Growth Hormone-Releasing Hormone Stimulates Mitogen-Activated ...

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 6 Printed in U.S.A.

Growth Hormone-Releasing Hormone Stimulates Mitogen-Activated Protein Kinase* CELIA M. POMBO, JUAN ZALVIDE, BRUCE D. GAYLINN,

AND

´ GUEZ CARLOS DIE

Department of Physiology, University of Santiago de Compostela School of Medicine, 15705 Santiago de Compostela, Spain; and Division of Endocrinology and Metabolism, University of Virginia Health System (B.D.G.), Charlottesville, Virginia 22908-0746 ABSTRACT GH-releasing hormone (GHRH) can induce proliferation of somatotroph cells. The pathway involving adenylyl cyclase/cAMP/protein kinase A pathway in its target cells seems to be important for this action, or at least it is deregulated in some somatotroph pituitary adenomas. We studied in this work whether GHRH can also stimulate mitogen-activated protein (MAP) kinase. GHRH can activate MAP kinase both in pituitary cells and in a cell line overexpressing the GHRH receptor. Although both protein kinase A and protein kinase C could activate MAP kinase in the CHO cell line studied, neither protein kinase A nor protein kinase C appears to be required for GHRH activation of MAP kinase in this system. However, seques-

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ITUITARY SOMATOTROPH cells are stimulated to proliferate and secrete GH by GH-releasing hormone (GHRH) from the hypothalamus (1). Some of the mechanisms by which this regulatory peptide modulates pituitary function have been elucidated after the molecular cloning of the GHRH receptor (2– 4). This receptor is a member of the superfamily of seven-helix transmembrane proteins of G protein-coupled receptors (GPCRs). In cells transfected with the cloned human GHRH receptor, GHRH stimulates the accumulation of intracellular cAMP, an action that involves a Gs protein-linked receptor/adenylate cyclase pathway (3). GHRH receptor also relays mitogenic stimuli to the somatotroph cell. The relevance of this signaling is underscored by the finding that deregulation of some of the components of this pathway has been implicated in several clinical disorders. Some human pituitary tumors that lead to acromegaly are associated with a dominant activating mutation in a Gs␣-subunit that constitutively activates adenylate cyclase and is supposed to cause proliferation of pituitary cells (5). Nevertheless, only 10 – 40% of GH-producing pituitary tumors harbor mutations in this gene (6). It is very likely that other genes important in mitogenic signaling and growth regulation are mutated in the rest of somatotroph adenomas. Intense research in this field has led to the association of adenoma development with abnormalities in difReceived January 3, 2000. Address all correspondence and requests for reprints to: Department of Physiology, University of Santiago de Compostela School of Medicine, 15705 Santiago de Compostela, Spain. E-mail: [email protected]. * This work was supported by grants from Fondo de Investigaciones Sanitarias de la Seguridad Social, Spanish Ministry of Health, and Xunta de Galicia to Carlos Die´guez, and by NIH Grant RO1-DK-45350 (to B.D.G.).

tration of the ␤␥-subunits of the G protein coupled to the receptor inhibits MAP kinase activation mediated by GHRH. This pathway also involves p21ras and a phosphatidylinositol 3-kinase, probably phosphatidylinositol 3-kinase-␥. Despite the involvement of p21ras, the protein kinase Raf-1 is not hyperphosphorylated in response to GHRH, contrary to what usually occurs when the Ras-Raf-MAP kinase pathway is activated. In summary, this work describes for the first time the activation of MAP kinase by GHRH and outlines a path for this activation that is different from the cAMP-dependent mechanism that has been traditionally described as mediating the mitogenic actions of GHRH. (Endocrinology 141: 2113–2119, 2000)

ferent chromosomal locations and to the identification of several candidate genes in human pituitary tumors (7). Despite these advances, no clear model on how cell growth might be affected in GH-secreting adenomas has been proposed. GHRH is a very important factor in the regulation of normal somatotroph cell growth. Therefore, study of the pathways involved in growth stimulation by this hormone may be useful in the identification of proteins important in the pathological growth of GH-producing adenomas. The ubiquitous mitogen-activated protein (MAP) kinases comprise a family of serine/threonine kinases that are involved in the transduction of externally derived signals regulating cell growth, division, and differentiation. Upon activation, MAP kinases phosphorylate and activate nuclear transcription factors involved in DNA synthesis and cell division (8). Activation of MAP kinases is an important event in cell growth. Most, if not all, mitogenic stimuli activate MAP kinases in some degree, and inhibition of this activation leads to the arrest of cell division (9). The mechanisms that regulate MAP kinase activation have been elucidated over the past years. Growth factor receptors of the tyrosine kinase class (RTKs) activate MAP kinases in a multistep process. Binding of the ligand to the receptors leads to the tyrosine phosphorylation in the receptor tail of a docking site for the adapter protein Grb2/Sem-5 (10). This causes recruitment of the exchange factor Sos and the following activation of p21ras; this initiates the activation of a linear cascade of protein kinases, including c-Raf, MAP kinase (MEK-1), and MEK-2, that ultimately phosphorylate and activate MAP kinases (11, 12). G protein-coupled receptors can also activate MAP kinases (12). The mechanism by which these receptors mediate MAP kinase activation were not studied in detail until recently.

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GHRH STIMULATES MAP KINASE

The mechanism employed by each receptor is determined by the G protein(s) with which it interacts and the available effectors in the cell in which it is acting (13). In GPCRs coupled to a Gs protein, Gs␣ has a dual effect on MAP kinase activity. In some situations, Gs␣ appears to have a growth inhibitory effect through its negative regulation of the Ras-Raf signaling pathway (14, 15). Protein kinase A (PKA) activated by Gs␣ through cAMP inhibits Raf activity through direct phosphorylation, thus also inhibiting its downstream MEK-MAP kinase cascade. On the other hand, Gs␣ can have a growth-stimulating activity through this same G␣/adenylate cyclase/cAMP pathway, although it is not clear what proliferation-related events are activated by this path (16). Recent reports suggest that PKA inhibition of MAP kinase involves c-Raf-1, and PKA stimulation involves B-Raf. The relative expression of these Raf isoforms in a specific cell line is suggested to determine whether PKA inhibits or stimulates MAPK (17). ␤␥-Subunits can stimulate Ras through a phosphatidylinositol 3-kinase (PI3K)-dependent path that includes stimulation of Src and the Shc/Grb2/ Sos complexes. After p21ras activation, the traditional RafMEK-MAP kinase core is stimulated (18). The receptor for GHRH is coupled to a Gs protein. As stated above, this receptor stimulates somatotroph cell growth. This stimulation seems to be mediated at least in part through the Gs␣/cAMP/PKA pathway, or at least deregulation of this path can result in abnormal growth in some adenomas. It is not clear, however, whether GHRH receptor also mediates MAP kinase activation as a part of its mitogenic signaling and, if this the case, through which mechanism it does so. Materials and Methods Materials GHRH-(1–29) was purchased from Peninsula Laboratories, Inc. Europe Ltd. H-89 (Merseyside, UK), dihydrochloride, was obtained from Calbiochem (La Jolla, CA). Forskolin, 12-O-tetradecanoyl phorbol 13acetate, and myelin basic protein (MBP) were obtained from Sigma (St. Louis, MO). Antibodies directed toward Erk-2, Raf-1, and p-Erk were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Western blot reagents and the alkaline phosphatase-conjugated goat antimouse antibody were obtained from Tropix (Bedford, MA). Cell culture medium and serum were obtained from Life Technologies, Inc. (Gaithersburg, MD). All other chemicals were purchased from Sigma unless otherwise specified.

Cell culture Primary cells from adenohypophysis were obtained and cultured as previously described (19). Specifically, they were cultured in defined medium, which consisted of Ham’s F-12/DMEM/BGJb medium in a ratio of 6:3:1 supplemented with (per liter) BSA (2 g), HEPES (2.38 g), hydrocortisone (100 ␮g), T3 (0.4 ␮g), transferrin (10 mg), glucagon (10 ng), epidermal growth factor (0.1 ␮g), and fibroblast growth factor (0.2 ␮g). Chinese hamster ovary cells (CHO) overexpressing the human GHRH receptor were grown to confluence in Ham’s F-12 medium supplemented with glutamine (2 mm) and 10% FBS. Twenty-four hours before treatments cells were changed to defined medium with 0.5% FBS. All pretreatments and treatments were performed in this medium at 37 C in a 95% air-5% CO2 incubator. Then cells were washed with cold Tris-buffered saline and lysed in 20 mm HEPES (pH 7.4), 50 mm ␤-glycerophosphate, 1 mm sodium orthovanadate, 1% Triton X-100, 10% glycerol, 1 mm EGTA, 1 mm dithiothreitol, 400 ␮m phenylmethylsulfonylfluoride, 2 ␮m leupeptin, and 10 U/ml Trasylol.

Plasmids and transfections Human GHRH receptor complementary DNA (cDNA) in the expression plasmid pCDM8 (4) and the G418 resistance plasmid pSV2neo were cotransfected by the calcium phosphate method into CHO cells (CHOK1 strain), as previously described (20). Briefly, these cells were selected with 400 ␮g/ml G418, and clonal cell lines were established. Clone CHO-4 was confirmed to express receptor by GHRH binding (B. Gaylinn, unpublished data), and GHRH receptor photoaffinity cross-linking (20). The plasmid pMT3 HA-MAP kinase (Erk-1) has been previously described (21). The plasmids encoding pcDNA3-RasN17 and pcDNA1-Gt (22) were provided by Dr. Crespo (Centro Superior Investigaciones Cientificas, Santander University, Sontonder, Spain). Subconfluent CHO-4 cells were transfected using the calcium phosphate precipitation technique. One to 10 ␮g expression plasmid DNA were used per plate and adjusted to 26 ␮g DNA with the appropriate empty vector. After 4 h of exposure to the DNA-containing cocktail, cells were shocked with 14% glycerol in PBS, washed twice, and refed with complete medium. Eighteen hours later, cells were changed to defined medium with 0.5% FBS and after 24 h in this medium, cells were exposed to the stimuli, and cell extracts were prepared as previously described.

cAMP determination Intracellular cAMP was determined by RIA of acid extracts from cells grown in 24-well cluster plates and pretreated with isobutylmethylxanthine as previously described (4).

Immune complex kinase assays and Western blot Extracts were exposed to the appropriate antibody (anti-Erk 2 or the murine monoclonal antibody 12CA5) for 3 h, and the immune complexes were collected with protein G-agarose beads (Pharmacia Biotech, Uppsala, Sweden). Beads were washed three times in the corresponding lysis buffer, three times in LiCl buffer (500 mm LiCl, 2 mm dithiothreitol, and 100 mm Tris-HCl, pH 7.6), and three times in assay buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 1 mm DTT, 0.1% Triton X-100). Reactions were performed in a 50-␮l volume of the corresponding assay buffer containing 100 ␮m [␥-32P]ATP (1–5000 cpm/pmol) and 10 ␮g MBP as substrate for the MAP kinases. The assay time was 20 min at 30 C. Reactions were stopped by the addition of 6 ⫻ Laemmli sample buffer. Samples were boiled and resolved in a SDS-PAGE. Once the substrate band was visualized by autoradiography, the band was cut out of the gel, and radioactivity was determined by liquid scintillation counting. The expression of the kinases was confirmed by Western blot after transferring the proteins resolved in a 12% polyacrylamide gel to a nitrocellulose membrane and incubating with the appropriate antibody.

Results

We first determined whether GHRH could stimulate MAP kinase activity through its receptor. To this end, primary cell cultures from anterior pituitary were serum starved for 24 h in defined medium with 0.5% FBS. Then cells were stimulated with 10 nm GHRH for 5 and 10 min. As shown in Fig. 1, GHRH induced an increase in MAP kinase activity at both times. This shows that GHRH can induce MAP kinase activity in its normal target organ, the pituitary. We wanted to study the activation of MAP kinase by GHRH in more detail and to assess whether this activation occurred in the same cells that harbored the GHRH receptor and was not the result of an indirect paracrine effect. Although some stable pituitary cell lines are available, none was suitable for this work due to lack of a functionally coupled GHRH receptor (GH3, GC cells) or constitutive elevation of cAMP (MtT/S cells). We therefore used CHO-4 cells, a cell line stably transfected with the human GHRH receptor. This provided us with a homogeneous cell population in which levels of GHRH receptor are constant.


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FIG. 1. MAP kinase activation by GHRH in primary cell cultures from anterior pituitary. Cells maintained in defined medium with 0.5% FBS for 24 h were treated for the indicated times with 10 nM GHRH. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Results in duplicate of a representative experiment of three performed are shown.

FIG. 2. Dose-dependent accumulation of cAMP in response to His1,Nle27-human GHRH-(1–32)NH2, forskolin (Forsk), or ethanol (EtOH) treatment for 15 min in stable clonal CHO cell lines transfected with G418 resistance vector alone (CHO-13) or vector plus full-length human GHRH receptor (CHO-4). Intracellular cAMP was assayed by RIA of extracts from cells grown in 24-well cluster plates and pretreated with isobutylmethylxanthine as previously described (4). Each bar shows the average of six replicates ⫾ SEM.

We first evaluated whether CHO-4 cells expressed a functional GHRH receptor by studying whether GHRH could elicit a rise in cAMP levels in these cells. Figure 2 shows that CHO-4 cells had a clear elevation of cAMP when challenged with subnanomolar concentrations of GHRH. On the contrary, CHO-13 cells, a matched vector-transfected control, did not respond to GHRH even at high doses. Forskolin was used as a positive control to verify that equivalent numbers of functional cells were present for each clone. As shown in Fig. 3, A and C, MAP kinase activity was also stimulated by 10 nm GHRH in CHO-4 cells. The response was time dependent, reaching its maximum at 5 min, and returning almost to basal levels after 1 h. CHO-13 cells did not show an activation of MAP kinase in response to GHRH under the same conditions in which CHO-4 cells responded to the hormone (data not shown). This shows that the activation of MAP kinase by GHRH is acting through the transfected GHRH receptor. We also assessed the phosphorylation state of p44 (pERK1) and p42 (pERK2) MAP kinases using a specific phosphotyrosine 204 antibody to confirm the activation of these two members of the MAP kinase family.

FIG. 3. Activation of MAP kinase by GHRH in CHO-4 cells. A, Time course of MAP kinase activation after GHRH treatment in CHO-4 cells. CHO-4 cells maintained in defined medium with 0.5% FBS for 24 h were treated with 10 nM GHRH for the indicated times. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Results in duplicate of a representative experiment of three performed are shown. B, Time course of MAP kinase phosphorylation after GHRH treatment in CHO-4 cells. MAP kinase phosphorylation was detected with a specific phospho-ERK (pERK) antibody by Western blotting. C, MAP kinase activity in CHO-4 cells untreated (control) or treated for 5 min with 10 nM GHRH. Shown are the mean ⫾ SEM of six independent experiments. *, P ⬍ 0.05 vs. control.

As shown in Fig. 3B, the pattern of phosphorylation of both ERKs correlates with the pattern of MAP kinase activation. The activation of MAP kinase was a specific effect, as other kinases that can be activated by extracellular signals, stressactivated protein kinase/Jun kinase, were not activated by GHRH in CHO-4 cells (negative data not shown). These experiments show that GHRH can stimulate in CHO-4 cells the two pathways we were interested in studying in a very similar manner to pituitary primary cells. Consequently, we used these cells in the next experiments to study the mechanisms by which GHRH stimulates MAP kinase activity.

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We first examined whether the cAMP/PKA pathway, which is classically associated with the mitogenic effects of GHRH, is also responsible for the activation of MAP kinase by this hormone. CHO-4 cells were pretreated with H-89, a PKA-specific inhibitor (23). After 1 h of pretreatment, cells were stimulated for 5 min with 10 nm GHRH or 1 ␮m forskolin. As shown in Fig. 4, pretreatment with H-89 had no significant effect on the MAP kinase activation induced by GHRH. On the contrary, MAP kinase activation by forskolin was inhibited 75% by H-89 pretreatment. These data suggest that MAP kinase activation by GHRH is not dependent on the cAMP/PKA pathway. One way in which some G protein-coupled receptors can stimulate MAP kinase is through the activation of protein kinase C (PKC) (24). To examine the role of this enzyme on MAP kinase activation by GHRH, we compared the effect of GHRH on MAP kinase activation in cells in which PKC was or was not down-regulated by prolonged incubation with the PKC activator phorbol 12-myristate 13-acetate (1 ␮m, overnight). As shown in Fig. 5, PKC down-regulation completely blocked phorbol 12-myristate 13-acetate-induced stimulation of MAP kinase activity, but had no effect on MAP kinase activity in response to GHRH. These results suggest that GHRH stimulation of MAP kinase enzyme occurs independently of PKC. Taken together, these two results show that even though MAP kinase can be activated in CHO-4 cells by cAMP-PKA and also through a PKC-dependent path, GHRH activates MAP kinase in these cells through an additional mechanism as well. It has been reported that for some G protein-coupled receptors the signal from the receptor to MAP kinase goes through the G ␤␥ dimers of the G protein (22, 25–27). A way to assess the involvement of ␤␥-subunits in a signaling pathway is to transiently express the ␣-subunit of transducin (Gt)

FIG. 4. Effects of inhibition of PKA on MAP kinase activation by GHRH. CHO cells maintained in defined medium with 0.5% FBS for 24 h were pretreated with 30 ␮M H-89 for 30 min before stimulation with 10 nM GHRH for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Bands were excised from the gel, and their radioactivity was counted. Shown are the mean ⫾ SEM.

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FIG. 5. Effects of PKC down-regulation on MAP kinase activation by GHRH. CHO-4 cells maintained in defined medium with 0.5% FBS for 24 h; for the last 12 h cells were pretreated with 1 ␮M TPA or dimethylsulfoxide (vehicle), then were treated with either 10 nM GHRH or 1 ␮M TPA for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Results of a representative experiment of three performed are shown.

that can bind ␤␥-subunits and thus block ␤␥-mediated signal transduction (28). We cotransfected CHO-4 cells with an expression plasmid carrying Gt with the hemagglutinin (HA)-Erk-1 kinase expression plasmid. In this way, by specifically immunoprecipitating the exogenous Erk-1 with an anti-HA antibody we could determine MAP kinase activity in transfected cells in which the ␤␥-subunits were sequestered by Gt expression. As shown in Fig. 6A, expression of Gt in CHO-4 cells partially blocked GHRH stimulation of the exogenous MAP kinase, suggesting that G ␤␥ mediates at least in part the activation of MAP kinase by GHRH. Figure 6B shows that the expression of exogenous Erk-1 was similar in all transfections performed, and that differences in MAP kinase activity were not due to differences in MAP kinase expression. We next tried to define whether the connection between GHRH receptor and MAP kinase involves the well known MAP kinase-activating pathway comprising the proteins Ras and Raf. To study whether Ras activity is necessary for activation of MAP kinase by GHRH, we inhibited Ras activity with the Ras dominant negative mutant RasN17 (29). We cotransfected CHO-4 cells with an expression plasmid carrying RasN17, with the HA-Erk-1 kinase expression plasmid. As shown in Fig. 7, in cells expressing the RasN17 mutant, MAP kinase activity in response to GHRH was inhibited almost to basal levels. In contrast, cells transfected with RasN17 did not impair the activation of MAP kinase by serum. This result suggests that signaling from the GHRH receptor to MAP kinase requires the activity of Ras. Classically, Ras-dependent activation of Erk requires members of the Raf family of serine/threonine kinases. To examine the role of Raf in GHRH signaling, we assessed the Raf phosphorylation state by its mobility shift on a SDS-


FIG. 6. Effect of ␤␥ sequestration on MAP kinase activation by GHRH. A, CHO-4 cells were transfected with either an expression plasmid containing Gt or a control pcDNA vector together with pMT3HAErk1 kinase. Eighteen hours later medium was changed to defined medium with 0.5% FBS, and after 24 h, cells were stimulated with 10 nM GHRH for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Results in duplicate of a representative experiment of two performed are shown. B, Proteins from cell lysates of the same experiments were resolved in SDS-PAGE, and the levels of HA-Erk-1 expression were determined by Western blot analysis.

PAGE gel, based on the fact that after growth factor stimulation of many cell types, Raf-1 becomes markedly hyperphosphorylated, resulting in reduced electrophoretic mobility of the protein (30). Specifically, we examined the phosphorylation state of Raf-1 in 10 nm GHRH-treated cells at different time points after treatment. As is shown in Fig. 8, we did not observe changes in Raf-1 gel mobility from as early as 5 min up to as late as 1 h after GHRH treatment. On the contrary, 20% serum treatment of the cells for 20 min stimulated an increase in Raf-1 phosphorylation (Fig. 8, lane 7). These results suggest that MAP kinase activation by GHRH occurs without the Raf-1 hyperphosphorylation typically seen upon activation of the Raf-dependent cytoplasmic kinase cascade. Coupling of G protein-coupled receptors to MAP kinases through ␤␥ proteins has been reported to involve a novel class of PI3 kinase, namely PI3K␥. This is an enzyme that has been described to be activated directly by ␤␥-subunits independently of a regulatory p85 subunit. This enzyme is sensitive to general PI3K inhibitors (31). To study its possible involvement in MAP kinase activation by GHRH, we treated CHO-4 cells with the PI3K inhibitors wortmannin or LY294002 and then challenged them with GHRH. Figure 9 shows that MAP kinase stimulation was severely impaired in cells treated with either of these inhibitors, showing that

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FIG. 7. Effect of Ras inhibition on MAP kinase activation by GHRH. CHO-4 cells were transfected with either an expression plasmid containing the dominant negative mutant RasN17 or a control pcDNA vector together with pMT3-HAErk1 kinase. Eighteen hours later medium was changed to defined medium with 0.5% FBS, and after 24 h, cells were stimulated with 10 nM GHRH for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Bands were excised from the gel, and their radioactivity was counted. The average and SE of the mean (SEM) are shown.

FIG. 8. Effects of GHRH and serum treatment on Raf-1 phosphorylation. Cells treated with 10 nM GHRH or 20% serum for the indicated times were lysed, and their proteins were resolved in an 8% SDSPAGE and subjected to Western blot using anti-Raf-1 antibody. The experiment was repeated three times with identical results.

PI3K activity is involved in GHRH receptor-MAP kinase signaling. Discussion

Somatotroph cell proliferation in the pituitary is under the control of GHRH. There is strong experimental evidence supporting this. Transgenic mice in which GHRH is overexpressed show somatotroph hyperplasia that can progress to a pituitary adenoma (32, 33). Mice carrying the little mutation have a GHRH receptor that cannot bind its ligand (34). These mice show a clear somatotroph hypoplasia due to lack of mitogenic activity of GHRH in these cells (35). The stimulation of cell proliferation by GHRH seems to have a clear correlate in pituitary adenomas. A significant proportion of

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FIG. 9. Effect of PI3K inhibition on MAP kinase activation by GHRH. CHO-4 cells were pretreated with the PI3K inhibitors wortmannin (20 ␮M; Wn) or LY294002 10␮m (LY) for 30 min, and then treated with 10 nM GHRH for 5 min. MAP kinase activity was determined in immunoprecipitates using MBP as a substrate. MBP was separated by SDS-PAGE. Bands were excised from the gel, and their radioactivity was counted. The average and SEM are shown.

GH-producing adenomas have activating mutations in the Gs␣ subunit of the trimeric GTP-binding protein coupled to this receptor. Despite this activity of GHRH, there has been no detailed analysis to date on what mitogenic pathways are stimulated by GHRH. Specifically, it is not known whether the adenylyl cyclase/cAMP/PKA pathway that seems to be deregulated in many adenomas is the only path relevant to the modulation of cell proliferation by GHRH. Alternatively, GHRH may modulate a number of intracellular pathways to produce the final biological effect, in this case cell division. Activation of MAP kinase is a hallmark of mitogenic stimuli. MAP kinase can also be activated in settings other than mitogenesis, as is in terminal differentiation and cell senescence, but it is rare that a hormone or growth factor can produce cell division without activating the MAP kinase pathway. GHRH has not been reported to date to be able to activate MAP kinase. In the present study we have investigated the activation of MAP kinase by GHRH. GHRH can stimulate MAPK activity both in somatotroph cells and in a stable cell line overexpressing the human GHRH receptor. PKA is not involved in MAPK activation by GHRH, as inhibition of PKA did not result in any significant reduction of MAP kinase activation by GHRH. This is in agreement with other Gs-coupled receptors, in which activation of MAP kinase does not depend on cAMP elevation. Curiously enough, the elevation of cAMP by the inhibitor of the catalytic subunit of PKA forskolin could induce significant activation of MAP kinase in CHO-4 cells, and this activation was dependent on PKA activity. This is in contrast to some other cellular models in which MAP kinase activation by G protein-coupled receptors has been studied. In these models,

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cAMP has an inhibitory effect on MAP kinase activity that has been proposed to modulate the response of this kinase to different stimuli. The connection between cAMP and MAP kinase in CHO-4 cells will be important in future studies about the cross-talking between these two pathways in relation to cell proliferation. Another pathway that has been proposed to be important in MAPK activation by other GPCRs PKC does not seem to be involved in MAP kinase activation in this particular setting, as its down-regulation does not impair GHRH signaling to this kinase. In contrast, the ␤␥-subunits of the trimeric G protein-coupled to GHRH receptor do seem to be involved, as their sequestration by transducin inhibits MAP kinase activation by GHRH to a great extent. Signaling from ␤␥ complexes to MAP kinase appears to require Ras in mammalian cells (22, 36). To prove whether that is also true in our system, we transfected CHO-4 cells with a dominant negative mutant of p21ras, RasN17. We found that expression of this Ras mutant nearly abolished the activity of the exogenous Erk-1 in response to GHRH. That proves that Ras signaling is required for the MAP kinase activation by GHRH. This is a significant finding that links an oncogenic protein, p21ras, with signal transduction by GHRH, implicating that deregulation of its function can contribute to cellular transformation. In the most common sequence of events, activation of MAP kinase activity through p21ras occurs through stimulation of Raf-1 kinase activity by Ras. This activated Raf, in turn, phosphorylates MEK-1, the protein directly responsible for MAP kinase activation. When this pathway is on, Raf-1 is usually hyperphosphorylated, a state that can be identified by its migration in SDS-PAGE gels. The exact role of multisite phosphorylation in Raf regulation, however, remains under discussion. A late work of Wartmann et al. suggests that the mobility shift associated with hyperphosphorylation of Raf-1 represents a negative feedback mechanism contributing to the desensitization of the MAPK signaling cascade (37). In GHRH-treated cells, this hyperphosphorylation of Raf-1 does not occur, although Raf-1 is indeed phosphorylated in serum-treated cells. Although more work needs to be performed to define the exact role of the Raf-1 kinase in the GHRH activation of MAPK, the present data suggest that the sequence of events that normally occurs after growth factor stimulation of the Ras/Raf/MAPK pathway does not happen in our system. It is possible that effectors other than Raf-1 are involved in the activation of MAPK. In agreement with our data are those of Al-Alawi et al., which described the mitogenic effects of TSH linked to Ras but independent of Raf. Their results also show that stimulation does not result in the hyperphosphorylation of Raf. In this case, however, MAPK was also not activated by TSH (38). A PI3K activity seems necessary for MAP kinase activation mediated by GHRH. Albeit conclusive evidences are lacking at present, this PI3K might be PI3K␥. This isoform of PI3K has been described to be directly activated by ␤␥-subunits. Given that this seems to be the path through which GHRH stimulates MAP kinase, we think the most likely place for PI3K␥ in the signal between GHRH receptor and MAP kinase is immediately downstream of the ␤␥-subunits. However, more work will be needed to analyze whether the PI3K


involved in this pathway is PI3K␥ and to unequivocally place the components of this signaling pathway with respect to one another. In summary, we unambiguously demonstrate that GHRH stimulates MAP kinase activity. Although more data are needed, we also provide some preliminary evidence on the role played by different molecules on the mechanism by which this occurs. Thus, this activation appears to be independent of the Gs/cAMP/PKA pathway, being more likely mediated by the ␤␥-subunits of the G protein acting on a Ras-dependent pathway. In any event, the involvement of GHRH in the regulation of MAP kinase activity adds a further level of complexity to the study of cell division in somatotroph cells and opens a new perspective in the understanding of the pathophysiology of the pituitary adenomas. Specifically, it will be interesting to study whether somatotroph adenomas, either with or without a Gs␣ mutation, have elevated MAP kinase activity. Also, some adenomas might have alterations in some of the components of the pathway we have just described. Alternatively, in a pathological setting, MAP kinase might be activated through the classic AC/cAMP/PKA pathway, much the same way as forskolin can induce this activation in CHO-4 cells. Acknowledgments We thank Dr. P. Crespo for kindly providing the Gt and N17 expression plasmids. We also thank C. Gianzo for technical assistance.

References 1. Ruvkun G 1992 Cell signalling. A molecular growth industry. Nature 360:711–712 2. Mayo KE 1992 Molecular cloning and expression of a pituitary-specific receptor for growth hormone-releasing hormone. Mol Endocrinol 6:1734 –1744 3. Lin C, Lin SC, Chang CP, Rosenfeld MG 1992 Pit-1-dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. Nature 360:765–768 4. Gaylinn BD, Harrison JK, Zysk JR, Lyons CE, Lynch KR, Thorner MO 1993 Molecular cloning and expression of a human anterior pituitary receptor for growth hormone-releasing hormone. Mol Endocrinol 7:77– 84 5. Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar L 1989 GTPase inhibiting mutations activate the ␣ chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 340:692– 696 6. Shimon I, Melmed S 1997 Pituitary tumor pathogenesis. J Clin Endocrinol Metab 82:1675–1681 7. Prezant TR, Melmed S 1998 Pituitary oncogenes. In: Webb SM (ed) Pituitary Tumors: Epidemiology, Pathogenesis and Management. BioScientifica, Bristol, pp 81–93 8. Blenis J 1993 Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci USA 90:5889 –5892 9. Pages G, Lenormand P, Allemain G, Chambard JC, Meloche S, Pouyssegur J 1993 Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319 – 8323 10. Schlessinger J 1993 How receptor tyrosine kinases activate Ras. Trends Biochem Sci 18:273–275 11. Leevers SJ, Paterson HF, Marshall CJ 1994 Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369:411– 414 12. de Vries-Smits AM, Burgering BM, Leevers SJ, Marshall CJ, Bos JL 1992 Involvement of p21ras in activation of extracellular signal-regulated kinase 2. Nature 357:602– 604 13. van Biesen T, Luttrell LM, Hawes BE, Lefkowitz RJ 1996 Mitogenic signaling via G protein-coupled receptors. Endocr Rev 17:698 –714 14. Burgering BM, Pronk GJ, van Weeren PC, Chardin P, Bos JL 1993 cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12:4211– 4220

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15. Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, Krebs EG 1993 Protein kinase A antagonizes platelet-derived growth factorinduced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci USA 90:10300 –10304 16. Dhanasekaran N, Heasley LE, Johnson GL 1995 G protein-coupled receptor systems involved in cell growth and oncogenesis. Endocr Rev 16:259 –270 17. Gao Z, Chen T, Weber MJ, Linden J 1999 A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways. J Biol Chem 274:5972–5980 18. Gutkind JS 1998 Cell growth control by G protein-coupled receptors: from signal transduction to signal integration. Oncogene 17:1331–1342 19. Soto JL, Castrillo JL, Dominguez F, Dieguez C 1995 Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture. Endocrinology 136:3863–3870 20. Gaylinn BD, Lyons CE, Zysk JR, Clarke IJ, Thorner MO 1994 Photoaffinity cross-linking to the pituitary receptor for growth hormone-releasing factor. Endocrinology 135:950 –955 21. Pombo CM, Kehrl JH, Sanchez I, Katz P, Avruch J, Zon LI, Woodgett JR, Force T, Kyriakis JM 1995 Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature 377:750 –754 22. Crespo P, Cachero TG, Xu N, Gutkind JS 1995 Dual effect of ␤-adrenergic receptors on mitogen-activated protein kinase. Evidence for a ␤␥-dependent activation and a G␣s-cAMP-mediated inhibition. J Biol Chem 270:25259 –25265 23. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]5- isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272 24. van Biesen T, Hawes BE, Raymond JR, Luttrell LM, Koch WJ, Lefkowitz RJ 1996 G(o)-protein ␣-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism. J Biol Chem 271:1266 –1269 25. Hawes BE, van Biesen T, Koch WJ, Luttrell LM, Lefkowitz RJ 1995 Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem 270:17148 –17153 26. Hawes BE, Luttrell LM, van Biesen T, Lefkowitz RJ 1996 Phosphatidylinositol 3-kinase is an early intermediate in the G␤␥-mediated mitogen-activated protein kinase signaling pathway. J Biol Chem 271:12133–12136 27. Koch WJ, Hawes BE, Allen LF, Lefkowitz RJ 1994 Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G␤ gamma activation of p21ras. Proc Natl Acad Sci USA 91:12706 –12710 28. Federman AD, Conklin BR, Schrader KA, Reed RR, Bourne HR 1992 Hormonal stimulation of adenylyl cyclase through Gi-protein ␤␥ subunits. Nature 356:159 –161 29. Feig LA, Cooper GM 1988 Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol 8:3235–3243 30. Heidecker G, Kolch W, Morrison DK, Rapp UR 1992 The role of Raf-1 phosphorylation in signal transduction. Adv Cancer Res 58:53–73 31. Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B 1995 Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269:690 – 693 32. Mayo KE, Hammer RE, Swanson LW, Brinster RL, Rosenfeld MG, Evans RM 1988 Dramatic pituitary hyperplasia in transgenic mice expressing a human growth hormone-releasing factor gene. Mol Endocrinol 2:606 – 612 33. Asa SL, Kovacs K, Stefaneanu L, Horvath E, Billestrup N, GonzalezManchon C, Vale W 1992 Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. Endocrinology 131:2083–2089 34. Godfrey P, Rahal JO, Beamer WG, Copeland NG, Jenkins NA, Mayo KE 1993 GHRH receptor of little mice contains a missense mutation in the extracellular domain that disrupts receptor function. Nat Genet 4:227–232 35. Wilson DB, Wyatt DP, Gadler RM, Baker CA 1988 Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse. Acta Anat 131:150 –155 36. Crespo P, Xu N, Simonds WF, Gutkind JS 1994 Ras-dependent activation of MAP kinase pathway mediated by G-protein ␤␥ subunits. Nature 369:418 – 420 37. Wartmann M, Hofer P, Turowski P, Saltiel AR, Hynes NE 1997 Negative modulation of membrane localization of the Raf-1 protein kinase by hyperphosphorylation. J Biol Chem 272:3915–3923 38. al-Alawi N, Rose DW, Buckmaster C, Ahn N, Rapp U, Meinkoth J, Feramisco JR 1995 Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade. Mol Cell Biol 15:1162– 1168