Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells

Molecular and Cellular Endocrinology 339 (2011) 172–179

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Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells夽 Tselmeg Mijiddorj, Haruhiko Kanasaki ∗ , Indri N. Purwana, Aki Oride, Kohji Miyazaki Department of Obstetrics and Gynecology, Shimane University School of Medicine, 89-1 Enya Cho, Izumo City 693-8501, Shimane Prefecture, Japan

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Article history: Received 2 December 2010 Received in revised form 23 March 2011 Accepted 15 April 2011 Keywords: PACAP Prolactin TRH

a b s t r a c t In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3 ± 0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8 ± 0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Prolactin is secreted from lactotrophs and somatolactotrophs in the anterior pituitary gland. Of the pituitary hormone secreting cells, which also include corticotrophs, thyrotrophs, and gonadotrophs, it is the somatotrophs and lactotrophs that are acidophilic. This property suggests a common origin for the latter two cell types. Consistent with this, the acidophilic cells of the anterior pituitary gland of rats are approximately equally divided between somatotrophs, which secrete only growth hormone; lactotrophs, which secrete only prolactin; and somatolactotrophs, which secrete both prolactin and growth hormone (Frawley et al., 1985). GH3 cells are a clonal strain of rat pituitary tumor cells,

夽 This work was supported in part by Grants in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (H. K. and K. M.) and Grants from Uehara Memorial Foundation (H. K.). ∗ Corresponding author. Tel.: +81 853 20 2268; fax: +81 853 20 2264. E-mail address: [email protected] (H. Kanasaki). 0303-7207/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.04.010

which can synthesize and secrete both prolactin and growth hormone because the cells may be cultured as either somatotrophs or somatolactotrophs (Boockfor et al., 1985). As GH3 cells have many properties common to normal lactotrophs and somatolactotrophs, these cells are a valuable model for studying the regulation of prolactin-secreting cell function. Although it is well known that dopamine, a prolactin inhibiting factor, largely participates in the control of prolactin release (BenJonathan and Hnasko, 2001), prolactin secretion is also regulated by several hypothalamic hormones such as thyrotropin-releasing hormone (TRH). TRH, a primary secretagogue for thyrotropinstimulating hormone from thyrotrophs, also regulates prolactin synthesis and secretion, although it is unclear at present whether TRH is a physiological regulator of prolactin (Yamada et al., 1997, 2006). TRH stimulates inositol phospholipid metabolism by activating membrane receptors in prolactin-producing cells and accelerates its signaling cascade (Gershengorn, 1986). Using GH3 cells, we have previously reported that stimulation of GH3 cells with TRH increases the activity of extracellular signal-regulated kinase (ERK). ERK is a member of the mitogen-activated protein

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2. Materials and methods 2.1. Materials The following chemicals and reagents were obtained from the indicated sources: Fetal Bovine Serum, Trypsin (GIBCO, Invitrogen, Carlsbad, CA); Dulbecco’s modified eagle medium (DMEM), penicillin–streptomycin, TRH (Sigma Chemical Co., St. Louis, MO), PACAP (Peptide Institute, Osaka, Japan).

2.2. Cell culture GH3 cells were plated in 35-mm tissue culture dishes and incubated in highglucose DMEM containing 10% heat-inactivated FBS and 1% penicillin–streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. After 24 h, the culture medium was changed to high-glucose DMEM containing 1% heat-inactivated FBS and 1% penicillin–streptomycin and incubated without (control) or with test reagents for the indicated times.

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kinase families, which signals via protein kinase C. We have previously shown that TRH-induced ERK activation is involved in prolactin synthesis but not in prolactin secretion (Kanasaki et al., 1999). TRH also inhibits DNA synthesis and reduces growth hormone synthesis via ERK (Kanasaki et al., 1999, 2002). Pituitary adenylate cyclase-activating polypeptide (PACAP) was first isolated from an extract of ovine hypothalamus on the basis of its ability to stimulate cAMP formation in rat pituitary cells (Miyata et al., 1989). This peptide exists in two amidated forms, PACAP27 and PACAP38, with 27 and 38 amino acid residues, respectively (Miyata et al., 1990). PACAP is found in high concentrations in the hypothalamus (Arimura et al., 1991). PACAP immunostaining fibers are present in the median eminence (ME) and PACAP is detected in the hypophysial portal blood (Dow et al., 1994). These observations suggest that PACAP works as a hypophysiotropic factor. PACAP exerts its action via G-protein-linked receptors, such as the PACAP type I receptor (PAC1R) and the VPAC receptors (Vaudry et al., 2009). PACAP acts predominantly via PAC1R and stimulates both inositol phosphate turnover and cAMP accumulation, and is more potent than vasoactive-intestinal polypeptide (VIP) (Miyata et al., 1989). PACAP is widely distributed throughout the central nervous system, and plays an important role in the regulation of various physiological events including neuronal differentiation, behavior, and hormone secretion (Hashimoto, 2002). The involvement of PACAP in the regulation of pituitary gonadotropins is well established (McArdle and Counis, 1996; Winters and Moore, 2007; Heinzlmann et al., 2008). The studies aimed at investigating the effect of PACAP on prolactin synthesis and secretion by pituitary cells have led to controversial results. PACAP was initially believed to be devoid of prolactin-releasing activity in cultured rat adenohypophysial cells (Miyata et al., 1989; Hart et al., 1992). Additionally, PACAP was found to have no effect on prolactin release from cultured ovine (Sawangjaroen et al., 1997) and bovine (Hashizume et al., 1994) pituitary cells. Other studies, however, have shown that PACAP can either slightly increase or inhibit prolactin release from rat pituitary cells (Arbogast and Voogt, 1994; Jarry et al., 1992). PACAP has no effect on prolactin release in female lactating rats with actively nursing pups, but it stimulates prolactin release in lactating rats separated from their pups (Arbogast and Voogt, 1994). These results suggest that PACAP might regulate prolactin; however, the stimulatory effect of PACAP on prolactin synthesis and release in single prolactin-producing cells is inconclusive in experimental models. In this present study, we investigated the action of PACAP and the function of PAC1R in single clones of prolactin-producing cells using somatolactotroph, GH3 cells. Prolactin gene expression, and its association with TRH, and intracellular signaling systems in ERK activation were also examined in this study.

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Fig. 1. Effect of PACAP and TRH on prolactin promoter activity. GH3 cells were cotransfected with 0.1 ␮g of PRL-TK vector and 2.0 ␮g of luciferase vector linked with the prolactin promoter (Prolactin-Luc). After 48 h of culture, the cells were then treated with 100 nM of PACAP and 100 nM of TRH for 6 h. A luciferase assay was performed to examine prolactin promoter activities, which were then normalized to PRL-TK activity, and are expressed as the fold of activation over the unstimulated controls. Values are means ± SEM (three independent experiments done with triplicate samples). *P < 0.05; **P < 0.01 vs. control.

2.3. Transfections and luciferase assays The reporter constructs used were generated by fusing −609/+12 of the prolactin gene to the firefly luciferase cDNA in pGL3, as previously described (Kanasaki et al., 2002). GH3 cells were transiently transfected by electroporation (Harada et al., 2007) with 2.0 ␮g/dish of reporter construct and 0.1 ␮g of pRL-TK (Promega), and plated in 35-mm tissue culture dishes. When PAC1R expressed to GH3 cells, PAC1 receptor expressing vector (HA-tagged PAC1/pEF-BOS in pCAM17) which was kindly provided from Prof. A. Baba (Osaka University) was used. After stimulation, cells were washed with ice-cold PBS and lysed with PLB (Passive Lysis Buffer, Promega). Cell debris were pelleted by centrifugation at 14,000 × g for 10 min at 4 ◦ C, and firefly luciferase and Renilla luciferase activities were measured in the supernatants with the Dual-Luciferase Reporter Assay System, using a luminometer (TD-20/20) (Promega) according to the manufacturer’s protocol. Luciferase activity was normalized for Renilla luciferase activity to correct for transfection efficiency, and the results were expressed as the fold increase compared to the unstimulated control. All experiments were performed independently, three times, each in triplicate. 2.4. Western blot analysis GH3 cells were rinsed with PBS, then lysed on ice with RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 mg/ml Aprotinin, and 1 mM sodium orthovanadate, scraped for 20 s, and centrifuged at 14,000 × g for 10 min at 4 ◦ C. The protein concentration was measured in the cell lysates using the Bradford method of protein quantitation. Ten micrograms of denatured protein per well was separated on a 10% SDS-PAGE gel according to standard protocols. Protein was transferred onto polyvinylidene difluoride membranes (Hybond-P PVDF, Amersham Biosciences, Little Chalfont, UK), which were blocked for 2 h at room temperature in Blotto (5% milk in TBS). Membranes were incubated with phospho-ERK1/2 antibody (p-ERK) (1:250 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Blotto overnight at 4 ◦ C and washed 3 times for 10 min per wash with TBS/1% Tween. A subsequent incubation with monoclonal HRP-conjugated antibody was carried out for 1 h at room temperature in Blotto, and the appropriate additional washes were performed. Following chemiluminescence (ECL) detection (Amersham Biosciences, Little Chalfont, UK), membranes were exposed onto X-ray film (Fujifilm, Tokyo, Japan). After strip washing (Restore buffer, Pierce Chemical Co., Rockford, IL), membranes were reprobed with ERK1/2 antibody (1:10,000 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature, followed by incubation with HRPconjugated secondary antibody and continuation of the procedure as described above. For determination of PAC1R, membranes were incubated with PAC1R antibody (1:500 dilution) (Abcam Inc., Cambridge, MA) and were reprobed with ␤-actin antibody (1:200 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Films were analyzed by densitometry, and the intensities of p-ERK1/2 were normalized to that of total ERK1/2 to correct protein loading. The corrected results for ERK phosphorylation were expressed as the fold induction over the controls. 2.5. RNA preparation, reverse transcription, and real-time quantitative RT-PCR procedure Total RNA from untreated or treated GH3 cells was extracted using the commercially available extraction method Trizol-S (GIBCO BRL Life Technologies), according to the manufacturer’s instructions. To obtain cDNA, 1.0 ␮g of total RNA was reverse

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Fig. 2. Effect of increasing concentrations of PACAP on prolactin promoter activity. GH3 cells were transfected without (Mock) (A) or with PAC1R expression vector (1.0 ␮g) (B), together with both 2.0 ␮g of prolactin-Luc and PRL-TK (0.1 ␮g) vectors. 48 h after transfection, cells were treated with the indicated amount of PACAP for 6 h. A luciferase assay was performed to examine prolactin promoter activities, which were then normalized to PRL-TK activity, and are expressed as the fold of activation over the unstimulated controls. Values are means ± SEM (three independent experiments done with triplicate samples). *P < 0.05; **P < 0.01 vs. control. (C) Expression level of PAC1R protein in mock and 4.0 ␮g of PAC1R expression vector transfected GH3 cells were determined by Western blotting as described in Section 2. transcribed using an oligo-dT primer (Promega), and was prepared using a First Strand cDNA Synthesis Kit (Invitrogen) in reverse transcription (RT) buffer. The preparation was supplemented with 0.01 dithiothreitol (DTT) and 1 mM each of dNTP, and 200 units of RNAse inhibitor/human placenta ribonuclease inhibitor (Ribonuclease Inhibitor, Code No. 2310, Takara, Tokyo, Japan) in a final volume of 10 ␮l. The reaction was incubated at 37 ◦ C for 60 min. Quantification of prolactin, growth hormone and PAC1-R mRNA was obtained through real-time quantitative PCR (ABI Prism 7000, Perkin Elmer Applied Biosystems, Foster City, CA) following the manufacturer’s protocol (User Bulletin No. 2), and utilizing a Universal Probe Library Probe and Fast Start Master Mix (Roche Diagnostics, Mannheim, Germany). Using specific primers for prolactin, growth hormone (Kanasaki et al., 2002) and PAC1R (Kanasaki et al., 2009), the simultaneous measurement of mRNA and GAPDH permitted normalization of the amount of cDNA added per sample. For each set of primers, a no template control was included. The thermal cycling conditions were: 95 ◦ C, 10 min for denaturation, followed by 40 cycles of 95 ◦ C, 15 s, and 60 ◦ C, 1 min. The crossing threshold was determined using PRISM 7000 software and post amplification data were analyzed using delta–delta CT method with Microsoft Excel.

3.2. PAC1R expression potentiates the effect of PACAP on prolactin promoter activity Although the effect of PACAP on prolactin promoters was limited in GH3 cells, the effect was dose dependent (Fig. 2A); although 100 nM of PACAP achieved only a 1.63 ± 0.37-fold increase in prolactin promoter activity. This was thought to be the result of a deficiency in PAC1R in the GH3 cells. To test this hypothesis, we transiently transfected the expression vectors for PAC1R and examined the effect of PACAP. Although GH3 cells did not have much

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All experiments were independently repeated at least three times. Each experiment was performed with triplicate samples (luciferase assays) or duplicate samples (Western blot) in each experimental group. Values were expressed as means ± SEM. Statistical analysis was performed using the one-way ANOVA followed by Duncan’s multiple range test. P < 0.05 was considered statistically significant.

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3. Results 3.1. Activation of the prolactin promoter by PACAP and TRH Prolactin promoter activity following stimulation by PACAP and TRH was examined using GH3 cells. Both PACAP and TRH stimulated prolactin promoter activity; however, the effect of PACAP was limited. While PACAP increased prolactin promoter activity significantly (1.3 ± 0.1-fold), the magnitude of this increase was significantly less than the 3.6 ± 0.1-fold increase seen following TRH stimulation (Fig. 1).

0 Mock

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Fig. 3. Effect of PAC1R overexpression on prolactin promoter activities. GH3 cells were transfected with the indicated amount of PAC1R expression vector, together with either 2.0 ␮g of prolactin-Luc and PRL-TK (0.1 ␮g). 48 h after transfection, cells were treated with 100 nM PACAP for 6 h. A luciferase assay was performed to examine prolactin promoter activities which were then normalized to PRL-TK activity, and are expressed as the fold stimulation of the control. Values are means ± SEM of the fold induction taken from three independent experiments done with triplicate samples. *P < 0.05; **P < 0.01 vs. control.

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Fig. 4. Effect of combined treatment with TRH and PACAP on prolactin promoter activities in PAC1R overexpressing GH3 cells. GH3 cells were transfected without (A) or with 1.0 ␮g PAC1-receptor expression vector (B), together with either prolactin-Luc or PRL-TK (0.1 ␮g). 48 h after transfection, cells were treated with either 100 nM TRH or 100 nM PACAP, or both as indicated for 6 h A luciferase assay was performed to examine prolactin promoter activities which were then normalized to PRL-TK activity, and are expressed as the fold of activation over the unstimulated controls. Values are means ± SEM from three independent experiments done with triplicate samples. **P < 0.01 vs. control. In the PAC1R expressing cells, the differences between TRH and TRH + PACAP, and between PACAP and TRH + PACAP were statistically significant (P < 0.01 and P < 0.05).

PAC1R proteins in mock transfected cells, expression level of PAC1R was dramatically increased after transfection with PAC1R vectors (Fig. 2B). Following transfection of the PAC1R into the cells, the response of the prolactin promoter activity to PACAP stimulation was dramatically increased. In these transfected cells, 1 nM PACAP

increased prolactin promoter activity 4.1 ± 0.6-fold (Fig. 2C). To examine whether the number of PAC1R expressed in the cells influences the response to PACAP, GH3 cells were transfected with increasing amounts of PAC1R expression vector and stimulated with a constant concentration of PACAP. The response to PACAP in PAC1R expressing cells was significantly increased in comparison

Fig. 5. Effect of TRH and PACAP on ERK phosphorylation. GH3 cells were transfected without (Mock) (A) or with 1.0 ␮g PAC1R expression vector (B). 48 h after transfection, cells were treated with 100 nM GnRH, 100 nM PACAP or both for 10 min. After cells were harvested, cell lysates (10 ␮g) were subjected to SDS-PAGE followed by Western blotting and incubation with antibody against phosphorylated ERK and total ERK. The visualized bands were quantified by scanning densitometry using NIH Image and normalized to total ERK. Results were expressed as the fold increase over the non stimulated cells (control) and represent the mean ± SEM from three independent experiments done with triplicate samples. We performed three independent experiments and representative autoradiographs are shown. **P < 0.01 vs. control. In the PAC1R expressing cells, the differences between TRH and TRH + PACAP, and between PACAP and TRH + PACAP was statistically significant (P < 0.01 and P < 0.05).

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Fig. 6. Messenger RNA levels of prolactin and growth hormone by TRH and PACAP stimulations. GH3 cells were transfected without (Mock) and with 1.0 ␮g PAC1R expression vector. After cells were treated with 100 nM TRH (A and B) and 100 nM PACAP (C and D) for 48 h, respectively, messenger RNA was extracted and reverse transcripted. Prolactin (A and C) and growth hormone (B and D) mRNA levels were measured with quantitative real time PCR. Results are expressed as fold stimulation over unstimulated cells and present the mean ± SEM of three independent experiments, each performed in triplicate samples. *P < 0.05; **P < 0.01 vs. control.

to mock transfected cells, and the increases were dose dependent (Fig. 3).

expressing GH3 cells. The combination of TRH and PACAP had a synergistic effect on ERK phosphorylation (Fig. 5B).

3.3. Prolactin promoter activity by combined stimulation with TRH and PACAP

3.5. Effects of PACAP on mRNA expression levels of prolactin and growth hormone

Both TRH and PACAP stimulated prolactin promoter activity in prolactin-producing GH3 cells. Next, we examined the effect of combined treatment with TRH and PACAP on prolactin promoters. In mock transfected GH3 cells, 10 nM of PACAP failed to stimulate prolactin promoter significantly and did not modify the action of TRH (Fig. 4A). In PAC1R expressing GH3 cells, PACAP increased prolactin promoter activity more than did TRH. TRH and PACAP increased the activity of prolactin promoters up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively in these cells, and combined stimulation with PACAP and TRH further increased prolactin promoters to 6.8 ± 0.6-fold (Fig. 4B).

We have previously reported that TRH increases prolactin mRNA expression, and decreases the mRNA for growth hormone (Kanasaki et al., 2002). Indeed, TRH showed similar effects on prolactin and growth hormone mRNA levels in GH3 cells regardless of whether they expressed PAC1R (Fig. 6A and B). PACAP significantly increased prolactin mRNA expression in PAC1R expressing GH3 cells (2.8 ± 0.4-fold) (Fig. 6C). In contrast to the action of TRH, PACAP did not decrease growth hormone mRNA expression (Fig. 6D).

3.4. ERK activation by PACAP stimulation Previous studies have shown that TRH activates both ERK isoforms (ERK1 and ERK2), and that ERK activation is important for the induction of the prolactin promoter (Kanasaki et al., 1999, 2002; Oride et al., 2008). The effect of PACAP on ERK phosphorylation was next examined. In mock transfected cells, TRH increased ERK phosphorylation up to 3.9 ± 0.2-fold; however, PACAP failed to activate ERK. TRH-increased ERK phosphorylation was not modulated in the presence of PACAP (Fig. 5A). PACAP increased ERK phosphorylation to a similar extent as that achieved with TRH stimulation in PAC1R

3.6. Effects of PAC1R expression on TRH-induced prolactin promoter activity and ERK phosphorylation Adequate expression of PAC1R exerts the stimulatory effect of PACAP on prolactin expression, as described above. Next, we examined whether PAC1R expression affects the action of TRH in GH3 cell. By transfection of PAC1R to the cells, prolactin promoter activity induced by TRH was significantly increased compared to the mock transfected cells. Increasing amounts of PAC1R expression vector further stimulated TRH-induced prolactin promoter activity and significant difference was obtained when 4.0 ␮g of PAC1R vectors were transfected to the cells (Fig. 7A). Similar potentiation of TRH action was also observed on ERK phosphorylation. TRHinduced ERK phosphorylation was much more increased in the cells

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Fig. 8. PAC1R expression by TRH and PACAP stimulation. GH3 cells were treated with 100 nM TRH and 100 nM PACAP for 48 h. (A) Messenger RNA was extracted and reverse transcripted. PAC1R mRNA levels were measured with quantitative real time PCR. Results are expressed as fold stimulation over unstimulated cells and present the mean ± SEM of three independent experiments, each performed in triplicate samples. **P < 0.01 vs. control. (B) Expression level of PAC1R protein in GH3 cells treated by TRH and PACAP for 48 h was determined by Western blotting as described in Section 2.

strated that PACAP, but not TRH, increased the PAC1R protein expression in GH3 cells (Fig. 8). 4. Discussion Fig. 7. Effects of PAC1R expression on TRH-induced prolactin promoter activity and ERK phosphorylation. (A) GH3 cells were transfected with the indicated amount of PAC1R expression vector, together with either 2.0 ␮g of prolactin-Luc and PRL-TK (0.1 ␮g). Forty eight hours after transfection, cells were treated with 100 nM TRH for 6 h. A luciferase assay was performed to examine prolactin promoter activities which were then normalized to PRL-TK activity, and are expressed as the fold stimulation of the control in mock. Values are means ± SEM of the fold induction taken from three independent experiments done with triplicate samples. *P < 0.05 vs. control in mock. The difference between TRH stimulation in mock and in 4.0 ␮g/well PAC1R overexpressing cells was statistically significant (P < 0.05). (B) GH3 cells were transfected without (Mock) or with 4.0 ␮g/well PAC1R expression vector. Forty eight hours after transfection, cells were treated with 100 nM GnRH for 10 min. After cells were harvested, cell lysates (10 ␮g) were subjected to SDS-PAGE followed by Western blotting and incubation with antibody against phosphorylated ERK and total ERK. The visualized bands were quantified by scanning densitometry using NIH Image and normalized to total ERK. Results were expressed as the fold increase over the non-stimulated control in mock. Values are means ± SEM of the fold induction taken from three independent experiments done with triplicate samples. *P < 0.05; **P < 0.01 vs. control in mock. The difference between TRH stimulation in mock and in 4.0 ␮g/well PAC1R overexpressing cells was statistically significant (P < 0.01).

which overexpressed PAC1R (Fig. 7B). The basal level of ERK phosphorylation was increased in the cells transfected with PAC1Rs.

3.7. PAC1R expression by PACAP stimulation PACAP did not exert its stimulatory effect on prolactin promoter activity in GH3 cells without transfection of PAC1R expression vectors, probably due to the lack of adequate endogenous PAC1R. Next, we examined whether TRH or PACAP have a potency to induce PAC1R in GH3 cells. Incubation of the cells with TRH failed to increase PAC1R mRNA, however, PACAP significantly increased its own PAC1R gene expression. Western blotting analysis demon-

The intravenous injection of PACAP into rats induces an increase in plasma prolactin concentration (Murakami et al., 1995; Yamauchi et al., 1995), and prolactin levels are significantly reduced in PACAP knockout animals (Isaac and Sherwood, 2008). In cultured rat adenohypophysial cells, PACAP is devoid of prolactin-releasing activity (Hart et al., 1992). PACAP has also been reported to have no effect on prolactin release from cultured ovine pituitary cells (Sawangjaroen et al., 1997). Benter et al. (1995) described differential effects of PACAP on prolactin release depending on if rat pituitary cells were cultured in a monolayer or as an aggregate. In addition, PACAP has been shown to regulate prolactin through modulation of various hypothalamic factors (Tohei et al., 2001; Anderson and Curlewis, 1998). In this present study, we examined the function of PACAP and its PAC1R, on prolactin expression using pituitary prolactinproducing GH3 cells. As shown previously, PACAP stimulated prolactin transcriptional activity significantly over baseline; however, the absolute magnitude of this increase was minimal (Fig. 1). Consistent with the observation that a higher concentration of PACAP potentiated its effect on the prolactin promoter, prolactin promoter activity was further increased by overexpression of the PAC1R in GH3 cells (Figs. 2 and 3). These results suggest that the response to PACAP depends on the density of PAC1R on the cell surface. GH3 cells are a clonal strain of rat pituitary cells. Although these cells have many properties common to normal pituitary cells, they lack a functional dopamine D2 receptor (Missale et al., 1991). It is plausible that in the process of immortalization, or after multiple passages, the PACAP receptor may have also been lost. The GH3 cells overexpressing PAC1R responded to PACAP by increasing prolactin promoter activity, to a similar or greater degree than the increase obtained following TRH stimulation (Fig. 4B). In addition, ERK was phosphorylated to a greater extent following

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PACAP stimulation in PAC1R expressing GH3 cells (Fig. 5B). We have previously reported that TRH increased ERK and that ERK activation is involved in prolactin synthesis but not in prolactin secretion in GH3 cells (Kanasaki et al., 1999, 2002). Considering the observation that PACAP-induced prolactin promoter activity in PAC1R expressing GH3 cells is proportional to ERK activation, we again verified that ERK activation influenced prolactin gene expression. TRH is thought to stimulate inositol phospholipid by activating membrane TRH receptors, which in turn stimulate the protein kinase C (PKC) pathway and ultimately release calcium from intracellular storage sites (Gershengorn, 1986). ERK is phosphorylated and activated via PKC-dependent and PKC-independent pathways (Ohmichi et al., 1994), with the former pathway activating MEKkinase with PKC. The latter pathway is associated with tyrosine phosphorylation of Shc proteins, which are linked to Ras-dependent MEKK activation. MEKK activates MEK, which ultimately activates ERK (Winitz et al., 1993). In contrast to TRH, PACAP mainly stimulates cAMP accumulation by binding to the PAC1 receptor which couples with adenylate cyclase via Gs proteins. PAC1R also couples with Gq proteins in a manner similar to TRH, and activates phospholipase C/PKC pathways (Miyata et al., 1989; Martinez-Fuentes et al., 1998). Both TRH and PACAP increased ERK phosphorylation following prolactin gene expression in the setting of abundantly expressed PAC1R. In addition, the combination of TRH was synergistic with respect to ERK phosphorylation and prolactin gene expression (Figs. 4 and 5), which supports the notion that TRH and PACAP share signal transduction systems to regulate prolactin gene expression. Interestingly, the effects of TRH on prolactin promoters and ERK phosphorylation were further potentiated in the cells which have adequate PAC1R (Fig. 7). As we confirmed that TRH does not bind to the PAC1R (data not shown), the existence of PAC1R itself might augment the ability of TRH to stimulate prolactin synthesis. The mechanisms of how the existence of PAC1R potentiates the effect of TRH on prolactin gene expression still remain unknown. Endogenous PAC1R expression in GH3 cells was increased by PACAP, but not by TRH (Fig. 8). First, we expected that the TRH regulates PAC1R expression and modulates its ability to stimulate prolactin, similar to the phenomenon observed in other pituitary cell types (Kanasaki et al., 2009; Purwana et al., 2010). In the gonadotroph cell line, L␤T2, GnRH regulates PACAP gene expression as well as the PAC1R. PACAP also regulates GnRH receptor expression (Kanasaki et al., 2009), and increasing the amount of GnRH receptor expressed differentially modulates gonadotropin LH␤ and FSH␤ gene expression (Bedecarrats and Kaiser, 2003; Kaiser et al., 1995). However, as we did not confirm the ability of TRH to stimulate PACAP and PAC1R expression in GH3 cells (data not shown), it is deniable that such interaction exists between TRH and PAC1R through PACAP expression in this cell. From this current observation, it is conceivable that PACAP is necessary to maintain its own PAC1R expressions. Without constant PACAP stimulation, GH3 might lose sufficient PAC1R enough to respond to PACAP stimulation. The effect of PACAP on prolactin gene expression was similar to that of TRH; however, its effect on growth hormone expression was different. TRH stimulation increased the level of prolactin mRNA and decreased growth hormone mRNA expression (Fig. 6A and B). These results are similar to our previous observation (Kanasaki et al., 2002). In contrast, PACAP increased both the prolactin and growth hormone mRNA levels in PAC1R expressing GH3 cells (Fig. 6C and D). The stimulatory effect of PACAP on growth hormone mRNA expression was consistent with previous observations (Velkeniers et al., 1994; Wong et al., 2005). The TRH-induced prolactin expression and concomitant decrease in growth hormone partially affected ERK activation (Kanasaki

et al., 2002). It has previously been suggested that growth hormone mRNA levels could be elevated by PACAP via functional coupling of the calcium/calmodulin kinase II cascade with the adenylate cyclase/cAMP/PKA pathway (Wong et al., 2005). Although TRH and PACAP share signal-transduction systems in pituitary cells, it is clear that other, more complex mechanisms also play a role in regulating prolactin and growth hormone. These complex pathways have yet to be fully elucidated. In this present study, we found that PACAP had a stimulatory effect on prolactin gene expression in pituitary prolactin-producing cells, provided that the PAC1R was adequately expressed. Actions of TRH were also potentiated in the presence of PAC1R. As PACAP/PAC1R has a direct effect on prolactin-producing cells, it is likely a regulator of prolactin synthesis and secretion in physiological conditions.

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