Involvement of Mitogen-Activated Protein Kinase in Cyclic Adenosine ...

0013-7227/01/$03.00/0 Endocrinology Copyright © 2001 by The Endocrine Society

Vol. 142, No. 7 Printed in U.S.A.

Involvement of Mitogen-Activated Protein Kinase in Cyclic Adenosine 3ⴕ,5ⴕ-Monophosphate-Induced Hormone Gene Expression in Rat Pituitary GH3 Cells* TOSHIE YONEHARA, HARUHIKO KANASAKI, HIDEYUKI YAMAMOTO, KOHJI FUKUNAGA, KOHJI MIYAZAKI, AND EISHICHI MIYAMOTO Department of Pharmacology (T.Y., H.Y., K.F., E.M.), Kumamoto University School of Medicine, Kumamoto 860-0811; and Department of Obstetrics and Gynecology, Shimane Medical University (T.Y., H.K., K.M.), Izumo 693-8501, Japan ABSTRACT We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH3 cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-

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dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH. (Endocrinology 142: 2811–2819, 2001)

YNTHESIS AND SECRETION of hormones in pituitary cells are correctly regulated by several hormones/factors originated in hypothalamus, such as TRH (1) and pituitary adenylate cyclase-activating polypeptide (PACAP) isolated from bovine hypothalamus (2). Exposure of rat anterior pituitary cells to PACAP resulted in stimulation of both adenylate cyclase activity and release of PRL and GH (2). Cloning of PACAP complementary DNA (cDNA) revealed that PACAP is a member of the glucagon/secretin/vasoactive intestinal polypeptide family of peptides (2). It has been reported that PACAP is 1000-fold more potent than vasoactive intestinal polypeptide in stimulating adenylate cyclase in pituitary cells (2). PACAP is known to stimulate PRL gene expression via the cAMP-dependent protein kinase (cAMPkinase)-mediated pathway that is independent of the pathway employed by TRH (3, 4). However, the molecular mechanisms by which the cAMP kinase-mediated pathway stimulates the gene expression and secretion of PRL remain to be elucidated. Mitogen-activated protein kinase (MAP kinase) or extracellular signal-regulated kinase (ERK) was originally reported to be activated by growth factors and to be involved

in the proliferation and differentiation of cells through stimulation of gene expression (5). In addition, MAP kinase has been implicated in other cellular events involving hormone secretion. GH3 cells, which are a clonal strain of rat pituitary tumor cells, are a useful model system for study of the synthesis and secretion of both PRL and GH (6). MAP kinase has been reported to be activated by both TRH (7, 8) and estradiol (9) in GH3 cells. Recently, we precisely analyzed the relationship between the physiological functions of TRH and the activation of MAP kinase in GH3 cells. We found that PRL synthesis by TRH was mainly conducted by stimulation of the MAP kinase pathway (8). In a series of experiments we noticed that the treatment of GH3 cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), stimulated MAP kinase (8). These results prompted us to investigate whether the cAMP-kinase-mediated pathway stimulated the gene expression and secretion of PRL via MAP kinase pathway. In this study we examined the possible involvement of MAP kinase in the regulation of gene expression and secretion of PRL and GH by PACAP27 as well as CPT-cAMP and overexpression of the catalytic subunit of cAMP-kinase.

Received December 19, 2000. Address all correspondence and requests for reprints to: Eishichi Miyamoto, M.D., Department of Pharmacology, Kumamoto University School of Medicine, 2–2-1 Honjo, Kumamoto 860-0811, Japan. E-mail: [email protected]. * This work was supported in part by Grants-in-Aid for Scientific Research and for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan; a research grant from Human Frontier Science Program (to H.Y., K.F., and E.M.); and a grant from the Ministry of Health and Welfare (to K.M.).

Materials

Materials and Methods The following chemicals and reagents were obtained from the indicated sources: FCS, JRH Bioscience (Lenexa, KS); [␥-32P]ATP, NEN Life Science Products (Wilmington, DE); CPT-cAMP and antibody to the catalytic subunit of cAMP-kinase (anti-cAMP-kinase antibody), Sigma (St Louis, MO); PACAP27 amide (PACAP27), Nova Biochemical Co. (La¨ufelfingen, Switzerland); and Ham’s F-10 medium, ICN Biomedicals, Inc. (Tokyo, Japan). Myelin basic protein (MBP) was purified from bovine brain (10).

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SYNTHESIS OF PRL BY cAMP VIA MAP KINASE

Cell culture GH3 cells, a rat prolactinoma cell line, were cultured in Ham’s F-10 medium containing 15% horse serum, 2.5% FCS, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin and maintained at 37 C in an atmosphere of 95% air-5% CO2 (8, 11). Two or 3 days before an experiment, 2–3 ⫻ 105 cells were plated on a 35-mm petri dish (Nunc, Roskilde, Denmark). When test reagents were added, cultured cells were washed once with Krebs-Ringer HEPES buffer (KRH) containing 130 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm sodium phosphate, 1.2 mm MgSO4, 10 mm glucose, and 20 mm HEPES (pH 7.4) and preincubated in KRH at 37 C for 60 min. Cells were then incubated in KRH at 37 C for the indicated times with or without the test reagents in KRH. When we examined the inhibitory effects of PD098059 and H89, the cells were preincubated with each inhibitor for 60 min in KRH at 37 C (12). We chose the concentrations of H89 (10 ␮m) and PD098059 (50 ␮m) on the basis of previous reports (8, 12). We also confirmed that the lower concentrations of inhibitors did not completely abolish CPT-cAMP-induced MAP kinase activation and PRL promoter activation (data not shown). After preincubation, the cells were incubated with or without 1 mm CPT-cAMP or 100 nm PACAP27 in the presence or absence of each inhibitor in KRH. After incubation for the indicated times, the medium was quickly aspirated off, and the cells were frozen in liquid N2.

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mRNA (GenBank V01239) (18). For amplification of PRL cDNA, we used a sense primer (5⬘-AATGACGGAAATAGATGATTG-3⬘) that corresponds to nucleotides 29 – 49 and an antisense primer (5⬘CCAGTTATTAGTTGAAACAGA-3⬘) that corresponds to nucleotides 546 –566. For amplification of GH cDNA, we used a sense primer (5⬘-CTGCTGACACCTACAAAGA-3⬘) that corresponds to nucleotides 186 –204 and an antisense primer (5⬘-CAGTGTGTGCCTAGAAAGCA-3⬘) that corresponds to nucleotides 679 – 698. PCR amplification was performed using the GeneAmp PCR system 2400 (Perkin-Elmer Corp., Foster City, CA) for 30, 17, and 17 cycles for PRL, GH, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Products of PRL, GH, and GAPDH were separated by electrophoresis on a 1.0%, 1.5%, and 1.5% agarose gel, respectively, visualized by ethidium bromide staining, and quantified by scanning densitometry using NIH image (version 1.61) (19). The amount of PCR product was normalized to that of GAPDH in each sample. Before a quantitative RT-PCR analysis was carried out, the linear range of amplification was established by changing the number of PCR cycles and the amount of total RNA for reverse transcription. In pilot experiments, the amplification curves of PRL, GH, and GAPDH cDNAs were linear from 28 –32 cycles, 13–17 cycles, and 15–19 cycles, respectively. In addition, we confirmed a linear relationship between the relative signal of each PCR product and the amount of total RNA ranging from 0.5– 4.0 ␮g (data not shown).

Assay for MAP kinase activity Frozen GH3 cells were scraped from the dishes and solubilized in 0.2 ml of 50 mm HEPES (pH 7.4), 0.1% Triton X-100, 4 mm EGTA, 10 mm EDTA, 15 mm Na4P2O7, 100 mm ␤-glycerophosphate, 25 mm NaF, 0.1 mm leupeptin, 75 ␮m pepstatin A, 1 mm dithiothreitol, 1 mm (p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 1 mm Na3VO4, and 100 nm calyculin A (13). The procedures for treatment of cells were carried out at 4 C. After sonication (Sonifier 250, Branson, Danbury, CT), the insoluble materials were removed by centrifugation at 15,000 ⫻ g for 5 min. The protein concentration was determined by the method of Bradford (14) with BSA as standard. Extracts were treated with SDS sample buffer (15) and boiled for 1.5 min. Samples containing the same amount of proteins (15 ␮g protein) were assayed for MAP kinase by SDS-PAGE using MBP as a substrate by the method of Geahlen et al. (16) and Gotoh et al. (17). After the gel was dried, the amount of 32P incorporation into MBP phosphorylated by MAP kinase was quantified using a Bio-Imaging analyzer (FLA2000, Fujifilm, Tokyo, Japan).

Reporter plasmid constructs and luciferase assay Genomic DNA of GH3 cells was isolated using Genomic DNA Isolation Kit (5 Prime33 Prime, Boulder, CO). PCR was carried out to amplify the fragment containing PRL promoter. Using the PCR primers containing XhoI restriction site, which consisted of a sense primer (5⬘-TATCTCGAGGTCTGGTTGATT-3⬘) and an antisense primer (5⬘-ATACTCGAGAACCACTGCTTT-3⬘), the PRL promoter

Hormone measurement GH3 cells were seeded on Falcon 24-well plates and grown under the same conditions as described above. After the cells were preincubated at 37 C for 60 min in KRH, the media were removed, and the cells were incubated at 37 C for the indicated times in 300 ␮l KRH with or without CPT-cAMP. Appropriate inhibitors were added during preincubation and the CPT-cAMP treatment. After incubation, the media were collected and centrifuged at 12,000 ⫻ g for 10 min, and the supernatants were used for assay of PRL and GH. To measure intracellular hormone contents, cells in 35-mm dishes were scraped with 0.5% Triton X-100 in PBS. After sonication, insoluble materials were removed by centrifugation at 15,000 ⫻ g for 5 min, and the supernatants were used for assay of PRL and GH. The amounts of PRL and GH were determined by a double antibody RIA using the rat [125I]PRL assay system and rat [125I]GH assay system (Amersham Pharmacia Biotech, Little Chalfont, UK).

RT-PCR Total RNAs were prepared from GH3 cells using TRIzol LS reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Messenger RNA (mRNA) was reverse transcribed into single stranded cDNA using an oligo(deoxythymidine) primer (Promega Corp., Madison, WI) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). The reaction mixtures were diluted 20-fold and then subjected to PCR amplification of PRL or GH mRNA. The PCR primers were designed based on the published sequences of the PRL mRNA (GenBank AF022933) and GH

FIG. 1. Time course of MAP kinase activation by treatment with CPT-cAMP. A, An autoradiograph of the time course of CPT-cAMP (cAMP)-induced MAP kinase activation. Cell extracts (15 ␮g protein) were prepared and subjected to SDS-PAGE containing MBP as a substrate to assay for MAP kinase activity, as described in Materials and Methods. B, The activities of MAP kinase were quantified. The MAP kinase activity of the control without CPT-cAMP was taken as 100%, and from this value, other values were calculated. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01; *, P ⬍ 0.05 (vs. control).

SYNTHESIS OF PRL BY cAMP VIA MAP KINASE region (⫺609 to 12; the transcription initiation start site of PRL was numbered as 1) was amplified. Amplification of the GH promoter region (⫺563 to 30) was carried out with the PCR primers containing the NheI site, which consisted of a sense primer (5⬘-TATGCTAGCCAACAAAATGGC-3⬘) and an antisense primer (5⬘-ATAGCTAGCAGTTTGGAATCT-3⬘). The fragments of PRL and GH promoter regions were excised with XhoI and NheI restriction enzymes, respectively, and inserted into the XhoI and NheI sites of pGL3-basic luciferase reporter vector (Promega Corp.; termed pGL3-PRLp and pGL3-GHp). Both strands of PRL and GH promoter regions were sequenced with RV primer 3 and GL primer 2 (Promega Corp.), using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM 310 sequencer (Perkin-Elmer Corp.). GH3 cells were cotransfected with pGL3-PRLp or pGL3-GHp (1.0 ␮g of each DNA) and pRL-TK (0.1 ␮g of DNA; Promega Corp.), which contains Renilla luciferase under the herpes simplex virus thymidine kinase promoter using 10 ␮l Lipofectamine (Life Technologies, Inc.) in 1 ml serum-free medium (20). After incubation of the cells for 6 – 8 h, the culture medium was changed to standard medium, and the cells were cultured for an additional 48 h. After the cells were treated with chemicals for each experiment, the activities of firefly luciferase and Renilla luciferase were measured by the Dual Luciferase Reporter Assay System (Promega Corp.) with a luminometer (TD-20/20, Pro-

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mega Corp.) according to the manufacturer’s protocol. The ratio of luminescence signal by firefly luciferase to that by Renilla luciferase was determined.

Overexpression of the catalytic subunit of cAMP-kinase in GH3 cells GH3 cells were grown in the medium as described above, and 2–3 ⫻ 105 cells were plated on a 35-mm petri dish and cultured in standard medium for 24 h. The cells were transfected with the pCAGGSneo expression vector (a gift from Prof. J. Miyazaki, Osaka University, Osaka, Japan; mock-transfected cells) or pFC-PKA (Stratagene, La Jolla, CA), using 20 ␮l Lipofectamine (Life Technologies, Inc.) in 1 ml serum-free medium for 8 h. The culture medium was changed to standard medium, and the cells were cultured for an additional 48 h. Immunostaining of GH3 cells after transfection with the catalytic subunit of cAMP-kinase was carried out as previously reported (11).

Statistical evaluation Values were expressed as the mean ⫾ se. Statistical analysis was performed using one-way ANOVA plus Duncan’s multiple range test. P ⬍ 0.05 was considered statistically significant.

FIG. 2. Effects of CPT-cAMP on synthesis of PRL and GH. A, Effects of CPT-cAMP on intracellular PRL and GH contents. GH3 cells were incubated without (Control) or with 1 mM CPT-cAMP (cAMP). After 48 h, the medium was removed, and cells were washed three times with PBS. Intracellular PRL and GH contents were determined as described in Materials and Methods. B, Effects of CPT-cAMP on the amounts of PRL and GH mRNAs. GH3 cells were incubated without (Control) or with 1 mM CPT-cAMP (cAMP) for 24 h. Total RNA (1.5 ␮g) prepared from the cells was reverse transcribed, and PCR was carried out using PRL, GH, and GAPDH primers. C, The visualized PCR products were quantified by scanning densitometry using NIH Image. The amount was normalized to that of the PCR product of GAPDH in each sample. D, Reporter gene assay of PRL and GH promoters. GH3 cells were cotransfected with pRL-TK vector (0.1 ␮g) and with pGL3-PRLp (1.0 ␮g) or pGL3-GHp (1.0 ␮g) for 8 h. Then the medium was exchanged for the growth medium and incubated without (Control) or with 1 mM CPT-cAMP (cAMP), and cells were further cultured for 48 h. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01; *, P ⬍ 0.05 (vs. control).

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Results Activation of MAP kinase by stimulation with CPT-cAMP

Overexpression of the catalytic subunit of cAMP-kinase and its effects on MAP kinase activation and hormone synthesis

In previous work we reported that only the 42-kDa ERK2 occurred in GH3 cells among ERK family proteins (8). Therefore, we examined ERK2 activation as an indicator of MAP kinase activity in the following experiments. We previously reported that MAP kinase activity was increased slightly, but significantly, by brief treatment with CPT-cAMP, a membrane-permeable cAMP analog (8). It was interesting that the robust activation of MAP kinase was observed by the longer treatment with 1 mm CPT-cAMP (Fig. 1A). When the results shown in Fig. 1A were quantified, activation of MAP kinase reached a maximal peak of 530 ⫾ 12.3% at 10 min and gradually decreased within 60 min (Fig. 1B). The effects of CPT-cAMP were completely inhibited by the addition of 10 ␮m H89 (a cAMP kinase inhibitor) or 50 ␮m PD098059 [a specific MAP kinase kinase (MEK) inhibitor; data not shown]. These results indicate that CPT-cAMP activates MAP kinase via activation of cAMP-kinase and MEK.

To confirm the involvement of cAMP-kinase in the observed phenomena, we transiently overexpressed the catalytic subunit of cAMP-kinase in GH3 cells. To estimate the transfection efficiency of GH3 cells, we immunostained the cells with the anti-cAMP-kinase antibody. We found that 9.8 ⫾ 0.4% of the cells were immunostained among the cells examined (data not shown). Under the conditions used, this indicated that the transfection efficiency of GH3 cells was about 10%. Compared with mock-transfected cells, the activity of MAP kinase was increased to 138 ⫾ 2.0% by overexpression of cAMP-kinase (Fig. 4A). When we measured the intracellular content of hormone, the PRL content was increased to 131 ⫾ 1.7%, whereas the GH content was decreased to 85 ⫾ 3.5% by overexpression of cAMP-kinase (Fig. 4B). Figure 4C showed that overexpression of cAMP-kinase increased the PRL mRNA level to 213 ⫾ 0.3% and decreased the GH mRNA level to 60 ⫾ 2.4%. The activity of the PRL promoter was increased to 387 ⫾ 20.7% by overexpression of cAMP-kinase (Fig. 4D). From these results, we concluded that the effects of overexpression of cAMP-kinase were essentially the same as those of CPT-cAMP treatment. Furthermore, Fig. 5 showed that PD098059 as well as H89 completely inhibited the stimulatory effects of the overexpression of cAMP-kinase on intracellular PRL content. These results

Effects of CPT-cAMP on hormone synthesis in GH3 cells

We next examined the effects of CPT-cAMP on hormone synthesis. GH3 cells were treated with 1 mm CPT-cAMP for 48 h, and the contents of PRL and GH were determined (Fig. 2A). Treatment with CPT-cAMP increased the intracellular PRL content approximately 1.4-fold, but decreased the GH content approximately 0.6-fold. To investigate whether CPTcAMP affected the levels of mRNAs of PRL and GH, we performed quantitative RT-PCR analysis. Treatment of the cells with CPT-cAMP for 24 h significantly increased the level of PRL mRNA, but decreased that of GH mRNA (Fig. 2B). When the results were quantified, the level of PRL mRNA was increased to 425 ⫾ 10.0%, and that of GH mRNA was decreased to 55 ⫾ 1.5% (Fig. 2C). These results were consistent with the results from the measurement of hormone contents. We isolated the genomic DNAs from GH3 cells to obtain the promoter regions of PRL and GH. The sequencing of the obtained fragments revealed that the nucleotide sequences were over 97.5% identical to that of the rat sequences reported (data not shown). A luciferase-coding sequence in pGL3-basic vector was bound to the 3⬘-terminus of each fragment obtained. Figure 2D showed that stimulation of the cells with CPT-cAMP significantly increased the activity of PRL promoter by 2.2 ⫾ 0.1-fold. It was unexpected that the activity of GH promoter was increased 2.0 ⫾ 0.1-fold by CPT-cAMP treatment. Involvement of MAP kinase activation in PRL promoter activation

For the next step we considered the possibility that CPTcAMP may activate PRL promoter via MAP kinase activation. As shown in Fig. 3, the stimulatory effect of CPT-cAMP on PRL promoter was abolished by H89. Furthermore, PD098059 completely inhibited the effect of CPT-cAMP. These results strongly suggest that activation of MAP kinase by the CPT-cAMP treatment is necessary for activation of PRL promoter.

FIG. 3. Luciferase reporter gene assay showing the effects of protein kinase inhibitors on CPT-cAMP-induced activation of PRL promoter. GH3 cells were cotransfected with pGL3-PRLp (1.0 ␮g) and pRL-TK (0.1 ␮g) for 8 h. Then the medium was exchanged for the growth medium, and cells were further cultured for 48 h. The cells were preincubated without or with 10 ␮M H89 and 50 ␮M PD098059 in serum-free medium for 30 min and further treated for 6 h without (Control) or with 1 mM CPT-cAMP (cAMP) in the presence or absence of inhibitors. The activity is expressed as a percentage of the control. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01 vs. control. Differences between cAMP and cAMP plus H89 and between cAMP and cAMP plus PD098059 were statistically significant (P ⬍ 0.01).


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FIG. 4. Effects of overexpression of cAMP-kinase on MAP kinase activation and hormone content. GH3 cells were transfected with pFC-PKA (1.0 ␮g) or pCAGGSneo (1.0 ␮g; MOCK) for 8 h, then the medium was exchanged for the growth medium, and cells were further cultured for 48 h. A, MAP kinase activities were determined. Inset, An autoradiograph of the gel showing the activation of MAP kinase by overexpression of cAMP-kinase. B, Intracellular PRL and GH contents were determined. C, PRL and GH mRNAs were determined by quantitative RT-PCR. The amount of PCR product was normalized to that of GAPDH in each sample. D, PRL promoter activity was determined by luciferase reporter gene assay. GH3 cells were cotransfected with pFC-PKA (1.0 ␮g) or pCAGGSneo (1.0 ␮g; MOCK), and with pGL3-PRLp (1.0 ␮g) and pRL-TK (0.1 ␮g) for 8 h. Then the medium was exchanged for the growth medium, and cells were further cultured for 48 h. In all experiments the value of mock-transfected cells without any stimulant was taken as 100%, and from this value, other values were calculated. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01; *, P ⬍ 0.05 (vs. mock).

clearly demonstrated that the MAP kinase activation by overexpressed cAMP-kinase was necessary for the increase in PRL content. PACAP-induced PRL promoter activation via MAP kinase in GH3 cells

PACAP exists in two bioactive molecular forms. One consists of 38 residues (PACAP38), and another consists of the N-terminal 27 residues of PACAP38 (PACAP27) (2, 21). Both forms have been reported to occur at high concentrations in the hypothalamus and stimulate adenylate cyclase activity. In porcine somatotrophs, PACAP38 was reported to activate phospholipase C (PLC) as well as adenylate cyclase (22). In contrast, PACAP27 was reported to activate adenylate cyclase, but not PLC (22, 23). Therefore, we chose PACAP27 in the following experiments. We first examined the time

course of MAP kinase activation by PACAP27 (Fig. 6, A and B). The activation reached a maximal peak of 240 ⫾ 23.1% at 10 min after stimulation and gradually decreased within 60 min (Fig. 6B). As shown in Fig. 6C, H89 and PD098059 completely abolished the activation of MAP kinase by PACAP27. These results indicate that PACAP27 activates MAP kinase via activation of cAMP-kinase and MEK. Figure 6D showed that PACAP27 activated the PRL promoter 1.5 ⫾ 0.2-fold, and the PACAP27-induced PRL promoter activation was completely inhibited by H89 and PD098059 (Fig. 6D). These results suggest that activation of MAP kinase is necessary for PRL promoter activation by PACAP27. Effects of PACAP27 on hormone synthesis in GH3 cells

We next examined the effects of PACAP27 on hormone synthesis. When we examined the intracellular contents of

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PACAP27-induced PRL secretion was abolished by H89, but was not inhibited by PD098059. These results suggest that MAP kinase is not involved in hormone secretion induced by CPT-cAMP or PACAP27 in GH3 cells. Discussion

FIG. 5. Effects of protein kinase inhibitors on the cAMP-kinaseinduced increase in PRL content in GH3 cells. GH3 cells were transfected with pFC-PKA (1.0 ␮g) or pCAGGSneo (1.0 ␮g; MOCK) in the absence or presence of 2 ␮M H89 or 10 ␮M PD098059 for 5 h, as indicated. The medium was exchanged for the growth medium without or with 10 ␮M H89 or 50 ␮M PD098059, as indicated, and the cells were further cultured for 24 h. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments four times with reproducible results, and representative results are shown. *, P ⬍ 0.05 vs. mock. Differences between pFC-PKA and pFC-PKA plus H89 and between pFC-PKA and pFC-PKA plus PD098059 were statistically significant (P ⬍ 0.05).

PRL and GH, we found that treatment with PACAP27 increased intracellular PRL content 2.1 ⫾ 1.9-fold, but decreased GH content 0.5 ⫾ 4.2-fold (Fig. 7A). Furthermore, the quantitative RT-PCR analysis demonstrated that treatment of the cells with PACAP27 significantly increased the level of PRL mRNA, but decreased that of GH mRNA (Fig. 7B). Figure 7C shows that the PRL mRNA level was increased to 270 ⫾ 7.1%, and the GH mRNA level was decreased to 52 ⫾ 13%. These results indicated that the effects of PACAP27 on PRL and GH were essentially the same as those of CPT-cAMP. Effects of protein kinase inhibitors on cAMP- and PACAP27-induced hormone secretion in GH3 cells

Treatment of the cells with cAMP has been reported to activate the secretion of PRL and GH (24). Therefore, we examined whether the activation of MAP kinase was involved in CPT-cAMP-induced hormone secretion (Fig. 8A). The amounts of secreted PRL and GH increased 3.3- and 2.9-fold, respectively, by treatment of the cells with CPTcAMP (Fig. 8A). Pretreatment with 10 ␮m H89 almost completely inhibited the CPT-cAMP-induced secretion of both hormones. In contrast to the effects of H89, 50 ␮m PD098059 showed no inhibitory effect on the secretion of PRL or GH (Fig. 8A). Furthermore, we examined the PACAP27-induced PRL secretion (Fig. 8B). PRL secretion was increased approximately 1.4-fold by treatment with PACAP27. As expected,

It has been reported that cAMP stimulates the synthesis (25, 26) and secretion (27, 28) of PRL. However, the molecular mechanisms of the cAMP actions remain to be elucidated. We considered that these investigations would greatly contribute to understanding the molecular mechanisms by which the hormones/factors regulate the functions of pituitary cells via cAMP-kinase-mediated pathway. We previously reported that CPT-cAMP activated MAP kinase in GH3 cells (8). The additive effects on MAP kinase activation were observed by treatments with CPT-cAMP and TRH. Recently, we found that the long treatment of the cells with CPT-cAMP dramatically activated MAP kinase, and the activation level was comparable with the effect of TRH. As the stimulatory effect of TRH on PRL synthesis was dependent on the activation of MAP kinase (8), we considered the possibility that the stimulatory effect of CPT-cAMP may also depend on the activation of MAP kinase. Before we examine this possibility, we confirmed that CPT-cAMP increased PRL content, PRL mRNA, and PRL promoter activity. It was interesting that the activation of the PRL promoter was completely inhibited by PD098059 as well as H89. These results suggest that the increase in PRL synthesis by CPT-cAMP is mainly conducted by PRL promoter activation by MAP kinase. It is obvious that the effect of CPT-cAMP was mediated by cAMP-kinase from the following reasons: 1) H89 inhibited the activation of MAP kinase and PRL promoter by CPT-cAMP; and 2) overexpression of the catalytic subunit of cAMP-kinase imitated the effects of CPT-cAMP on MAP kinase activation, PRL content, PRL mRNA, and PRL promoter activity. Both H89 and PD098059 completely inhibited the increase in PRL content. We noticed that the PRL promoter activity was decreased by treatment with H89 or PD098059. The reasons are not clear at present. The long treatment of the cells with the inhibitors may inhibit endogenous activities of cAMP-kinase and MAP kinase under basal conditions, which may maintain the control level of the promoter activity. Inhibition of endogenous activity of cAMP-kinase may be one of the reasons why H89 was shown to inhibit PRL secretion in Fig. 8. The molecular mechanisms by which cAMP-kinase activates MAP kinase are not clear at present. A mitogenic action of cAMP analog was mediated by activation of MAP kinase in PC12 cells (29). In the study using COS-7 cells transfected with Gi-coupled receptor, cAMP stimulated the MAP kinase via the ␤- and ␥-subunits of Gi protein (30). It was also reported that Rap-1, a Ras homolog, was involved in cAMPinduced MAP kinase activation in neurons (31). One of these mechanisms may be involved in the activation of MAP kinase in GH3 cells. In contrast to PRL content, GH content was decreased by treatment of GH3 cells with CPT-cAMP. This result was unexpected, because cAMP has been reported to stimulate both GH secretion (32) and gene expression (33) in other somatotrophs. It should be noted that CPT-cAMP activated


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FIG. 6. PACAP27-induced MAP kinase activation and PRL gene expression. A, An autoradiograph showing the time course of MAP kinase activation by treatment with 100 nM PACAP27. B, The activity of MAP kinase was quantified during the time course. MAP kinase activity without PACAP27 was taken as 100%. C, PACAP27-induced MAP kinase activation without or with inhibitors was quantified. The cells were preincubated without or with 10 ␮M H89 and 50 ␮M PD098059 for 60 min in KRH, as indicated. After preincubation, cells were incubated for 10 min without (Control) or with 100 nM PACAP27 in the presence or absence of inhibitors in KRH, as indicated. D, GH3 cells were cotransfected with pGL3-PRLp (1.0 ␮g) and pRL-TK (0.1 ␮g) for 8 h. Then the medium was exchanged for growth medium, and the cells were further cultured for 48 h. GH3 cells were preincubated without or with 10 ␮M H89 and 50 ␮M PD098059 in serum-free medium for 60 min, as indicated. After preincubation, GH3 cells were incubated without (Control) or with 100 nM PACAP27 in the presence or absence of inhibitors in serum-free medium for 6 h, as indicated, and luciferase activity was measured. The activity is expressed as a percentage of the control. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01; *, P ⬍ 0.05 (vs. control). Differences between PACAP27 and PACAP27 plus H89 and between PACAP27 and PACAP27 plus PD098059 were statistically significant.

GH promoter as well as PRL promoter to the same levels. Therefore, the stability of the mRNA of GH may be decreased by CPT-cAMP in GH3 cells and may, in turn, result in the decrease in GH content. Reduction of GH content by CPTcAMP may be the specific phenomenon in GH3 cells. In previous work we reported that treatment of GH3 cells with TRH reduced the GH content (8). Recently, we found that GH promoter activity was not changed by TRH treatment (Kanasaki, H., T. Yonehara, and E. Miyamoto, unpublished observation). It has been reported that PACAP27 activated only adenylate cyclase and did not activate the PLC-protein kinase C pathway in porcine somatotrophs (22). Therefore, we stimulated GH3 cells with PACAP27, because PACAP38 was reported to activate MAP kinase via multiple protein kinase pathways (22, 23).

We confirmed that PACAP27 activated MAP kinase and PRL promoter and that these effects were completely abolished by the addition of H89 and PD098059. To our knowledge, our finding is the first report on the activation of MAP kinase by PACAP27 via the cAMP-kinase pathway. In previous work we reported that TRH activated MAP kinase mainly via the protein kinase C pathway (8). Recently, we found that TRH-induced PRL synthesis was not inhibited by H89 and confirmed that PD098059 and calphostin C (a protein kinase C inhibitor) inhibited the reactions (8). Furthermore, TRH stimulated PRL promoter activity via activation of MAP kinase (Kanasaki, H., T. Yonehara, and E. Miyamoto, unpublished observation). In this context, Watters et al. reported that estradiol stimulated PRL synthesis via tyrosine phosphorylation of c-Raf-1 and activation of MAP kinase (9). These results suggest that in addi-

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FIG. 8. Effects of protein kinase inhibitors on CPT-cAMP- and PACAP27-induced hormone secretion. The cells were preincubated without or with 10 ␮M H89 and 50 ␮M PD098059 for 30 min in KRH. A, After preincubation, cells were incubated for 60 min without (Control) or with 1 mM CPT-cAMP (cAMP) in the presence or absence of inhibitors in KRH, as indicated. The amounts of PRL and GH secreted in the incubation medium were determined. B, After preincubation as described above, cells were incubated for 2 h without (Control) or with 100 nM PACAP27 in the presence or absence of inhibitors in KRH, as indicated. The amount of PRL secreted in the incubation medium was determined. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. A: **, P ⬍ 0.01 vs. cAMP treatment. B: **, P ⬍ 0.01 vs. control. The difference between PACAP27 and PACAP27 plus H89 was statistically significant (P ⬍ 0.05). FIG. 7. Effects of PACAP27 on synthesis of PRL and GH. A, Effects of PACAP27 on intracellular PRL and GH contents. GH3 cells were incubated without (Control) or with 100 nM PACAP27. After 24 h, the medium was removed, and cells were washed three times with PBS. Intracellular PRL and GH contents were determined as described in Materials and Methods. B, Effects of PACAP27 on the amounts of PRL and GH mRNAs. GH3 cells were incubated without (Control) or with 100 nM PACAP27 for 24 h. Total RNA (1.5 ␮g) prepared from the cells was reverse transcribed, and PCR was carried out using PRL, GH, and GAPDH primers. C, The visualized PCR products were quantified by scanning densitometry using NIH Image. The amount was normalized to that of the PCR product of GAPDH in each sample. Values are the mean ⫾ SE (three wells per condition in a single experiment). We repeated the same experiments at least three times with reproducible results, and representative results are shown. **, P ⬍ 0.01; *, P ⬍ 0.05 (vs. control).

tion to the cAMP-kinase pathway, other pathways of MAP kinase activation are involved in PRL synthesis. cAMP is also known to have stimulatory effects on the secretion of PRL and GH (24). We confirmed that treatment of GH3 cells with CPT-cAMP induced the secretion of PRL and GH. Although these stimulatory effects were blocked by H89, PD098059 did not affect the secretion of PRL and GH. We reported that PD098059 did not show any effect on TRHinduced secretion of PRL and GH (8). Taken together, MAP kinase may not have a critical role in the hormone secretion induced by CPT-cAMP and TRH in GH3 cells. In contrast, we found that KN93, an inhibitor of Ca2⫹/calmodulin-dependent protein kinase II, inhibited CPT-cAMP-induced secretion


of PRL and GH (Yonehara, T., H. Kanasaki, and E. Miyamoto, unpublished observation). In previous work we reported that a high concentration of wortmannin, an inhibitor of myosin light chain kinase in high doses, inhibited TRH-induced secretion of PRL and GH. Therefore, it may be interesting to examine whether treatment with CPT-cAMP activates the Ca2⫹ signaling pathway, including calmodulin-dependent protein kinase II and myosin light chain kinase, followed by increases in hormone secretion. The promoter of PRL has been reported to include binding sites for Ets-1 and Pit-1/GHF-1. Overexpression of these transcription factors synergistically enhanced PRL synthesis in a Ras/Raf cascade-dependent manner (34). It was also reported that two cAMP response element (CRE) sites were involved in cAMP-stimulated-Pit-1/GHF-1 expression. CREbinding protein (CREB) has been reported to be phosphorylated by MAP kinase-activated protein kinase, possibly through p70S6K, as well as cAMP kinase (35, 36). Therefore, CREB may also have a critical role in PRL gene expression in the cascade of interaction of cAMP-kinase- and MAP kinase-induced signalings. In our experiments the reasons why the activation of PRL promoter by overexpression of cAMP-kinase was more pronounced than the activation of MAP kinase are not clear at present. One explanation may be that it is due to different assay conditions, such as the time course. It is likely that CREB is directly phosphorylated by overexpressed cAMP-kinase. However, as PD098059 completely abolished the increase in the intracellular content of PRL, the MAP kinase pathway would be critical for the regulation of PRL by cAMP-kinase. Acknowledgments We gratefully acknowledge Drs. H. Ohkubo, H. Oda, and T. Kawano (Kumamoto University) for technical support and critical comment on the manuscript.

References 1. Gershengorn MC 1986 Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. Annu Rev Physiol 48:515–526 2. Miyata A, Arimura A, Dahl DH, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH 1989 Isolation of a novel 38 residue hypothalamic peptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164:567–574 3. Coleman DT, Bancroft C 1993 Pituitary adenylate cyclase-activating polypeptide stimulates prolactin gene expression in a rat pituitary cell line. Endocrinology 133:2726 –2742 4. Coleman DT, Chen X, Sassaroli M, Bancroft C 1996 Pituitary adenylate cyclaseactivating polypeptide regulates prolactin promoter activity via a protein kinase A-mediated pathway that is independent of the transcriptional pathway employed by thyrotropin-releasing hormone. Endocrinology 137:1276 –1285 5. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling : transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179 –185 6. Armen H, Tashjian Jr AH 1979 Clonal strains of hormone-producing pituitary cells. Methods Enzymol 58:527–535 7. Ohmichi M, Sawada 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. Evidence of a role for early tyrosine phosphorylation. J Biol Chem 269:3783–3788 8. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E 1999 Mitogen-activated protein kinase activation by stimulation with thyrotropinreleasing hormone in rat pituitary GH3 cells. Biol Reprod 61:319 –325 9. Watters JJ, Chun TY, Kim YN, Bertics PJ, Gorski J 2000 Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells. Mol Endocrinol 14:1872–1881 10. Deibler GE, Martenson RE, Kies MW 1972 Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem 2:139 –165 11. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E 2000 Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells. Biol Reprod 62:1486 –1494

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12. Munir I, Fukunaga K, Miyazaki K, Okamura H, Miyamoto E 1999 Mitogenactivated protein kinase activation and regulation of cyclooxygenase 2 expression by platelet-activating factor and hCG in human endometrial adenocarcinoma cell line HEC-1B. J Reprod Fertil 117:49 –59 13. Kurino M, Fukunaga K, Ushio Y, Miyamoto E 1995 Activation of mitogenactivated protein kinase in cultured rat hippocampal neurons by stimulation of glutamate receptors. J Neurochem 65:1282–1289 14. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 –254 15. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680 – 685 16. Geahlen RL, Anostario M Jr, Low PS, Herrison ML 1986 Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 153:151–158 17. Gotoh Y, Nishida E, Yamashita T, Hoshi M, Kawakami M, Sasaki H 1990 Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells : identity with the mitogenactivated protein MAP kinase of fibroblastic cells. Eur J Biochem 193:661– 669 18. Barta A, Richards RI, Baxter JD, Shine J 1981 Primary structure and evolution of rat growth hormone gene. Proc Natl Acad Sci USA 78:4867– 4871 19. Takeuchi Y, Yamamoto H, Miyakawa T, Miyamoto E 2000 Increase of brainderived neurotrophic factor gene expression in NG108 –15 cells by the nuclear isoforms of Ca2⫹/calmodulin-dependent protein kinase II. J Neurochem 74:1913–1922 20. Tabuchi H, Yamamoto H, Matsumoto K, Ebihara K, Takeuchi Y, Fukunaga K, Hiraoka H, Sasaki Y, Shichiri M, Miyamoto E 2000 Regulation of insulin secretion by overexpression of Ca2⫹/calmodulin-dependent protein kinase II in insulinoma MIN6 cells. Endocrinology 141:2350 –2360 21. Miyata A, Dahl RD, Jiang L, Kitada C, Kubo K, Fujino M, Minamino N, Arimura A 1990 Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). Biochem Biophys Res Commun 170:643– 648 22. Martinez-Fuentes AJ, Castano JP, Gracia-Navarro F, Malagon MM 1998 Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 activate common and distinct intracellular signaling pathways to stimulate growth hormone secretion from porcine somatotropes. Endocrinoligy 139:5116 –5124 23. Spengler D, Waeber C, Pontaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L 1993 Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170 –175 24. Sletholt K, Hang E, Gautvik KM 1987 On the involvement of cyclic AMP and extracellular Ca2⫹ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture. Bioscience Reports 7:93–105 25. Maurer RA 1981 Transcriptional regulation of the prolactin gene by ergocryptine and cyclic AMP. Nature 294:94 –97 26. Liang J, Kim KE, Schoderbek WE, Manuer RA 1992 Characterization of a nontissue-specific, 3⬘,5⬘-cyclic adenosine monophosphate-responsive element in the proximal region of the rat prolactin gene. Mol Endocrinol 6:885– 892 27. Swennen L, Denef C 1982 Physiological concentrations of dopamine decrease adenosine 3⬘,5⬘-monophosphate levels in cultured rat anterior pituitary cells and enriched populations of lactotrophs: evidence for a causal relationship to inhibition of prolactin release. Endocrinology 111:398 – 405 28. Delbeke D, Scammell JG, Dannies PS 1984 Difference in calcium requirements for forskolin-induced release of prolactin from normal pituitary cells and GH4C1 cells in culture. Endocrinology 114:1433–1440 29. Frodin M, Peraldi P, Van Obberghen E 1994 Cyclic AMP activates mitogenactivated protein kinase cascade in PC12 cells. J Biol Chem 269:6207– 6214 30. Faure M, Voyno-Yasenetskaya TA, Bourne HR 1994 cAMP and ␤␥ subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 269:7851–7854 31. Vossler MR, Yao H, York RD, Pan M-G, Rim CS, Stork PJS 1997 cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap-1-dependent pathway. Cell 89:73– 82 32. Brain CE, Chomczynski P, Downs TR, Frohman LA 1991 Impaired generation of cyclic adenosine 3⬘,5⬘-monophosphate in a somatomammotroph cell line derived from dwarf (dw) rat anterior pituitaries. Endocrinology 129:3410 –3416 33. Dannies PS, Tashjian AH Jr 1980 Action of cholera toxin on hormone synthesis and release in GH cells: evidence that adenosine 3⬘,5⬘-monophosphate does not mediate the decrease in growth hormone synthesis caused by thyrotropin-releasing hormone. Endocrinology 106:1532–1536 34. Bradford AP, Conrad KE, Wasylyk C, Wasylyk B, Gutierrez-Hartmann A 1995 Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain. Mol Cell Biol 15:2849 –2857 35. Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959 –962 36. Pende M, Fisher TL, Simpson PB, Russell JT, Blenis J, Callo V 1997 Neurotransmitter- and growth factor-induced cAMP response element binding protein phosphorylation in glial cell progenitors: role of calcium ions, protein kinase C, and mitogen-activated protein kinase/ribosomal S6 kinase pathway. J Neurosci 17:1291–1301