HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 28, 21639-21647, July 14, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/28/21639    most recent M910252199v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yokoi, T. Articles by Murata, Y. Search for Related Content PubMed PubMed Citation Articles by Yokoi, T. Articles by Murata, Y. Social Bookmarking                      What's this?

Activation of the Luteinizing Hormone  Promoter by Gonadotropin-releasing Hormone Requires c-Jun NH₂-terminal Protein Kinase^*

Takeshi Yokoi, Masahide Ohmichi, Keiichi Tasaka, Akiko Kimura, Yuki Kanda, Jun Hayakawa, Masahiro Tahara, Koji Hisamoto, Hirohisa Kurachi, and Yuji Murata

From the Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan

Received for publication, December 20, 1999, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca²⁺, whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca²⁺. To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone  (LH) promoter activity, we transfected the rat LH (156 to +7)-luciferase construct into LT2 cells. GnRH activated the rat LH promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LH promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LH promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LH promoter by GnRH. In addition, GnRH did not induce either the rat LH promoter activity in LT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LH promoter activity in a c-Jun-dependent mechanism in LT2 cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GnRH,¹ a hypothalamic decapeptide, serves as a key regulator of the reproductive system. GnRH acts on anterior pituitary gonadotropes to stimulate the synthesis and release of the pituitary gonadotropins LH and FSH. The gonadotropins are subunit hormones, each containing noncovalently linked - and -subunits (1, 2). Within a species, the -subunits are identical, while the -subunits differ and confer the physiological specificity of the heterodimeric hormone. Each -subunit as well as the common -subunit is encoded by different genes on separate chromosomes. When GnRH binds to its seven-transmembrane receptor (3), it induces interaction of the receptor with the heterotrimeric G_q protein, which leads to activation of phospholipase C and formation of inositol 1,4,5-triphosphate and diacylglycerol, leading to elevation of intracellular Ca²⁺ and activation of protein kinase C (PKC) (4-6).

Intracellular transmission of extracellular signals is mediated in large part by several groups of sequentially activated protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) cascades. In growth factor signaling, the key elucidated MAPK cascade is the extracellular signal-regulated kinase (ERK). Recent evidence indicates that many G protein-coupled receptors can activate the ERK cascade (7-11). The signals transmitted through the ERK cascade lead to activation of a set of regulatory molecules that eventually initiates cellular responses such as growth and differentiation (12-14). Recently, it has been shown that GnRHa is capable of activating ERK in pituitary organ culture (15) and the T3-1 gonadotrope cell line (16, 17). However, the ERK cascade is not the only link between membrane receptors and their intracellular targets, and in the past few years several other ERK-like cascades have been identified (13). One of the most studied of these cascades is the Jun NH₂-terminal kinase (JNK; also known as stress-activated protein kinase (SAPK); Refs. 18 and 19) cascade, which is known to be activated in response to cellular stresses such as apoptosis (18, 20). ERK, JNK, and p38 (21) constitute the MAPK family. Recent data suggest that GnRH is capable of activating JNK (22) and p38 (23) in the T3-1 gonadotrope cell line.

It was reported that GnRH induction and basal control of the -subunit gene seem to occur through the PKC/ERK pathway, while induction of the LH gene is dependent on calcium influx in the T3-1 gonadotrope cell line, suggesting the differential stimulation of transcription of rat LH subunit genes by GnRH (24). However, the T3-1 gonadotrope cell line does not express the LH gene. Mellon and co-workers (25), using targeted oncogenesis in transgenic mice, have recently generated an immortal gonadotrope cell line (LT2). The cells of this line express the mRNA of GnRH receptor and of both the - and -subunits of LH (26, 27).

Taken together, these facts led us to examine whether GnRH stimulates the activity of ERK and/or JNK, and whether the respective cascades play a role in the transcriptional activation of the rat LH gene in LT2 cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Phorbol 12-myristate 13-acetate (PMA) and myelin basic protein were purchased from Sigma. Bisindolylmaleimide (GF 109203X) was purchased from Calbiochem (Laufelfingen, Switzerland). GnRH was obtained from Peninsula Laboratories (Belmont, CA). GnRH agonist, [D-Leu⁶,Por⁹-NHEt]leuprolide, was a gift from Takeda Chemical Industries (Japan). ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech. [-³²P]ATP (3000 Ci/mmol) was obtained from NEN Life Science Products. Erk1 rabbit polyclonal anti-ERK antibody, anti-Myc antibody, and anti-HA antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059 and the SAPK/JNK assay kit, including NH₂-terminal c-Jun fusion protein bound to glutathione-Sepharose beads and a phosphospecific c-Jun antibody (Ser⁶³), were obtained from New England Biolabs (Beverly, MA).

Cell Cultures-- LT2 cells (26) were generously provided by P. Mellon (La Jolla, CA). Cells were cultured at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a water-saturated atmosphere of 95% O₂ and 5% CO₂.

Construction of Expression Plasmids-- The wild type 156 to +7 LH promoter construct, cloned upstream of luciferase in PL(KS)b-Luc vector, was a kind gift from Dr. Y. Sadovsky (Washington University School of Medicine, St. Louis, MO) (28). The plasmid pLNCX-MAPK (KM) (29) was a kind gift from Dr. A. Gutierrez-Hartmann (University of Colorado Health Sciences Center, Denver, CO). Plasmid encoding the dominant negative form of Ets-2 (30) was a kind gift from Dr. K. E. Boulukos (Center de Biochimie, Faculté des Sciences, Nice, France). pAPr-etsZ, encoding the consensus DNA-binding domain of Ets-2, was a kind gift from Dr. M. Ostrowski (Ohio State University, Columbus, OH) (31). The plasmids encoding the dominant negative c-Jun (dnJun), pLHCc-Jun (S63A, S73A) (32, 33), and TAM-67 (34) were kind gifts from Dr. D. Mercola (University of California, San Diego, CA). The plasmids encoding the dominant negative SAPK/JNK (pcDL-SR-SAPK-VPF) and the wild type SAPK/JNK (pcDL-SR-wt-SAPK) were kind gifts from Dr. E. Nishida (Kyoto University, Kyoto, Japan). Myc-tagged p42^MAPK expression plasmid (pEXV-Erk2-tag) was a kind gift from Dr. C. J. Marshall (Institute of Cancer Research, London, United Kingdom; Ref. 35).

Clone Selection-- The dominant negative c-Jun (dnJun) expression plasmid pLHCc-JUN (S63A, S73A) was constructed as described previously (32). LT2 cells were transfected for 12 h in six-well tissue culture plates with 2 µg of pLHCdnc-JUN (S63A, S73A) using LipofectAMINE Plus (Life Technologies, Inc.) (36). Clone selection was performed by adding hygromycin to the medium at 200 µg/ml final concentration 2 days after the transfection. After 3 weeks, several clones were isolated using cloning rings. Selected clones were then maintained in medium supplemented with hygromycin (100 µg/ml), and only low passage cells (p < 10) were used for the experiments described here.

Assay of ERK Activity-- Cells were incubated overnight in the absence of serum and then treated with various substances. They were then washed twice with phosphate-buffered saline and lysed in ice-cold HNTG buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EDTA, 10 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 100 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (37). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad protein assay reagent. Erk1 rabbit polyclonal antibody was bound to protein A-Sepharose beads, and 300 µg of protein from the lysate samples was immunoprecipitated at 4 °C for 2 h. The immunoprecipitated products were washed once in HNTG buffer, twice in 0.5 M LiCl, 0.1 M Tris, pH 8.0, and once in kinase assay buffer (25 mM HEPES, pH 7.2-7.4, 10 mM MgCl₂, 10 mM MnCl₂, and 1 mM dithiothreitol), and samples were resuspended in 30 µl of kinase assay buffer containing 10 µg of myelin basic protein and 40 µM [-³²P]ATP (1 µCi) as described previously (16). The kinase reaction was allowed to proceed at room temperature for 5 min and stopped by the addition of Laemmli SDS sample buffer (38). Reaction products were resolved by 15% SDS-PAGE.

Assay of 42-kDa ERK Activity Using a Transient Expression System-- This activity was assayed as described (9, 10, 39). Briefly, LT2 cells cultured in 100-mm dishes were transfected with Myc-tagged p42^MAPK expression plasmid (1 µg of pEXV-Erk2-tag) in combination with 9 µg of pLNCX, pLNCX-MAPK (KM), pcDL-SR, or pcDL-SR-SAPK-VPF using LipofectAMINE Plus. At 72 h after transfection, serum-deprived cells were incubated with 1 µM GnRHa for 5 min, and the expressed Myc-tagged p42^MAPK was immunoprecipitated with 1 µg of antibody 9E10. The ERK activity in the immunoprecipitate was measured as described above.

Assay of JNK Activity-- JNK activity was precipitated from 250 µg of whole cell lysates by incubation with 2 µg of GST-c-Jun-(1-89) fusion protein/GSH-Sepharose beads for 18 h at 4 °C (New England Biolabs; Ref. 19). c-Jun-(1-89) contains a high affinity binding site for JNK close to the NH₂ terminus; this site contains two phosphorylation sites at Ser⁶³ and Ser⁷³. The beads were washed and resuspended in 50 µl of kinase buffer containing 100 µM ATP for 30 min at 30 °C as described (39). The solid-phase kinase reaction was terminated by addition of Laemmli sample buffer, and phosphorylation of GST-c-Jun on Ser⁶³ was examined after SDS-PAGE and immunoblotting with anti-phospho(Ser⁶³) c-Jun antibody.

Assay of JNK Activity Using a Transient Expression System-- LT2 cells cultured in 100 mm dishes were transfected with HA-tagged wild type SAPK/JNK expression plasmid (1 µg of pcDL-SR-wt-SAPK) or HA-tagged dominant negative SAPK/JNK expression plasmid (1 µg of pcDL-SR-SAPK-VPF) using LipofectAMINE Plus. At 72 h after transfection, serum-deprived cells were incubated with 1 µM GnRHa for 30 min, and cell lysates were immunoprecipitated with anti-HA antibody. The expressed HA-tagged wild type SAPK/JNK or dominant negative SAPK/JNK was eluted with 1% SDS, and the JNK activity was measured as described above.

Rat LH Promoter Assay-- LT2 cells cultured in 24-well plates were transfected with the rat 156 to +7 LH-luciferase construct and CMV--galactosidase plasmid (to normalize for cell viability and transfection efficiency) in combination with the indicated plasmids using LipofectAMINE Plus. At 48 h after transfection, serum-deprived cells were incubated with 100 nM GnRH for the indicated times. In some of the experiments, cells were treated with 20 µM PD98059 for 15 min before the addition of 100 nM GnRH. Cell extracts were prepared by lysing the cells with three sequential freeze-thaw cycles in a buffer containing 100 mM potassium phosphate, pH 7.8, and 10 mM dithiothreitol. Vigorous vortexing was used to enhance cell lysis. Unlysed cells and insoluble material were pelleted at 10,000 rpm for 10 min at 4 °C. The supernatant volume was measured, and aliquots of the supernatant were used in the subsequent luciferase and -galactosidase assays.

Luciferase was assayed as described previously (40). Briefly, the luciferase assay mixture contained 100 mM KPO₄, pH 7.8, 1 mM dithiothreitol, 3.7 mM MgSO₄, 530 µM ATP, and 470 µM luciferin plus 20 µl of cell extract in a final volume of 100 µl. Luciferin was added just before measuring light units, which were measured in duplicate during the first 40 s of the reaction at 25 °C in a luminometer (41).

-Galactosidase was assayed as described previously (40). The -galactosidase buffer contained 60 mM sodium phosphate, pH 7.5, 1 mM MgCl₂, 0.80 mg/ml O-nitrophenyl--galactopyranoside, and 40 mM -mercaptoethanol. A standard curve for 100 microunits to 2 milliunits of -galactosidase was made with each assay. A 30-µl aliquot of cell extract was incubated with assay buffer until color developed (30-120 min), and the reaction was then stopped by adding Na₂CO₃ to a final concentration of 625 mM. Absorbance was then read at 405 nm.

Luciferase light units were normalized relative to the activity of -galactosidase. The control value was set at 1 and the data expressed as -fold stimulation relative to control. Data are expressed as the mean ± S.E.

Statistics-- Statistical analysis was performed by Student's t test, and p < 0.01 was considered significant. Data are expressed as the mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activation of ERK and JNK by GnRH-- To evaluate whether ERK is activated by GnRH in LT2 cells, cultured cells were exposed to GnRHa for the indicated times (Fig. 1A). Cell lysates were immunoprecipitated with anti-ERK antibody and examined for ERK activity by assaying the incorporation of ³²P into MBP. The GnRHa-dependent increase in ERK activity reached a plateau from 5 min through 10 min and rapidly declined thereafter. We next examined the effect of GnRH on the activation of JNK, which is a member of the MAP kinase family. Cultured cells were exposed to GnRHa for the indicated times and cell lysates were incubated with GST-c-Jun fusion protein, followed by precipitation and Western analysis using anti-phospho-c-Jun antibody (Fig. 1B). The activation of JNK by GnRHa in LT2 cells was detectable at 5 min, reached a broad plateau from 30 min through 3 h, and declined thereafter. These results indicate that JNK activation by GnRHa was slower than ERK activation. We also found that GnRH activated both ERK and JNK, and that the time courses for ERK and JNK activation by GnRH were similar to that of GnRHa (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of GnRHa on the activity of ERK (A) and JNK (B). LT2 cells were grown in 100-mm dishes. A, cells were treated with 1 µM GnRHa for the indicated times (lanes 2-6). Cell lysates were prepared and immunoprecipitated with anti-ERK antibody (A-ERK), and the immunoprecipitates were incubated with [-32P]ATP in the presence of MBP, as described under "Experimental Procedures." After the reactions were stopped with Laemmli sample buffer, samples were subjected to SDS-PAGE and autoradiography. B, cells were treated with 1 µM GnRHa for the indicated times (lanes 2-5). Cell lysates were prepared and incubated with GST-c-Jun fusion protein/GSH-Sepharose beads, followed by SDS-PAGE and Western blot analysis with anti-phospho(Ser63) c-Jun antibody, as described under "Experimental Procedures." Autoradiograms of phosphorylated GST-c-Jun are shown.

Effect of Pertussis Toxin on GnRH-induced ERK and JNK Activation-- We compared the mechanisms of ERK and JNK activation induced by GnRH. It has been shown that the receptor for GnRH (3-6) is a member of the superfamily of G protein-coupled receptors. To determine what type of G protein is coupled to the GnRH receptor, we pretreated LT2 cells with 100 ng/ml pertussis toxin (PTX) for 4 h in order to inactivate G_i and G_o proteins, and then treated the cells with 1 µM GnRHa for 5 min (Fig. 2A) or 30 min (Fig. 2B, upper panel). Whereas PTX clearly caused a decrease in GnRHa-induced ERK activation (Fig. 2A, lane 7), PTX did not have a detectable effect on GnRHa-induced JNK (Fig. 2B, upper panel, lane 7) activation. Thus, although PTX-sensitive G proteins are partly involved in the effect of GnRHa on ERK activity, as previously reported (42), PTX-sensitive G proteins are not involved in the effect of GnRHa on JNK activity, as was also previously reported (22).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of PTX, the down-regulation of PKC, or the PKC inhibitor GF109230X on GnRHa-induced ERK (A) or JNK (B). Cells were grown in 100-mm dishes. In A and B (upper panel), cells were pretreated with 1 µM PMA for 16 h (lanes 4-6) or 100 ng/ml PTX for 4 h (lane 7), followed by treatment with 1 µM PMA for 5 min (lanes 1 and 5), 1 µM GnRHa for 5 min (A, lanes 3, 6, and 7), or 1 µM GnRHa for 30 min (B, upper panel, lanes 3, 6, and 7). In B (lower panel), cells were pretreated with 10 µM GF109230X (GF) for 10 min (lanes 2 and 4) followed by treatment with 1 µM GnRHa for 30 min (lanes 3 and 4) or 1 µM PMA for 30 min (lane 5). Lysates of cells were assayed for ERK (A) or JNK (B) activity as described in the legend for Fig. 1. Autoradiograms of 32P-labeled MBP (A) and phosphorylated GST-c-Jun (B) are shown.

Role of PKC in Activation of ERK and JNK-- Many G protein-linked receptors can mediate stimulation of ERK activity via the phospholipase C-dependent activation of PKC (43-46). Activation of ERK (16, 17) or JNK (22) by GnRH requires PKC in T3-1 cells. Therefore, the role of PKC in GnRH-induced ERK (Fig. 2A) or JNK (Fig. 2B) activation in LT2 cells was examined. Exposure of LT2 cells to PMA caused stimulation of ERK activity (Fig. 2A, lane 1). However, the ability of PMA to induce the activation of ERK does not necessarily mean that the PKC pathway is involved in GnRHa-induced ERK activation, as has been shown in the case of norepinephrine-induced ERK activation in both adipocytes (47) and GT-1 GnRH neuronal cell lines (10). Whether PKC is indeed involved in GnRH signaling was determined using PKC depletion. Pretreatment with 1 µM PMA for 16 h to deplete most PKC isoforms completely abolished the GnRHa-induced ERK activation (Fig. 2A, lane 6). On the other hand, treatment with 1 µM PMA for 5 min (Fig. 2B, upper panel, lane 1) or for 30 min (Fig. 2B, lower panel, lane 5) did not induce JNK activation. Moreover, neither pretreatment with 1 µM PMA for 16 h (Fig. 2B, upper panel, lane 6) nor pretreatment with the selective PKC inhibitor GF 109203X (48) at 10 µM (Fig. 2B, lower panel, lane 4) had any effect on the GnRHa-induced JNK activation. These results suggest that activation of ERK by GnRH was mediated by PKC, whereas activation of JNK by GnRH was not mediated by PKC.

Role of Extracellular and Intracellular Ca²⁺ in ERK and JNK Activation-- It has been reported that elevated Ca²⁺ is necessary for GnRH-induced ERK activation in T3-1 cells (16, 17). We therefore evaluated the role of extracellular and intracellular Ca²⁺ in the GnRH- induced ERK (Fig. 3A) and JNK (Fig. 3B) activation in LT2 cells. Elimination of extracellular Ca²⁺ by treatment with 3 mM EGTA for 1 min or with 1 µM nifedipine for 10 min clearly attenuated GnRHa-induced ERK activation (Fig. 3A, lanes 3 and 5), indicating that Ca²⁺ influx is required for GnRHa-induced ERK activation. Moreover, treatment with either 50 µM 1,2-bis(o-aminophenoxy)ethane-N,N,N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) for 20 min to eliminate intracellular Ca²⁺ (Fig. 3A, lane 6) or 3 mM EGTA for 15 min to eliminate extracellular and intracellular Ca²⁺ (49) (Fig. 3A, lane 4) clearly attenuated GnRHa-induced ERK activation, indicating that intracellular Ca²⁺ is also required for GnRHa-induced ERK activation. On the other hand, elimination of extracellular Ca²⁺ by either treatment with 3 mM EGTA for 1 min or with 1 µM nifedipine for 10 min or elimination of intracellular Ca²⁺ by treatment with 50 µM BAPTA-AM had no effect on GnRHa-induced JNK activation (Fig. 3B). These results suggest that GnRH-induced ERK activation was dependent on extracellular and intracellular Ca²⁺, whereas GnRH-induced JNK activation was independent of extracellular and intracellular Ca²⁺ in LT2 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Role of Ca2+ in the activation of both ERK (A) and JNK (B) by GnRHa. Cells were grown in 100-mm dishes. In A, cells were pretreated with 3 mM EGTA for 1 min (lane 3), 3 mM EGTA for 15 min (lane 4), 1 µM nifedipine for 10 min (lane 5), or 50 µM BAPTA-AM for 20 min (lane 6), and then treated with 1 µM GnRHa (lanes 2-6) for 5 min. In B, cells were pretreated with 3 mM EGTA for 1 min (lane 3), 1 µM nifedipine for 10 min (lane 4), or 50 µM BAPTA-AM for 20 min (lane 5), and then treated with 1 µM GnRHa (lanes 2-5) for 30 min. Lysates of cells were assayed for ERK (A) or JNK (B) activity as described in the legend for Fig. 1. Autoradiograms of 32P-labeled MBP (A) and phosphorylated GST-c-Jun (B) are shown.

Stimulation of LH Promoter Activity by GnRH-- We sought to determine whether the ERK and/or JNK cascades are involved in the regulation of LH synthesis induced by GnRH. A rat LH promoter (156 to +7 bp)-luciferase reporter construct was transiently transfected into LT2 cells. As shown in Fig. 4, addition of 100 nM GnRH enhanced the luciferase activity in a time-dependent fashion. To examine whether the stimulation of the LH promoter by GnRH is the result of activation of the ERK cascade, either PD98059, an inhibitor of MEK, or an expression vector, pLNCX-MAPK (KM), encoding a catalytically inactive form of MAPK (iMAPK), was used. PD98059 is relatively specific for MEK, with no inhibitory activity against a number of other serine/threonine and tyrosine kinases (50-52). Although pretreatment with 20 µM PD98059 for 15 min completely abolished the GnRHa-induced ERK activation (Fig. 5B), pretreatment with 20 µM PD98059 had no effect on GnRH-induced LH promoter activation (Fig. 5A). In addition, cotransfection with pLNCX-MAPK (KM) at doses up to 2.4 µg had no effect on GnRH-induced LH promoter activation (Fig. 5A), whereas cotransfection with pLNCX-MAPK (KM) completely abolished the GnRHa-induced ERK activation (Fig. 5C). These results suggest that the ERK cascade is not involved in the GnRH-induced LH promoter activation. We next examined the involvement of the JNK cascade in the stimulation of the LH promoter by GnRH. An expression plasmid that encodes a dominant negative SAPK/JNK (pcDL-SR-SAPK-VPF) was used to inhibit the JNK cascade (53). GnRHa-induced JNK activation in cells transfected with pcDL-SR-SAPK-VPF was clearly attenuated compared with that in cells transfected with pcDL-SR-wt-SAPK (Fig. 6B), confirming the negative effects of the expression of a dominant negative SAPK/JNK on the endogenous kinase in LT2 cells. Moreover, cotransfection with pcDL-SR-SAPK-VPF did not interfere with GnRHa-induced ERK activation (Fig. 6C), suggesting the specificity of action of the SAPK-VPF vector in LT2 cells. Cotransfection with pcDL-SR-SAPK-VPF significantly attenuated the GnRH-induced LH promoter activation dose-dependently, whereas cotransfection with pcDL-SR had no effect (Fig. 6A), suggesting that the JNK cascade is involved in the GnRH-induced LH promoter activation.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Stimulation of the rat LH promoter activity by GnRH. LT2 cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal. After transfection, cells were treated with 100 nM GnRH for the indicated times prior to harvesting. Luciferase activity was normalized relative to -galactosidase activity, and the basal activity was set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six transfections. The activities at 4, 12, and 24 h were significantly different from that of the control (**, p < 0.01).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Role of ERK cascade in the GnRH-dependent stimulation of the rat LH promoter. A, LT2 cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal, with or without 1.2 or 2.4 µg of iMAPK vector (pLNCX-MAPK (KM)), as indicated. After transfection, cells were incubated with or without 20 µM PD98059 for 15 min as indicated, and then treated with 100 nM GnRH for 24 h prior to harvesting. B, cells grown in 100-mm dishes were pretreated with 20 µM PD98059 for 15 min (lane 3), and then treated with 1 µM GnRHa for 5 min (lanes 2 and 3). Lysates of cells were assayed for ERK activity as described in the legend for Fig. 1A. C, cells were transfected with pLNCX (lanes 1 and 2) or pLNCX-MAPK (KM) (lanes 3 and 4) together with Myc-tagged p42MAPK expression plasmid (pEXV-Erk2-tag) and, after 72 h, were stimulated with 1 µM GnRHa for 5 min (lanes 2 and 4). Cell lysates were immunoprecipitated with anti-Myc antibody (A-Myc), and the immunoprecipitates were incubated with [-32P]ATP in the presence of MBP, as described under "Experimental Procedures." After the reactions were stopped with Laemmli sample buffer, samples were subjected to SDS-PAGE and autoradiography.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Role of JNK cascade in the GnRH-dependent stimulation of the rat LH promoter. A, LT2 cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal, with or without 0.4, 1.2, or 2.4 µg of pcDL-SR or pcDL-SR-SAPK-VPF, as indicated. After transfection, cells were treated with 100 nM GnRH for 24 h prior to harvesting. Luciferase activity was normalized relative to -galactosidase activity, and the basal activity was set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six transfections. ** indicates p < 0.01 as compared with the respective control. B, cells were transfected with pcDL-SR-wt-SAPK (lanes 1 and 2) or pcDL-SR-SAPK-VPF (lanes 3 and 4) and, after 72 h, were stimulated with 1 µM GnRHa for 30 min (lanes 2 and 4). Cell lysates were immunoprecipitated with anti-HA antibody (A-HA), and the expressed HA-tagged wild-type SAPK/JNK or dominant negative SAPK/JNK was eluted with 1% SDS, and the JNK activity was measured as described under "Experimental Procedures." C, cells were transfected with pcDL-SR (lanes 1 and 2) or pcDL-SR-SAPK-VPF (lanes 3 and 4) together with Myc-tagged p42MAPK expression plasmid (pEXV-Erk2-tag) and, after 72 h, were stimulated with 1 µM GnRHa for 5 min (lanes 2 and 4). Cell lysates were immunoprecipitated with anti-Myc antibody (A-Myc), and the immunoprecipitates were incubated with [-32P]ATP in the presence of MBP, as described in the legend for Fig. 5C.

Role of PKC and Ca²⁺ in GnRH-induced LH Promoter Activation-- Since PKC and Ca²⁺ are not involved in GnRHa-induced JNK activation (Figs. 2 and 3), we next examined the effect of PKC and Ca²⁺ on GnRH-induced LH promoter activation. Pretreatment with 10 µM GF 109203X for 10 min, 3 mM EGTA for 15 min, 1 µM nifedipine for 10 min, or 50 µM BAPTA-AM for 20 min had no effect on GnRHa-induced LH promoter activation (Fig. 7). Thus, neither PKC nor Ca²⁺ is involved in GnRHa-induced LH promoter activation, just as they are not involved in GnRHa-induced JNK activation.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Roles of PKC and Ca2+ in the GnRH-dependent stimulation of the rat LH promoter. LT2 cells were pretreated with 10 µM GF109230X for 10 min, 3 mM EGTA for for 15 min, 1 µM nifedipine for 10 min, or 50 µM BAPTA-AM for 20 min, as indicated. Cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal. After transfection, cells were treated with 100 nM GnRH for 24 h prior to harvesting. Luciferase activity was normalized relative to -galactosidase activity, and the basal activity was set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six transfections. ** indicates p < 0.01 as compared with the respective control.

A c-Jun Transcription Factor Is a Nuclear Acceptor of the JNK Signaling Cascade-- It has been demonstrated that JNK phosphorylates c-Jun and ATF-2 at the putative regulatory amino-terminal serine residues and thereby increases their transcriptional activities (18, 19). Moreover, JNK has been reported to activate Elk-1, resulting in an increase in c-fos gene expression (54). The Ets domain transcription factor Elk-1 is a substrate for three distinct classes of MAP kinase family members (55-58). In addition, Ets family binding sites have been identified in the rat LH promoter between 156 and +7. Therefore, we first examined whether Ets transcription factors are the nuclear acceptors for GnRH signaling. Members of the Ets transcription factor family contain a transactivation domain at the amino terminus and a highly conserved DNA-binding domain at the carboxyl terminus, and this latter domain defines the Ets family of transcription factors since it lacks homology to other DNA-binding motifs (59, 60). To examine the functional role of Ets transcription factors in GnRH-induced activation of the LH promoter, the effect of an expression plasmid that encodes a dominant negative Ets construct (pAPr-etsZ) was examined. The pAPr-etsZ construct encodes the highly conserved DNA-binding domain of c-Ets-2 protein devoid of the transactivation domain, and inhibits both Ets-1- and Ets-2-mediated responses (31) since Ets-1 and Ets-2 recognize the same DNA sequence motif (31, 59). Cotransfection with pAPr-etsZ at doses up to 2.4 µg had no effect on GnRH-induced transcriptional stimulation (Fig. 8A). We also examined the effect of an expression plasmid that encoded a truncated Ets-2 with dominant-negative activity (pRK5-ets-21-328). Cotransfection with pRK5-ets-21-328 also had no effect on GnRH-induced transcriptional stimulation (Fig. 8B). These results suggest that the nuclear acceptor for the stimulation of LH promoter activity by GnRH is not a member of the Ets transcription factor family. Since the LH promoter does not contain a consensus ATF/CREB site, we next examined whether c-Jun is involved as a nuclear acceptor of a JNK signal. A dominant negative inhibitor (61, 62) of the JNK cascade, dnJun, was used to inhibit the phosphorylation of c-Jun. The dnJun mutant cannot be phosphorylated at the NH₂-terminal serine residues owing to substitution of serines 63 and 73 by alanine, and the mutant consequently blocks the enhanced transactivation promoted by JNK-dependent phosphorylation of these sites (61, 62). Thus, dnJun blocks c-Jun phosphorylation-dependent events of the JNK cascade (32, 61, 62). Cotransfection with a dnJun expression vector significantly attenuated the GnRH-induced LH promoter activation (Fig. 9A). In addition, cotransfection of TAM-67, a well characterized transdominant negative inhibitor of AP-1 owing to a deletion of residues 2-122 (34), significantly attenuated the GnRH-induced LH promoter activation in a dose-dependent manner (Fig. 9B). We further examined whether the LH promoter was activated by GnRH in clonal lines of LT2 cells, which stably expressed a dominant negative inhibitor (61, 62) of the JNK cascade, dnJun (dnJun-LT2). GnRH-induced ERK activation was not attenuated in dnJun-LT2 cells, suggesting that there is no cross-talk between the ERK and JNK cascades (Fig. 9C). Expression of dnJun significantly attenuated the GnRH-induced LH promoter activation (Fig. 9D). These results suggest that c-Jun is involved in the GnRH-induced LH transcriptional activation.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Ets is not involved in the GnRH-dependent stimulation of the rat LH promoter. LT2 cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal, with or without 1.2 or 2.4 µg of pAPr or pAPr-etsZ (A) or 1.2 µg of pRK5 or pRK5-ets-21-328 (B), as indicated. After transfection, cells were treated with 100 nM GnRH for 24 h prior to harvesting. Luciferase activity was normalized relative to -galactosidase activity, and the basal activity was set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six transfections. ** indicates p < 0.01 as compared with the respective control.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   dnJun inhibits GnRH activation of the rat LH promoter. LT2 cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control, pCMVgal, with or without 1.2 µg of pLHCX or pLHCdnJun (A) or 0.4, 0.8, or 1.2 µg of TAM-67 (B), as indicated. In D, LT2 cells or dnJun-expressing LT2 (dnJun-LT2) cells were transiently cotransfected with 0.4 µg of the rat LH (156 to +7)-luciferase construct and 0.04 µg of an internal control. After transfection, cells were treated with 100 nM GnRH for 24 h prior to harvesting. Luciferase activity was normalized relative to -galactosidase activity, and the basal activity was set at 1.0. Data are expressed as the mean -fold activation ± S.E. of six transfections. ** indicates p < 0.01 as compared with the respective control. In C, LT2 and dnJun-LT2 cells were treated with (lanes 2 and 4, respectively) or without 1 µM GnRHa for 5 min (lanes 1 and 3, respectively). Lysates of cells were assayed for ERK activity as described in the legend for Fig. 1A. Autoradiograms of 32P-labeled MBP are shown.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both the biosynthesis and the secretion of the gonadotropins are under the regulation of GnRH. Previous studies indicated that the GnRH receptor couples the G proteins of the G_q/11 family with phosphoinositide turnover and a resultant increase in intracellular calcium concentration and PKC activation, to stimulate secretion of LH and FSH (4-6). However, the molecular mechanisms by which GnRH mediates its transcriptional effects remain largely unknown. It was reported that GnRH-induced activation and basal control of the -subunit gene seem to occur through the PKC/ERK pathway, while induction of the LH gene is dependent on calcium influx using T3-1 cells, which do not express the LH gene (24). The -subunits confer the physiological specificity of the heterodimeric hormone. Thus, a systematic approach to identifying mechanisms of hormonal regulation of gonadotropin subunit gene expression has been hampered by the lack of an available cell line that expresses the , LH, and FSH genes in a regulated manner. An immortal gonadotrope cell line (LT2) which expresses mRNA for GnRH receptor and for both the - and -subunits of LH, has recently been generated (26, 27). Therefore, we examined the mechanism by which GnRH induces the biosynthesis of LH using LT2 cells in this study. As in T3-1 cells (17, 22), GnRHa caused both ERK and JNK activation in LT2 cells. Although it was reported that activation of ERK by GnRH contributes to stimulation of the -subunit promoter (16, 24, 63), the role of JNK activation by GnRH has not been clarified hitherto. We present here the first evidence that a JNK cascade is necessary to elicit LH promoter activity in a c-Jun-dependent mechanism.

Activation of ERK is induced by phosphorylation of both threonine and tyrosine residues of the enzyme as a result of successive stimulation of Ras, ERK kinase kinase which may be Raf-1, MEK kinase, or an alternative kinase, and MEK (12-14). Protein kinase C activates Raf-1 by direct phosphorylation (64). Distinct pathways of G_i- and G_q-mediated ERK activation have been reported (65). The activation of G_i- coupled receptors, such as oxytocin (8), prostaglandin F2 (9), endothelin-1 (11), and prolactin-releasing peptide (40), appears to be PTX-sensitive and PKC-independent. However, in the case of receptors coupled to G_q, such as bombesin (43) and thyrotropin-releasing hormone (7), activation is thought to be secondary to stimulation of phosphatidylinositol 4,5-bisphosphate-phospholipase C, leading to production of inositol phosphate and diacylglycerol, with subsequent PKC-mediated stimulation of ERK. In the present study, pretreatment with PTX detectably blocked the GnRHa-induced ERK activation (Fig. 2) and apparent down-regulation of PKC by prolonged incubation with PMA attenuated the stimulation of ERK activity by GnRHa (Fig. 2), suggesting the involvement of both G_i and G_q protein in GnRHa-induced ERK activation.

One important downstream biochemical event that occurs after ligand binding to many growth-promoting receptors is the activation of members of the MAP kinase family, including ERK and JNK (21). The ERK cascade is strongly activated by growth and differentiation factors, and sustained activation is thought to be an important signal for promoting cell proliferation and differentiation (12-14). The JNK cascade is also activated by cellular stresses (18, 19). These observations suggest the existence of parallel cascades leading to activation of either ERK or JNK. Is the mechanism of GnRHa-induced ERK activation different from that of GnRHa-induced JNK activation? In most cases (7, 10), PKC and Ca²⁺ have been shown to stimulate ERK activity. However, in endothelin-1-stimulated Rat-1 cells, JNK but not ERK activation was inhibited by chelation of Ca²⁺ and by down-regulation of PKC (67). Similarly, in cardiac myocytes, activation of JNK by angiotensin II was strongly suppressed by down-regulation of PKC or by chelation of intracellular Ca²⁺ (68). On the other hand, in GN4 rat liver epithelial cells, angiotensin II activated JNK in a Ca²⁺-dependent, PKC-independent manner (69). In the present study, GnRHa-induced JNK but not ERK activation was independent of both extracellular Ca²⁺ and intracellular Ca²⁺ (Fig. 3). Moreover, GnRHa-induced ERK activation was PKC-dependent, whereas JNK activation was PKC-independent (Fig. 2). The time course of JNK activation (Fig. 1B) in response to GnRHa was slower than that of ERK activation (Fig. 1A). Thus, the regulation of the JNK activation by GnRHa appeared to be different from that of the ERK activation.

Ca²⁺ is a critical mediator of the induction of gonadotropin secretion by GnRH (5, 70, 71). Studies have shown that calcium ionophores and calcium channel agonists can stimulate gonadotropin secretion. The stimulatory actions of GnRH on LH and FSH secretion can be inhibited by calcium channel antagonists and culturing the secretory cells in calcium-free medium. Elimination of extracellular Ca²⁺ by treatment with 3 mM EGTA for 1 min or 1 µM nifedipine for 10 min or elimination of intracellular Ca²⁺ by treatment with 50 µM BAPTA-AM for 20 min did not abolish the GnRHa-induced activation of JNK (Fig. 3B). These results suggest that GnRHa-induced activation of JNK is independent of extracellular and intracellular Ca²⁺ and does not seem to involve gonadotropin secretion.

Although little is known regarding the role of the activation of the MAP kinase family in the biosynthesis of hormones, we recently showed that both ERK and JNK are necessary to elicit rat prolactin promoter activity by prolactin-releasing peptide in an Ets-dependent mechanism (40). GnRH-induced activation of the LH promoter was not attenuated by either pretreatment with MEK inhibitor PD98059 or by cotransfection with an iMAPK construct (Fig. 5A). On the other hand, GnRH-induced activation of the LH promoter was attenuated by cotransfection with a dominant negative SAPK/JNK construct (Fig. 6A). Moreover, the lack of involvement of PKC and Ca²⁺ in GnRHa-induced LH promoter activation (Fig. 7) is similar to that in GnRHa-induced JNK activation (Figs. 2 and 3). Thus, although GnRH induces the activation of both ERK and JNK, the JNK cascade seems to be required for the GnRH-induced LH promoter activation, as in the case of tumor necrosis factor- production in mast cells (72). In addition, since it was reported that stimulation of JNK by GnRH was mediated by c-Src in T3-1 cells (22), we examined the effect of the Src-selective tyrosine kinase inhibitor herbimycin A on GnRHa-induced JNK activation and LH promoter activation. Pretreatment with 5 µM herbimycin A for 18 h had no effect on GnRHa-induced JNK activation or LH promoter activation (data not shown), suggesting that the mechanism of JNK activation might be different in other cell lines. The possible mechanisms that cause activation of the JNK cascade in LT2 cells are under investigation.

No transcription factors that bind to a GnRH-responsive region of the LH promoter have been identified yet. JNKs phosphorylate two sites of the NH₂-terminal transactivating domain of c-Jun (Ser⁶³ and Ser⁷³), ATF-2/CREB, and Elk-1, thereby increasing their transcriptional activities (18, 19, 54). Although Ets family binding sites have been identified in the rat LH promoter between 156 and +7, cotransfection with either pAPr-etsZ or pRK5-ets-21-328 had no effect on the GnRH-induced LH promoter activation (Fig. 8). The proximal LH promoter does not contain ATF/CREB sites. Since the proximal LH promoter between 95 and 86 contains 75.3% homology with the consensus AP-1 sites (TGA(C/G)TCA) (73), it is conceivable that c-Jun could be involved as a nuclear acceptor of a JNK signal. Therefore, we used dnJun to examine whether c-Jun might be involved as a nuclear acceptor of the JNK signal. dnJun has been characterized and successfully used in a number of studies (32, 61, 62). In addition, overexpression of dnJun did not alter the enzyme activity of JNK, showing that the derivative acts at a point distal to JNK in the JNK signal transduction cascade, consistent with the inhibition of the AP-1 transactivation function as previously shown (61, 62). Moreover, independent studies using well characterized antisense reagents complementary to the JNK-1 and JNK-2 isoforms confirm that dnJun specifically blocks the JNK cascade (74). Cotransfection of a dnJun expression vector significantly attenuated the ability of GnRH to induce LH promoter activation (Fig. 9A), suggesting that c-Jun is a substrate for JNK in the GnRH-induced LH promotor activation. In addition, cotransfection of TAM-67, a well characterized transdominant negative inhibitor of AP-1 owing to a deletion of residues 2-122 (34), significantly attenuated the GnRH-induced LH promoter activation in a dose-dependent manner (Fig. 9B). Moreover, GnRH did not induce either the rat LH promoter activity (Fig. 9D) in dnJun-LT2 cells. These results suggest that c-Jun seems to be a substrate for JNK, as in the case of the ceramide-induced cyclooxygenase-2 gene expression (75), and to be involved in the GnRH-induced LH transcriptional activation.

The signaling cascades that couple the activation of GnRH receptor to transcription are not yet fully defined. Although the JNK cascade appeared to be necessary for the GnRH-induced LH promoter activation, it is not clear whether the activation of the JNK cascade is sufficient to induce the activation. It remains to be determined whether other MAP kinase family members such as p38 or the newly described SAPK3 (21) are also involved in GnRH-induced LH promoter activation. Although it was reported that activation of ERK by GnRH contributes to stimulation of the -subunit promoter (16, 24, 63), the ERK cascade did not appear to be involved in the GnRH-induced LH transcriptional activation in this study. What is the role of the ERK cascade in the biological response to GnRH in LT2 cells? Thus, the complete role of the MAP kinase family in the action of GnRH in gonadotrope remains to be explored. It was reported that GnRH induces pituitary cell differentiation (66). Therefore, apart from a contribution to mediating transcriptional responses to GnRH, either ERK or JNK activation may be associated with other yet unknown cellular responses to GnRH, such as effects on long term maintenance of the gonadotrope phenotype.

	ACKNOWLEDGEMENTS

We thank Dr. P. Mellon for the gift of LT2 cells, Dr. Y. Sadovsky for the gift of the reporter construct PL(KS)b-156LHluc, Dr. E. Nishida for the gift of the pcDL-SR-SAPK-VPF and pcDL-SR-wt-SAPK plasmids, Dr. D. Mercola for the gift of the plasmids encoding the dominant negative c-Jun and TAM-67, Dr. A. Gutierrez-Hartmann for the gift of the plasmid pLNCX-MAPK (KM), Dr. K. E. Boulukos for the gift of the plasmids encoding Ets-2 and its dominant negative form, and Dr. M. Ostrowski for the gift of pAPr-etsZ.

	FOOTNOTES

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

To whom all correspondence and reprint requests should be addressed: Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3354; Fax: 81-6-6879-3359; E-mail: masa@gyne.med.osaka-u.ac.jp.

Published, JBC Papers in Press, April 27, 2000, DOI 10.1074/jbc.M910252199

	ABBREVIATIONS

The abbreviations used are: GnRH, gonadotropin-releasing hormone; GnRHa, gonadotropin-releasing hormone agonist; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated (protein) kinase; JNK, c-Jun NH₂-terminal protein kinase; SAPK, stress-activated protein kinase; iMAPK, a catalytically inactive form of MAPK; dnJun, dominant negative c-Jun; MBP, myelin basic protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PTX, pertussis toxin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; LH, luteinizing hormone; FSH, follicle-stimulating hormone; CMV, cytomegalovirus; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES