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Endocrinology Vol. 140, No. 6 2850-2858
Copyright © 1999 by The Endocrine Society

ARTICLES

Mitogen-Activated Protein Kinase-Dependent Stimulation of Proliferation of Rat Lactotrophs in Culture by 3',5'-Cyclic Adenosine Monophosphate1

Shinichi Suzuki, Isao Yamamoto and Jun Arita

Department of Physiology (S.S., J.A.), Yamanashi Medical University, Yamanashi 409-3898; and Department of Neurosurgery (I.Y.), Yokohama City University School of Medicine, Yokohama 236-0004, Japan

Address all correspondence and requests for reprints to: Dr. J. Arita, Department of Physiology, Yamanashi Medical University, Tamaho, Yamanashi 409-3898, Japan. E-mail: jarita{at}res.yamanashi-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracellular cAMP regulates cell proliferation as a second messenger of extracellular signals in a number of cell types. We investigated, by pharmacological means, whether an increase in intracellular cAMP levels changes proliferation rates of lactotrophs in primary culture, whether there are interactions between signal transduction pathways of cAMP and the growth factor insulin, and where the dopamine receptor agonist bromocriptine acts in the cAMP pathway to inhibit lactotroph proliferation. Rat anterior pituitary cells, cultured in serum-free medium, were treated with cAMP-increasing agents, followed by 5-bromo-2'-deoxyuridine (BrdU) to label proliferating pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for PRL and BrdU. Treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) increased BrdU-labeling indices of lactotrophs in a dose- and incubation time-dependent manner. The cAMP-increasing agents were also effective in increasing BrdU-labeling indices in populations enriched for lactotrophs by differential sedimentation. The stimulatory action of forskolin was observed, regardless of concentrations of insulin that were added in combination with forskolin. Inhibition of the action of endogenous cAMP by H89 or KT5720, a protein kinase A inhibitor, attenuated an increase in BrdU-labeling indices by insulin treatment. On the other hand, the specific mitogen-activated protein kinase inhibitor PD98059, which was effective in blocking the mitogenic action of insulin, markedly suppressed the forskolin-induced increase in BrdU-labeling indices. (Bu)2cAMP antagonized not only inhibition of BrdU labeling indices but also changes in cell shape induced by bromocriptine treatment, although forskolin did not have such an antagonizing effect. These results suggest that: 1) intracellular cAMP plays a stimulatory role in the regulation of lactotroph proliferation; 2) cAMP and insulin/mitogen-activated protein kinase signalings require each other for their mitogenic actions; and 3) the antimitogenic action of bromocriptine is, at least in part, caused by inhibition of cAMP production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLIFERATION OF LACTOTROPHS in the anterior pituitary gland is regulated by interactions of extracellular signals derived from three distinct levels. First, hormones secreted by peripheral endocrine glands affect lactotroph proliferation. Estradiol, in particular, has a remarkable mitogenic action in vivo and in vitro on lactotrophs (1, 2, 3, 4, 5). Second, substances that are secreted by hypothalamic neurons and transported via hypophysial portal blood influence lactotroph proliferation. Dopamine and its agonist bromocriptine inhibit proliferation of normal lactotrophs and prolactinoma cells in experimental animals and humans (6, 7, 8, 9), whereas TRH and vasoactive intestinal peptide (VIP) have been reported to stimulate lactotroph proliferation (10, 11, 12). Third, intrapituitary growth factors, such as epidermal growth factor, insulin-like growth factor-1 (IGF-1), and nerve growth factor, alter lactotroph proliferation in an autocrine or paracrine manner (11, 13, 14, 15, 16, 17). Although extracellular signals regulating lactotroph proliferation and their interactions have thus been studied, little is known about the intracellular signal transduction pathways leading to DNA synthesis and proliferation after binding of these extracellular signals to receptors.

cAMP is a ubiquitous second messenger implicated in negative and positive modulation of cell proliferation (for review, see Refs. 18, 19). cAMP inhibits cell proliferation in a number of cell types (including fibroblasts, smooth muscle cells, adipocytes, and T lymphocytes) and stimulates it conversely in certain cells (such as Swiss 3T3 cells, thyroid cells, and somatotrophs). Although the mechanism of protein kinase A (PKA) activation by cAMP is well characterized, the mechanism by which PKA then induces arrest or progression of cell cycle is poorly understood. Possible candidates responsible for mediation of the mitogenic action of cAMP/PKA could be cAMP response element binding protein (CREB) (20) and p70S6K (21), in somatotrophs and Swiss 3T3 cells, respectively. In contrast, based on the findings that, in fibroblasts and adipocytes, cAMP inhibits the activity of a protein kinase cascade (22, 23, 24), which consists of Ras, Raf 1, mitogen-activated protein kinase (MAPK) kinase, and MAPK, in order (for review, see Ref. 25) and mediates mitogenic actions of many growth factors (26, 27, 28), it has been suggested that the antimitogenic action of cAMP is mediated by the MAPK cascade.

The present study was undertaken to investigate whether agents that increase intracellular cAMP levels affect proliferation rates of lactotrophs in primary culture, and whether there are interactions between signal transduction pathways of cAMP and insulin, a growth factor that stimulates potently cell proliferation. Furthermore, the site in the cAMP signaling pathway at which bromocriptine inhibits lactotroph proliferation was determined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Experiments were conducted under the guideline of the Ethical Committee of Animal Experiments in Yamanashi Medical University. Six-week-old female Wistar rats, purchased from Japan SLC (Shizuoka, Japan), were used for primary pituitary cell cultures. Anterior pituitary cells were dispersed as described previously (9). Briefly, after decapitation, five anterior pituitaries were minced in MEM for suspension (Sigma Chemical Co., St. Louis, MO), containing NaHCO3 (0.35 g/liter), penicillin G (100 U/ml), streptomycin (100 µg/ml), BSA (0.1%), and HEPES (20 mM) (S-MBH) and were incubated at 37 C in S-MBH containing 0.01% trypsin (TRL-3, Worthington Biochemical Corp., Freehold, NJ) and 0.005% deoxyribonuclease, in a siliconized Spinner suspension flask, with constant stirring for a total of 80–100 min. The dispersed pituitary cells were treated with 0.1% trypsin inhibitor and 0.025% deoxyribonuclease and were subjected to cell counting and viability test by trypan blue exclusion. A 100-µl aliquot of cell suspension, containing 1.5 x 105 cells in DMEM, supplemented with 20 mM HEPES, was placed on poly-D-lysine-coated 35-mm culture dishes (Falcon, Becton Dickinson and Co., Bedford, MA), so that it spread to form a circle of approximately 10 mm in diameter. The cells were subsequently allowed to attach to the surface of the dishes in a humidified CO2 incubator for 1 h. The pituitary cells were flooded with 2 ml DMEM containing HEPES, 8.3% horse serum, 1.7% FBS, penicillin, and streptomycin and were cultured at 37 C in a humidified atmosphere of 5% CO2-95% air for 1–2 days. After the culture with medium containing serum, the cultured cells were washed with a 1:1 mixture of DMEM and Ham’s Nutrient Mix F-12 without phenol red and containing 15 mM HEPES, 100 U/ml penicillin G, 100 µg/ml streptomycin, 1.2 mg/ml NaHCO3 (DMEM/F-12) and were cultured for another 3 days with serum-free, chemically defined experimental medium. The serum-free medium was freshly changed 1 day after initiation of serum-free culture. The serum-free experimental medium was originally derived from medium described elsewhere (29), with some modifications (DMEM/F-12 supplemented with varying concentrations of bovine insulin, depending upon experiments, 10 µg/ml bovine transferrin, 40 nM sodium selenite, 30 µM ethanolamine, 100 pM triiodothyronine, and 5 mM ethanol).

For experiments using enriched lactotrophs, enzymatically dissociated rat anterior pituitary cells were subjected to differential sedimentation on a discontinuous Percoll gradient, as described elsewhere (30). The gradient was constructed of four concentrations of Percoll (Sigma Chemical Co.) with densities of 1.045, 1.065, 1.080, and 1.090. Two milliliters of Percoll solution were layered on top of each other, starting with the highest density, in a 15-ml conical centrifuge tube. Freshly dispersed pituitary cells in 1 ml S-MBH were loaded on the top of the gradient and centrifuged at 400 x g in a swing-out rotor for 20 min at room temperature. A lactotroph-enriched pituitary cell layer at the interface between 1.045 and 1.065 was carefully collected with a Pasteur pipette and washed twice with S-MBH. The enriched lactotrophs were plated and cultured in the same manner as unenriched pituitary cells.

Treatment of lactotrophs in culture
Forskolin (Sigma Chemical Co.) and bromocriptine methylate (Sigma Chemical Co.) were initially dissolved with ethanol at concentrations of 20 mM and 1 mM, respectively. N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide (H89) (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) was dissolved with 50% ethanol at a concentration of 25 mM. PD98059 (New England Biolabs, Inc., Beverly, MA) and KT5720 (Biomol Research Laboratories, Inc.) were dissolved with dimethyl sulfoxide at concentrations of 20 mM and 1.2 mM, respectively. These agents were diluted immediately before use, the final concentrations of ethanol and dimethyl sulfoxide in medium being 0.06–0.31% and 0.25%, respectively. (Bu)2cAMP and 8-bromo-cAMP (Sigma Chemical Co.) were dissolved with the serum-free medium. Cultured pituitary cells were treated with the above regents for the indicated times and with 200 µM 5-bromo-2'-deoxyuridine (BrdU) for 3–24 h before the end of culture, to label proliferating cells.

After BrdU labeling, cultured pituitary cells were detached from culture dishes and redispersed with trypsin, as described elsewhere (9). Cells suspended with Earle’s balanced salt solution, containing 20 mM HEPES, were attached to poly-D-lysine-coated glass slides by centrifugation with a cytocentrifuge (SC-2, Tomy, Tokyo, Japan) at 105 x g for 2 min. The cells attached on glass slides were fixed with ice-cold methanol for 30 min, washed with distilled water, and stored in PBS at 4 C until immunostaining.

Immunostaining
The anterior pituitary cells, attached to glass slides, were double immunostained for BrdU and PRL, as described previously (9). Briefly, the slides were treated with 3 M HCl for 30 min and 10% normal donkey serum in PBS for 15 min. Double-labeling immunofluorescence staining was performed by three steps using the following reagents: 1) a mixture of mouse monoclonal anti-BrdU (Sigma Chemical Co.) at 1:200 dilution and rabbit antirat PRL (NIDDK IC-5) at 1:4000 dilution; 2) biotinylated horse antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) at 1:50 dilution; and 3) a mixture of Texas Red-labeled streptavidin (Amersham, Arlington Heights, IL) at 1:50 dilution and fluorescein-isothiocyanate-labeled donkey antirabbit IgG (Amersham) at 1:50 dilution. The cells were incubated with 100 µl of each reagent diluted with PBS containing 10% normal donkey serum for 1 h, followed by a 20-min washing.

The immunostained slides were mounted with PermaFluor (Immunon, Pittsburgh, PA) and were observed with a fluorescence microscope (BX50-FLA, Olympus Corp., Tokyo, Japan) equipped with a dual-band mirror unit for fluorescein-isothiocyanate and Texas Red (U-DM-FI/TX).

Statistical analysis
A minimum of 1000 PRL-immunoreactive cells were examined, in randomly chosen fields for each slide, to determine the BrdU-labeling index, which was the percentage of pituitary cells immunoreactive for both PRL and BrdU of total PRL-immunoreactive cells counted. Three slides were analyzed for each treatment group derived from the same cell preparations. Experiments were replicated three times with separate batches of cell preparations. Differences between individual groups were statistically analyzed using one-way or two-way ANOVA followed by Fisher’s PLSD test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of forskolin and (Bu)2cAMP on lactotroph proliferation
The dose responses of BrdU-labeling indices of lactotrophs to two cAMP-related agents that increase intracellular cAMP levels by distinct mechanisms were determined. The mean BrdU-labeling index of lactotrophs that were cultured in medium containing 10 ng/ml insulin and labeled with BrdU for 24 h was 3.4 ± 0.8% (mean ± SEM, based on six independent experiments). BrdU-labeling indices of lactotrophs were increased 40 h after treatment with forskolin, an adenylate cyclase activator (Fig. 1Go, left). Treatment with 1–3 µM forskolin induced a 4.2-fold maximal increase in BrdU-labeling indices, compared with vehicle treatment. Similarly, (Bu)2cAMP, a membrane-permeable cAMP analog, was effective in increasing BrdU-labeling indices with less potency than forskolin (Fig. 1Go, right). (Bu)2cAMP at 0.3 mM induced a 2.6-fold maximal rise in BrdU-labeling indices, compared with vehicle treatment. The increased BrdU-labeling indices, by (Bu)2cAMP treatment, were found to be caused by cAMP moiety itself, because butyrate, a potential degradation product of (Bu)2cAMP, did not change BrdU-labeling indices at 0.1 mM or less, and it significantly decreased rather than increased them, at 0.3 mM (P < 0.05) (Fig. 2Go). This was further supported by the result that another cAMP analog 8-bromo-cAMP, at doses of 0.3 and 1 mM, was also effective in increasing BrdU-labeling indices (P < 0.01).



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  		Figure 1. Dose responses of lactotroph proliferation to forskolin and (Bu)2cAMP (db-cAMP). Anterior pituitary cells that had been cultured in serum-free medium containing 10 ng/ml insulin were treated with either vehicle or varying concentrations of forskolin (left) or (Bu)2cAMP (right) for 40 h and were labeled with BrdU for 24 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to vehicle-treated control groups. Data are the mean ± SEM of triplicate determinations from a representative experiment that was replicated three times with separate batches of cell preparations.

 


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  		Figure 2. Effects of (Bu)2cAMP, 8-bromo-cAMP (8b-cAMP), and butyrate on lactotroph proliferation. Anterior pituitary cells that had been cultured in serum-free medium containing 10 ng/ml insulin were treated with vehicle, (Bu)2cAMP, 8-bromo-cAMP, or sodium butyrate for 40 h and were labeled with BrdU for 24 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to vehicle-treated control groups. Data are the mean ± SEM of triplicate determinations from a representative experiment that was replicated twice with separate batches of cell preparations.

 
To minimize influences of other cell types that contaminate lactotroph populations, we examined the effects of the cAMP-increasing agents on BrdU-labeling indices of enriched lactotrophs. Differential sedimentation with a Percoll gradient successfully enriched pituitary cells for lactotrophs, from 45–55% to 75–81%. Forskolin and (Bu)2cAMP, at 1 µM and 0.3 mM, respectively, induced significant increases in BrdU-labeling indices of the enriched lactotrophs that were cultured in medium containing 10 or 100 ng/ml insulin (P < 0.01) (Fig. 3Go). There was no significant difference in magnitudes of the forskolin-induced increases in BrdU-labeling indices, between enriched and unenriched lactotrophs (data not shown).



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  		Figure 3. Mitogenic actions of forskolin and (Bu)2cAMP on enriched lactotrophs. Anterior pituitary cells that had been enriched for lactotrophs by differential sedimentation and cultured in serum-free medium containing 10 or 100 ng/ml insulin were treated with either vehicle, 0.3 mM (Bu)2cAMP, or 1 µM forskolin for 40 h and were labeled with BrdU for 24 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to a vehicle-treated control group cultured with 10 ng/ml insulin. *, Significantly different from the corresponding vehicle-treated groups.

 
To see the time course of the action of forskolin, BrdU-labeling indices were determined in lactotrophs treated with 1 µM forskolin, for varying times from 6–36 h. Because BrdU incubation time was shortened to 6 h to improve time resolution, BrdU-labeling indices in Fig. 4Go reflect the percent of lactotrophs that were labeled with BrdU during periods of the preceding 6 h before the treatment times indicated in the abscissa. Forskolin treatment did not affect indices until 12 h after the initiation of treatment, but increased BrdU-labeling indices at 12–18 h (P < 0.01, 2-way ANOVA). BrdU-labeling indices were thereafter increased progressively, up to 36 h tested.



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  		Figure 4. Time course of the mitogenic action of forskolin on lactotrophs. Anterior pituitary cells that had been cultured in serum-free medium containing 10 ng/ml insulin were treated with either vehicle or 1 µM forskolin for the indicated times, ranging from 6–36 h, and were labeled with BrdU for 6 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to the corresponding vehicle-treated control groups.

 
Dose response of lactotroph proliferation to insulin in combination with or without forskolin
As insulin concentrations in medium were increased, BrdU-labeling indices rose remarkably in a dose-dependent manner (Fig. 5Go). BrdU-labeling indices of lactotrophs cultured with insulin at 5000 ng/ml, a maximal concentration tested, were 10.4-fold higher than those of lactotrophs without insulin (P < 0.01). Treatment with 1 µM forskolin for 40 h, in combination with insulin, increased BrdU-labeling indices to significantly higher levels than did insulin alone, at any concentrations (P < 0.01, 2-way ANOVA).



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  		Figure 5. Dose response of lactotroph proliferation to insulin in combination with or without forskolin. Anterior pituitary cells that had been cultured in serum-free medium containing varying concentrations of insulin were treated with either vehicle or 1 µM forskolin for 40 h and were labeled with BrdU for 12 h before the end of treatment, except 5000 ng/ml insulin-cultured groups with 3-h labeling with BrdU. BrdU-labeling indices of lactotrophs are expressed relative to vehicle-treated control group cultured without insulin. *, Significantly different from the corresponding vehicle-treated groups, based on two-way ANOVA.

 
Inhibitory effects of PKA inhibitors on insulin-induced lactotroph proliferation
To determine the optimum doses of PKA inhibitors for sufficiently blocking the action of cAMP/PKA, we preliminarily examined the effects of pretreatment with varying concentrations of H89 and KT5720, on an increase in BrdU-labeling indices of lactotrophs induced by 1 µM forskolin. The effective doses of H89 and KT5720 in blocking the action of forskolin were found to be 1 and 0.5 µM, respectively (Figs. 6Go and 7Go). The dose of H89 also blocked the effect of 0.3 mM (Bu)2cAMP (data not shown). Treatment for 28 h with H89 or KT5720 at these concentrations had no effect on basal levels of BrdU-labeling indices when given into medium without insulin. H89 did not change an increase in BrdU-labeling indices induced by 100 ng/ml insulin but significantly attenuated that induced by 5000 ng/ml insulin, by 56% (P < 0.01) (Fig. 6Go). KT5720 was effective in suppressing increases in BrdU-labeling indices induced by 100 and 5000 ng/ml insulin, by 53 and 58%, respectively (P < 0.01) (Fig. 7).



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  		Figure 6. Inhibition of insulin-induced lactotroph proliferation by the PKA inhibitor H89. Anterior pituitary cells that had been cultured without or with 100 or 5000 ng/ml insulin were treated with either vehicle or 1 µM H 89 for 28 h and were labeled with BrdU for 12 h before the end of treatment, except 5000 ng/ml insulin-cultured groups with 3-h labeling with BrdU. Some groups cultured with 100 ng/ml insulin were treated with 1 µM forskolin 30 min after the H89 treatment. BrdU-labeling indices of lactotrophs are expressed relative to a vehicle-treated control group cultured without insulin. *, Significantly different from the corresponding vehicle-treated groups.

 


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  		Figure 7. Inhibition of insulin-induced lactotroph proliferation by the PKA inhibitor KT5720. Anterior pituitary cells that had been cultured without or with 100 or 5000 ng/ml insulin were treated with either vehicle or 0.5 µM KT5720 for 28 h and were labeled with BrdU for 12 h before the end of treatment, except 5000 ng/ml insulin-cultured groups with 3-h labeling with BrdU. Some groups cultured with 100 ng/ml insulin were treated with 1 µM forskolin 30 min after the KT5720 treatment. BrdU-labeling indices of lactotrophs are expressed relative to a vehicle-treated control group cultured without insulin. *, Significantly different from the corresponding vehicle-treated groups.

 
Inhibitory effects of a MAPK kinase inhibitor on forskolin-induced lactotroph proliferation
Based on the results of preliminary studies, a PD98059 concentration of 50 µM was chosen to block the activity of MAPK kinase. Indeed, pretreatment with 50 µM PD98059, 30 min earlier, completely abolished a 3.1-fold increase in BrdU-labeling indices by 200 ng/ml insulin (P < 0.01) (Fig. 8Go). Treatment for 28 h with 1 µM forskolin, in the absence of insulin, increased BrdU-labeling indices to high levels similar to those achieved by 200 ng/ml insulin. Pretreatment with PD98059 had no effect on basal levels of BrdU-labeling indices but significantly attenuated the forskolin-induced increase in BrdU-labeling indices (by 59%).



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  		Figure 8. Inhibition of forskolin-induced lactotroph proliferation by the MAPK kinase inhibitor PD98059. Anterior pituitary cells that had been cultured in serum-free medium without insulin were pretreated with either vehicle or 50 µM PD98059 30 min before treatment with either vehicle, 1 µM forskolin, or 200 ng/ml insulin for 28 h and were labeled with BrdU for 12 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to a vehicle-treated basal group. *, Significantly different from the corresponding vehicle-treated groups.

 
Effects of (Bu)2cAMP and forskolin on proliferation and cell shape of bromocriptine-treated lactotrophs
We have previously shown that treatment with dopamine and its agonist bromocriptine suppresses proliferation of cultured lactotrophs and changes cell shape from flat to round (9). We investigated whether treatment with (Bu)2cAMP and forskolin reverses these actions of bromocriptine on lactotrophs. BrdU-labeling indices of lactotrophs, cultured in medium containing 50 ng/ml insulin, revealed a 74% and 182% increase, 30 h after treatment with 1 mM (Bu)2cAMP and 1 µM forskolin, respectively (P < 0.01) (Fig. 9Go). Treatment with 1 nM bromocriptine suppressed basal levels of BrdU-labeling indices to 40% (P < 0.01). These bromocriptine-treated lactotrophs showed a significant increase in BrdU-labeling indices in response to (Bu)2cAMP (P < 0.01), which was similar in magnitude to that caused by bromocriptine-untreated control lactotrophs, but no significant increase in response to forskolin.



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  		Figure 9. Mitogenic actions of (Bu)2cAMP and forskolin on bromocriptine-treated lactotrophs. Anterior pituitary cells that had been cultured in serum-free medium containing 50 ng/ml insulin were treated without or with 1 nM bromocriptine in combination with either vehicle, 1 mM (Bu)2cAMP, or 1 µM forskolin for 30 h and were labeled with BrdU for 12 h before the end of treatment. BrdU-labeling indices of lactotrophs are expressed relative to a vehicle-treated control group. *, Significantly different from the corresponding vehicle-treated groups.

 
Approximately 60% of pituitary cells cultured in medium containing 50 ng/ml insulin were phase-dark, flat, and polygonal (Fig. 10AGo). Treatment with (Bu)2cAMP or forskolin alone slightly increased the percents of these cells and was accompanied by appearance of cells with extended fiber-like projections (Fig. 10BGo). Bromocriptine treatment decreased percents of the dark and flat cells and increased percents of refractive and round cells (Fig. 10CGo), as described previously (9). These changes, induced by bromocriptine, were not altered by forskolin treatment but were reversed considerably by (Bu)2cAMP treatment (Fig. 10DGo). The effects of bromocriptine alone and of a combination of bromocriptine and (Bu)2cAMP on cell shape were more remarkably observed in pituitary cells that had been cultured in medium containing 5000 ng/ml insulin than 50 ng/ml insulin (data not shown).



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  		Figure 10. Photomicrographs showing the action of (Bu)2cAMP on changes in cell shape induced by bromocriptine treatment of lactotrophs. Anterior pituitary cells that were used in experiments shown in Fig. 8Go were photographed under a phase-contrast microscope before redispersion. Pituitary cells were treated with vehicle alone (A), (Bu)2cAMP alone (B), bromocriptine alone (C), and a combination of bromocriptine and (Bu)2cAMP (D). Magnification, x156.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies have provided evidence for an important role of cAMP in the regulation of lactotroph functions, such as secretion and synthesis of PRL (31, 32, 33, 34, 35). Because intracellular cAMP regulates cell proliferation in a variety of cell types (18, 19), it was quite possible that cAMP is involved in the regulation of not only secretion and synthesis of PRL but also proliferation of lactotrophs. We therefore tested this possibility in lactotrophs in serum-free culture using pharmacological means. Increased intracellular cAMP levels, by treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) raised the BrdU labeling index of lactotrophs, which is a cell-specific and reliable parameter for DNA synthesis and, therefore, proliferation of lactotrophs (36). Maximal stimulation of lactotroph proliferation in response to forskolin and (Bu)2cAMP was obtained at 1 µM and 0.3 mM, respectively, with less potency of (Bu)2cAMP. These maximally effective doses in the present study are comparable with those reported in other studies (23, 31, 33, 34). The mitogenic action of (Bu)2cAMP was not caused by butyrate moiety of (Bu)2cAMP but to cAMP itself. Time course experiments showed that the mitogenic action of forskolin appeared at 12–18 h after the initiation of treatment and became progressively marked up to 36 h.

Forty-five percent of populations of cultured cells used in the present study were lactotrophs, the remaining being other cell types, including fibroblasts. Because the other cell types that contaminate lactotroph populations may mediate or influence the actions of forskolin and (Bu)2cAMP in a paracrine manner, their actions were examined in populations of lactotrophs enriched by differential sedimentation. Forskolin and (Bu)2cAMP were still effective in stimulating lactotroph proliferation in the enriched populations, suggesting little involvement, if any, of cell types other than lactotrophs in their actions.

Insulin, one of the essential supplements used in serum-free cultures, is a potent growth factor. Because the highest concentration of insulin used in the present study is supraphysiological, and insulin receptors are saturated at much lower concentrations, presumably the mitogenic action of insulin found in the present study is mediated mostly through the IGF-1 receptor (for review, see Ref. 37). Although insulin and IGF-1 are known to be mitogenic in a variety of cell types, including the PRL/GH-secreting cell line GH3 cell (11, 17, 38), the mitogenic action of insulin on lactotrophs in primary culture was confirmed for the first time in the present study. There has been growing evidence suggesting a crucial role of the MAPK cascade in mediating the mitogenic actions of growth factors, including insulin. It has been well established that growth factors activate the MAPK cascade in a variety of cell types (25) and that inhibition of the activation of the MAPK cascade blocks the mitogenic actions of the growth factors (26, 27, 28). Indeed, in the present study, PD98059, a specific inhibitor of MAPK kinase (MEK1) (39), was effective in totally abolishing lactotroph proliferation in response to insulin. Using this inhibitor of MAPK kinase, we investigated the involvement of MAPK cascade in the mitogenic action of forskolin on lactotrophs. PD98059 treatment suppressed markedly forskolin-induced lactotroph proliferation at a dose sufficient to abolish insulin-induced lactotroph proliferation with a similar magnitude to that by forskolin treatment. These results suggest that stimulation of lactotroph proliferation by increased intracellular levels of cAMP is highly dependent on the activity of the MAPK cascade. This is the first report demonstrating MAPK-dependent cell proliferation stimulated by cAMP signaling. It remains to be investigated whether cAMP/PKA may induce activation of MAPK cascade that, in turn, leads to stimulation of lactotroph proliferation. Consistent with this view are the findings that cAMP-increasing agents activate the MAPK cascade at a level upstream of MAPK kinase in cell lines of PC12, insulin-secreting ß cells, and preadipocytes, although the causal relationship between the activation of the MAPK cascade and cell proliferation has been denied or not clarified in these cells (40, 41, 42).

We next investigated the involvement of cAMP/PKA signaling pathway in the mitogenic action of insulin on lactotrophs using PKA inhibitors (43, 44). H89 and the more specific inhibitor KT5720 suppressed lactotroph proliferation in response to forskolin, confirming PKA activation in the cAMP-mediated stimulation of proliferation. The inhibitors at the same doses had no effect on proliferation of lactotrophs cultured without insulin but attenuated proliferation in response to intermediate and high concentrations of insulin. These results suggest that endogenous cAMP or activity of PKA is involved somewhere in the signaling pathway that mediates the mitogenic action of insulin via the IGF-1 receptor and MAPK cascade. Although it is not elucidated how cAMP/PKA is involved in the insulin/MAPK signaling pathway, it seems likely that the cAMP/PKA signaling pathway interacts at a level downstream of the MAPK cascade. Hypothesizing the existence of a convergence molecule that requires signals from both cAMP/PKA- and insulin/MAPK-mediated pathways for stimulation of proliferation may help explain not only the effects of the PKA inhibitors on lactotroph proliferation in response to insulin but also the effects of the MAPK kinase inhibitor on proliferation in response to forskolin. A possible candidate for such a target molecule of interactions of the cAMP/PKA and insulin/MAPK signalings is CREB. CREB was originally isolated as a nuclear protein that is phosphorylated and activated by PKA, leading to stimulation of transcription of a cAMP-responsive gene (45) and has been implicated in the regulation of proliferation of somatotrophs by studies using transgenic mice expressing a nonphosphorylatable CREB mutant (20). The finding that CREB is phosphorylated and activated not only by PKA but also indirectly by MAPK (46, 47) is consistent with possible CREB involvement in the interactions. Further studies are needed to determine whether CREB plays a critical role in convergence of interactions of the cAMP/PKA and insulin/MAPK signalings on lactotroph proliferation.

The extracellular signal that stimulates lactotroph proliferation, via an increase in intracellular levels of cAMP, remains to be determined. Among hypothalamus-derived substances, VIP and pituitary adenylate cyclase-activating polypeptide may be potential extracellular signals responsible for the cAMP-mediated mitogenesis. VIP and pituitary adenylate cyclase-activating polypeptide, which increase intracellular levels of cAMP in anterior pituitary cells, have been shown to stimulate not only PRL secretion (48, 49, 50, 51) but also proliferation of lactotrophs (12, 51). Conversely, intracellular cAMP may be negatively involved in mediation of a well-known inhibitory action of dopamine on lactotroph proliferation (6, 7, 8, 9), given that a decrease in intracellular levels of cAMP caused by adenylate cyclase inhibition has been reported in pituitary cells treated with dopamine or its agonists (32, 52). To determine the site of the antimitogenic action of dopamine in the cAMP/PKA system, we compared the effects of the dopaminergic agonist bromocriptine on the mitogenic actions of (Bu)2cAMP and forskolin. If bromocriptine inhibits a signaling pathway downstream of PKA, leading to attenuated response of cell cycle-regulating machinery to an increase in intracellular cAMP levels, the mitogenic actions of both (Bu)2cAMP and forskolin would be suppressed to the same extent. However, in bromocriptine-treated lactotrophs, a mitogenic response was still observed by (Bu)2cAMP but not by forskolin that is capable of more potently stimulating proliferation than (Bu)2cAMP in bromocriptine-untreated cells. Therefore, these results suggest that inhibition by the dopaminergic agonist of lactotroph proliferation induced by cAMP/PKA signaling is mainly caused by an action at the level of adenylate cyclase to suppress cAMP production.

We have previously shown that bromocriptine treatment of lactotrophs in serum-free culture causes a remarkable morphological change from flat phase-dark polygonal cell shape to round phase-bright cell shape, and that D2 receptor-mediated inhibition of lactotroph proliferation in serum-free culture is closely related to the changes in actin organization and cell shape (9). Treatment with (Bu)2cAMP that could reverse the antimitogenic action of bromocriptine was also effective in antagonizing the bromocriptine-induced changes in cell shape, although forskolin treatment was not. These results suggest that the bromocriptine action on cell shape of lactotrophs is attributable to a decrease in intracellular levels of cAMP that is capable of changing cell shape of lactotrophs.

In conclusion, cAMP is involved as a stimulatory second messenger in the regulation of proliferation of lactotrophs in primary culture. The present study suggests that interactions of cAMP/PKA and insulin/MAPK signalings are required for lactotroph proliferation. Suppression of cAMP production may be responsible, at least in part, for both inhibition of lactotroph proliferation and changes in cell shape that are induced by bromocriptine.


   Acknowledgments
 
The authors thank Dr. A. F. Parlow and the NIDDK for providing PRL antiserum for immunocytochemistry. We are also grateful to Dr. S. Atsumi and Ms. W. Takahashi for their expert technical assistance.


   Footnotes
 
1 This work was supported by the Ministry of Education, Science and Culture of Japan (Grants-in-Aid for Scientific Research 08670081 and 10670060). Back

Received December 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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