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

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

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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

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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 G_q/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 G_q/11 as well as to G_s, 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 -subunit complementary DNAs (cDNAs) showed that the GnRHR was able to couple to G_q/11 as well as to G_s and G_i 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 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 -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

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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

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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. 1). PMA also stimulated GnRHR-Luc activity in a time-dependent manner, with a significant stimulation of GnRHR-Luc by 6 h (Fig. 1); 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. 1). 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.

View larger version (44K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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.

View larger version (43K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   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.

View larger version (32K): [in this window] [in a new window]   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

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 -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 G_q/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 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 -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.

Received June 11, 1998.

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