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Regular Article |
a Department of Obstetrics and Gynecology, Shimane Medical University, Izumo 693-8501, Japan
b Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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growth hormone, pituitary hormones, prolactin, signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GH3 cells can synthesize and secrete prolactin and growth hormone because the cells exist as either somatotrophs or somatolactotrophs [4, 5]. Because the amounts of growth hormone and prolactin in GH3 cells are similar to those of normal pituitary cells, the cells are considered to be a valuable model for the study of normal pituitary acidophilic cell function. Previous work has demonstrated that TRH stimulates secretion of both prolactin and growth hormone, and causes reciprocal shifts in the proportion of prolactin and growth hormone in GH3 cells [2, 4–6]. Although the effect of TRH on the proportion of the hormones is obvious, the detailed mechanism of this phenomenon has not yet been elucidated.
Thyrotropin-releasing hormone is secreted from the hypothalamus of the brain, reaches the pituitary gland through blood flow, and stimulates synthesis and secretion of prolactin. Thyrotropin-releasing hormone is believed to stimulate inositol phospholipid metabolism by activating membrane receptors in lactotrophs, which in turn stimulate the protein kinase C (PKC) pathway and Ca2+ release from intracellular Ca2+ storage sites [7]. Ohmichi et al. [8] first reported that TRH induced activation of MAP kinase via PKC-dependent and PKC-independent pathways in GH3 cells. The former pathway activates MAP kinase kinase (MEK) kinase (MEKK) with PKC, and the latter induces tyrosine phosphorylation of Shc proteins, which are linked to Ras-dependent MEKK activation. MEKK activates MEK, which ultimately activates MAP kinase [9]. MAP kinase was originally reported to be activated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor, and to regulate cell proliferation and differentiation. Furthermore, we recently reported that another endogenous secretagogue, pituitary adenylate cyclase-activating polypeptide, stimulates both gene expression and secretion of prolactin and growth hormone in GH3 cells via activation of MAP kinase through the cAMP pathway [10].
In this study we investigated the effect of TRH on secretion and gene expression of both prolactin and growth hormone and examined possible involvement of MAP kinase activation in the proportional changes in the synthesis of both hormones in GH3 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetal calf serum was obtained from JRH Biosciences (Lenexa, KS) and [
-32P]ATP was obtained from DuPont-New England Nuclear (Wilmington, DE). Thyrotropin-releasing hormone and PD098059 were from Sigma Chemical Company (St. Louis, MO). U0126 was from Promega (Madison, WI), wortmannin was from Wako Pure Chemical Industries (Osaka, Japan), and KN93 was from Seikagaku Co. (Tokyo, Japan). Lipofectamin was from Life Technologies (Tokyo, Japan), and Ham F10 medium was from ICN Biomedicals (Tokyo, Japan). Myelin basic protein was purified from bovine brain [11].
Cell Culture
GH3 cells, a rat prolactinoma cell line, were cultured in Ham F10 medium containing 15% horse serum, 2.5% fetal calf serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin, and maintained at 37°C in an atmosphere of 95% air and 5% CO2. Two or three days before experiments, 2–3 x 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 at 37°C for the indicated times without (control) or with the test reagents in KRH. After incubation for the indicated times, the medium was quickly aspirated off, and the cells were frozen in liquid nitrogen.
Hormone Measurement
GH3 cells were seeded on Falcon 24-well plates and grown under the same conditions described above. After the cells were preincubated at 37°C for 60 min in KRH, the medium was removed. The cells were then incubated at 37°C for the indicated times in 200 µl of KRH with or without TRH. Appropriate inhibitors were added during preincubation and incubation. After incubation, the media were collected and centrifuged at 12 000 x g for 10 min, and the supernatants were used for the hormone assay. To measure hormone content, 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 x g for 5 min, and the supernatants were used for the hormone assay. The concentrations of prolactin and growth hormone were determined by the double-antibody radioimmunoassay using the rat prolactin [125I]assay system and the rat growth hormone [125I]assay system (Amersham Life Science Ltd., Buckinghamshire, England).
Reverse Transcriptase-Polymerase Chain Reaction of Prolactin and Growth Hormone
Total RNAs were prepared from GH3 cells using Trizol LS reagent (Life Technologies) according to the manufacturer's protocol. Messenger RNA was reverse transcribed into single-stranded cDNA using 1.0 µg of total RNA, an oligo(dT) primer (Promega) and Moloney murine leukemia virus reverse transcriptase (RT; Gibco BRL, Gaithersburg, MD). The reaction mixtures were diluted 20-fold and then subjected to polymerase chain reaction (PCR) amplification of prolactin and growth hormone cDNAs as previously described [12]. The PCR primers were designed on the basis of the published sequences of rat prolactin [13] and rat growth hormone [14].
To amplify cDNAs, each of the following sense primers were used: 5'-prolactin primer, 5'-AATGACGGAAATAGATGATTG-3'; 3'-prolactin primer, 5'-CCAGTTATTAGTTGAAACAGA-3'; 5'-growth hormone primer, 5'-CTGCTGACACCTACAAAGA-3'; and 3'-growth hormone primer, 5'-CAGTGTGTGCCTAGAAAGCA-3'. Polymerase chain reaction (PCR) amplification was carried out using the Gene Amp PCR system 2400 (Perkin-Elmer, Norwalk, CT) after denaturation of samples for 10 min. Each cycle consisted of denaturation at 94°C for 45 sec, annealing at 58°C for 45 sec, and extension at 72°C for 40 sec. After amplification, the final 5-min extension step was carried out at 72°C. When a quantitative RT-PCR analysis was carried out, the PCRs for prolactin and growth hormone were constructed for 30 and 17 cycles, respectively. The PCR products were separated by electrophoresis on a 1.0% agarose gel, visualized with ethidium bromide staining, and quantified by scanning densitometry using NIH Image software (version 1.61). The amounts of PCR products for prolactin and growth hormone were normalized to that of the PCR product of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample.
Transfection of GH3 Cells with pFC-MEKK Plasmid Vector
GH3 cells (10 x 105 cells/dish) were plated in a 35-mm dish (Nunc) and cultured in the standard medium for 3 days. The cells were transfected with pFC-MEKK plasmid vector (pFC-MEKK) (Stratagene, La Jolla, CA) or pCAGGSneo plasmid vector (a kind gift from Prof. J. Miyazaki; Osaka University, Japan) (i.e., mock-transfected cells) using 15 µl of lipofectamine (Life Technologies) in 1 ml of the serum-free medium for 6 h. The culture medium was changed to the standard medium, and the cells were cultured for an additional 40 h. After culture, the cells were washed with PBS 3 times, and were frozen in liquid nitrogen.
Reporter Plasmid Construct and Luciferase Assay
Genomic DNA of GH3 cells was isolated using the Genomic DNA Isolation Kit (5 Prime 
3 Prime, Boulder, CO). Polymerase chain reaction was carried out to amplify the fragment containing the prolactin promoter region (-609 to +12) with a sense primer (5'-TATCTCGAGGTCTGGTTGATT-3') and an antisense primer (5'-ATACTCGAGAACCACTGCTTT-3'), both of which contain an XhoI restriction site. For the growth hormone promoter region (-563 to +30), PCR was carried out with a sense primer (5'-TATGCTAGCCAACAAAATGGC-3') and an antisense primer (5'-ATAGCTAGCAGTTTGGAATCT-3'), both of which contain an NheI restriction site. The fragments were excised with XhoI or NheI restriction enzymes, and inserted into the pGL3-basic luciferase reporter vector (Promega), and were labeled, respectively, pGL3-PRLp and pGL3-GHp. GH3 cells were cotransfected with pGL3-PRLp or pGL3-GHp (0.5 µg of DNA) and pRL-TK (0.05 µg of DNA) (Promega), which contains Renilla luciferase under the herpes simplex virus thymidine kinase promoter, using 15 µl of lipofectamine in 2 ml of serum-free medium for 6 h, as previously described [10]. The culture medium was changed to the standard medium, and the cells were cultured for 40 h. Transfected cells were treated with 1 µM TRH for 6 h in the presence or absence of PD098059. The activities of firefly luciferase and Renilla luciferase were measured with the Dual-Luciferase Reporter Assay System (Promega) with a luminometer (TD-20/20) (Promega). The ratio of luminescence signal by firefly luciferase to that by Renilla luciferase was determined.
Other Procedures
Protein concentration was determined according to the method of Bradford [15] with BSA as the standard.
Statistical Evaluation
Each experiment was conducted in at least 3 independent determinations for cells of separate dishes. The same experiments were repeated 2 to 3 times. Similar results were obtained in subsequent experiments. Values were expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA plus the Duncan multiple range test. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The time course of TRH-stimulated hormone secretion from GH3 cells was examined. After a 30-min incubation with 1 µM TRH, both prolactin (Fig. 1A) and growth hormone (Fig. 1C) obtained from the same culture medium were significantly increased compared with levels in nonstimulated cells. To determine the possible involvement of protein kinase pathways in TRH-induced hormone secretion, GH3 cells were preincubated with PD098059 (a specific MEK inhibitor), KN93 (a calcium/calmodulin-dependent protein kinase II; CaM kinase II inhibitor) or wortmannin for 60 min, and stimulated with TRH for an additional 60 min. Prolactin secretion was not affected by preexposure to 50 µM PD098059 (Fig. 1B) or 200 nM wortmannin (data not shown), but it was completely inhibited by 10 µM KN93 and 1 µM wortmannin (Fig. 1B). Similar to prolactin secretion, TRH-induced growth hormone secretion was not inhibited by treatment with PD098059 (Fig. 1D) or 200 nM wortmannin (data not shown), but was significantly inhibited by KN93 and 1 µM wortmannin (Fig. 1D). It has been reported that 200 nM wortmannin specifically inhibits phosphatidylinositol 3-kinase (PI3-kinase) and that 1 µM wortmannin inhibits myosin light chain kinase (MLCK) as well as PI3-kinase [16–18]. These results suggest that TRH-stimulated secretion of both prolactin and growth hormone is correlated with CaM kinase II activation, MLCK activation, or both, but not with PI3-kinase or MAP kinase activation.
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Effects of TRH on Prolactin and Growth Hormone Gene Expression
We evaluated the effect of TRH on the level of mRNA of both prolactin and growth hormone using RT-PCR methods. The quantitative RT-PCR analysis yielded a linear relationship between the relative signal and the amount of total RNA ranging from 0.5 to 4.0 µg (data not shown). Treatment of GH3 cells with TRH significantly increased the amount of prolactin mRNA 24 and 48 h after TRH stimulation (Fig. 2, A and B), whereas growth hormone mRNA was dramatically inhibited by exposure to TRH (Fig. 2, A and C). TRH elevated prolactin mRNA and decreased growth hormone mRNA at 6 h and 12 h after treatment, but these effects were not statistically significant (data not shown). The maximal effects of TRH on the increase in prolactin mRNA (Fig. 3, A and B) and on inhibition of growth hormone mRNA (Fig. 3, A and C) were observed at approximately 100 and 10 nM, respectively. These results suggest that prolactin and growth hormone gene expressions are differently regulated by TRH, although secretion of both hormones is stimulated by TRH.
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Effects of MEK Inhibitors on TRH-Induced Prolactin and Growth Hormone Gene Expression
We have previously demonstrated by using a specific MEK inhibitor, PD098059, that activation of MAP kinase by TRH stimulation is involved in prolactin synthesis [3]. Maximal activation of MAP kinase (200%–400% increase over control) in these experiments was obtained by stimulation with 1 µM TRH, therefore, we used 1 µM TRH in the following experiments. To examine the effect of PD098059 on TRH-induced regulation of prolactin and growth hormone gene expression, the amounts of prolactin and growth hormone mRNA were measured in GH3 cells cultured with 1 µM TRH in the presence or absence of PD098059. Treatment of GH3 cells with 50 µM PD098059 completely abolished the increase in prolactin mRNA expression observed after 48 h of TRH stimulation (Fig. 4, A and B). Treatment of GH3 cells with PD098059 alone decreased prolactin mRNA levels in the control sample, suggesting that MAP kinase is partially activated without addition of TRH, and that endogenous stimulation of the mRNA is inhibited by PD098059 (Fig. 4B). In contrast, TRH-induced inhibition of growth hormone mRNA was prevented by addition of PD098059 (Fig. 4, A and C). It is interesting that addition of PD098059 alone increased growth hormone mRNA, suggesting that partial activation of MAP kinase without TRH treatment is inhibited with PD098059, thereby increasing the level of growth hormone mRNA (Fig. 4C).
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A separate experiment using another MEK inhibitor, U0126, showed similar effects to PD098059 on expression of prolactin and growth hormone mRNA (data not shown). Taken together with previous studies [3], these results suggest that TRH-induced MAP kinase activation is involved in induction and inhibition of prolactin and growth hormone mRNA synthesis, respectively.
Effects of Overexpression of MEK Kinase on Expression of Prolactin and Growth Hormone mRNA
To further examine the role of MAP kinase in prolactin and growth hormone mRNA expression, GH3 cells were transfected with a pFC-MEKK plasmid vector to induce expression of constitutively active MEKK and to subsequently activate MAP kinase. Compared with mock-transfected cells, the activity of MAP kinase was increased 2-fold to 3-fold by MEKK overexpression (data not shown). Overexpression of MEKK increased the amount of intracellular prolactin (Fig. 5A) and mRNA (Fig. 5D), whereas both the intracellular content (Fig. 5B) and mRNA (Fig. 5E) levels of growth hormone were significantly decreased compared with those of mock-transfected cells. These results suggest that the MAP kinase signaling cascade is involved in stimulating prolactin synthesis and inhibiting growth hormone synthesis in GH3 cells.
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Effects of TRH on Prolactin and Growth Hormone Promoter Activities
Thus far we have provided strong evidence that TRH-induced MAP kinase activation is correlated with the TRH-induced intracellular increase in prolactin and decrease in growth hormone. Next, we sought to determine the effects of TRH on prolactin and growth hormone promoter activities. We isolated the genomic DNA from GH3 cells to obtain the promoter regions of prolactin and growth hormone. Sequencing of the obtained fragments revealed that the proximal region of the 609-base pair (bp) nucleotides upstream of the transcription initiation site in the prolactin promoter was 97.5% identical to that of the rat sequence, and that the proximal region of 563-bp nucleotides in the growth hormone promoter was 99% identical to that of the rat sequence (data not shown). The obtained fragments of the prolactin and growth hormone promoter regions were each placed with a luciferase coding sequence in pGL-3-basic vectors. As shown in Figure 6A, the firefly luciferase activity of prolactin was increased 3.7 ± 0.6-fold by stimulation with TRH. Fifty-micromolar PD098059 alone significantly inhibited the basal level of prolactin promoter activity, and the effect of TRH on prolactin promoter activity was largely inhibited in the presence of PD098059. In contrast, no significant change was observed in growth hormone promoter activity by TRH stimulation (Fig. 6B). These results suggest that TRH stimulates prolactin expression by accelerating its promoter activity via activation of MAP kinase, whereas inhibition of growth hormone expression is not dependent on its promoter activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We recently reported that activation of MAP kinase by TRH stimulation is not related to prolactin secretion, but is involved in prolactin synthesis and inhibition of proliferation and morphological changes in GH3 cells [3]. Wang and Maurer [20] have also shown that the ability of TRH to stimulate the prolactin promoter depends on MAP kinase activation. Furthermore, Kievit et al. [21] reported the involvement of MAP kinase activation in cAMP-induced prolactin transcription via activation of Rap-1. That the cAMP signaling pathway also regulates prolactin gene expression through MAP kinase activation agrees well with our recent reports that cAMP increases the levels of the protein, mRNA, and transcription of prolactin in GH3 cells [10]. In the present study we further elucidated the role of MAP kinase in prolactin and growth hormone gene expression, and concluded that the observed decreases in growth hormone at the protein and mRNA levels are regulated by different mechanisms than those that mediate increased prolactin synthesis.
GH3 cells are a clonal strain of rat pituitary cells that synthesize and secrete both prolactin and growth hormone [4]. Analyses of GH3 cells by fixed sequential plaque assays have demonstrated that GH3 cells consist of 2 types of hormone secreting cells: somatotrophs, which secrete only growth hormone; and somatolactotrophs, which secrete both growth hormone and prolactin [1, 2, 4–6]. Treatment of GH3 cells with TRH induces shifts in the relative proportions of hormone secreting cells, such that the number of prolactin secreting cells increases and the number of growth hormone secreting cells decreases [4–6]. Pituitary glands of adult rats have 3 types of cells involved in prolactin and growth hormone secretion: somatotrophs, somatolactotrophs, and lactotrophs. Both the number and size of lactotrophs increase during pregnancy [22]. Considering previous evidence, TRH may act not only as a hormone secretagogue, but also as a factor to stimulate prolactin synthesis. The possibility cannot be excluded, however, that TRH promotes cell differentiation in which the proportion of somatotrophs decreases and the proportion of lactotrophs increases. It has been shown that exposure of GH4Cl cells, a subclone of GH3 cells, to EGF decreases the rate of cell proliferation in association with marked morphological changes, and exhibits a concomitant increase in prolactin synthesis and a decrease in growth hormone synthesis [6]. In pituitary cell primary culture, EGF treatment greatly increases the proportion of classical lactotrophs from about 0.5% to 8% [23]. Because EGF is one of the most effective activators of MAP kinase, MAP kinase may be involved in promotion of the lactotroph phenotype.
It has been reported that prolactin gene expression is regulated by several factors [24–26]. The prolactin promoter contains binding sites for transcription factors such as Ets-1 and GHF-1/Pit-1, both of which synergistically enhance prolactin gene expression by activating the Ras-MAP kinase signaling cascade. Although the growth hormone promoter is homologous to the prolactin promoter and contains both GHF-1 binding and several potential Ets sites, it does not respond to Ras-MAP kinase activation because of the lack of a composite GHF-1/Ets-1 response element [26]. In the present study, overexpression of the constitutively active form of MEKK increased the expressions of both prolactin mRNA and protein. Furthermore, activation of the prolactin promoter with TRH was completely inhibited in the presence of PD098059. These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis by increasing the promoter activity in association with activation of transcription factors.
However, we cannot rule out the possibility that TRH induced the differentiation of GH3 cells to lactotrophs, and as a result, the number of cells expressing prolactin, or expression of prolactin in individual cells (or both) were increased. Growth hormone mRNA and protein levels were significantly decreased by stimulation with TRH, as was overexpression of MEKK. However, the promoter activity of growth hormone showed no significant change in the presence of TRH. These results may suggest that the expression of growth hormone and its mRNA is not regulated by MAP kinase, and that the decrease in the hormone level is due to the instability of the mRNA. Alternatively, because the decrease was recovered by inhibition of MAP kinase and transfection of MEKK, some other mechanisms via the MAP kinase pathway may be involved. We used a growth hormone reporter construct that contained nucleotides -563 to +30 of the promoter upstream of luciferase. It may be possible that some unknown transcriptional repressor elements exist upstream of this sequence, and that these elements are involved in inhibition of gene expression by MAP kinase. We tried to amplify the longer fragment to examine this possibility, but could not obtain any fragments for unknown reasons (data not shown).
The previous [3] and present studies indicate that TRH-induced prolactin and growth hormone secretion are not regulated by MAP kinase, suggesting that MAP kinase activation is not required for exocytosis (Fig. 1). Instead, TRH-induced prolactin and growth hormone secretion is completely blocked by either wortmannin at a concentration required to inhibit MLCK [17, 18] or by a CaM kinase II inhibitor, KN93 [27], in GH3 cells (Fig. 1). These results suggest that regulation of hormone secretion differs from that of hormone synthesis.
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FOOTNOTES |
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1 This work was supported in part by Grants-in-Aid for Scientific Research form the Ministry of Education, Science, Sports and Culture of Japan; by a research grant from the Human Frontier Science Program (to H.Y., K.F., and E.M.); and by a grant from the Ministry of Health and Welfare (to K.M.). 
2 Correspondence: Eishichi Miyamoto, Department of Pharmacology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan. FAX: 81 96 373 5078; emiyamot{at}gpo.kumamoto-u.ac.jp
Accepted: January 24, 2002.
Received: December 7, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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