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Endocrinology Vol. 139, No. 5 2235-2239
Copyright © 1998 by The Endocrine Society

ARTICLES

Luteinizing Hormone-Releasing Hormone-Signal Transduction and Stathmin Phosphorylation in the Gonadotrope {alpha}T3–1 Cell Line1

Sophia V. Drouva, Benoit Poulin, Valérie Manceau and André Sobel

CNRS UMR 6544 (S.V.D., B.P.), Université de la Méditerranée, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France; and Institut National de la Santé et de la Recherche Médicale, Unité 440 (V.M., A.S.), 75005 Paris, France

Address all correspondence and requests for reprints to: A. Sobel, Institut National de la Santé et de la Recherche Médicale, Unité 440, 17 Rue du Fer à Moulin, 75005 Paris, France. E-mail: sobel{at}infobiogen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the effects of GnRH (LHRH) and of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate on stathmin phosphorylation in the gonadotrope {alpha}T3–1 cell line. Stathmin expression and its phosphorylation were maximal during the exponential phase of cell growth. LHRH stimulated stathmin phosphorylation through a specific receptor in a dose- and time-dependent manner, and TPA induced a similar extensive stathmin phosphorylation. Their effects were inhibited either in PKC-depleted {alpha}T3–1 cells, or by the PKC inhibitor staurosporine. In the context of the known implication of PKC in LHRH-induced signal transduction, our results show that stathmin phosphorylation is involved in LHRH transduction, either as a result of direct activation of specific PKC isoforms or through a pathway involving kinases downstream to PKC activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH (LHRH), secreted from hypothalamic neurons, acts on pituitary gonadotropes and regulates the synthesis and release of LH and FSH through specific receptors (1). The signaling events underlying LHRH action on gonadotropes have been investigated and seem to include phospholipase C (PLC) activation via a specific G{alpha}q/11 protein, generation of inositol 1,4,5-trisphosphate and diacylglycerol, a rise in cytoplasmic Ca2+ [mobilization from intracellular stores (first phase), extracellular influx after activation of voltage-dependent Ca2+ channels (second phase)], and activation of protein kinase C (PKC) isoenzymes (1, 2). The LHRH-induced PLC stimulation is followed by a phospholipase A2-mediated arachidonic acid release and by a sustained phospholipase D activation (1, 2, 3). Some of the cell responses to LHRH stimulation require increases of intracellular Ca2+ and Ca2+/calmodulin-dependent pathways, and/or activation of PKC, whereas others seem to be Ca2+- and PKC-independent (1).

Much less is known about the intracellular processes affected by the various second messengers in gonadotropes. In most biological systems, these various second messengers act through intracellular pathways in which protein phosphorylation-dephosphorylation reactions are known to play a major regulatory role (4, 5). Several phosphoproteins, related to the multihormonal regulation of normal pituitary cells in culture, have been identified (6, 7), among which stathmin has been thoroughly characterized over the past years (8, 9). Stathmin [also referred to as p19 (10), prosolin (11), or Op18 (12)] is a 19-kDa cytoplasmic protein, highly expressed in the nervous and neuroendocrine systems (13, 14) and whose phosphorylation could be related to cell regulation by diverse extracellular factors such as hormones (6), neurotransmitters (15), or growth and differentiation factors (16, 17). It possesses an N-terminal regulatory domain containing four phosphorylation sites (18) that can be phosphorylated, respectively, by Ca2+/calmodulin-dependent kinases (19) or cell cycle-dependent kinases (18) in vitro, and by mitogen-activated protein kinases (MAPKs) (20) or PKA (14, 18) in vitro and in vivo. The phosphorylation of stathmin on these four sites accounts for all its diverse molecular forms observed in vivo (18), possibly reflecting an integrated signal that can be further relayed toward molecular targets/partners of stathmin through its C-terminal "interaction" domain (21). Several target/partners candidates for stathmin have been identified recently, such as the protein kinase KIS (21, 22), the tumor suppressor gene product CC2/tsg101 (21, 23), and tubulin (24, 25, 26, 27, 28).

The development of a gonadotrope cell line ({alpha}T3–1), obtained by targeted tumorigenesis in transgenic mice (29), provided an homogeneous cell population model, appropriate for detailed investigations related to LHRH-associated cell signaling biochemical processes. In the present study, we therefore characterized in {alpha}T3–1 cells the expression of the intracellular phosphoprotein stathmin during cell growth, and its phosphorylation, in response to LHRH or to direct activation of PKC by the phorbol ester TPA. Altogether, our results indicate that LHRH specifically induces the phosphorylation of stathmin through a PKC-dependent pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
{alpha}T3–1 cells were grown and passaged routinely, as previously described (2, 3), in DMEM [with 4.5 mg/ml glucose (Gibco Life Technologies Cergy-Pontoise, France), supplemented with 10% FCS and penicillin/streptomycin (100 and 75 U/ml respectively, Sigma, St-Quentin Fallavier, France)]. Cultures were maintained at 37 C in a humidified air atmosphere of 5.8% CO2. When cells reached 50–60% confluency, the culture medium was changed to DMEM containing antibiotics and 1% FCS to avoid further divisions and thus obtain cells in a homogeneous developing state for the experiments.

Pharmacological treatments
Pharmacological reagents used and their sources were: TPA (Sigma); LHRH antagonist D-pGlu1, D-Phe2, [D-Trp3,6]LHRH (Sigma); and staurosporine (Fluka Biochemika, St-Quentin Fallavier, France). Each treatment was applied to cells for 30 min, unless indicated otherwise. Each series of experiments was repeated two to six times independently and provided similar results, relative to stathmin expression and/or phosphorylation, as analyzed by PAGE.

PAGE, protein detection, and immunoblot
About 50 µg of total cell lysate protein were analyzed by one- or two-dimensional PAGE. One-dimensional gel electrophoresis was performed on 13% polyacrylamide gels, according to Laemmli (30). Two-dimensional gels were performed as described (31): the isoelectric focusing gel contained ampholines, pH 3.5–10.5 (0.5%)/pH 5–7 (2%), and the second dimension was run on 13% polyacrylamide gels. Proteins were either silver-stained, as described (8), or immunoblotted as follows. The gels were transferred to nitrocellulose in a semidry electroblotting apparatus, in a buffer containing 48 mM Tris, 39 mM glycine, and 20% methanol. Membranes were blocked with 2.5% casein in buffer A (12 mM Tris-HCl, pH 7.4, 160 mM NaCl, 0.1% Triton, Serva, Heidelberg, Germany), and probed with antistathmin C (1:10,000) (13) in buffer A containing 1% casein. Bound antibodies were detected by a second goat antirabbit antiserum coupled to peroxidase (1:10,000) (Dako, A/S, Glostrup, Denmark), revealed by the chemiluminescent ECL (enzyme chemiluminescence) kit protocol (Amersham, Les Ulis, France). As a control for protein loading, the same blot was then reprobed with an antiactin mouse monoclonal antibody (1:3,000) (Amersham) with an appropriate secondary antibody coupled to peroxidase for ECL.

Quantification of stathmin phosphorylation
Spots corresponding to unphosphorylated (N1) or phosphorylated (P1 and P2) forms of stathmin, visualized by silver-staining on two-dimensional polyacrylamide gels, were quantified with a CCD camera and image analysis system (Bio-1D, Vilbert-Lourmat, Marne-la-Vallée, France). Because the overall staining of each gel could vary from sample to sample, the absolute staining intensities of spots did not faithfully reflect the absolute amounts of phosphorylated and nonphosphorylated forms. The extent of stathmin phosphorylation in each sample was therefore evaluated as the ratio of its phosphorylated forms (P1+P2) vs. its unphosphorylated form (N1) on the corresponding gel or as the percentile of stathmin phosphorylation, yielding values not depending on the variability of the silver-staining procedure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stathmin expression during {alpha}T3–1 cell growth
Stathmin was detected in {alpha}T3–1 cells by either immunoblotting with specific antibodies or silver-staining (Fig. 1Go, A and B). One-dimensional immunodetection revealed a high expression of stathmin, comparable with the level in a whole brain extract, with the highest expression during the exponential growth phase, at 2–4 days of culture, followed by a decrease in later cultures when cells have reached confluency (Fig. 1AGo). The level of actin immunoreactivity was used as an internal standard for protein loading in these experiments. Although two-dimensional silver-stained gels do not reflect absolute levels of protein expression, which are more accurately reflected on one-dimensional immunoblots, they allow the separate detection and relative quantification of the nonphosphorylated (major N1 and minor N2) and phosphorylated (P1) forms of stathmin (18), which could indeed be revealed in untreated {alpha}T3–1 cells in culture (Fig. 1BGo). Comparison of the relative intensity of spots P1 and N1/N2 on each individual gel shows that the degree of phosphorylation of stathmin was, like its expression level, maximal during the exponential phase of cell growth (2–4 days) and diminished at confluency (Fig. 1Go), which also explains the apparently higher intensity of spot N1 at day 7 than at day 4 in culture.



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  		Figure 1. Expression of stathmin in the {alpha}T3–1 gonadotrope cell line. A, One-dimensional immunoblot with an antistathmin antiserum (see Materials and Methods) of a neonatal mouse brain sample used as control (b) and of {alpha}T3–1 cells grown in culture for the indicated times (2, 4, and 7 days). The same blot was reprobed with an antiactin monoclonal antibody as a control for protein loading. B, The same {alpha}T3–1 samples were analyzed by two-dimensional gel electrophoresis, followed by silver-staining, to reveal the various nonphosphorylated (N1 and the minor form N2) and phosphorylated (P1) forms of stathmin. The area shown within the entire 2D-gel (left) is presented in more detail (right) for the three samples analyzed. Note that silver-staining of separate two-dimensional gels does not directly reflect the absolute expression level of stathmin, but comparison of phosphorylated vs. nonphosphorylated spots on each gel allows the evaluation of the degree of phosphorylation of stathmin in the corresponding sample.

 
LHRH-induced phosphorylation of stathmin
LHRH, applied to {alpha}T3–1 cell line cultures at 50% confluency, stimulated stathmin phosphorylation in a time- and concentration-dependent manner, as revealed by the increased relative detection of its P1 (phosphorylated on one site) and P2 forms (phosphorylated on two sites), as compared with its nonphosphorylated form N1 (Figs. 2Go and 3Go). The degree of stathmin phosphorylation progressively increased up to 30 min (maximum time tested), and half-maximal stimulation was observed at about 10-9 M LHRH (Fig. 2Go). In addition, the LHRH-induced phosphorylation of stathmin is clearly a specific response, because the LHRH antagonist D-pGlu1-D-Phe2-D-Trp3,6-LHRH (10-8 M), although devoid of effect by itself (data not shown), extensively blocked the stimulatory effect of LHRH (Fig. 3Go).



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  		Figure 2. Dose-response and kinetics of LHRH-induced stathmin phosphorylation in {alpha}T3–1 cells. Cells treated with the indicated concentrations of LHRH, for the indicated times, were analyzed for their stathmin form distribution, as in Fig. 1Go. The stathmin areas of silver-stained two-dimensional electrophoresis gels are shown (top). N1 refers to the nonphosphorylated form of stathmin; P1 and P2, to its forms phosphorylated on one and two sites, respectively. As indicated in Fig. 1Go, the relative intensities of the phosphorylated (P1 and P2) vs. the nonphosphorylated (N1) forms of stathmin reflect its degree of phosphorylation within each sample. Stathmin phosphorylation, in response to LHRH, was evaluated by quantitating spot intensities from replicate dose-response experiments (bottom), such as that shown above (see Materials and Methods).

 


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  		Figure 3. Specificity of stathmin phosphorylation in response to LHRH. {alpha}T3–1 gonadotrope cells were treated without (control, C) or with LHRH (LHRH, L), or with LHRH in the presence of its specific antagonist D-pGlu1-D-Phe2-D-Trp3,6-LHRH (LHRH + antagonist, L+A), at the concentrations indicated. The cells were then collected and analyzed by two-dimensional gel electrophoresis; as in Fig. 1Go, the separated proteins were revealed by silver-staining, and (A) whole gels, or (B) details of the stathmin area, together with the corresponding quantification of stathmin phosphorylation, are shown. N1 refers to the nonphosphorylated form of stathmin; P1 and P2 refer to its forms phosphorylated on one and two sites, respectively. As indicated in Fig. 1Go, the extent of phosphorylation of stathmin in control conditions and in response to each treatment was evaluated by comparing the intensities of phosphorylated vs. nonphosphorylated forms of stathmin on each individual gel of the corresponding sample (see Materials and Methods). The gels presented are representative of several comparable experiments.

 
PKC implication in LHRH-induced stathmin phosphorylation
Like LHRH, the phorbol ester TPA (10-7 M), when administered to {alpha}T3–1 cells for 30 min, highly stimulated stathmin phosphorylation (Fig. 4Go), suggesting that PKC activation was able to induce stathmin phosphorylation in these cells. This was further supported by the fact that staurosporine (10-6 M), a relatively specific inhibitor of PKC-dependent phosphorylations, counteracted the TPA-induced phosphorylation of stathmin (Fig. 4Go). In addition, staurosporine (10-6 M) also reduced the LHRH-induced stathmin phosphorylation in these cells, suggesting that the effect of LHRH on stathmin phosphorylation might involve PKC activation. However, the inhibitor at 10-7 M was less effective (data not shown).



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  		Figure 4. Pharmacological analysis of PKC-dependence of stathmin phosphorylation. {alpha}T3–1 cells were treated with staurosporine (10-6 M) (S), TPA (10-7 M) (T), LHRH (10-7 M) (L) alone or in combination (T+S: staurosporine + TPA/L+S: staurosporine + LHRH). Stathmin phosphorylation was evaluated as in Fig. 3Go (see Materials and Methods), after two-dimensional electrophoretic separation of its phosphorylated and nonphosphorylated forms. The results (•) from several replicate experiments and the corresponding mean value ({diamond}) are reported for each pharmacological treatment.

 
To further investigate whether PKC stimulation is implicated in the LHRH effect on stathmin phosphorylation, {alpha}T3–1 cells were exposed to TPA (10-7 M) for a 24-h period, a treatment inducing an almost complete disappearance of both soluble and membrane fractions of TPA-sensitive PKC isoforms present in these cells, resulting in a highly reduced TPA responsiveness (2, 32, 33). Interestingly, whereas the 24-h TPA treatment did not result in any change of basal stathmin phosphorylation, the resulting PKC-depleted {alpha}T3–1 cells showed no induction of stathmin phosphorylation in response to LHRH (10-8 M) (Fig. 5Go). This suggests that the LHRH-induced effect on stathmin is most likely mediated through activation of a PKC-dependent transduction pathway.



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  		Figure 5. Involvement of PKC-dependent pathways in LHRH-induced stathmin phosphorylation. {alpha}T3–1 cells were treated, or not, with TPA for 24 h to deplete endogenous PKCs. LHRH was applied for 30 min on control or TPA-treated cells, and the phosphorylation of stathmin was then analyzed, as in Figs. 1–3GoGoGo, through comparison of its form distribution on silver-stained two-dimensional electrophoresis gels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously demonstrated that in normal anterior pituitary cells in culture, the phosphorylation of a set of proteins designated as proteins 1–15 is associated with multihormonal regulations occurring in the intact pituitary (6, 7). In particular, stathmin (originally described as proteins 7 and 8) is part of the normal intracellular mechanisms involved in the control of cell functions by different neuropeptides and steroid hormones (6, 7).

In the present study, using a homogeneous cell population of gonadotropes, we demonstrated that in spite of their tumoral origin, the {alpha}T3–1 gonadotrope cell line expressed stathmin and that both its expression and phosphorylation reached a maximum during the exponential phase (2–4 days) of cell growth. This result is similar to observations in other cell lines where the expression of stathmin was shown to be controlled by cell density (34). When applied to {alpha}T3–1 cells, LHRH highly stimulated stathmin phosphorylation through a specific receptor in a time- and dose-dependent manner, and the LHRH antagonist blocked the effect.

In experiments performed in parallel and under the same experimental conditions, the neuropeptide stimulated (sequentially) PLC, phospholipase A2, and phospholipase D activities through pertussis toxin-insensitive G proteins (2, 3).

The tumor-promoting phorbol ester TPA, a relatively specific activator of PKC isoenzymes (4), when acutely administered to the cells, induced a degree of stimulation of stathmin phosphorylation similar to that obtained after LHRH stimulation. Both TPA- and LHRH-induced stathmin phosphorylation were abolished in previously PKC-depleted {alpha}T3–1 cells. This might suggest that PKC isoforms sensitive to long TPA treatment are either directly or indirectly (see below) implicated in LHRH-evoked stathmin phosphorylation. In addition, staurosporine (a relatively specific inhibitor of PKC), although without effect by itself, inhibited the TPA and LHRH-induced stathmin phosphorylation. It must be noticed, however, that at 10-7 M, although staurosporine diminished TPA-induced stathmin phosphorylation, it was unable to completely counteract the LHRH effect (data not shown). The PKC inhibitor may exhibit different potencies on the various PKC isoforms (35) that are present in {alpha}T3–1 cells (32, 33, 36). It is possible, therefore, that the isoforms implicated in LHRH action are less sensitive to staurosporine administration. Evidence from our (2) and other studies (32) shows that in the {alpha}T3–1 gonadotrope cell line, several PKC isoforms are present and are differentially activated or down-regulated by TPA or LHRH (2, 32, 33). This might explain the slight difference observed between the LHRH- and TPA-induced extents of stathmin phosphorylation.

We have previously shown that, under our experimental conditions, LHRH did not induce cAMP production (3). This might exclude the possibility that the stathmin phosphorylation after LHRH application implicates an adenylate cyclase-cAMP system.

Although stathmin has been shown not to be a direct substrate for PKC in vitro (18), it is attractive to speculate that specific PKC isoforms stimulated by LHRH might be directly implicated in the process of stathmin phosphorylation. Alternatively, phosphorylation of stathmin might be mediated by a PKC-dependent MAPK activation, in response to LHRH stimulation (20, 37).

It was recently demonstrated that stathmin may interact with KIS, a protein kinase proposed to be involved in the control of protein synthesis (21, 22), and that stathmin forms 2:1 complexes with tubulin in a way depending on its phosphorylation status (27, 28) and, hence, participates in the control of the dynamic instability of microtubules (24, 25, 26). These results stress the importance of stathmin in the control of major physiological processes, which might be of importance in endocrine cells, where protein synthesis and the dynamics of the cytoskeleton are indeed major components of the synthesis and release of peptide hormones.

In conclusion, our results demonstrate, for the first time, a link between stathmin phosphorylation and the transduction pathway triggered by LHRH in a pure gonadotrope cell line. Stathmin further appears as a detector of LHRH-induced signal transduction, and analysis of its phosphorylation gives important clues for a PKC-dependent pathway involved in LHRH action. Further analysis of phosphorylated stathmin sites could allow the dissection of the kinase(s) involved downstream of various PKC isoforms, toward control of the resulting biological response to LHRH.


   Acknowledgments
 
Thanks are given to Dr. R. I. Weiner for providing us with {alpha}T3–1 cell line.


   Footnotes
 
1 This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Association Française contre les Myopathies, the Association pour la Recherche contre le Cancer, and the Ligue Nationale Française contre le Cancer. Back

Received September 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals