF? type II receptor and form a stable complex between receptor and ligand (23). Findlay et al. (3) indicated that betaglycan might play an important role in the growth and differentiation of follicles, supported by the result that betaglycan mRNA was detected in the postnatal rat ovary. Moreover, recent reports also show that betaglycan, with the activin type II receptor, becomes an inhibin coreceptor and antagonize activin action (2). In this study, we investigated the regulation of betaglycan mRNA expression in the granulosa cell culture system. Our results show that estradiol, in the presence of FSH, induce both the promoter activity and the level of betaglycan mRNA through increasing mRNA stability. These accumulated data indicate a possibility that betaglycan regulates follicular development inside the ovary.
Previously we reported activin induces FSH as well as LH receptor expression in the primary granulosa cell culture from DES-treated rats (17, 18), consistent with other reports (24, 25). In the study, we showed that FSH receptor levels reached maximum at 24 h of culture. However, the effect of activin was terminated for a short time due to the reduction in activin level and the increase of follistatin, binding to activin and antagonizing its effect (26). As shown in Fig. 1, without estrogen, betaglycan mRNA peaked at time 0, followed by a decline as incubation continued. A similar result was seen with estradiol treatment alone, whereas a concomitant treatment with FSH and estradiol increased betaglycan mRNA more than double, compared with the control level. This result suggests FSH and estradiol synergistically up-regulate betaglycan mRNA level in rat granulosa cell culture. Additionally, FSH induced the secretion of both inhibin A and B, especially inhibin A, in immature granulosa cell culture (27). Collectively Lewis et al. (2) found betaglycan protein in follicular granulosa cells, thecal cells, and oocytes in rat ovary through immunohistochemistry. Thus, we speculate that betaglycan plays an important role for both follicular growth and oocyte maturation in the rat ovary.
Luciferase assay was performed to elucidate the mechanism of betaglycan mRNA regulation by FSH and estradiol. To investigate the hormonal regulation of the 5'-flanking region of betaglycan, we examined the clone –2013 to –253 bp of the 5'-flanking region. The sequencing of betaglycan promoter region showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding motif such as GC box, activator protein-2, and specificity protein 1 (Sp 1) existed in this region (22). The cAMP-responsive element (28) and activator protein-2 (29) are well known as most characterized cAMP-responsive elements. This is consistent with the data that the granulosa cells, transfected with a luciferase reporter construct containing this region of the 5'-flanking region of the betaglycan, were responsive to FSH and 8-Br-cAMP in Fig. 6B. In a few cases, estrogen can bind to the half-site of estrogen-responsive elements (EREs) (ggtca) when assisted by the binding of Sp 1 to nearby GC-rich motifs (30). In addition, within the 5'-flanking region of betaglycan, there is no ERE, but there are two half-sites of ERE, which is considered to function when located near Sp1 and GC-rich motif. This indicates that estradiol may not affect the betaglycan promoter.
The basal promoter activity was induced most in cells transfected 24 h after plating, followed by a gradual decline toward 72 h, as shown in Fig. 6A. Combined with the data in Fig. 1, the effect of DES treatment for rats remains in the granulosa cells, especially at the beginning of the culture, supporting that estradiol is an important factor for betaglycan induction. Despite the persistent effect by DES treatment, the addition of estradiol into the culture medium did not induce an additive increase of the promoter activity by FSH. This raises the possibility that the ER pathway does not play a role in the transcription of betaglycan. However, because the promoter region we cloned might not include ERE, we cannot exclude the involvement of the ER pathway in the transcription of betaglycan. Further experiments are required to complete our understanding of the transcriptional mechanisms activating betaglycan in rat granulosa cells. It is well accepted that the signaling cascade of FSH receptor is mainly intermediated by cAMP after subsequent activation of adenylate cyclase. As shown from results in Fig. 4, the protein kinase A pathway consistently plays an important role in the induction of betaglycan mRNA. In the primary cultures of human granulosa-luteal cells from in vitro fertilization patients, Liu et al. (21) suggested that the protein kinase C (PKC) signal transduction pathway is involved in betaglycan induction because the PKC inhibitor, staurosporine, increased the betaglycan mRNA level. However, they failed to demonstrate that the PKC activator, tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate, increases betaglycan mRNA. The study using a stable cell line permanently overexpressing FSH receptor showed that FSH receptor coupled to the PKC pathway with a very high FSH dosage (100 ng/ml or more) (31). The capability of inducing inositol phosphate release remains unclear in both Sertoli and granulosa cells. Thus, we believe that there is no interaction between the PKC signaling pathway and the induction of betaglycan mRNA. Recently several emerging reports in the literature (32) have reported that cAMP intermediates the signaling pathway by FSH not only for PKA, but also for exchange protein activated by cAMP (Epac). In rat granulosa cell culture, Gonzalez-Robayna et al. (33) reported that FSH and 8-Br-cAMP stimulate phosphorylation of protein kinase B through Epacs. To date, there is no clear evidence of Epacs intermediating the regulation of betaglycan expression, and we need to examine this further.
To ascertain the effect of estradiol on betaglycan mRNA through ER, we first used ICI, which can antagonize both isoforms of ER (ER and ER?). In Fig. 5, A and B, ICI 182780 reduced the mRNA level of betaglycan in a dose-dependent manner. ER is known to be expressed in universal tissues (i.e. ovary, uterus, lung, neural tissue) (34). Inside the ovary, ER is expressed in thecal cells and interstitial cells, whereas ER? is thought to be mainly located in granulosa cells (35). Thus, R. R-THC, a weak agonist for ER and an antagonist for ER?, consistently diminished the mRNA level of betaglycan, as in the case of ICI. This suggests that ER? mainly intermediates estradiol signal transduction to increase betaglycan mRNA in the granulosa cells.
The data observed in Fig. 7 suggest FSH and estrogen, when administered together, alters betaglycan mRNA stability in rat granulosa cells. According to the promoter assay, the concomitant treatment with both FSH and estradiol just increased the promoter activity by 1.5-fold. This indicates that the mRNA stability dominantly controls the level of betaglycan mRNA rather than its transcription. The results above provide us one of the mechanisms underlying the regulation of betaglycan expression. However, questions concerning the limitations of this method, the measurement betaglycan mRNA decay, still remain. This assay may not truly reflect the physiological transcript stability, and important elements, modulating the function of gonadotropin receptors, ER, and signal transduction, which can be blocked by the actinomycin-D treatment because there are still limitations to this method.
Previously we have shown that activin, IGF-I, and TGF? increased the level of LH receptor mRNA by changing its stability (18, 36, 37). In the LH receptor, an mRNA binding protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH receptor mRNA stability in the rat ovary (38, 39). To date, there is no evidence supporting the same mechanism, as in the case of LH receptor mRNA. However, this might be one of pivotal roles in controlling mRNA expression of betaglycan.
In this report, we demonstrated that betaglycan is markedly induced by estradiol, assisting FSH synergistically in rat granulosa cell culture although the possibility of other hormones and growth factors is not eliminated (i.e. activin, IGF-I, etc.). Ginther et al. (40) reported that future dominant follicle showed a transient elevation of activin A and estradiol in follicular fluid. Given that betaglycan opposes activin action in the presence of inhibin A, we are inspired to speculate that estradiol is abundant in the dominant follicle, inducing betaglycan, and consequently antagonizing activin in the granulosa cells of the preovulatory follicle.
Here we have first demonstrated the regulatory mechanism of betaglycan mRNA in the granulosa cells. Although the physiological role of betaglycan in the ovary has not been cleared to date, it is likely that betaglycan plays a major role in ovarian functions. The autocrine/paracrine actions of activin inside ovary have been well accepted. Similarly, betaglycan could be an important factor in controlling the ovarian function to modulate the effects of activin. Further investigations are obviously necessary to address this issue.
Acknowledgments
We thank Dr. Yumiko Abe for her excellent technical assistance. We also thank the National Hormone and Pituitary Distribution Program (National Institute of Diabetes and Digestive and Kidney Disease) for supplying the rat FSH assay kit.
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