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Endocrinology Vol. 143, No. 5 1651-1659
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Transcriptional Activation of the Ovine Follicle-Stimulating Hormone-ß Gene by Gonadotropin- Releasing Hormone Involves Multiple Signal Transduction Pathways

Vyacheslav V. Vasilyev, Flavia Pernasetti, Suzanne B. Rosenberg, Mark J. Barsoum, Darrell A. Austin, Nicholas J. G. Webster and Pamela L. Mellon

Departments of Reproductive Medicine (F.P., V.V.V., S.B.R., M.J.B., P.L.M.), Neuroscience (V.V.V., P.L.M.), and Medicine (N.J.G.W.), University of California-San Diego, La Jolla, California 92093-0674; and Medical Research Service (N.J.G.W., D.A.A.), San Diego Veterans Affairs Healthcare System, San Diego, California 92161

Address all correspondence and requests for reprints to: Pamela L. Mellon, Ph.D., Department of Reproductive Medicine 0674, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674. E-mail: . pmellon{at}ucsd.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHß, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LßT2 cell line expresses FSHß mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHß promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHß promoter involves at least two elements, present between -4152/-2878 and -2550/-1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHß response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that calcium influx itself is not sufficient to confer the response, but it is necessary for both 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and GnRH induction of the FSHß gene. Moreover, we show that GnRH regulation of FSHß gene expression is mediated by PKC and establish the presence of multiple PKC isozymes in LßT2 cells. Interestingly, GnRH and TPA induce activity of the FSHß promoter through different, although possibly overlapping, pools of PKC isoforms. This is further supported by the use of a MAPK inhibitor, which abolishes the induction of FSHß by GnRH, but not by TPA. In conclusion, we have demonstrated that calcium, PKC, and MAPK signaling systems are all involved in the induction of FSHß gene expression by GnRH in the LßT2 mouse gonadotrope cell model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE REGULATION of mammalian sexual maturation and reproductive function requires the integration of hormonal regulation at the hypothalamus, pituitary, and gonadal levels. GnRH, a decapeptide secreted into the hypothalamic portal vessels, stimulates gonadotrope cells in the anterior pituitary to synthesize and secrete both gonadotropin hormones, LH and FSH. The gonadotropins, in turn, act on the ovaries and testes to stimulate steroidogenesis and gametogenesis. LH and FSH are members of the glycoprotein hormone family that also includes TSH and CG. These hormones are heterodimeric in structure, composed of a common {alpha}-subunit noncovalently linked to a hormone-specific ß-subunit, which confers physiological specificity (1).

Not only is GnRH critical for the synthesis and secretion of gonadotropins, the pattern in which it is secreted has a profound impact on gonadotrope function. GnRH is released in a pulsatile fashion that varies in frequency and amplitude as a function of hormonal status and the reproductive cycle (2). The intermittent pattern of release is critical for normal sexual development and gametogenesis, as interruption of GnRH pulses or administration of long-acting GnRH analogs and antagonists produces suppression of both gonadotropin and sex steroid production, resulting in infertility (3). Moreover, it has been shown that faster GnRH pulses selectively induce LHß expression, whereas slower GnRH pulses are more effective in the induction of FSHß expression (4). This independent regulation of FSH vs. LH synthesis by GnRH is crucial to the estrous cycle. Such differential regulation of LHß and FSHß suggests that distinct mechanisms are involved in GnRH regulation of these genes. These mechanisms may involve activation of different transcription factors and/or preferential sensitivity to distinct second messenger pathways.

GnRH acts on gonadotropin gene expression through the GnRH receptor (GnRH-R), a G protein-coupled, seven-transmembrane receptor that activates several signal transduction pathways. This receptor activates L-type calcium channels, allowing extracellular calcium into the cell (5). PLC is also activated upon GnRH binding to its receptor, leading to cleavage of phosphatidylinositol-diphosphate, located in the cell membrane, into IP3, which mediates calcium release from intracellular stores, and produces diacylglycerol (DAG). Increased concentrations of intracellular calcium together with DAG production lead to activation of PKC, which, in turn, leads to activation of other protein kinases, such as MAPK. Such signaling cascades can then regulate transcription through phosphorylation of DNA-binding proteins.

Little is known about the mechanisms involved in transcriptional regulation of the FSHß gene by GnRH due to the lack of a gonadotrope cell model in which to perform these studies. Recently, it has been shown that the LßT2 gonadotrope cell line, established by targeted tumorigenesis in transgenic mice (6), expresses endogenous FSHß mRNA (7, 8). Furthermore, we have demonstrated that the ovine FSHß (oFSHß) gene responds to GnRH in these cells, and that this response is both promoter and cell specific (7). Here, we employed this novel FSHß-expressing cell model to study the mechanisms involved in GnRH transcriptional regulation of the oFSHß gene. We show that the activating protein-1 (AP-1) sites located in the proximal promoter are not involved in GnRH responsiveness in this gonadotrope cell model. The FSHß GnRH response is ultimately mediated by at least two elements present between -4152/-2878 and -2550/-1089 bp in association with one or several elements within the proximal region of the promoter. Furthermore, we report that GnRH responsiveness of the FSHß gene is dependent on PKC activation of MAPK, and that calcium influx is necessary, but not sufficient, for GnRH induction. Finally, we found that GnRH and 12-O-tetradecanoyl-phorbol-13- acetate (TPA) employ distinct PKC isoforms to stimulate FSHß gene expression. These studies further our understanding of the actions of GnRH in regulation of the gonadotropin genes, a key issue in the control of reproductive function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
GnRH (des-Gly10,[D-Ala6]LH-releasing hormone ethylamide), ionomycin, TPA, and EGTA were obtained from Sigma (St. Louis, MO). Bisindolylmaleimide I hydrochloride (BMM I), Go 6976, Go 6983, Ro-31-8425, and U0126 were obtained from Calbiochem (La Jolla, CA).

Construction of plasmids for transfection analysis
A 5.5-kb region of the oFSHß gene encompassing 4741 bp of the promoter and 759 bp downstream from the +1 transcription start site, including the first intron of the gene (the oFSHß translation start site was inactivated to permit usage of the start site of the reporter gene sequence) (9) was subcloned into the KpnIB and XbaI restriction sites of the pGL3-Basic luciferase (Luc) reporter plasmid (Promega Corp., Madison, WI), and the resulting plasmid was named oFSHß-Luc.

The {Delta}AP1oFSHß-Luc plasmid contains two AP-like mutated sites located in the proximal region of the oFSHß promoter (-83 and -120 bp, respectively), constructed as described by Strahl et al. (10). This plasmid was provided by Dr. W. Miller.

The -2878 bp truncation was generated by digestion of the oFSHß-Luc with Asp718 and MscI. The Asp718 site was filled in using Klenow polymerase to generate a blunt end, and the plasmid was then religated to itself. The -1444 bp truncation was obtained by digestion of oFSHß-Luc with KpnI and BstxI. After digestion, the 7.2-kb fragment was religated to itself, including a linker containing the KpnI and BstxI complementary sequences: 5'-CCGGGATCCGCCAATGCC-3'.

The -1822 truncation of the oFSHß regulatory region was generated by digesting the oFSHß-Luc plasmid with PmlI and BglII, followed by isolation of the 2581-bp promoter fragment and cloning of the fragment into pGL3 digested with SmaI and BglII. The -984 truncation was obtained by digesting the oFSHß-Luc plasmid with PpuMI, followed by Klenow polymerase fill-in and BglII digestion. The resulting 1743-bp fragment was cloned into pGL3 basic vector digested with SmaI and BglII.

The -750FSH-Luc plasmid (provided by Dr. William Miller) (11) containing 751 bp of the oFSHß promoter and 759 bp downstream from the +1 transcription start site (inactivated to permit usage of the start site of the reporter gene sequence), including the first exon and the first intron, was digested with SalI and SacI to create the -751 truncation plasmid in pGL3 vector backbone. After the digestion, the 1510-bp fragment was cloned into the pGL3 basic vector digested with XhoI and SacI.

The -4152/-2878{Delta}oFSHß-Luc plasmid was obtained by digesting the oFSHß-Luc plasmid with MscI, which cuts at -4152 and -2878 bp. The 1274-bp MscI-MscI fragment was discarded, and the vector was religated to itself. The -2550/-1089{Delta}oFSHß-Luc plasmid, with deletion between -2550 and -1089 bp, was created by digesting the oFSHß-Luc vector with SpeI, followed by religation of the vector, as described above.

The thymidine kinase (TK)-Luc plasmid contains the -81 bp region of the herpes simplex I TK promoter cloned in pXP2 (provided by Dr. Sylvia Evans). This region of the TK promoter is missing both the CCAAT box and the distal GC box present in the 109-bp fragment of the promoter.

The plasmid containing the region from -4152 to -2878 bp of the FSHß regulatory region fused to TK-Luc was created by digesting FSHß-Luc plasmid with MscI and subcloning the 1274 bp MscI-MscI fragment into TK-Luc plasmid digested with Asp718 and blunt-ended with Klenow. The plasmid containing the region from -2550 to -1089 bp of the FSHß regulatory region fused to TK-Luc was created by digesting the oFSHß-Luc plasmid with SpeI and subcloning the 1461-bp SpeI-SpeI fragment into TK-Luc plasmid digested with XhoI and blunt-ended with Klenow. The plasmid containing both -4152 to -2878 bp and -2550 to -1089 bp of the FSHß regulatory region fused to TK-Luc was created by digesting the oFSHß-Luc plasmid with SpeI and subcloning the blunt-ended 1.4-kb SpeI-SpeI fragment into the XhoI blunted site of the plasmid containing the fragment -4152 to -2878 bp fused to the minimal TK promoter as described above.

Plasmid DNA was prepared from overnight bacterial cultures using DNA plasmid columns according to the supplier’s protocol (QIAGEN, Chatsworth, CA) or a cesium chloride protocol adapted from Sambrook et al. (12).

Cell culture and transient transfections
Cells were grown in 60-mm diameter dishes to 60–70% confluence in DMEM (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with 10% FBS (Omega Scientific, Inc., Tarzana, CA) at 37 C with 5% CO2. Transient transfections were performed using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN), following the manufacturer’s protocol. Twenty-four hours after transfection, the medium was changed to 1% FBS/DMEM, and 24 h after the medium change, the treatments were performed for the amount of time indicated in each figure before harvest. The cells were then harvested, and Luc, chloramphenicol acetyltransferase (CAT), or ß-galactosidase (ßgal) assays were performed. All experiments were conducted using 0.44 pmol of the reporter plasmids unless noted otherwise. One-half microgram of cytomegalovirus (CMV)-ßgal, or 1 µg TK-CAT plasmids was used as the internal control. All transfection experiments were performed at least three times.

Luc, ßgal, and CAT assays
Cells were washed twice in 1x PBS, and then 1 ml harvesting buffer (0.15 M NaCl, 1 mM EDTA, and 40 mM Tris-HCl, pH 7.4) was added to each dish. Cells were scraped, transferred to microcentrifuge tubes, and collected by centrifugation at 14,000 rpm for 10 sec. The supernatant was discarded, and the cells were resuspended in 50–100 µl lysis solution (Galacto-light assay system, Tropix, Bedford, MA).

Luc activity was measured using an E.G.&G. Berthold Microplate Luminometer (Nashua, NH) by injecting 100 µl of a buffer containing 100 mM Tris-HCl (pH 7.8), 15 mM MgSO4, 10 mM ATP, and 65 µM luciferin/well. ßgal assays were performed using the Galacto-light assay system following the manufacturer’s protocol. Before each ßgal assay, cell extracts were heat-inactivated at 48 C for 50 min. CAT assays were performed following the protocol described by Seed et al. (13).

Immunoblotting for PKC isoforms
LßT2 cells were grown to confluence in 10-cm dishes, washed once with PBS, and incubated overnight in the presence or absence of 1 µM TPA. Thereafter, cells were washed with ice-cold PBS, lysed on ice in SDS sample buffer (50 mM Tris, 5% glycerol, 2% SDS, 0.005% bromophenol blue, and 84 mM dithiothreitol, pH 6.8), boiled for 5 min to denature proteins, and sonicated for 5 min to shear the chromosomal DNA. Equal volumes of lysates were separated by SDS-PAGE on 10% gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA). The membranes were blocked with 5% nonfat dried milk in TBS-Tween [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20]. Antibodies to individual PKC isoforms were obtained from BD Transduction Laboratories, Inc. (San Diego, CA). Blots were incubated with primary antibodies at a 1:1000 dilution in blocking buffer for 60 min at room temperature in a Hoefer Deca-Probe incubation manifold (Amersham Pharmacia Biotech, Piscataway, NJ), washed in Tween-TBS, and then incubated with horseradish peroxidase-linked secondary antibodies, followed by chemiluminescent detection. The polyvinylidene difluoride membranes were immediately stripped by placing the membrane in stripping buffer (0.5 M NaCl and 0.5 M acetic acid) for 10 min at room temperature. The membrane was then washed once for 10 min in TBS-Tween, reblocked, and blotted with antibodies to ERK1 and -2 to control for equal protein loading.

Statistical analysis
The results were analyzed using one-way ANOVA, with the exception of the data presented in Fig. 2Go, for which multiway ANOVA was performed. Fisher’s protected least significant difference test was used as the post hoc test, with P < 0.05 considered statistically different.



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  		Figure 2. The induction of oFSHß by GnRH requires multiple regions of the gene. LßT2 cells were transiently transfected with either 3 µg oFSHß-Luc or an equimolar amount of the wild-type or mutated oFSHß-Luc plasmids or with TK promoter-based plasmids, as indicated. The CMVßgal (0.5 µg) plasmid was employed as an internal control. Forty hours after transfection, cells were treated with GnRH (1 nM) for 6 h before harvest. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each plasmid in each experiment was set at 100 to allow direct comparison of the magnitude of the GnRH induction. Results are presented as the mean ± SEM of three to five independent experiments, each performed in duplicate. Values marked with asterisk are statistically different from the corresponding untreated value within the same plasmid transfected (P < 0.05).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The FSHß regulatory region responds to GnRH in LßT2 cells
LßT2 cells have previously been shown to synthesize GnRH-R (6) and to respond to GnRH (14). To characterize the responsiveness of the oFSHß 5'-flanking region to GnRH in these cells, we performed transient transfections with the oFSHß-Luc plasmid in LßT2 cells, testing for the optimal GnRH dose-response and time-course conditions. A TK-CAT plasmid was used as an internal control in these experiments. We tested 3-, 6-, 9-, and 24-h treatments, each with 1, 10, or 100 nM GnRH analog. We observed that the response of the promoter is maximal at 1 nM GnRH for 6 h, compared with other time points and hormone concentrations tested. This stimulatory effect increases reporter values to approximately 2-fold above the control level (Fig. 1Go). We then performed all subsequent experiments under these established optimal conditions (1 nM GnRH for 6 h).



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  		Figure 1. The GnRH response of the oFSHß-regulatory region is dose and time dependent. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 1.0 µg of a TK-CAT plasmid as an internal control. Forty hours after transfection, cells received GnRH treatment at 1, 10, or 100 nM (final concentration), each for 3, 6, 9, and 24 h before harvest. Luc and CAT assays were performed, and the Luc activity of each sample was normalized to the internal control CAT activity. The values of the untreated samples for each time point were set at 100 to allow direct comparison of the magnitude of the GnRH induction. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Values marked with an asterisk (1 and 10 nM GnRH, 6 h) are statistically different from the untreated value at the given time point (P < 0.05).

 
Proximal AP-1 binding sites are not necessary for induction of the FSHß regulatory region by GnRH
Recently, Strahl et al. (15) showed that two functional AP-1 binding sites in the proximal region (located at -120 and -83 bp) of the oFSHß regulatory region are involved in the GnRH response of this gene in HeLa cells cotransfected with the GnRH-R. As the pool of G proteins, kinases, and transcription factors in HeLa cells probably differs from that present in the LßT2 gonadotrope cells, we investigated the role of these AP-1 sites in GnRH regulation of oFSHß in the gonadotrope LßT2 cell. A plasmid containing the two AP-1 sites mutated by site-directed mutagenesis ({Delta}AP-1oFSHß-Luc) (15) was transfected into LßT2 cells and compared with the wild-type oFSHß-Luc plasmid, treated with 1 nM GnRH for 6 h (Fig. 2Go). We observed that both wild-type and mutated AP-1 regulatory regions are induced by GnRH, at 230 ± 24% and 170 ± 16% of the untreated values, respectively. Luc values for both GnRH-treated plasmid groups were statistically different from the values for the untreated groups (P < 0.01) and were not significantly different from each other. These data indicate that the proximal AP-1 sites are not involved in induction of the oFSHß gene by GnRH in the context of a gonadotrope-derived LßT2 cells under the 6-h, 1-mM GnRH conditions tested.

Interaction of multiple elements in the FSHß regulatory region is required for induction by GnRH
To determine which section(s) of the oFSHß regulatory region plays a role in the GnRH stimulatory response, we performed transient transfection experiments in LßT2 cells with a series of plasmids containing progressive 5'-deletions as well as internal deletions of this region (Fig. 2Go). As expected, the full-length oFSHß-Luc plasmid was induced 230 ± 24% by 1 nM GnRH during a 6-h long treatment compared with the untreated cells. The -2878 oFSHß 5'-deletion was only partially induced in the presence of GnRH (150 ± 18% compared with the untreated cells). Values between the two GnRH-treated groups were statistically different (P = 0.0001), indicating that at least one element involved in the GnRH response of oFSHß was lost with this -2878 deletion. Additional 5'-deletions of the oFSHß regulatory region containing -1822, -1444, -984, or -751 bp from the transcription start were not significantly induced by 1 nM GnRH treatment for 6 h, suggesting that the region downstream of -1822 was not capable of conferring a response to GnRH. The oFSHß regulatory region with an internal deletion between -4152 and -2878 bp was partially induced (160 ± 19%) by GnRH (P = 0.0001). Internal deletion between -2550 and -1089 bp exhibited a similar partial loss of the GnRH response, leading to a 170 ± 18% induction of the reporter gene (P = 0.002). These results suggest that each of these deleted regions might contribute to the GnRH response of the oFSHß promoter.

We then performed transient transfections in LßT2 cells using plasmids containing each of these upstream regions (-4152 to -2878 bp, -2550 to -1089 bp) or both of them combined, cloned upstream of a minimal (-81) TK promoter, to determine whether these elements are capable of conferring a GnRH response to a heterologous promoter. As shown in Fig. 2Go, neither of these oFSHß upstream regions was capable of conferring a GnRH response to the heterologous TK promoter either alone or in combination. This indicates that the response of the FSHß regulatory region to GnRH involves multiple interacting elements, and that there is apparently no single element through which activation occurs.

Activation of the PKC signaling system, but not calcium, induces the FSHß regulatory region in LßT2 cells
GnRH-R activation by ligand binding leads to an increase in the intracellular calcium concentration as well as activation of the PKC signaling system (16). To determine whether activation of calcium and/or PKC systems recapitulates GnRH stimulation of the FSHß regulatory region, we performed transient transfections in LßT2 cells, as shown in Fig. 3Go. Activation of the PKC system by TPA (100 nM) for 6 h resulted in a 2.3-fold induction of oFSHß-driven Luc reporter activity, comparable to the induction observed with 1 nM GnRH, whereas ionomycin, a calcium ionophore (0.5 µM, 6 h), did not cause any significant change in oFSHß reporter activity. These results indicate that the PKC system could be involved in GnRH stimulation of oFSHß and that calcium influx alone is not sufficient to induce FSHß transcription.



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  		Figure 3. Activation of PKC with TPA induces FSHß promoter activity. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 0.5 µg of the CMVßgal plasmid as an internal control. Forty hours after transfection, cells were treated with 1 nM GnRH, 500 nM ionomycin, or 100 nM TPA for 6 h before harvest. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each experiment was set at 100 to allow direct comparison of the result of the treatments. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Values marked with an asterisk (GnRH and TPA treatments) are statistically different from the value of untreated cells (P < 0.05).

 
TPA down-regulation of PKC prevents induction of the FSHß regulatory region by GnRH
As TPA treatment stimulated oFSHßLuc activity similarly to GnRH treatment, we hypothesized that TPA-activated PKC isoforms could be involved in the GnRH signaling pathway. To investigate this, we treated LßT2 cells with 1 µM TPA for 24 h to down-regulate the TPA-dependent PKC signaling system. Thus, we added a 10-fold higher dose of TPA than that used for the 6-h induction in the other figures, and we treated for 18 h before GnRH treatment and for the last 6 h of the 24 h; both TPA and GnRH treatment were concurrent (Fig. 4Go). After 24-h treatment with TPA alone, the activity of the oFSHß regulatory region returned to the equivalent of the untreated control value. As expected, 6 h of GnRH treatment alone induced Luc activity 2.3-fold compared with that in untreated cells. In contrast, 24-h TPA treatment completely abolished the capacity of a 6-h GnRH treatment to stimulate FSHß regulatory region activity, indicating that the TPA-down-regulated PKCs are necessary for induction of the oFSHß regulatory region by GnRH.



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  		Figure 4. TPA down-regulation of PKC prevents GnRH induction of oFSHß. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 0.5 µg of the CMVßgal plasmid as an internal control. Sixteen hours after transfection, cells were treated with 1 µM TPA (final concentration). Eighteen hours later, without changing the medium, GnRH (1 nM) was added to half of the plates treated with TPA and to half of plates in the untreated group. Cells were harvested 6 h after the addition of GnRH. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each experiment was set at 100 to allow direct comparison of the results of the treatments. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Value marked with an asterisk (group treated with GnRH and TPA) is statistically different from the GnRH alone group (P < 0.05).

 
PKC {alpha}, ß, {delta}, {epsilon}, and {theta} isozymes are present in LßT2 cells and are down-regulated by TPA pretreatment
To characterize the PKC isoforms present in the gonadotrope-derived LßT2 cells and to demonstrate that they are efficiently down-regulated by TPA treatment, we performed Western blots using antibodies specific for each PKC isoform (Fig. 5AGo). We observed that the DAG-dependent conventional PKC isoforms {alpha} and ß; the DAG-dependent novel isoforms {delta}, {epsilon}, and {theta} isoforms; and the DAG-independent atypical {lambda} isoform are present in LßT2 cells, whereas {eta} and {zeta} are absent. Treatment of cells for 16 h with 1 µM TPA causes a greater than 90% decrease in the levels of {alpha}, ß, {delta}, {epsilon}, and {theta} isoforms, whereas expression of the DAG-independent {lambda} isoform is unchanged (Fig. 5BGo). From these data we conclude that multiple PKC isoforms are present, and several may be responsible for induction of oFSHß-driven reporter gene expression by GnRH.



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  		Figure 5. Expression of PKC isoforms in LßT2 cells and down-regulation by chronic TPA treatment. LßT2 cell lysates were separated by SDS-PAGE, immunoblotted with antibodies to individual PKC isoforms, then detected by enhanced chemiluminescence. The conventional PKC isoforms {alpha} and ß; the novel isoforms {delta}, {epsilon}, and {theta}; and the atypical PKC isoform {lambda} were detected in the cell lysates. Membranes were stripped and reblotted for ERK1 and -2 to control for protein loading. A, Untreated cells; B, cells that had been treated with 1 µM TPA for 16 h.

 
Calcium is necessary, but not sufficient, for induction of the FSHß regulatory region by GnRH
One of the main differences between different classes of PKC is their ability to respond to calcium. The PKC isoforms {alpha}, ß, and {gamma} contain the Ca2+-binding domain (denominated C2) and are regulated by calcium (17). To investigate the role of the calcium signaling system and further delineate which PKC classes are involved in the oFSHß response to GnRH, we performed transient transfections with the oFSHß-Luc plasmid in LßT2 cells and treated with a calcium chelator, EGTA (2 mM) alone or in combination with GnRH (1 nM), ionomycin (0.5 µM), or TPA (100 nM), for 6 h (Fig. 6Go). We observed that EGTA alone does not have an effect on oFSHß activity, whereas GnRH alone induces reporter activity to 180 ± 19% of untreated cells, as expected. However, addition of EGTA results in a dramatic reduction of the GnRH stimulatory effect (P = 0.0093), reducing it to 110 ± 23% of control. TPA alone induces the activity of the oFSHß regulatory region to approximately 250 ± 27% of control, whereas the combination of EGTA with TPA results in partial reduction of the TPA stimulatory effect, to 160 ± 26% of the control value. These results indicate that although calcium influx is not sufficient to stimulate oFSHß transcription (Fig. 6Go), it is necessary for GnRH stimulation of this promoter and is partially involved in the TPA response. The complete abolishment of the GnRH response in the absence of calcium suggests that the classical PKC isoforms ({alpha}, ß, and {gamma}) are most likely involved in GnRH signaling. Additional PKC isoforms are implicated in the TPA response, as the absence of calcium does not completely block the stimulatory effect of TPA.



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  		Figure 6. The calcium signaling system is necessary, although not sufficient, for the induction of FSHß gene expression by GnRH. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 0.5 µg of a CMVßgal plasmid as an internal control. Forty hours after transfection, cells were treated with 2 mM EGTA (final concentration) with or without GnRH (1 nM), ionomycin (500 nM), or TPA (100 nM) for 6 h before harvest. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each experiment was set at 100 to allow direct comparison of the results of the treatments. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Values marked with an asterisk (groups treated with GnRH, TPA, and combination of EGTA and TPA) are statistically different from untreated cells. Values marked with # (within the groups treated with GnRH or TPA) are statistically different from the values of the cells treated with GnRH and EGTA (P = 0.009) or TPA and EGTA (P = 0.009), respectively.

 
GnRH and TPA activate the FSHß regulatory region through different PKC isoforms
BMM I has previously been shown to down-regulate FSHß induction by GnRH in HeLa cells (15). To investigate whether this is true in LßT2 cells, we performed transient transfection assays with the oFSHß-Luc plasmid and treated cells with specific inhibitors for different PKC isoforms, alone or in combination with GnRH (1 nM) or TPA (100 nM). As expected, GnRH alone induced the reporter activity by 200 ± 39% (Fig. 7AGo), and this induction was completely abolished by Go 9676, Go 6983, and Go 8425, PKC isoform-specific inhibitors. Interestingly, the combination of BMM I with GnRH resulted in only partial inhibition of the GnRH stimulatory effect. It did not reach statistical difference from the control or individual treatments with BMM I or GnRH. These data indicate that GnRH induces FSHß through multiple PKC isoforms, not all of which are sensitive to BMM I. When the PKC inhibitors were combined with TPA, Go 6986 (but not the other inhibitors tested) prevented induction of the FSHß regulatory region by TPA (Fig. 7BGo). The fact that Go 6976 and Ro-31-8425 completely inhibited GnRH, but did not affect TPA stimulation of the oFSHß regulatory region suggests that the GnRH and TPA signaling pathways are not the same, although they may overlap.



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  		Figure 7. The effects of inhibitors of different PKC isoforms on the induction of FSHß gene expression by GnRH and TPA. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 0.5 µg of a CMVßgal plasmid as an internal control. Forty hours after transfection, cells were treated with 1 nM GnRH (A) or 100 mM TPA (B), with or without PKC inhibitors BMM I (100 nM), Go 6983 (100 nM), Go 6976 (200 nM), or Ro-31-8425 (400 nM), for 6 h before harvest. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each experiment was set at 100 to allow direct comparison of the results of the treatments. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Values marked with an asterisk (GnRH) are statistically different from the value of the untreated cells (P < 0.05). Values marked with # (GnRH) are statistically different from the value of cells treated with Go 6983, Go 6976, or Ro-31-8425 with GnRH (P < 0.05). Values marked with an asterisk (groups treated with TPA and either BMM I or Go 6976 or Ro-31-8425) are statistically different from the value of cells treated with BMM I or Go 6976 or Ro-31-8425 without GnRH (P < 0.05).

 
Inhibition of the MAPK pathway abolishes the effect of GnRH, but not the effect of TPA
At least two possible pathways downstream of PKC have been identified as being involved in GnRH responsiveness. One pathway results in activation of AP-1, and the other results in activation of ERK (18, 19). As we have shown that the two AP-1-binding sites located in the oFSHß-proximal promoter are not important for the GnRH response, we examined a specific MAPK kinase (MEK) inhibitor, U0126, to determine the role of the MAPK pathway in GnRH induction of FSHß gene expression. We performed transient transfections in LßT2 cells treated with GnRH (1 nM), ionomycin (0.5 µM), or TPA (100 nM) in combination with or without U0126 (750 nM; Fig. 8Go). As shown previously (Figs. 3Go and 4Go), GnRH and TPA, but not ionomycin, induce reporter activity compared with untreated cells. The MEK inhibitor U0126 alone does not affect basal reporter activity. Combination of U0126 with GnRH results in a dramatic reduction in the GnRH response to 112 ± 16%, whereas combination of U0126 with TPA does not affect the TPA stimulatory effect. Thus, these results indicate that the MAPK pathway is involved in the induction of FSHß by GnRH, but not by TPA, demonstrating that GnRH and TPA act via distinct pathways to regulate the oFSHß gene in the LßT2 gonadotrope cell line.



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  		Figure 8. Inhibition of MEK prevents the activation of FSHß by GnRH, but not by TPA. LßT2 cells were transiently transfected with 3 µg of the oFSHß-Luc reporter plasmid and 0.5 µg of a CMVßgal plasmid as an internal control. Forty hours after transfection, cells were treated with 750 nM U0126 (final concentration) with or without GnRH (1 nM), ionomycin (500 nM), or TPA (100 nM) for 6 h before harvest. Luc and ßgal assays were performed, and the Luc activity of each sample was normalized to the internal control ßgal activity. The value of the untreated samples for each experiment was set at 100 to allow direct comparison of the results of the treatments. Results are the mean ± SEM of three independent experiments, each performed in duplicate. Values marked with an asterisk (groups treated with GnRH, TPA, or combination of U0126 and TPA) are statistically different from the value of untreated cells. Groups marked with # (GnRH) are statistically different from the value of cells treated with the combination of U0126 and GnRH (P = 0.029).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have demonstrated that multiple signaling systems are involved in the induction of FSHß gene expression by GnRH in the LßT2 mouse gonadotrope cell model. Our transient transfection experiments show that continuous GnRH treatment for 6 h induces oFSHß gene expression, with a maximal 2-fold increase in reporter activity at 1 nM GnRH. Previous studies of GnRH induction of the oFSHß gene using heterologous HeLa and COS-7 cells cotransfected with the GnRH-R, in which the 5.5-kb regulatory region of oFSHß is stimulated 3.1- and 2.7-fold, respectively, by treatment with 100 nM GnRH for 12 h (15), showed comparable induction levels. Furthermore, in primary rat pituitary cells, Haisenleder et al. (20) showed that even 24 h of GnRH pulses (60 min apart) produce only a 1.5-fold induction of FSHß mRNA.

We observed that the optimal GnRH concentration is 1 nM, whereas 100 nM GnRH produces less than half of this induction in gonadotrope-derived LßT2 cells. This difference in optimal GnRH concentration between cell types may be due to desensitization of GnRH receptors at high doses of hormone. McArdle et al. (21) have shown that treatment for 60 min with 100 nM GnRH reduces cell surface GnRH receptor number by 48% in the gonadotrope precursor cell line, {alpha}T3–1. The cotransfection experiments in HeLa and COS-7 cells were performed with high concentrations of hormone (100 nM) and for a relatively long incubation period (12 h). Moreover, as those cells were cotransfected with the GnRH-R, it is possible that the level of receptors expressed was higher than in the LßT2 gonadotrope cells and therefore required a higher concentration of hormone to attain desensitization.

The GnRH response was lost after a 9-h incubation in our experiments regardless of the GnRH concentration applied, with a slight recovery after 24-h treatment. A reciprocal relationship has been shown between FSHß and follistatin mRNAs, an FSHß inhibitory factor, in response to different patterns of GnRH treatment using perifused male rat pituitary cells (22). In those studies continuous incubation with 10 nM GnRH for 4 h stimulated follistatin and FSHß mRNA approximately 2-fold, whereas a 10-h incubation stimulated follistatin mRNA 4-fold, with no significant increase in FSHß mRNA. GnRH has also been shown to suppress activin ßB mRNA after a 6-h treatment in primary rat pituitary cells (23). As LßT2 cells are known to synthesize follistatin, activin, and activin receptors (7), this provides a possible mechanism for the lack of induction of oFSHß transcription by longer incubation with GnRH. In this model, longer-term exposures to GnRH may induce endogenous follistatin and/or decrease endogenous activin, leading to a decrease in FSHß transcription, overriding transcriptional stimulation of the oFSHß regulatory region by GnRH.

Interestingly, although GnRH induction of FSHß is observed in both LßT2 gonadotrope-derived and heterologous HeLa cell lines, our results indicate that different regulatory elements are involved. Two AP-1-like elements located in the proximal region of the oFSHß regulatory region have been previously shown to play a role in the GnRH response of this region in HeLa cells (15). However, here we show that mutation of these sites does not affect GnRH responsiveness of the oFSHß regulatory region in the gonadotrope-derived LßT2 cell line. This difference is most likely due to the fact that distinct sets of transcription factors, kinases, G proteins, receptors, and other classes of molecules are expressed in different cell types. Interestingly, Huang et al. (24) recently showed that the proximal AP-1 sites in the oFSHß promoter are important for the synergistic effect of activin and GnRH in transgenic mouse primary pituitary cultures while not affecting the response of the promoter to GnRH alone. The researchers also reported that mutation of these AP-1 sites had no effect on the expression and regulation of the transgene driven by oFSHß 5'-regulatory region in vivo with regard to basal activity, castration, down-regulation of GnRH action by Lupron, or GnRH immunoneutralization. Thus, the AP-1-like sites located in the proximal region of the oFSHß promoter are not involved in GnRH response in the gonadotrope cell context, emphasizing the idea that heterologous cells do not accurately reflect cell-specific responses.

Our search for DNA elements implicated in GnRH stimulation of the oFSHß regulatory region revealed that this response involves multiple regions of the promoter, and that these regions contribute to different degrees to the response. Progressive 5'-deletions of the oFSHß regulatory region revealed that deletion of the region between -4741 and -2878 bp leads to partial loss of the response, whereas deletion of the region between -4741 and -1822 bp leads to its complete loss. These data, taken together, indicate that there are at least two elements located between -4741 and -1822 bp involved in GnRH response of this promoter. Deletions of large regions of the promoter between coordinates -4152 and -2878 bp and between -2550 and -1089 bp both resulted in a partial decrease in the GnRH response, further narrowing the location of these two responsive elements to within each of the two deleted regions. Interestingly, although these regions play a role in the GnRH response in the context of the oFSHß proximal promoter, they are not capable of conferring a GnRH response to a heterologous TK promoter, suggesting that an element(s) present within the proximal promoter of oFSHß is also required for GnRH responsiveness of the gene. As the proximal region of oFSHß alone is not sufficient for the GnRH response, this region most likely acts as an anchoring element, important for the interaction(s) between proximal and distal regions of the promoter, thereby leading to GnRH stimulation.

Activation of the calcium system by ionomycin does not up-regulate FSHß gene expression, whereas activation of the PKC signaling system by TPA leads to FSHß induction, similar to GnRH. These data suggest that both the TPA and GnRH responses could involve PKC activation. This was further supported by our observation that GnRH induction of FSHß is completely abolished by depletion of PKC during long-term TPA pretreatment. We have shown that multiple PKC isoforms are present in LßT2 cells, and most of these are down-regulated by sustained TPA treatment. Our studies to determine which PKC classes are involved in the oFSHß gene response to GnRH revealed that although calcium influx induced by ionomycin is not sufficient to produce a stimulatory response, calcium chelation by EGTA affects GnRH and TPA responses differently. EGTA completely abolishes GnRH induction of the gene, whereas it only partially blocks the TPA effect, suggesting that calcium-dependent PKC isoforms are involved in the GnRH signal transduction pathway, but TPA most likely recruits additional PKC isoforms. This is further supported by our results with inhibitors of different PKC isoforms. Although the TPA response is only affected by one inhibitor, GnRH induction is inhibited by most inhibitors tested. Moreover, inhibition of MAPK activity by the MEK inhibitor, U0126, completely abrogates the GnRH stimulatory effect, whereas no effect is observed on TPA stimulation of oFSHß, suggesting that although PKC stimulation is involved in both TPA and GnRH responses, the intermediate messengers participating in these responses are distinct. GnRH stimulation of oFSHß through MAPK is in agreement with recent studies on regulation of MAPK family members by GnRH. Heisenleder et al. (20) showed that in primary pituitary cultures, inhibition of MAPK with PD98059 abolished the induction of FSHß mRNA by GnRH. Yokoi et al. (25) observed that GnRH-induced ERK activation is dependent on PKC or on extracellular and intracellular calcium and demonstrated that both GnRH and TPA induce ERK1 and ERK2 in LßT2 cells. Moreover, Saunders et al. (26) showed that inhibition of PKC by GF109203X abolishes GnRH induction of rat FSHß promoter activity, using the GGH3 somatotrope cell line stably transfected with GnRH-R.

In summary, over the past several years enormous progress has been made in our understanding the molecular mechanisms underlying GnRH regulation of {alpha}-subunit, LH ß-subunit, and GnRH-R. In contrast, GnRH regulation of the FSH ß-subunit gene has remained relatively unexplored due to the lack of a gonadotrope cell model in which to perform these studies. Results from the present study show that the AP-1 sites located in the proximal promoter of the oFSHß gene do not play a role in GnRH responsiveness in the gonadotrope cell model despite their involvement in the response of the oFSHß gene to GnRH in heterologous cells. GnRH regulation of oFSHß gene expression is mediated by PKC/MAPK activation, involving at least two areas of the regulatory region. In addition, calcium influx by itself is not sufficient to confer the response, but it is necessary for both TPA and GnRH induction of the FSHß gene. Furthermore, our experiments with PKC and MAPK inhibitors demonstrate that GnRH and TPA induce the activity of the oFSHß promoter through different, although possibly overlapping, pools of PKC isoforms. Elucidation of the specific signal transduction pathways used by GnRH to induce the gonadotropin genes is important to understanding the mechanisms by which GnRH, acting through a single receptor, can differentially regulate the LH vs. FSH ß-subunit genes during the estrous cycle.


   Acknowledgments
 
We thank William Miller and Huey-Jing Huang for kindly providing both the wild-type and the AP-1 mutant oFSHß-Luc plasmids. We thank Djurdjica Coss and Mark Lawson for helpful discussions and reading of the manuscript. We also thank Rachel White for assistance with the cell cultures.


   Footnotes
 
This work was supported by NICHHD/NIH through Cooperative Agreement U54-HD-12303 as part of the Specialized Cooperative Centers Program in Reproduction Research (to P.L.M. and N.J.G.W.), NIH Grant R37-HD-20377 (to P.L.M.), a V.A. Merit Review Grant (to N.J.G.W.), NIH Training Grant T32-DK-07541 (to V.V.V. and S.B.R.), and an American Heart Association Postdoctoral Fellowship and a Fellowship from the Lalor Foundation (to F.P.).

Abbreviations: AP-1, Activating protein-1; BMM I, bisindolylmaleimide I hydrochloride; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; DAG, diacylglycerol; ßgal, ß-galactosidase; GnRH-R, GnRH receptor; Luc, luciferase; MEK, MAPK kinase; oFSHß, ovine FSHß; TK, thymidine kinase; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.

Received October 10, 2001.

Accepted for publication January 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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