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Endocrinology Vol. 140, No. 1 358-364
Copyright © 1999 by The Endocrine Society

ARTICLES

Transcriptional Activation of Gonadotropin-Releasing Hormone (GnRH) Receptor Gene by GnRH: Involvement of Multiple Signal Transduction Pathways1

Xinwei Lin and P. Michael Conn

Oregon Regional Primate Research Center (X.L., P.M.C.), Beaverton, Oregon 97006; and the Department of Physiology and Pharmacology, Oregon Health Sciences University (P.M.C.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: connm{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH STIMULATES the synthesis and release of pituitary gonadotropins (LH and FSH), acting through the GnRH receptor (GnRHR) on the plasma membrane (1). The GnRHR is a member of the G protein-coupled receptor (GPCR) family (2). The GnRHR couples to multiple G proteins, including Gq/11, which activates phospholipase C, leading to production of diacylglycerol and activation of protein kinase C (PKC) (3, 4, 5, 6, 7). Activation of GnRHR also stimulates Ca2+ influx and an increase in the intracellular Ca2+ concentration (7) as well as an increase in cAMP levels (8, 9), suggesting that multiple signal transduction pathways may mediate GnRH action.

Rat pituitary GGH3 cells, a GH3-derived cell line (10) stably expressing rat GnRHR (11), is a useful model system for study of GnRH action (12). GGH3 cells transfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells (13). In GGH3 cells, the GnRHR is coupled to Gq/11 as well as to Gs, which activates adenylate cyclase, leading to production of cAMP and activation of protein kinase A (PKA) (12, 14, 15, 16). A recent study relying on palmitoylation of G proteins and overexpression of G protein {alpha}-subunit complementary DNAs (cDNAs) showed that the GnRHR was able to couple to Gq/11 as well as to Gs and Gi in pituitary gonadotropes and GGH3 cells (17), suggesting that similar signal transduction pathways are employed to mediate GnRH action in GGH3 cells and pituitary cells.

Mitogen-activated protein kinase (MAPK), also designated extracellular signal-regulated kinases (ERKs), comprise a family of serine/threonine kinases that are involved in the transduction of externally derived signals regulating cell growth, division, and differentiation (18). Upon activation, MAPKs translocate to the nucleus, where they phosphorylate and activate nuclear transcription factors involved in DNA synthesis and cell division (18, 19, 20). MAPK was identified in primary pituitary cells as well as in pituitary-derived cell lines, including GH3 and {alpha}T3–1 cells (21, 22, 23). GnRH was shown to activate MAPK activity, probably through the PKC pathway (22, 23, 24, 25, 26). GnRH also activates the MAPK cascade, which, in turn, contributes to stimulation of gonadotropin {alpha}-subunit gene promoter activity (25, 26, 27). Recent studies revealed that GnRH regulates transcriptional activities of LH and FSH subunits in part through differential use of PKC and Ca2+ pathways (28) or PKC/MAPK and Ca2+ signal transduction pathways (27).

Several studies showed that pituitary GnRHR number (7) and the levels of GnRHR messenger RNA (29, 30) change during the estrous cycle and are associated with changes in the sensitivity of gonadotropes to GnRH and levels of serum gonadotropins, suggesting that GnRHR is an important site for the regulation of gonadotropin release, and GnRH is involved in the regulation of its own receptor (7). In addition, our previous study using GGH3 cells showed that GnRH activates transcriptional activity of its own receptor gene in part through the cAMP signal transduction pathway (31). In the present study we explored the possibility of involvement of multiple signal transduction pathways in regulation of GnRHR gene transcription by GnRH in the GGH3 cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Natural sequence GnRH was provided by the National Pituitary Agency. A GnRH agonist (GnRH-A), buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH), was a gift from Hoechst-Roussel Pharmaceuticals (Somerville, NJ). (Bu)2cAMP (Sigma Chemical Co., St. Louis, MO), phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.), EGTA (Sigma Chemical Co.), PD 98059 (2'-amino-3'-methoxyflavone; Calbiochem, La Jolla, CA), GF 109203X (bisindolylmaleimide I; Calbiochem), and D600 (methoxyverapamil; Knoll, Whippany, NJ) were obtained from the sources indicated. DMEM, OPTI-MEM, and lipofectamine were purchased from Life Technologies (Grand Island, NY). Restriction enzymes, modified enzymes, and competent cells for subcloning were purchased from Promega Corp. (Madison, WI). Other reagents were of the highest degree of purity available from commercial sources.

Reporter plasmids and expression vectors
Luciferase reporter gene vector (GnRHR-pXP2) with a 1226-bp promoter fragment (-1164 to +62 relative to the major transcription start site) of the mouse GnRHR (mGnRHR) gene (32) was provided by Dr. W. W. Chin. Promoterless pXP2 vector was generated by digestion of the GnRHR-pXP2 construct with BamHI and BglII to delete the GnRHR gene fragment and religation of the vector. An expression vector (pCIS-lacZ) expressing ß-galactosidase driven by the cytomegalovirus promoter was provided by Dr. Tae H. Ji and used as an internal control (33). Expression vectors for kinase-deficient forms of human ERK1 (pCEP4L/Erk1 K71R, ERK1-Mut) and ERK2 (pCEP4L/Erk2 K52R, ERK2-Mut) were provided by Dr. Melanie Cobb (34, 35). An expression vector for wild-type human Raf-1 (pUSE Raf-1) (36) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY), and an empty expression vector, pcDNA3.1, was obtained from Invitrogen (Carlsbad, CA) and used as a control plasmid.

Transient transfection of GGH3 cells
GnRHR-pXP2 reporter gene vector or control vector pXP2 was transiently expressed in GGH3 cells (GGH3-1' line) (11). GGH3 cells were maintained in growth medium [DMEM containing 10% FCS (HyClone Laboratories, Inc., Logan, UT) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)] in a humidified atmosphere (37 C) containing 5% CO2. Cells (5 x 105/well) were seeded in six-well plates (Costar, Cambridge, MA). Twenty-four hours after plating, the cells were transfected with 1.5 µg GnRHR-pXP2 or promoterless pXP2 plus 0.5 µg pCIS-lacZ/well using 5 µl lipofectamine in 1 ml OPTI-MEM. Five hours later, 1 ml DMEM containing 20% FCS was added to each well. Twenty-four hours after the start of transfection, the medium was replaced with fresh growth medium, and the cells were allowed to grow for another 24 h before treatment and functional assays (luciferase assay and ß-galactosidase assay). For transfection of kinase-defective expression vectors, GGH3 cells were transfected with 1.5 µg GnRHR-pXP2 or promoterless pXP2 plus 1 µg ERK1-Mut, 1 µg ERK2-Mut, or 0.5 µg ERK1-Mut plus 0.5 µg ERK2-Mut/well. An equal amount (1 µg) of empty expression vector pcDNA3.1 was used in the control transfection. For transfection of Raf-1 expression vector, GGH3 cells were transfected with 1.5 µg GnRHR-pXP2 or promoterless pXP2 plus different amounts (0, 0.2, 1, or 5 µg) of Raf-1 vector per well. The total amount of expression vector was maintained at a constant value (6.5 µg) for each transfection by adding appropriate amounts of empty expression vector (pcDNA3.1). pCIS-lacZ (0.5 µg/well) was used as an internal control.

Luciferase and ß-galactosidase assays
After treatment of GGH3 cells with GnRH-A or other compounds for the indicated times, cells were washed twice with PBS and lysed in 150 µl reporter lysis buffer (Promega Corp.). Luciferase activity in 20 µl cell lysate was determined using the luciferase assay system (Promega Corp.) in a LuciCount microplate luminometer (Packard, Meriden, CT). ß-Galactosidase activity in 30 µl cell lysate was also measured using a ß-galactosidase enzyme assay system (Promega Corp.) in a SpectraCount photometric microplate counter (Packard) and was used as an internal control. The luciferase activity was normalized for the transfection efficiency of each well by dividing luciferase activity by ß-galactosidase activity.

Data analysis
Transfections were performed in triplicate for each experiment. The data shown are the means of triplicate wells for the same treatment in each experiment and are presented as the mean ± SEM of replicates (n = 3) in each experiment. The SEM was typically less than 10% of the mean. The data were analyzed by one-way ANOVA followed by Duncan’s multiple range test. P < 0.05 was considered significant. Each experiment was repeated at least three times with similar results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Involvement of the PKC pathway in GnRH activation of GnRHR gene transcriptional activity
The transcriptional activity of the GnRHR promoter in the transiently transfected GGH3 cells was assessed by expression of the GnRHR promoter-controlled reporter gene (luciferase gene). Transient transfection of GGH3 cells with GnRHR-pXP2, a luciferase reporter gene vector containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mGnRHR gene, resulted in a 10- to 25-fold increase in GnRHR-luciferase reporter gene (GnRHR-Luc) activity compared with that in the GGH3 cells transfected with the promoterless pXP2 vector.

To examine the contribution of the PKC pathway in GnRH regulation of GnRHR gene transcriptional activity, GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or pXP2 plus pCIS-lacZ were treated with medium alone, a metabolically stable GnRH agonist buserelin (GnRH-A; 10-7 M), PMA (an activator of PKC; 162 nM or 100 ng/ml), or PMA (162 nM) plus GnRH-A (10-7 M) for 3 h or 6 h before harvesting. The cells were harvested, and luciferase and ß-galactosidase were measured. GnRH-A significantly stimulated GnRHR-Luc activity at both 3 and 6 h, with higher stimulation by 6 h (Fig. 1Go). PMA also stimulated GnRHR-Luc activity in a time-dependent manner, with a significant stimulation of GnRHR-Luc by 6 h (Fig. 1Go); however, the responses to PMA treatment were much lower than the stimulation in response to GnRH-A. Treatment with GnRH-A and PMA together did not cause additive stimulation of GnRHR-Luc activity. Instead, GnRHR-Luc activity in response to treatment with PMA and GnRH-A was lower than that in response to treatment with GnRH-A alone (Fig. 1Go). These data indicate that activation of the PKC pathway stimulated GnRHR promoter activity, but did not potentiate stimulation of GnRHR promoter activity by GnRH-A.



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  		Figure 1. Effect of a PKC activator (PMA) on GnRH stimulation of GnRHR transcriptional activity. Forty-eight hours after transfection of GGH3 cells with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ, the cells were incubated with medium alone, a GnRH agonist buserelin (GnRH-A; 10-7 M), PMA (162 nM or 100 ng/ml), or PMA (162 nM) plus GnRH-A (10-7 M) for 3 or 6 h before harvesting. The cells were then washed twice with PBS and lysed in reporter lysis buffer. Luciferase activity in 20 µl cell lysate and ß-galactosidase activity in 30 µl cell lysate were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity assayed from each well. The luciferase activity was then normalized as the fold induction of luciferase activity from GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ divided by that from GGH3 cells transfected with promoterless pXP2 plus pCIS-lacZ. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
GF 109203X is a highly selective inhibitor of PKC{alpha}, -ßI, -ßII, -{gamma}, -{delta}, and -{epsilon} isozymes, with an inhibitory potency that is approximately 200 times greater for PKC than for PKA. GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ were treated with medium alone, GnRH-A (10-7 M), PMA (162 nM), or (Bu)2cAMP (5 mM) in presence or absence of GF 109203X (5 µM) for 6 h before harvesting. GF 109203X caused a significant decrease in basal GnRHR-Luc activity (Fig. 2Go). In addition, GF 109203X abolished GnRH-A- or PMA-stimulated GnRHR-Luc activity (Fig. 2Go). The nearly complete blockade of PMA- or GnRH-A-stimulated GnRHR-Luc activity by the PKC inhibitor indicated that the PKC-dependent pathway participated in the GnRH regulation of GnRHR promoter activity. GF 109203X did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity (Fig. 2Go), thereby demonstrating the specificity of GF 109203X in inhibiting the PKC-dependent pathway.



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  		Figure 2. Effect of a PKC inhibitor (GF 109203X) on GnRH stimulation of GnRHR transcriptional activity. Forty-eight hours after transfection of GGH3 cells with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ, the cells were incubated with medium alone, GnRH-A (10-7 M), PMA (162 nM), or (Bu)2cAMP (5 mM) in the presence or absence of GF 109203X (5 µM) for 6 h before harvesting. The cells were lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as the fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
PMA is a pharmacological activator of most isoforms of PKC. Short term treatment of primary cultures of pituitary cells (or other types of cells) with PMA activates PKC activity, whereas longer term treatment of PMA results in a depletion of PMA-sensitive isoforms of PKC, indicated by a loss of responsiveness to exogenous PKC activators, a loss of extractable PKC activity, and a loss of phorbol ester-binding sites (37). To assess the effect of PKC depletion on GnRH regulation of GnRHR promoter activity, GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ were pretreated with medium or PMA (1 µM) for 24 h. The cells were then stimulated with medium alone, GnRH-A (10-7 M), PMA (162 nM), or (Bu)2cAMP (5 mM) for 6 h before harvesting. Pretreatment with PMA resulted in a modest, but insignificant, increase in basal GnRHR-Luc activity (Fig. 3Go). Pretreatment with PMA blunted the stimulation of GnRHR-Luc activity by GnRH-A or PMA and did not affect the stimulation of GnRHR-Luc activity by (Bu)2cAMP (Fig. 3Go). These results further support the view that the PKC-dependent pathway is involved in the mechanism of GnRH activation of GnRHR transcriptional activity.



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  		Figure 3. Effect of PKC depletion on activation of GnRHR transcriptional activity by GnRH, PMA, and cAMP. Forty-eight hours after transfection of GGH3 cells with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ, the cells were pretreated with medium (dimethylsulfoxide) or PMA (1 µM) for 24 h. The cells were then stimulated with medium alone, GnRH-A (10-7 M), PMA (162 nM), or (Bu)2cAMP (5 mM) for 6 h before harvesting. The cells were lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
Involvement of Ca2+ pathway in GnRH activation of GnRHR gene transcriptional activity
The role of Ca2+ in the regulation of GnRHR promoter activity by the releasing hormone was assessed by preventing external Ca2+ influx with the external Ca2+ chelator EGTA and the Ca2+ ion channel antagonist, D600. GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ were treated with medium alone, GnRH-A (10-7 M), EGTA (2 mM), or EGTA (2 mM) plus GnRH-A (10-7 M) for 6 h before harvesting. EGTA did not affect basal GnRHR-Luc activity, but significantly attenuated GnRH-A-stimulated GnRHR-Luc activity (Fig. 4AGo). In the next experiment, GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or pXP2 plus pCIS-lacZ were treated with medium alone or GnRH-A (10-7 M) in the absence or presence of D600 (0.01 mM or 0.1 mM) for 6 h before harvesting. D600 was applied 30 min before GnRH-A. Treatment with D600 resulted in a decrease in basal GnRHR-Luc activity in a dose-dependent manner, with a significant reduction at 0.1 mM (Fig. 4BGo). D600 also blocked GnRH-A-stimulated GnRHR-Luc activity in a dose-dependent manner, with complete inhibition of GnRH-A-stimulated GnRHR-Luc activity at 0.1 mM (Fig. 4BGo). These data suggest that the Ca2+ signal transduction pathway is involved in the GnRH regulation of GnRHR promoter activity.



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  		Figure 4. Effects of Ca2+ ion chelator (A) and Ca2+ channel antagonist (B) on GnRH activation of GnRHR transcriptional activity. Forty-eight hours after transfection of GGH3 cells with GnRHR-pXP2 plus pCIS-lacZ or pXP2 plus pCIS-lacZ, the cells were incubated with medium alone, GnRH-A (10-7 M), EGTA (2 mM), or EGTA (2 mM) plus GnRH-A (10-7 M) for 6 h (A) or treated with medium alone or GnRH-A (10-7 M) in the absence or presence of D600 (0.01 and 0.1 mM) for 6 h before harvesting (B). The cells were lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as the fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
Involvement of the MAPK pathway in GnRH activation of GnRHR gene transcriptional activity
Raf-1 (MAPK kinase kinase or MAPKKK) is a proximal kinase in the mammalian ERK1/2 pathway that phosphorylates and activates MEK1 and MEK2 (MAPK kinase or MAPKK). MEKs, in turn, mediate the activation of ERK1 and ERK2 (18). The role of the MAPK cascade in the GnRH regulation of GnRHR promoter activity was assessed by overexpression of Raf-1. GGH3 cells were cotransfected with GnRHR-pXP2 or pXP2 and different amounts of human wild-type Raf-1 expression vector (pUSE Raf-1) for 48 h. The cells were harvested, and the luciferase and ß-galactosidase activities were measured. Overexpression of Raf-1 resulted in concentration-dependent inhibition of the GnRHR-Luc activity (Fig. 5AGo), with a significant decrease in GnRHR-Luc activity at 1 and 5 µg/well Raf-1 expression vector. In the next experiment, GGH3 cells cotransfected with GnRHR-pXP2 or pXP2 and 1 µg/well pUSE Raf-1 vector or control vector (pcDNA3.1) were treated with GnRH-A (10-7 M) or PKC inhibitor GF 109203X (5 µM) for 6 h before harvesting. Overexpression of Raf-1 partially blocked GnRH-A-stimulated GnRHR-Luc activity. On the other hand, GF 109203X inhibited basal GnRHR-Luc activity and did not affect the inhibition of GnRHR-Luc activity by the overexpression of Raf-1 (Fig. 5BGo). These results suggested that the Raf-1-mediated MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR-Luc activity and that the PKC pathway is not involved in the regulation of GnRHR-Luc activity by Raf-1.



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  		Figure 5. Effect of overexpression of Raf-1 on basal and GnRH-A-stimulated GnRHR transcriptional activity. A, GGH3 cells were cotransfected with GnRHR-pXP2 or pXP2 and different amounts (0, 0.2, 1, or 5 µg) of human wild-type Raf-1 expression vector (pUSE Raf-1) for 48 h. B, GGH3 cells cotransfected with GnRHR-pXP2 and 1 µg/well pUSE Raf-1 vector or control vector (pcDNA3.1) were treated with GnRH-A (10-7 M) or PKC inhibitor GF 109203X (5 µM) for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
PD 98059 selectively blocks the activity of MEK by inhibiting the activation of MAPK and subsequent phosphorylation of MAPK substrates both in vitro and in intact cells (38, 39). GGH3 cells transfected with GnRHR-pXP2 plus pCIS-lacZ or with pXP2 plus pCIS-lacZ were treated with medium alone or GnRH-A (10-7 M) in the absence or presence of PD 98059 (10 or 50 µM) for 6 h before harvesting. PD 98059 was applied 30 min before GnRH-A. Treatment with 50 µM PD 98059 significantly stimulated basal GnRHR-Luc activity. PD 98059 also potentiated GnRH-A-stimulated GnRHR-Luc activity (Fig. 6Go).



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  		Figure 6. Effect of MAPK kinase inhibitor on activation of GnRHR transcriptional activity by GnRH. Forty-eight hours after transfection of GGH3 cells with GnRHR-pXP2 plus pCIS-lacZ or pXP2 plus pCIS-lacZ, the cells were treated with medium alone or GnRH-A (10-7 M) in the absence or presence of PD 98059 (10 or 50 µM) for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 
Kinase-deficient mutants of protein kinases, such as Raf-1, Ras, and ERKs, have been used for interference with endogenous protein kinases and inhibiting the activity of endogenous protein kinases (34, 40, 41). Two mutants of ERKs (K71R ERK-1 and K52R ERK-2) possess less than 1% and less than 5% wild-type kinase activity in vitro, respectively, and have been successfully used for inhibition of ERK activities (25, 26, 34, 35). GGH3 cells cotransfected with pXP2-GnRHR or pXP2 and ERK1-Mut expression vector (1 µg/well), ERK2-Mut expression vector (1 µg/well), or ERK1-Mut (0.5 µg/well) and ERK2-Mut vectors (0.5 µg/well) together were treated with medium alone or GnRH-A (10-7 M) for 6 h before harvesting. Overexpression of ERK1-Mut or ERK2-Mut alone did not significantly affect the basal GnRHR-Luc activity; the combination of ERK1-Mut and ERK2-Mut significantly stimulated GnRHR-Luc activity. On the other hand, either overexpression of ERK1-Mut or ERK2-Mut alone or overexpression of ERK1-Mut and ERK2-Mut together significantly increased GnRH-A-stimulated GnRHR-Luc activity. ERK1-Mut had higher potentiation of GnRH-A-stimulated GnRHR-Luc activity compared with ERK2-Mut, and ERK1-Mut and ERK2-Mut together showed an additive effect of ERK1-Mut and ERK2-Mut alone (Fig. 7Go). These results indicated that inhibition of the MAPK pathway with MEK inhibitors or expression vectors for MAPK inhibitor (kinase-deficient MAPKs) activated basal and GnRH-A-stimulated GnRHR-Luc activities. This supports the view that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity.



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  		Figure 7. Effect of overexpression of kinase-deficient ERK mutants on activation of GnRHR transcriptional activity by GnRH. GGH3 cells cotransfected with pXP2-GnRHR or pXP2 and ERK1-Mut expression vector (1 µg/well), ERK2-Mut expression vector (1 µg/well), or ERK1-Mut (0.5 µg/well) plus ERK2-Mut vectors (0.5 µg/well) were treated with medium alone or GnRH-A (10-7 M) for 6 h before harvesting. The cells were then lysed, and luciferase and ß-galactosidase activities were measured. Luciferase activity was calculated as luciferase activity/ß-galactosidase activity and then normalized as the fold induction divided by that of pXP2. The data shown are the means of triplicate determinations. Error bars show the SEM. Significant differences at P < 0.05 between groups are designated by different lowercase letters above the bars.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The GGH3 cell line has been demonstrated to be a useful model for study of the molecular and cellular mechanisms of GnRH action. Recent studies from our laboratory using G protein palmitoylation and overexpression of different G protein {alpha}-subunit cDNAs indicated that GnRHR couples to similar G proteins (Gq/11, Gs, and Gi) in GGH3 cells and in pituitary primary cells, suggesting that similar signal transduction pathways are employed to mediate GnRH action in both cell types (17). In addition, GGH3 cells have been used for the study of the regulation of the gonadotropin subunit genes by GnRH. Corroborative experiments have also been performed in these cell lines previously, which demonstrated similarities in GnRH-mediated responses, compared with that in primary pituitary cells, including the degree of stimulation of {alpha}, LHß, and FSHß gene promoter activities and differential regulation of the gonadotropin subunit gene promoter activities by GnRH (13, 42, 43). In this study, we examined the regulation of GnRHR gene transcription by GnRH using GGH3 transiently transfected with a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment of the mGnRHR gene (32).

GnRH has been shown to activate transcriptional activities of LH and FSH subunits in part through differential use of the PKC and Ca2+ pathways (28) or the PKC/MAPK and Ca2+ pathways (27). In addition, the frequency of Ca2+ pulses regulates GnRHR gene expression, suggesting that GnRH-stimulated intracellular Ca2+ signals may also be involved in mediation of the transcriptional regulation of the GnRHR gene by GnRH (44). In the present study, activation of PKC by PMA significantly stimulated GnRHR-Luc activity; inhibition of PKC by GF 109203X or by depletion of PKC blocked PMA- or GnRH-A-stimulated GnRHR-Luc activity. Furthermore, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with Ca2+ chelator EGTA or Ca2+ ion channel antagonist, D600. These results suggest that GnRH activates GnRHR transcriptional activity through multiple signal transduction pathways, including PKC- and Ca2+-dependent pathways as well as through the cAMP-PKA-dependent pathway as previously shown. The present results show that either GnRH-A or PMA alone significantly stimulated GnRHR-Luc activity, although stimulation by PMA was much lower than that by GnRH-A. However, no additional stimulation of GnRHR-Luc activity was observed with cotreatment by GnRH-A and PMA compared with the stimulation by GnRH-A alone. Similarly, a recent report showed that the {alpha}-subunit gene promoter activity in response to treatment with PMA and GnRH agonist together was significantly lower than that in response to the treatment with GnRH agonist alone (28). The mechanism for the reduction in stimulation of GnRHR-Luc by cotreatment with GnRH-A and PMA is unclear. A possible interpretation is that activation of PKC isoforms by PMA could compete or down-regulate activation of the PKC pathway by GnRH-A. Our previous study showed that GnRH activates GnRHR gene transcription in part through the cAMP signal transduction pathway. In the present study, use of a PKC inhibitor or depletion of PKC did not affect (Bu)2cAMP stimulation of GnRHR-Luc activity. However, the PKC inhibitor or depletion of PKC did abolish PMA- or GnRH-A-stimulated GnRHR-Luc activity. In addition, the PKC inhibitor also inhibited basal GnRHR-Luc activity. Reduction of the basal GnRHR-Luc activity by the PKC inhibitor may conceal the remaining GnRH-A-stimulated GnRHR-Luc activity in the presence of PKC inhibitor, which may be caused by another signal transduction pathway (e.g. cAMP). On the other hand, although our previous study showed that PKC activator did not stimulate an increase in cAMP (11), some studies showed that activation of PKC affects PKA subunit levels in the absence of cAMP elevation, suggesting cross-talk between PKA and PKC pathways (45).

Recent studies indicate that signaling pathways connecting GPCRs to nuclear events regulating gene expression occur through divergent MAPK cascades (19). MAPK was potently activated upon ligand addition by GPCRs. The Gq/11-coupled receptor activation of MAPK has been shown to be PKC dependent, fully PKC independent, or partially PKC dependent (18, 19). GnRH was shown to activate MAPK activity in {alpha}T3–1 cells and primary cultures of pituitary cells, which probably involved PKC and Ca2+ (22, 23, 24, 26). In addition, GnRH activates the MAPK cascade, which, in turn, contributes to stimulation of gonadotropin {alpha}-subunit gene promoter activity (25, 26, 27). Recent studies revealed that GnRH regulates the transcription of LH and FSH subunits in part through differential use of PKC/MAPK and Ca2+ signal transduction pathways (27). In the present study, activation of MAPK activity by overexpression of MAPK kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by the MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activities. These unexpected results suggest that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR promoter activity. One of the mechanisms by which the MAPK cascade mediates transduction of externally derived signals regulating cell growth, division, and differentiation is by phosphorylation and activation of nuclear transcription factors by MAPK (18, 20). Phosphorylation of transcriptional factors by MAPK usually results in increased gene expression (20). However, MAPK was shown to phosphorylate the transrepression domain of c-Fos and c-Myb, suggesting participation of MAPK in the repression of gene expression (46, 47). Indeed, a recent report showed that the phosphorylation of heat shock factor-1 by MAPK represses transcriptional activation of heat shock gene by heat shock factor-1 (48). The mechanism of negative regulation of GnRHR gene transcriptional activity by MAPK is unknown. It is possible that MAPKs activated by GnRH phosphorylate transcriptional factors, which, in turn, repress GnRHR promoter activity through their action on the 1226-bp promoter fragment of the mGnRHR gene. Although recent unpublished results in our laboratory showed that GnRH can activate MAPK activity in GGH3 cells comparable to that observed in other cell lines, the role of MAPK in regulation of GnRHR gene transcription by GnRH is apparently needed to be confirmed using pituitary cells or gonadotrope cell line. Studies in several other GPCR showed that GPCR-mediated activation of PKC directly or indirectly activates Raf kinase via a poorly understood mechanism (18, 19). In the present study, GF 109203X inhibited basal GnRHR-activity, but did not affect the inhibition of GnRHR-Luc activity by overexpression of Raf-1. Although these results did not rule out the potential involvement of PKC pathway in the activation of MAPK, they suggest that the PKC pathway is not involved in the regulation of GnRHR gene transcription by Raf-1.


   Acknowledgments
 
We are grateful to Drs. W. W. Chin, Tae H. Ji, and M. H. Cobb for providing mouse GnRHR reporter gene vector, pCIS-lacZ vector, and kinase-deficient ERKs expression vectors, respectively. We thank Jo Ann Janovick for her help.


   Footnotes
 
1 This study was supported by NIH Grants HD-19899, RR-00163 and HD-18185. Back

Received June 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Conn PM 1994 The molecular mechanism of gonadotropin-releasing hormone action in the pituitary. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1815–1832
  2. Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205[Abstract/Free Full Text]
  3. Hawes BE, Barnes S, Conn PM 1993 Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology 132:2124–2130[Abstract]
  4. Hsieh K-P, Martin TFJ 1992 Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11. Mol Endocrinol 6:1673–1681[Abstract]
  5. Shah BH, Milligan G 1994 The gonadotropin-releasing hormone receptor of {alpha}T3–1 pituitary cells regulates cellular levels of both of the phosphoinositidase C-linked G proteins, Gq{alpha} and G11{alpha}, equally. Mol Pharmacol 46:1–7[Abstract]
  6. Stanislaus D, Janovick JA, Brothers S, Conn PM 1997 Regulation of Gq/11{alpha} by the gonadotropin-releasing hormone receptor. Mol Endocrinol 11:738–746[Abstract/Free Full Text]
  7. Conn PM, Janovick JA, Stanislaus D, Kuphal D, Jennes L 1995 Molecular and cellular basis of gonadotropin-releasing hormone action in the pituitary and central nervous system. In: Litwack G (ed) Vitamins and Hormones. Academic Press, New York, vol 50:151–214
  8. Bourne GA 1988 Cyclic AMP indirectly mediates the extracellular Ca2+-independent release of LH. Mol Cell Endocrinol 58:155–160[CrossRef][Medline]
  9. Borgeat P, Chavancy G, Dupont A, Labrie F, Arimura A, Schally AV 1972 Stimualtion of adenosine 3':5'-cyclic monophosphate accumulation in anterior pituitary gland in vitro by synthetic luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 69:2677–2681[Abstract/Free Full Text]
  10. Tashjian AH JR, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352[Medline]
  11. Stanislaus D, Janovick JA, Jennes L, Kaiser UB, Chin WW, Conn PM 1994 Functional and morphological characterization of four cell lines derived from GH3 cells stably transfected with gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid. Endocrinology 135:2220–2227[Abstract]
  12. Kaiser UB, Conn PM, Chin WW 1997 Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev 18:46–70[Abstract/Free Full Text]
  13. Kaiser UB, Sabbagh E, Saunders BD, Chin WW 1998 Identification of cis-acting deoxyribonucleic acid elements that mediate gonadotropin-releasing hormone stimulation of the rat luteinizing hormone ß-subunit gene. Endocrinology 139:2443–2451[Abstract/Free Full Text]
  14. Janovick JA, Conn PM 1994 Gonadotropin-releasing hormone (GnRH)-receptor coupling to inositol phosphate and prolactin production in GH3 cells stably transfected with rat GnRH receptor complementary deoxyribonucleic acid. Endocrinology 135:2214–2219[Abstract]
  15. Kuphal D, Janovick JA, Kaiser UB, Chin WW, Conn PM 1994 Stable transfection of GH3 cells with rat gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid results in expression of a receptor coupled to cyclic adenosine 3',5'-monophosphate-dependent prolactin release via a G-protein. Endocrinology 135:315–320[Abstract]
  16. Lin X, Janovick JA, Brothers S, Blomenröhr M, Bogerd J, Conn PM 1998 Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellualr carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation. Mol Endocrinol 12:161–171[Abstract/Free Full Text]
  17. Stanislaus D, Ponder S, Ji TH, Conn PM 1998 GnRH receptor couples to multiple G-proteins in gonadotropes and in GGH3 cells: evidence from palmitoylation and overexpression of G-proteins. Biol Reprod 59:579–586[Abstract/Free Full Text]
  18. Van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ 1996 Mitogenic signaling via G protein-coupled receptors. Endocr Rev 17:698–714[CrossRef][Medline]
  19. Gutkind JS 1998 The pathways connecting G protein-coupled receptors to the nucleus through devergent mitogen-activated protein kinase cascades. J Biol Chem 273:1839–1842[Free Full Text]
  20. Campbell JS, Seger R, Graves JD, Graves LM, Jensen AM, Krebs EG 1995 The MAP kinase cascade. Recent Prog Horm Res 50:131–159
  21. Ohmichi M, Sawasa T, Kanda Y, Koike K, Hirota K, Miyake A, Saltiel AR 1994 Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. J Biol Chem 269:3783–3788[Abstract/Free Full Text]
  22. Mitchell R, Sim PJ, Leslie T, Johnson MS, Thomson FJ 1994 Activation of MAP kinase associated with the priming effect of LHRH. J Endocrinol 140:R15–R18
  23. Sim PJ, Wolbers WB, Mitchell R 1995 Activation of MAP kinase by the LHRH receptor through a dual mechanism involving protein kinase C and a pertussis toxin-sensitive G protein. Mol Cell Endocrinol 112:257–263[CrossRef][Medline]
  24. Reiss N, Llevi LN, Shacham S, Harris D, Seger R, Naor Z 1997 Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary {alpha}T3–1 cell line: differential roles of Ca2+ and protein kinase C. Endocrinology 138:1673–1682[Abstract/Free Full Text]
  25. Roberson MS, Misra-Press A, Laurance ME, Stork PJS, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone a-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
  26. Sundaresan S, Colin IM, Pestell RG, Jameson JL 1996 Stimualtion of mitogen-activated protein kinase by gonadotropin-releasing hormone: evidence for the involvement of protein kinase C. Endocrinology 137:304–311[Abstract]
  27. Weck J, Fallest PC, Pitt LK, Shupnik MA 1998 Differential gonadotropin-releasing hormone stimualtion of rat luteinizing hormone subunit gene transcription by Ca2+ influx and mitogen-activated protein kinase-signaling pathways. Mol Endocrinol 12:451–457[Abstract/Free Full Text]
  28. Saunders BD, Sabbagh E, Chin WW, Kaiser UB 1998 Differential use of signal transduction pathways in the gonadotropin-releasing hormone-mediated regulation of gonadotropin subunit gene expression. Endocrinology 139:1835–1843[Abstract/Free Full Text]
  29. Bauer-Dantoin AC, Hollenberg AN, Jameson JL 1993 Dynamic regulation of gonadotropin-releasing hormone receptor mRNA levels in the anterior pituitary gland during the rat estrous cycle. Endocrinology 133:1911–1914[Abstract]
  30. Brooks J, Taylor PL, Saunders PTK, Eidne KA, Struthers WJ, McNeilly AS 1993 Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle. Mol Cell Endocrinol 94:R23–R27
  31. Lin X, Conn PM 1998 Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH and cyclic adenosine monophosphate. Endocrinology 139:3896–3902[Abstract/Free Full Text]
  32. Albarracin CT, Kaiser, UB, Chin WW 1994 Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. Endocrinology 135:2300–2306[Abstract]
  33. Offermanns S, Iida-Klein A, Segre GV, Simon MI 1996 G{alpha}q family members couple parathyroid hormone (PTH)/PTH-related peptide and calcitonin receptors to phospholipase C in COS-7 cells. Mol Endocrinol 10:566–574[Abstract]
  34. Frost JA, Geppert TD, Cobb MH, Feramisco JR 1994 A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum. Proc Natl Acad Sci USA 91:3844–3848[Abstract/Free Full Text]
  35. Robbins DJ, Zhen E, Owaki H, Vanderbilt CA, Ebert D, Geppert TD, Cobb MH 1993 Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J Biol Chem 268:5097–5106[Abstract/Free Full Text]
  36. Beck TW, Huleihel M, Gunnell M, Bonner TI, Rapp UR 1987 The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus. Nucleic Acids Res 15:595–609[Abstract/Free Full Text]
  37. McArdle CA, Conn PM 1989 Use of protein kinase C-depleted cells for investigation of the role of protein kinase C in stimulus-response coupling in the pituitary. In: Conn PM (ed) Methods in Enzymology. Academic Press, San Diego, vol 168:287–301
  38. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR 1995 A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689[Abstract/Free Full Text]
  39. Aless DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR 1995 PD 98059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J Biol Chem 270:27489–27494[Abstract/Free Full Text]
  40. Kolch W, Heidecker G, Lloyd P, Rapp UR 1991 Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 349:426–428[CrossRef][Medline]
  41. Pagés G, Lenormand P, L’Allemain G, Chambard J-C, Meloche S, Pouysségur J 1993 Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319–8323[Abstract/Free Full Text]
  42. Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM, Chin WW 1995 A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 92:12280–12284[Abstract/Free Full Text]
  43. Kaiser UB, Katzenellenbogen RA, Conn PM, Chin WW 1994 Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct. Mol Endocrinol 8:1038–1048[Abstract]
  44. Haisenleder DJ, Yasin M, Marshall JC 1997 Gonadotropin subunit and gonadotropin-releasing hormone receptor gene expression are regulated by alterations in the frequency of Ca2+ pulsatile signals. Endocrinology 138:5227–5230[Abstract/Free Full Text]
  45. Garrel G, McArdle CA, Hemmings BA, Counis R 1997 Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3–1 cells: evidence for cross-talk between PKA and protein kinase C pathways. Endocrinology 138:2259–2266[Abstract/Free Full Text]
  46. Chen R-H, Abate C, Blenis J 1993 Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci USA 90:10952–10956[Abstract/Free Full Text]
  47. Aziz N, Wu J, Dubendorff JW, Lipsick JS, Sturgill TW, Bender TP 1993 c-Myb and v-Myb are differentially phosphorylated by p42mapk in vitro. Oncogene 8:2259–2265[Medline]
  48. Chu B, Soncin F, Price BD, Stevenson MA, Calderwood SK 1996 Sequential phosphorylation by mitogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by heat shock factor-1. J Biol Chem 271:30847–30857[Abstract/Free Full Text]




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