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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitogen-activated protein kinases (MAP kinases), also called extracellular regulated kinases (ERKs), mainly respond to mitogens and growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor, and regulate cell proliferation and differentiation. Recently, we demonstrated that thyrotropin-releasing hormone (TRH)-stimulated MAP kinase activation is involved in inhibition of proliferation as well as stimulation of prolactin biosynthesis [6]. In contrast, the p38 MAP kinase pathway, like the c-Jun N-terminal kinase (JNK) pathway, is activated in response to cellular stress and inflammation and is involved in many fundamental biological processes. For example, it has been proposed that p38 MAP kinase functions in the regulation of cytokine production [7–9], cell cycle control [10, 11], and apoptosis [12–15]. The subfamily of p38 MAP kinase consists of p38
, p38ß, p38
, and p38
. It has been reported that p38 and p38ß MAP kinase isoforms are involved in apoptosis [14, 15].
Rat pituitary cell line GH3 cells, which synthesize and secrete both prolactin and GH, are a useful model system for the study of hormonal dysfunctions. These hormones have been implicated as having a significant role in human reproduction by modulating gonadotropin secretion [16, 17]. In the present study, we found that bromocriptine induced apoptosis in GH3 cells, with concomitant activation of p38 MAP kinase. The effect of bromocriptine on apoptosis was not mediated through dopamine D2 receptors. In addition, treatment of the cells with EGF or TRH inhibited bromocriptine-induced p38 MAP kinase activation and in turn rescued cells from bromocriptine-induced apoptosis.
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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); 2-bromo--ergocryptine and TRH (Sigma Chemical Co., St. Louis, MO); recombinant EGF (Bachem, Budendorf, Switzerland); SB202190 and SB203580 (Calbiochem, San Diego, CA); S(-)-eticropride hydrochloride (Research Biochemical International, Natick, MA); anti-p38 MAP kinase antibody and anti-phospho p38 MAP kinase antibody (New England BioLabs, Beverly, MO); and Ham's F-10 medium (ICN Biomedicals, Tokyo, Japan). Myelin basic protein was purified from bovine brain [18].
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 and 5% CO2 [6]. Two or three days before experiments, 2–3 x 105 cells were plated on a 35-mm Petri dish (Nunc, Roskilde, Denmark). Then the cells were incubated at 37°C for the indicated times without (control) or with the test agents in the serum-containing medium.
Assay for p38 MAP Kinase and ERK Activity
For p38 MAP kinase assay, after GH3 cells were cultured for 3 days they were incubated for the indicated times without (control) or with 10 µM bromocriptine in the medium containing serum. Test agents such as 20 µM SB202190, 20 µM SB203580, and 20 µM S(-)-eticropride hydrochloride (eticropride) were added in the presence and absence of bromocriptine. In the case of ERK assay, GH3 cells that had been cultured for 3 days were preincubated for 60 min at 37°C in Krebs-Ringer Hepes buffer (KRH buffer). After preincubation, the cells were incubated for 10 min with 10 µM bromocriptine, 10 ng/ml EGF, and 1 µM TRH in KRH buffer. After incubation, the medium was aspirated off, and cells were washed three times with PBS and frozen in liquid N2. Frozen GH3 cells were scraped off 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, 5 µM pepstatin A, 1 mM dithiothreitol, 1 mM (p-amidinophenyl) methanesulfonyl fluoride hydrochloride (solubilization solution), 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. The extracts were treated with SDS sample buffer [19] and boiled for 1.5 min. Samples containing the same amount of proteins (10–15 µg of protein) were assayed for p38 MAP kinase and ERK by SDS-PAGE containing myelin basic protein (0.5 mg/ml) as a substrate. After electrophoresis, SDS was removed by washing the gel with 20% 2-propanol. Then, after denaturation with 6 M guanidine/HCl and renaturation in a 0.04%-Triton X-100-containing buffer, the gel was preincubated at 30°C for 1 h with 30 ml of 40 mM Hepes (pH 8.0) containing 2 mM dithiothreitol, 10 mM MgCl2, and 0.1 mM sodium orthovanadate. Protein kinase reaction was then performed by incubating the gel at 30°C for 1 h with 30 ml of 40 mM Hepes (pH 8.0) containing 0.5 mM EGTA, 10 mM MgCl2 and 0.1 mM sodium orthovanadate, and 25 µM [-32P]ATP (25 µCi) [20, 21]. After the gel was dried, the amount of 32P incorporation into myelin basic protein phosphorylated by ERK or p38 MAP kinase was quantified by a Bio-Imaging analyzer (BA100; Fujifilm, Tokyo, Japan).
Western Immunoblotting
Cells plated in a 35-mm dish were scraped off and solubilized with 100 µl of the solution used in the kinase assays. Aliquots were examined for protein concentration, and the same amount of protein was subjected to SDS-PAGE in 10% acrylamide and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with the anti-p38 MAPK antibody and anti-phospho p38 MAPK antibody, each diluted 1:250. Immunoreactive proteins were visualized with 125I-labeled protein A and horseradish peroxidase-conjugated anti-rabbit antibody, respectively [22].
Immunostaining Procedures
GH3 cells were cultured without (control) or with 10 µM bromocriptine in the serum-containing culture medium. After 48 h, the cells were washed with PBS and fixed at -20°C for 10 min with cold methanol. After 10 min of dehydration at 25°C, the cells were treated with 0.05% Triton X-100 in PBS for permeabilization. Nonspecific antibody binding was blocked by preincubation with 10% goat serum in PBS (blocking solution), followed by incubation overnight with normal rabbit IgG or the rabbit anti-phospho p38 MAP kinase (Thr180/Tyr182) antibody in the blocking solution. The cells were then washed five times with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG in PBS containing 0.01% Triton X-100 for 1 h. Next the cells were stained with propidium iodide for 2 min before mounting and were examined under a confocal laser scanning light microscope (Olympus, Tokyo, Japan) [23].
Quantitation of Apoptosis (TUNEL Procedure)
Three days after plating, the GH3 cells were exposed to 10 µM bromocriptine in serum-containing F-10 medium to examine bromocriptine-induced apoptosis. SB202190 (20 µM) and SB203580 (20 µM) were added as indicated. After 48 h, cells were washed three times with PBS and fixed with 4% formaldehyde in PBS for 25 min at 4°C. Apoptotic cells were detected using the apoptosis detection system, fluorescein (Promega, Madison, WI), which measures the fragmented DNA of apoptotic cells by incorporating fluorescein-12-dUTP at the 3'-OH ends of the DNA by means of the enzyme terminal deoxynucleotidyl transferase, which forms a polymeric tail using the principle of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay [24]. The total number of cells counted was at least 500 per sample in three independent experiments.
Other Procedures
Protein concentration was determined by the method of Bradford [25] with BSA as the standard.
Statistical Evaluation
Each experiment was conducted in at least three independent determinations for cells of separate dishes. The same experiments were repeated at least two to three times. Similar results were obtained from experiment to experiment. Values were expressed as means ± SE. Statistical analysis was performed using one-way ANOVA followed by 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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Western blot analysis with anti-p38 MAP kinase antibody indicated an immunoreactive band with a molecular mass of 38 kDa in the extract of rat pituitary adenoma cell line GH3 cells, indicating that p38 MAP kinase was present in these cells. There was no quantitative change in the amount of p38 MAP kinase between the control and bromocriptine-treated cells (Fig. 1A). When the p38 MAP kinase activity was assayed by in-gel kinase assay using myelin basic protein as substrate, increased activities of p38 MAP kinase were observed 48 and 72 h after treatment with 10 µM bromocriptine without significant changes in ERK kinase activities. Under the basal conditions, the p38 MAP kinase activity gradually increased during culture. Treatment with 10 µM bromocriptine significantly potentiated p38 MAP kinase 3- to 5-fold compared to the value in controls (Fig. 1, B and C).
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Immunostaining Analysis of GH3 Cells with Anti-Phospho p38 MAP Kinase Antibody
We further examined the localization of p38 MAP kinase activated with bromocriptine using anti-phospho p38 MAP kinase antibody, which recognizes phosphorylated Thr180/Tyr182. Western blotting analysis with the phospho-specific antibody revealed that the increase in the phosphorylation of Thr180/Tyr182 was correlated with the bromocriptine-induced p38 MAP kinase activation (Fig. 2A). After treatment of GH3 cells with 10 µM bromocriptine for 48 h, images from the confocal laser scanning light microscope were examined (Fig. 2B). Phase-contrast microscopy showed that apoptotic cells were round and loosely attached to the culture plates of dishes and therefore easily detached from plates (Fig. 2B, a and e). These cells were stained using the TUNEL procedure. Immunocytochemical analysis showed that apoptotic cells were strongly immunostained with the anti-phospho p38 MAP kinase antibody, especially in the cytoplasms (Fig. 2B, b and f). The nuclei of the apoptotic cells were morphologically irregular (data not shown), condensed, and fragmented (Fig. 2B, c and g). Pseudocolored confocal imaging indicated that the margins around the condensed and fragmented nuclei (Fig. 2B, g and h) were in yellow. Therefore, it is not clear that activated p38 MAP kinase was actually localized in the nuclei of apoptotic cells, but the localization of the activated kinase seemed to be observed in the nuclei by pseudocolored confocal image.
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Effects of Specific p38 MAP Kinase Inhibitors on Bromocriptine-Induced p38 MAP Kinase Activation
To further confirm that the increased activities measured by in-gel kinase assay were related to p38 MAP kinase activation, we tested effects of two specific p38 MAP kinase inhibitors, SB202190 and SB203580, on the bromocriptine-induced p38 MAP kinase activation. Bromocriptine induced activation of p38 MAP kinase up to 300% compared to the value in nontreated cells. Both SB202190 and SB203580 completely inhibited bromocriptine-induced p38 MAP kinase activation to control levels (Fig. 3).
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Induction of Apoptosis by Stimulation with Bromocriptine and Effects of p38 MAP Kinase Inhibitors
To address whether the bromocriptine-induced p38 MAP kinase activation was related to apoptosis, we assessed apoptotic cells by measurement of DNA fragmentation using TUNEL staining. Under basal conditions, a few cells were TUNEL positive. Treatment with bromocriptine for 48 h significantly increased the number of apoptotic cells (Fig. 4, B and F) compared to the value in nontreated cells (Fig. 4, A and E). High magnification by microscopic analysis revealed that nuclear fragmentation and condensation occurred in cells in which apoptosis was induced by bromocriptine (Fig. 4, C and G). Inclusion of 20 µM SB202190 or SB203580 in the medium significantly reduced the number of apoptotic cells (Fig. 4, D and H). We summarized the ratio of apoptotic cells by TUNEL staining by counting a total of more than 500 cells in different microscopic fields. The percentage of apoptotic cells increased 4-fold following bromocriptine treatment compared to the value in nontreated cells, and the effects of bromocriptine were largely prevented by treatment with either SB202190 or SB203580 (Fig. 5).
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Effects of a Dopamine D2 Receptor Antagonist on Bromocriptine-Induced p38 MAP Kinase Activation
Inhibition of prolactin release by bromocriptine is known to occur through the dopamine D2 receptor. Thus, we tested the effects of a selective D2 receptor antagonist, eticropride, on p38 MAP kinase activation. Inclusion of 20 µM eticropride in the medium failed to inhibit the p38 MAP kinase activation induced by bromocriptine (Fig. 6). This suggested that the activation of p38 MAP kinase by bromocriptine is not mediated through the dopamine D2 receptor in GH3 cells. In a separate experiment, the effects of 10 µM dopamine on activation of p38 MAP kinase were examined. Incubation of the cells with dopamine for 48 h did not activate p38 MAP kinase (data not shown). These results also suggest that the effect of bromocriptine on p38 MAP kinase activation is not mediated through the dopamine D2 receptor in GH3 cells.
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Effects of EGF and TRH on p38 MAP Kinase Activation and Apoptosis Induced by Bromocriptine
It has been reported that apoptosis is regulated by opposing actions between the ERK and the JNK/p38 MAP kinase pathways [12]. Therefore, we examined the effects of the ERK signaling pathway on bromocriptine-induced apoptosis as well as p38 MAP kinase activation. Recently, we reported that stimulation with TRH and EGF potentially activates ERK, especially ERK2, in GH3 cells and in turn stimulates prolactin biosynthesis [6]. Consistent with the previous results, treatment with either 10 ng/ml EGF or 1 µM TRH increased ERK2 activity by about 500% and 200%, respectively (Fig. 7C). Under the basal conditions, treatment with EGF or TRH alone significantly attenuated the p38 MAP kinase activity (Fig. 7B). The bromocriptine-induced p38 MAP kinase activation was abolished completely by EGF in the incubation medium and partly by TRH (Fig. 7B). The potencies of EGF and TRH with respect to inhibition of p38 MAP kinase activation induced by bromocriptine were related to their abilities with regard to ERK activation. Finally, we tested the effects of EGF and TRH on bromocriptine-induced apoptosis at 48 h. Consistent with the results of their inhibitory effects, treatment with EGF and TRH significantly attenuated the number of apoptotic cells increased by bromocriptine treatment. In contrast to findings with EGF, under basal conditions, treatment with TRH alone significantly reduced the number of apoptotic cells (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we demonstrated bromocriptine-induced apoptosis in GH3 cells by using TUNEL staining methods. These results were consistent with previous observations [28–30]. The bromocriptine-induced apoptosis was closely associated with activation of p38 MAP kinase. It has been proposed that the p38 MAP kinase pathway functions in the regulation of cytokine production [7, 8], proliferation and differentiation of B and T cells [31–33], and apoptosis [12–15, 34–37]. Among these functions, bromocriptine-induced p38 MAP kinase in GH3 cells was possibly implicated in apoptosis induced by bromocriptine, since the selective inhibitors for p38 MAP kinase could rescue the cells from apoptosis. Although bromocriptine-induced p38 MAP kinase activation was totally abolished by the inhibitors, the bromocriptine-induced apoptosis was only partly inhibited with the same concentration of the inhibitors. This suggests that, in addition to the p38 MAP kinase pathway, other unknown pathways are also involved in bromocriptine-induced apoptosis. The subfamily of p38 MAP kinase consists of , ß, , and isoforms. Among these, the and ß isoforms were inhibited by both SB202190 and SB203580, specific p38 MAP kinase inhibitors. Therefore, it was reported that inhibitors for p38 MAP kinase inhibit apoptosis [35–37]. On the other hand, Nemoto et al. [15] reported that SB202190 induces apoptosis. According to current reports, p38 isoform induces apoptosis, while p38ß isoform induces hypertrophic responses [14] or prevents apoptosis of cells [15]. Taken together, differences in the effects of inhibitors of p38 MAP kinase on apoptosis may reflect differences in the amounts of the isoforms in the tissues.
There is a controversy about the presence of functional dopamine receptors in GH3 cells [38, 39]. In our experiments, bromocriptine-induced p38 MAP kinase activation was not elicited through the dopamine D2 receptor. This was confirmed by the fact that dopamine itself had no effect on p38 MAP kinase activation or on hormone secretion (data not shown). Dopamine has been reported to have neurotoxic effects and to induce apoptosis in cultured neurons [40–44]. The oxidative stress caused by reactive oxygen species generation has been implicated in the mechanism of neurotoxicity and apoptosis [41–43]. In future studies, we should address whether oxidative stress is underlying the bromocriptine-induced apoptosis.
Recently, Xia et al. [12] reported an opposing effect between ERK and p38 MAP kinase signaling in apoptosis. Here, we confirmed that the ERK signaling elicited by EGF and TRH rescued cells from p38 MAP kinase-induced apoptosis with concomitant inhibition of p38 MAP kinase activity in the bromocriptine-treated cells. We recently demonstrated that TRH-induced ERK activation has functions in the production of prolactin but not in the secretion process [6]. TRH elicited differentiation of GH3 cells, possibly by regulating the expression of prolactin and GH genes. In addition, TRH can inhibit proliferation of GH3 cells [6]. In contrast to EGF treatment, treatment with TRH promoted cell survival under basal conditions (Fig. 8), and inhibitory effects of TRH on the bromocriptine-induced p38 MAP kinase activation were not correlated with potency with regard to the prevention of apoptosis (Figs. 7B and 8). Phosphatidylinositol 3-kinase, which is activated by both the heterotrimeric GTP binding proteins and tyrosine kinase pathways, has been implicated in the survival signals [45, 46]. In this context, in addition to the ERK signaling, other anti-apoptotic signaling such as phosphatidylinositol 3-kinase pathway are possibly implicated in the anti-apoptotic effects elicited by TRH. These findings suggest that TRH physiologically regulates the production and secretion of hormones in pituitary cells and promotes survival in these hormone-secreting cells.
Addition of TRH or EGF increased the secretion of prolactin and decreased the secretion of GH in the medium as previously described [6, 47]. Bromocriptine decreased the secretion of both prolactin and GH in the medium (data not shown). These results were also consistent with earlier observations [26, 27]. Effects of bromocriptine on the secretion were not recovered by addition of SB202190, a specific inhibitor for p38 MAP kinase (data not shown). On the other hand, effects of bromocriptine on apoptosis were inhibited by addition of both EGF and TRH. This would suggest inhibitory and stimulatory effects of prolactin and GH, respectively, on apoptosis. However, effects of prolactin on apoptosis are still controversial [48–51], while those of GH are rather inhibitory [52, 53]. These results suggest that bromocriptine-induced apoptosis is mediated through activation of p38 MAP kinase, but not through secretion of prolactin or GH.
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FOOTNOTES |
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1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan; by a Research Grant from the Human Frontier Science Program (K.F. and E.M.); and by the Grant from the Ministry of Health and Welfare (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: December 31, 1999.
Received: August 3, 1999.
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REFERENCES |
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