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a Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
b Department of Obstetrics and Gynecology, Shimane Medical University, Izumo 693-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These results suggest that TRH-induced MAP kinase activation is involved in prolactin synthesis and differentiation of GH3 cells, but not in prolactin secretion.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thyrotropin releasing hormone (TRH), which is secreted from the hypothalamus of the brain, reaches the pituitary gland through blood flow and stimulates synthesis and secretion of prolactin. TRH is thought to stimulate inositol phospholipid metabolism in lactotrophs [1].
Mitogen-activated protein kinase (MAP kinase) or extracellular signal-regulated kinase (ERK) is widely distributed in eukaryotes as a family of serine/threonine protein kinases. MAP kinase was originally reported to be activated by growth factors and to be involved in proliferation and differentiation of cells through stimulation of gene expression. We recently reported that stimulation of glutamate receptors in primary cultures of hippocampal neurons activates MAP kinase through Ca2+-dependent and protein kinase C (PKC)-dependent pathways [2]. The Ca2+-dependent pathway may involve a novel tyrosine kinase (PYK2) [3]. The ß
subunits of the trimeric G subunit may also activate the MAP kinase cascade [4]. Ohmichi et al. [5] first reported that stimulation by TRH activates MAP kinase in GH3 cells, although they did not analyze the relationship between the activation of MAP kinase and prolactin secretion. The pathways involved included PKC-dependent raf-1 activation and tyrosine phosphorylation of Shc proteins linked to Ras-dependent raf-1 activation. In addition, MAP kinase has been implicated in other cellular events involving secretion. These include serotonin secretion from rat basophilic leukemia (RBL-2H3) cells [6], catecholamine secretion from bovine adrenal chromaffin cells [7, 8], bile acid secretion from rat liver [9], and pepsinogen secretion from guinea pig gastric chief cells [10]. Using GH3 cells, Kanada et al. [11] reported that both TRH-stimulated tyrosine phosphorylation of MAP kinase and prolactin secretion were strongly inhibited by treatment with the tyrosine kinase inhibitor ST638. Ohmichi et al. [12] reported that dopamine, a physiological prolactin inhibitory factor, inhibits TRH-induced MAP kinase activation in primary cultures of rat anterior pituitary cells.
In this study, we focused on MAP kinase activation induced by TRH and examined the role of MAP kinase activation in secretory processes and prolactin synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following chemicals and reagents were obtained from the indicated sources: fetal calf serum (JRH Biosciences, Lenexa, KS); [-32P]ATP (DuPont-New England Nuclear, Wilmington, DE); TRH, phorbol 12-myristate 13-acetate (PMA), and 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (CPTcAMP; Sigma Chemical Co., St. Louis, MO); genistein (GIBCO BRL, Gaithersburg, MD); calphostin C, (Kyowa Medex Co. Ltd., Tokyo, Japan); wortmannin (Wako Pure Chemical Industries, Osaka, Japan); KN93 (Seikagaku Co., Tokyo, Japan); anti-rat pan-ERK antibody and anti-mouse ERK2 antibody (Transduction Laboratory, Lexington, KY); [methyl-3H]thymidine (Amersham Life Science Ltd., Buckinghamshire, England); and Ham's F-10 medium (ICN Biomedicals, Tokyo, Japan). Myelin basic protein (MBP) was purified from bovine brain [13].
Cell Culture
GH3 cells, a rat prolactinoma cell line, were cultured in Ham's F-10 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:5% CO2. Two or three days before an experiment, 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 buffer) 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 N2.
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 ß-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 75 µM pepstatin A, 1 mM dithiothreitol, 1 mM (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, 1 mM Na3VO4, and 100 nM calyculin A. The procedures for treatment of cells were carried out at 0–4°C. After sonication (Sonifier 250; Branson, Danbury, CT), the insoluble materials were removed by centrifugation at 15 000 x g for 5 min. Extracts were treated with SDS sample buffer [14] and boiled for 1.5 min. Samples containing the same amount of proteins (10–15 µg of protein) were assayed for MAP kinase by SDS-PAGE using MBP as a substrate by the method of Geahlen et al. [15] and Gotoh et al. [16]. After the gel was dried, the amount of 32P incorporation into MBP phosphorylated by MAP kinase was quantified using a Bio-Imaging analyzer (BA100; Fujifilm, Tokyo, Japan).
Immunoblotting
Cells plated in a 35-mm dish were scraped and solubilized with 100 µl of the solution used in MAP kinase assays. Aliquots were assayed for protein concentration, and the same amount of protein was subjected to SDS-PAGE in 10% acrylamide and transferred to a nitrocellulose membrane. The membrane was incubated with the anti-rat pan-ERK antibody or anti-mouse ERK2 antibody, each diluted 1:500. Immunoreactive proteins were detected using 125I-labeled protein A.
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 buffer, the media were removed, and the cells were incubated at 37°C for the indicated times in 200 µl KRH with or without TRH. Appropriate inhibitors were added during preincubation and with TRH stimulation. After incubation, the media were collected and centrifuged at 12 000 x g for 10 min, and the supernatants were used for a prolactin 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 a hormone assay. The concentrations of prolactin and growth hormone were determined by a double-antibody RIA using the rat prolactin [125I] assay system and rat growth hormone [125I] assay system (Amersham Life Science Ltd.).
Cell Proliferation Assay
For DNA synthesis determination, GH3 cells were cultured in serum-free Ham's F-10 medium for 24 h. Cells were then stimulated for 8 h without or with test reagents in serum-free culture medium containing [3H]thymidine (1 µCi/ml). [3H]Thymidine-incorporated trichloroacetic acid-precipitable materials were solubilized in 0.5 M NaOH and quantified by liquid scintillation counting [17].
Other Procedures
Protein concentration was determined by the method of Bradford [18] with BSA as the standard.
Statistical Evaluation
Values were expressed as means ± SE. Statistical analysis was performed using one-way ANOVA plus Duncan's 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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We performed immunoblotting analysis using the anti-pan-ERK antibody, which recognizes ERK family proteins, and the anti-ERK2 antibody, which recognizes only the 42-kDa ERK2. Western blot analysis indicated an immunoreactive band of only 42 kDa in GH3 cells (Fig. 1A). Therefore, we examined only ERK2 activation as an indicator of MAP kinase activity in the following experiments. In the assay for MAP kinase using the MBP-containing gel, TRH-induced MAP kinase activation was observed for components of ERK (Fig. 1B). The results shown in Figure 1B were quantitated by a Bio-Imaging analyzer, plotted as a time course (Fig. 1C), and analyzed statistically. TRH induced rapid activation of MAP kinase, which reached a maximal peak of 300% at 5–10 min and was sustained at 200% 60 min after stimulation (Fig. 1C).
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A separate assay method for MAP kinase using a synthetic epidermal growth factor (EGF) receptor peptide as substrate showed similar results in terms of quantitative determination and the time course (data not shown). The maximal activation of MAP kinase was obtained with approximately 1 µM TRH with a 200–400% increase over the control (Fig. 1D).
Effects of Protein Kinase Inhibitors and Removal of Extracellular Ca2+ on TRH-Induced MAP Kinase Activation
We examined the effects of protein kinase inhibitors such as PD098059 (a specific MAP kinase [MEK] inhibitor), calphostin C (a relatively specific PKC inhibitor), and genistein (a tyrosine kinase inhibitor), and the effects of removal of extracellular Ca2+ on TRH-induced MAP kinase activation (Fig. 2). We chose the doses used in this experiment on the basis of previous reports. Fifty micromolar PD098059 [19] and 100 nM calphostin C [20] inhibited MAP kinase activation completely and by 80%, respectively. To further examine the interaction of TRH stimulation with the PKC pathway, PKC was down-regulated by prolonged incubation with 100 nM PMA for 24 h. Inactivation of PKC inhibited MAP kinase activation by TRH to the same extent as did calphostin C (data not shown). Two hundred micromolar genistein [21] and removal of extracellular Ca2+ also partially inhibited MAP kinase activation by 30% and 40%, respectively (Fig. 2).
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Effects of CPTcAMP on MAP Kinase Activation
To analyze the role of cAMP in MAP kinase activation, GH3 cells were treated with CPTcAMP, a membrane-permeable cAMP analogue. One millimolar CPTcAMP increased MAP kinase activity significantly by approximately 30%, and the effects of CPTcAMP were inhibited by addition of PD098059 (Fig. 3). Additive effects on MAP kinase activation were obtained by a combination of CPTcAMP and TRH, and these were completely abolished by addition of PD098059 (Fig. 3).
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Role of MAP Kinase Activation in TRH-Induced Prolactin Secretion
Figure 4A shows the time course of TRH-stimulated prolactin secretion from GH3 cells. The amounts of secreted prolactin increased significantly at 30 and 60 min after TRH stimulation compared to controls. To determine whether MAP kinase is involved in prolactin secretion induced by TRH, GH3 cells were preincubated with 50 µM PD098059 for 60 min and stimulated with TRH for 60 min. Prolactin secretion was not affected by pre-exposure to PD098059. Prolactin secretion with TRH was not inhibited by 100 nM calphostin C or 200 nM wortmannin, which at this dose specifically inhibits phosphatidylinositol 3-kinase (PI3-kinase) [22]. On the other hand, 1 µM wortmannin, which also inhibits myosin light chain kinase (MLCK) in higher doses [22–24], and 10 µM KN93 [25], an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), significantly inhibited prolactin secretion induced with TRH (Fig. 4B).
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Inhibition of DNA Synthesis by TRH and CPTcAMP
We next determined the effect of TRH and CPTcAMP treatment on DNA synthesis in GH3 cells. Incubation with 1 µM TRH significantly reduced DNA synthesis as determined by [3H]thymidine uptake during 8 h, and the inhibitory effects were completely blocked by coincubation with 50 µM PD098059 (Fig. 5). Although 1 mM CPTcAMP also significantly reduced DNA synthesis, the inhibition was not blocked by PD098059. Treatment with TRH together with CPTcAMP inhibited DNA synthesis to a greater extent than treatment with either reagent alone (Fig. 5).
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Treatment with TRH or CPTcAMP Altered Cellular Morphology
Figure 6 shows GH3 cells cultured in the presence or absence of either 1 µM TRH or 1 mM CPTcAMP. Control cells were predominantly spherical in shape (Fig. 6A). When cells were cultured with 1 µM TRH for 24 h, the morphology of the cells markedly changed. TRH-stimulated cells were larger, more elongated, and flatter than controls, and displayed angular borders (Fig. 6B). Addition of CPTcAMP produced similar morphological changes without the angular borders seen after TRH stimulation (Fig. 6C). The addition of PD098059 to the culture medium blocked the morphological changes induced by TRH (Fig. 6D) but not those induced by CPTcAMP (Fig. 6E). Figure 7 summarizes this data, showing the percentages of morphologically changed cells in which the cellular diameter was twice that of control cells.
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Effects of TRH on Hormone Content of GH3 Cells
To examine the effect of TRH on hormone synthesis, GH3 cells were cultured with 1 µM TRH for 48 h, and the hormone content was determined. Treatment with TRH significantly increased the intracellular prolactin content to approximately 3-fold but decreased growth hormone content. Treatment of cells with PD098059 completely abolished the increase in prolactin content induced by TRH but did not alter the inhibition of growth hormone synthesis (Fig. 8). The results were confirmed by the observation that down-regulation of PKC or treatment with wortmannin (200 nM) blocked the increase in prolactin content induced by TRH (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence indicate that MAP kinase is involved in release of both hormones [11, 12] and catecholamines [7, 8] and in secretion of digestive fluid [9] and enzymes [10]. However, in GH3 cells, TRH-induced MAP kinase activation was correlated not with prolactin secretion (Fig. 4) but with synthesis of prolactin (Fig. 8). This suggests that MAP kinase activation affects gene expression rather than secretion. In contrast, prolactin secretion induced by TRH was completely blocked by either wortmannin at a concentration required to inhibit MLCK [23, 24] or the CaM kinase II inhibitor, KN93 [25]. The data suggest that MLCK and CaM kinase II mediate prolactin secretion in GH3 cells and are consistent with previous reports showing that MLCK and CaM kinase II are involved in secretory processes in bovine adrenal chromaffin cells [24] and rat pancreatic ß cells [25], respectively.
A cAMP analogue also activated MAP kinase in GH3 cells and showed additive effects with TRH (Fig. 3). In general, cAMP inhibits MAP kinase activation induced by growth factors, as in Rat1 fibroblast cells stimulated with EGF [26, 27] and rat cortical astrocytes stimulated with basic fibroblast growth factor (bFGF) [28]. On the other hand, in PC12 cells [29], COS-7 cells [30], and rat ovarian granulosa cells [31], cAMP activates MAP kinase. Here we have shown that cAMP activates MAP kinase, although its effect is small. The effect of cAMP on MAP kinase activation was additive with TRH but inhibited with PD098059. On the other hand, although DNA synthesis was inhibited by cAMP as well as by TRH, the effect of cAMP was not blocked by PD098059, as was that of TRH (Fig. 5). Furthermore, morphological changes induced by TRH were also blocked by PD098059, whereas similar changes induced by cAMP were unaffected by PD098059 (Figs. 6 and 7). These results suggest that the effects of cAMP on GH3 cells differ from those of TRH.
GH3 cells are a clonal strain of rat pituitary cells that secrete prolactin and growth hormone. Analysis of GH3 cells by a fixed sequential plaque assay revealed the presence of cells that secrete only growth hormone and cells that secrete both growth hormone and prolactin [32]. There are three types of pituitary cells: somatotrophs, which secrete only growth hormone; somatolactotrophs, which secrete both growth hormone and prolactin; and lactotrophs, which secrete only prolactin [33]. Schonbrunn et al. [34] reported that chronic treatment of the GH3 cells with TRH or EGF increased prolactin synthesis and inhibited growth hormone production. They also observed that treated cells underwent morphological changes, becoming flatter, larger, and more angular. Boockfor and Schwarz [32] reported that chronic treatment of GH3 cells with TRH alters the proportion of cell types. That is, cells secreting only growth hormone or both growth hormone and prolactin changed to cells secreting only prolactin. Similar effects of TRH on secretory pattern were previously reported [35]. Furthermore, treatment of GH3 cells with EGF induced effects similar to those stimulated by TRH, including an increase in the proportion of cells secreting prolactin, a decrease in cells secreting growth hormone, and morphological changes [36, 37]. EGF activated MAP kinase and inhibited DNA synthesis in GH3 cells in the present study (data not shown). Taken together, treatment of GH3 cells with TRH induced the differentiation of somatolactotrophs into lactotrophs, on the basis of the increase in prolactin synthesis and the decrease in growth hormone synthesis (Fig. 8). The stimulative effects of TRH on prolactin synthesis were inhibited by PD098059, but the inhibitory effects on growth hormone were not. This suggests that MAP kinase is involved in both differentiation of the cells and in the increase in prolactin synthesis through TRH stimulation.
In this connection, regulators of pituitary cell-specific gene expression have been reported [38–40]. The promoter of the prolactin gene includes binding sites for transcription factors such as Ets-1 and GHF-1/Pit-1, both of which synergistically enhance prolactin synthesis in a Ras/Raf response element of the prolactin promoter [39]. Furthermore, it was proposed that the promoter of the growth hormone, which is also GHF-1-dependent, does not exhibit a Ras response because it lacks a composite Ets-1/GHF-1 binding element [40]. Since the Ets-1/GHF-1 composite element is necessary for targeting by the Ras pathway, growth hormone may not be induced by activation of MAP kinase as seen in the present study. Taken together, this study suggests that TRH is involved in prolactin synthesis and morphological changes through activation of MAP kinase and subsequent phosphorylation of Ets-1. This may partially induce cellular differentiation.
Here we have shown that TRH induces MAP kinase activation in GH3 cells. Prolactin secretion and synthesis were also induced by TRH. TRH may be involved in prolactin secretion through activation of MLCK and CaM kinase II, and in prolactin synthesis and differentiation of GH3 cells through activation of MAP kinase. Studies of the role of TRH in prolactin gene expression induced by MAP kinase activation are now ongoing.
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FOOTNOTES |
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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; emiyamoto{at}gpo.kumamoto-u.ac.jp
Accepted: March 15, 1999.
Received: January 5, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mediated MAP kinase activation by a common signaling pathway. Nature 1995; 376:781–784.[CrossRef][Medline] subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem 1994; 269:7851–7854.This article has been cited by other articles:
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