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Endocrinology, doi:10.1210/en.2006-1273
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Endocrinology Vol. 148, No. 6 2973-2983
Copyright © 2007 by The Endocrine Society

Conditional Overexpression of the Wild-Type Gs{alpha} as the gsp Oncogene Initiates Chronic Extracellularly Regulated Kinase 1/2 Activation and Hormone Hypersecretion in Pituitary Cell Lines

D. Romano1, K. Magalon1, M. Pertuit, R. Rasolonjanahary, A. Barlier, A. Enjalbert and C. Gerard

Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13916 Marseille cedex 20, France

Address all correspondence and requests for reprints to: C. Gerard, Unité Mixte de Recherche 6544, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille cedex 20, France. E-mail: corinne.gerard{at}univmed.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In pituitary cells, activation of the cAMP pathway by specific G protein-coupled receptors controls differentiative functions and proliferation. Constitutively active forms of the {alpha} subunit of the heterotrimeric Gs protein resulting from mutations at codon 201 or 227 (gsp oncogene) were first identified in 30–40% of human GH-secreting pituitary adenomas. This rate of occurrence suggests that the gsp oncogene is not responsible for initiating the majority of these tumors. Moreover, there is a large overlap between the clinical phenotypes observed in patients with tumors bearing the gsp oncogene and those devoid of this oncogene. To explore the role of Gs{alpha} in GH-secreting adenomas, we obtained somatolactotroph GH4C1 cell lines by performing doxycycline-dependent conditional overexpression of the wild-type Gs{alpha} protein and expression of the gsp oncogene. Although the resulting adenylyl cyclase and cAMP levels were 10-fold lower in the wild-type Gs{alpha}-overexpressing cell line, a sustained MAPK ERK1/2 activation was observed in both cell lines. Overexpression of the wild-type Gs{alpha} protein as the gsp oncogene initiated chronic activation of endogenous prolactin synthesis and release, as well as chronic activation of ERK1/2-sensitive human prolactin and GH promoters.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE cAMP PATHWAY is a conserved signaling pathway that regulates a plethora of cell functions in response to activated G protein-coupled receptors (GPCRs). During the last two decades, it has emerged that cAMP has cell type-specific effects, and the outcome in terms of proliferation or differentiation has been mostly attributed to crosstalk with the ERK1/2 cascade (for review, see Refs. 1 , 2). These pathways must be intact for normal homeostasis to be maintained, and several proto-oncogenes, such as the tyrosine kinase Src, the monomeric guanosine triphosphatase (GTPase) Ras, and the serine threonine kinase Raf, have been identified and found to be associated with tumor formation.

In pituitary cells, GHRH and vasoactive intestinal polypeptide (VIP) bind to specific GPCRs that bind in turn to the heterotrimeric Gs protein, resulting in activation of the adenylyl cyclase (AC) and generation of the intracellular second-messenger cAMP, which stimulates the cAMP-dependent protein kinase (3, 4). Activation of this cascade has been correlated with the synthesis and secretion of the GH and prolactin (PRL) (5, 6, 7, 8). It was recently established that the regulation of the PRL gene transcription by cAMP requires crosstalk with the ERK1/2 cascade and activation of the Ras and Rap1 monomeric GTPases (9, 10, 11). Besides being involved in hormone regulation processes, cAMP has been reported to promote somatotroph cell proliferation upon being activated by GHRH (12, 13).

During the last few years, impaired heterotrimeric G protein-coupled signal transduction has been identified as one of the causes of endocrine diseases (14). Activated forms of the {alpha} subunit of the heterotrimeric Gs protein, with mutations at codon 201 or 227 (which cause constitutive activation of the cAMP pathway by inhibiting the intrinsic GTPase activity of Gs{alpha}) were first identified in human (h) GH-secreting pituitary adenomas (15, 16) and have subsequently been described in other endocrine tumors (for review, see Ref. 17). These so-called gsp oncogenes are the only mutations found to occur with a significant prevalence (30–40%) in GH-secreting adenomas. However, there is no clearly visible relationship between the presence (gsp+) or absence (gsp–) of the gsp oncogene and the tumoral phenotype (18). This lack of correlation has been ascribed to several mechanisms liable to counteract the effects of cAMP activation in gsp+ GH-secreting adenomas: 1) gsp+ adenomas express low levels of Gs{alpha} protein (19); 2) the mRNA levels and activities of the cAMP-specific phosphodiesterases 4C and 4D are higher in gsp+ than in gsp– adenomas (20, 21); and 3) gsp+ adenomas are more sensitive to inhibition by somatostatin analogs (22). Although the gsp oncogene has been identified, its rate of occurrence in GH-secreting tumors suggests that it is not responsible for initiating the majority of these tumors. It is worth noting that wild-type Gs{alpha} subunit (WtGs{alpha}) overexpression has been observed in GH-secreting adenomas and associated with resistance to GHRH (23). The levels of expression of the WtGs{alpha} have been also correlated with various diseases. Because pseudohypoparathyroidism type Ia has been attributed to haploinsufficiency of the Gs{alpha} gene, the protein produced by a single normal allele does not suffice to sustain normal function (24), whereas a 3-fold increase in the Gs{alpha} expression level induces cardiomyopathy in mice (25). Transfection of the gsp oncogene in the GH3 pituitary cell line suggested that this oncogene may affect hormone regulation and cell proliferation (26, 27, 28).

To understand more clearly the role of Gs{alpha} overexpression compared with that of the gsp oncogene in the molecular mechanisms underlying GH-secreting adenoma initiation and progression, we produced GH4C1 cell lines by performing conditional overexpression of the WtGs{alpha} and expression of the gsp oncogene (R201C mutant of Gs{alpha}), using the inducible tetracycline (tet)-off system (29). Despite the great difference observed in the activation of the cAMP pathway, induction of both transgenes initiated a similar sustained activation of the MAPK ERK1/2 cascade, along with a chronic increase in hPRL and hGH promoter activities. In addition, endogenous PRL synthesis and release were chronically increased, whereas WtGs{alpha} overexpression and gsp oncogene did not promote GH4C1 cell proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Bacitracin and Nonidet P-40 were from VWR International (Fontenay-sous-Bois, France). U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylthio)butadiene], TransFast transfection reagent, and Dual-Luciferase reporter assay system were from Promega (Charbonnières, France), and all other reagents were purchased from Sigma (St. Quentin Fallavier, France).

Plasmid constructs
The plasmid pSV2 neo was from Clontech (Saint-Germain-en-Laye, France). The plasmid pUHD15-1 (kindly provided by H. Bujard, Zentrum für Molekulare Biologie Heidelberg, Heidelberg, Germany) encoded the hybrid tetracycline-dependent transactivator (tTA) described by Gossen and Bujard (29). The pPur{Delta}MtetO5-luc (kindly provided by L. Journot, Centre National de la Recherche Scientifique, Montpellier, France) encoded five copies of the heptameric tetO sequence described by Hoffmann et al. (30). The plasmids pcDNAI{alpha}sWt and pcDNAI{alpha}sR201C (a generous gift from C. Berlot, Yale University, New Haven, CT) encoded, respectively, the rat WtGs{alpha} protein and the rat {alpha}sR201C mutation and an epitope EE described by Grishina and Berlot (31). Both inserts WtGs{alpha} and R201CGs{alpha} excised from their respective plasmids by performing HindIII digestion were blunted and subcloned into the blunt pPur{Delta}Mtet05 vector cut by NotI restriction enzyme to obtain pPur{Delta}Mtet05-WtGs{alpha} and pPur{Delta}Mtet05-R201CGs{alpha} plasmids, respectively. The reporter plasmid PRL-164-Luc containing the (–164/–1) hPRL was kindly provided by J. Martial (University of Liège, Liège, Belgium). The reporter plasmid Pa3-GHp-Luc containing the –493-bp hGH gene promoter was kindly provided by N. L. Eberhardt (Mayo Clinic, Rochester, MN).

Generation of stable GH4C1 cell lines and cell culture procedures
Activating mutations of Gs{alpha} at codons 201 and 227 in the parental GH4C1 cell line (a generous gift from Dr. A. Sobel, Institut National de la Santé et de la Recherche Médicale, Paris, France) were excluded by sequencing as described previously (22). GH4C1 cells were cotransfected by electroporation with the pUHD 15-1 and pSV2neo plasmids. At 48 h after being transfected, the culture medium was supplemented with 250 µg/ml G418 (Invitrogen, Cergy Pontoise, France). Single transfected cell colonies were isolated by performing ring cloning and expanded into cell lines. The first generation of cell lines were screened by measuring the luciferase activity, after performing transient transfection with the pPur{Delta}MtetO5-luc plasmid using Transfast reagent in line with the instructions of the manufacturer, and cell cultures were incubated for 36 h in the presence or absence of 20 ng/ml doxycycline (Sigma). Among the 50 clones screened, the GH4C1 tTA clone with a doxycycline-dependent luciferase activity of 230 ± 65-fold was selected to establish the second generation of cell lines. The GH4C1 tTA clone, which stably expressed the tTA transactivator, was transfected with the pPur{Delta}MtetO5-WtGs{alpha} or the pPur{Delta}MtetO5-R201CGs{alpha} as described above. At 48 h after the transfection step, the culture medium was supplemented with 250 µg/ml G418, 10 µg/ml puromycine, and 20 ng/ml doxycycline. Twenty resistant clones for each construct were expanded into cell lines and screened to determine their doxycycline-dependent levels of expression of {alpha}s transgene. The various clones grown in Ham’s F10 medium supplemented with 15% horse serum (Eurobio, Les Ulis, France), 2.5% fetal calf serum (Invitrogen), penicillin (50 U/ml), streptomycin (50 µg/ml), G418 (250 µg/ml), puromycine (10 µg/ml), and doxycycline (20 ng/ml), maintained at 37 C in a water-saturated atmosphere containing 7% CO2 were subcultured weekly. The transgenes were induced by withdrawing doxycycline during the time(s) indicated. Experiments were done 4–5 d after plating the cells at the same density, without G418 and puromycine. Cells were serum starved in Ham’s F10 medium for the last 15 h before each experiment. Each experiment was done with cells at the same passage.

Immunoblotting of Gs{alpha} transgenes and total Gs{alpha}
GH4C1 cell lines grown 5 d in six-well tissue culture plates were washed in PBS (pH 7.4) and broken in a Teflon-lined glass Potter Elvehjem homogenizer in 10 mM Tris/1 mM EGTA buffer (pH 7.4), 5 µg/ml soybean trypsin inhibitor, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride and then centrifuged for 7 min at 500 x g and 4 C, after adding 5% sucrose. The supernatant was then centrifuged for 15 min at 12,500 x g at 4 C. The resulting pellet was resuspended in 50 mM Tris (pH 7.4) and sonicated. Fifty and 2 µg membrane proteins [determined using the Bio-Rad (Marnes-la-Coquette, France) DC protein assay] were resuspended in Laemmli’s sample buffer, separated on 10% SDS-PAGE, transferred onto polyvinylidene fluoride membrane (PerkinElmer, Courtaboeuf, France) for immunodetection of Gs{alpha} transgenes and total Gs{alpha}. Gs{alpha} transgenes were detected using a monoclonal anti-EE antibody (Eurogentec, Angers, France) and an antimouse IgG coupled to alkaline phosphatase as the secondary antibody. Total Gs{alpha} was detected using a polyclonal anti-Gs{alpha} antibody (Calbiochem via VWR International) and an antirabbit IgG coupled to alkaline phosphatase as the secondary antibody. ß-Actin content was routinely monitored by reprobing the membrane using a monoclonal anti-ß-actin antibody (Sigma). Blots were developed with the enhanced chemiluminescence Western-Star detection system (Tropix, Applera, Courtaboeuf, France) and quantified with a GeneGnome (Ozyme, St. Quentin Yvelines, France).

Gs{alpha} transgene mRNA and endogenous Gs{alpha} mRNA determination by real-time PCR
GH4C1 cell lines grown 5 d in six-well tissue culture plates were washed in PBS buffer. RNAs were extracted with the RNeasy kit (Qiagen, Courtaboeuf, France). The RNA samples were subsequently treated with 30 U ribonuclease-free deoxyribonuclease I (Qiagen) to prevent any contamination by genomic or plasmid DNA. cDNA was obtained from 2 µg total RNA using random-primed RT and superscript II reverse transcriptase (Invitrogen). Endogenous Gs{alpha} and Gs{alpha} transgene mRNA were measured by real-time quantitative PCR based on SYBR Green methods using an ABI 7700 apparatus (PerkinElmer). Forward primers included the EE sequence specifically recognizing either the Gs{alpha} transgenes (5'-AGGCCGAGTACATGCCGAC-3') or the endogenous Gs{alpha} gene (5'-ACGTGCCAAGTGACCAGGA-3'). A common reverse primer (5'-ACATCGAACATGTGGAAGTTGACT-3') was used. Standard curves were drawn up using dilutions of the plasmids used in the transfection step for transgene mRNA quantification (pPur{Delta}MtetO5-WtGs{alpha} and pPur{Delta}MtetO5-R201CGs{alpha}), and a plasmid named "endogenous Gs{alpha} plasmid" coding for WtGs{alpha} not bearing an EE sequence [cDNA from GH4C1 was subcloned in PCRscript (Stratagene Europe, Amsterdam, The Netherlands)] for endogenous Gs{alpha} mRNA quantification. The annealing extension temperature was 66 C. Amplification efficiencies of Gs{alpha} plasmids (pPur{Delta}MtetO5-WtGs{alpha}, pPur{Delta}MtetO5-R201CGs{alpha}, and endogenous Gs{alpha} plasmid) were identical. To check the specificity of each PCR, increasing concentrations of endogenous Gs{alpha} plasmid (up to five 107 copies) were measured with primers recognizing EE-Gs{alpha} mRNA and vice versa. No amplification was observed, nor was any amplification detected using the cDNA from GH4C1 and primers recognizing EE-Gs{alpha} plasmid. The mRNA levels were normalized to the 18S (32).

Membrane AC assay and cAMP level determination
AC assay was performed on membrane preparations from GH4C1 cell lines grown 5 d in 100 mm culture dishes as described previously (9). Intracellular cAMP levels were determined in GH4C1 cell lines grown in six-well tissue culture plates as described previously (33) in the absence of phosphodiesterase inhibitor.

ERK1/2 activation assay
GH4C1 cell lines grown 5 d in six-well tissue culture plates were incubated for 30 min in Ham’s F10 medium supplemented with 20 mM HEPES (pH 7.4). Cells were solubilized at 4 C for 20 min in a lysis buffer [25 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EGTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, and 10 µg/ml leupeptin and aprotinin]. Immunodetection of phosphorylated forms of ERK1/2 was performed as described previously (11). The total ERK1/2 content was systematically monitored by reprobing the membrane using a rabbit polyclonal ERK1 antiserum (Santa Cruz Biotechnology, Tebu, France).

Determination of endogenous rat PRL mRNA and PRL release
To determine the endogenous rat PRL mRNA levels, GH4C1 cell lines were plated 4 d into 35-mm dishes. Serum-starved cells were incubated for 6 h in Ham’s F10 medium containing 10–5 M Bacitracin. Northern blotting was performed with 10 µg total RNA samples as described previously (9). To determine the PRL release, GH4C1 cell lines were grown 4 d in 24-well tissue culture plates. Serum-starved cells were incubated for 6 h in fresh Ham’s F10 medium containing 10–5 M Bacitracin. At the end of the incubation period, the culture medium was recovered and centrifuged at 400 x g. The supernatant was stored at –80 C before the PRL RIA (34). PRL RIA reagents were a generous gift from Dr. A. Parlow and the National Hormone and Peptide Program Harbor–University of California Los Angeles Medical Center (Torrance, CA).

hPRL and hGH promoter assay
GH4C1 cell lines, grown in 24-well tissue culture plates for 3 d, were cotransfected with 100 ng of the reporter plasmid PRL-164Luc or the Pa3-Ghp-Luc and 4 ng of the renilla luciferase reporter vector phRL-TK (Promega) as an internal standard, using Transfast reagent. At 48 h after the transfection step, serum-starved cells were incubated in the same fresh medium containing 10–5 M Bacitracin for an additional 6 h, after which cells were washed, lysed, and analyzed to determine the luciferase activities in line with the instructions of the manufacturer (Promega).

Determination of [3H]thymidine incorporation and cell number
For [3H]thymidine incorporation, GH4C1 cell lines were grown 4 d in 24-well tissue culture plates. Fresh medium containing 0.5 µCi [3H]thymidine was added to each well during the last 4 h of incubation. Wells were then washed with cold PBS, and DNA was precipitated twice with cold 5% trichloroacetic acid for 15 min. Precipitates were solubilized in 0.25 N NaOH, and the radioactivity was quantified by scintillation counting. To determine the number of cells, GH4C1 cell lines were initially seeded at 5 x 106 cells into 100-mm-diameter dishes and grown for 7 d. Cells recovered by adding trypsin, were centrifuged at 400 x g, resuspended in culture medium, and counted using a hemocytometer.

Flow cytometry analysis
GH4C1 cell lines grown 4 d in six-well tissue culture plates were treated with trypsin, fixed in 70% ethanol for 30 min at 4 C, and treated with ribonuclease A (1 mg/ml) for 30 min at room temperature as described previously (35). DNA was stained with 25 µg/ml propidium iodide for 15 min at 4 C. Fluorescence-activated cell sorting (FACS) analysis was performed on an FACSCanto (Becton Dickinson, Pont-de-Claix, France) using FacsVantage Software.

Data and statistical analysis
Experiments were performed at least three times. The data (expressed as the percentage of noninduced cells) shown in the figures and table are from representative experiments or means ± SEM of several independent experiments or three to four determinations, as stated in the legends. Statistical analysis was performed using Mann-Whitney and Student’s t tests, and a P value of <0.05 was taken as the significance level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of doxycycline-dependent Gs{alpha}-expressing pituitary cells
To investigate the biological consequences of WtGs{alpha} overexpression and gsp oncogene expression in pituitary cells, we produced doxycycline-dependent Gs{alpha}-expressing clonal lines, using rat pituitary GH4C1 cells. GH4C1 cells constitutively expressing the tTA transactivator (GH4C1 tTA cells) were stably transfected with the plasmid pPur{Delta}MtetO5 encoding either EE-WtGs{alpha} or EE-R201CGs{alpha}. Subclones were prescreened to detect the expression of the transgene proteins 4 d after doxycycline withdrawal (induced cells). As can be seen from Fig. 1Go, transgene proteins were not detected in the presence of doxycycline (noninduced cells), whereas high levels of EE-tagged protein expression occurred after removing doxycycline in the various clones analyzed, except in the parental GH4C1 tTA cells (clone 1). The mRNA levels of both transgenes, as measured by performing real-time PCR, were barely detectable in the presence of doxycycline and amounted to less than 1–2% of the mRNA of the induced cells. Despite the relatively large dispersion that occurred as a result of the cloning, no significant differences were found to exist between the mRNA levels of the transgenes in the WtGs{alpha} clones and the R201CGs{alpha} clones after the induction process, whereas the level of Gs{alpha} transgene protein expression was higher in the WtGs{alpha} clones (Table 1Go). Inducing WtGs{alpha} or R201CGs{alpha} expression enhanced the AC activity, which suggests that the transgenes were functionally expressed. A greater level of AC activity was observed in the R201CGs{alpha} clones (Table 1Go).


Figure 1
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  		FIG. 1. Doxycycline-dependent expression of Gs{alpha} transgenes in pituitary cell lines. Different clones of GH4C1 established by stably transfecting the GH4C1 cell line tTA (clone no. 1) with the pPur{Delta}Mtet05-WtGs{alpha} (clone nos. 2–5) or with the pPur{Delta}Mtet05-R201CGs{alpha} (clone nos. 6–9) were grown in vitro for 4 d in the presence (+) or absence (–) of 20 ng/ml doxycycline. Expression of the tagged transgenes in 50 µg membrane proteins was detected by immunoblotting using an anti-EE antibody. Two representative experiments were given in the left and right, respectively.

 

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  		TABLE 1. Levels of transgene expression and AC activity in different doxycycline-dependent Gs{alpha}cell lines

 
Time course of Gs{alpha} transgene expression
Two prescreened clones with a functional high level of doxycycline-dependent WtGs{alpha} or R201CGs{alpha} transgene expression were selected for additional analysis. Expression of the mRNA of both transgenes showed the same time course and similar levels of expression. Maximum expression was observed after 4–8 d of induction, and these values decreased to 50% after 30 d of induction (Fig. 2AGo). Maximum expression of the transgene proteins was found to occur after 8 d of induction and then decreased to 50–60% from 20 d onward (Fig. 2Go, B–D). Although the mRNA levels of both transgenes were similar during the induction, the mutated protein levels were consistently lower than those of the wild-type transgene, which suggests that a posttranscriptional regulation of the gsp oncogene was involved (Fig. 2DGo). Total Gs{alpha} expression (endogenous WtGs{alpha} plus induced transgene), detected with an anti-Gs{alpha} antibody, is clearly enhanced in the Wt cell line during induction of the Wt transgene (Fig. 2Go, B and E) and represented 232 ± 37% (n = 4) of noninduced cells after 8 d of induction. Gs{alpha} expression was not significantly affected by 8 d of gsp oncogene induction [112 ± 9% of noninduced cells; n = 4 (Fig. 2Go, C and E)].


Figure 2
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  		FIG. 2. Time course of Gs{alpha} expression. Two selected GH4C1 cell lines transfected with the pPur{Delta}Mtet05-WtGs{alpha} (Wt cell line) or the pPur{Delta}Mtet05-R201CGs{alpha} (R201C cell line) were grown in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. A, Total mRNAs were extracted with the RNeasy kit, and transgene mRNA levels were measured by performing SYBR Green real-time PCR on the Wt cell line ({square}) and the R201C cell line ({blacksquare}). B, Immunoblots of tagged transgene (detected using the anti-EE antibody), ß-actin in 50 µg membrane proteins, and total {alpha}s in 2 µg membrane proteins of the Wt cell line grown in the presence (+) or absence (–) of 20 ng/ml doxycycline (DOX). C, Immunoblots of tagged transgene, ß-actin in 50 µg membrane proteins, and total {alpha}s in 2 µg membrane proteins of the R201C cell line grown in the presence (+) or absence (–) of 20 ng/ml doxycycline. D, Quantification of the EE transgene expression in the Wt cell line ({square}) and the R201C cell line ({blacksquare}). E, Quantification of total {alpha}s expression in the Wt cell line ({square}) and the R201C cell line ({blacksquare}). A representative experiment of at least three independent determinations under each experimental condition is shown. Variations of ß-actin level were 18 and 13%, respectively, in the Wt and R201C cell lines.

 
WtGs{alpha} overexpression and gsp oncogene enhance AC activity and increase the intracellular cAMP levels
As was to be expected, induction of the gsp oncogene was correlated with a large increase in the AC activity. The activity peaked after 8 d of induction (27.5 ± 7.5-fold over non-induced cells; n = 5), before decreasing gradually, and stabilized at 5- to 8-fold vs. that recorded in non-induced cells (Fig. 3AGo). Activation of the AC in the presence of the gsp oncogene was associated with an increase in the intracellular cAMP level, which also decreased gradually thereafter (Fig. 3BGo). A small but sustained activation of the AC activity (2.6 ± 0.9-fold over noninduced cells; n = 4) was also observed during overexpression of the WtGs{alpha} protein (Fig. 3AGo), which contributed to a small increase in the intracellular cAMP levels, which decreased gradually and stabilized at 1.4- to 1.8-fold over noninduced cells (Fig. 3BGo, inset).


Figure 3
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  		FIG. 3. Overexpression of the WtGs{alpha} and expression of the gsp oncogene increase the AC activity and cAMP levels. Inducible GH4C1 cell lines were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. A, AC activity was measured in Wt cell line ({square}) and R201C ({blacksquare}) cell line membrane preparations. B, Serum-starved GH4C1 Wt cell line ({square}) and R201C cell line ({blacksquare}) were incubated for 2 h in Ham’s F10 20-mM HEPES medium (pH 7.4) containing 5 µCi/ml [3H]adenine, washed, and further incubated for 15 min in adenine-free medium. Intracellular cAMP levels were determined as described in Materials and Methods. B, Inset, Diagram of cAMP levels in the Wt cell line ({square}). In the same experiment, the cAMP level of noninduced cells exposed 15 min to 0.1 µM VIP was increased 109 ± 4-fold and 32 ± 2-fold, respectively, in the R201C cell line and the Wt cell line. Each assay was performed in triplicate, and the values given are representative of at least three independent determinations under each experimental condition.

 
WtGs{alpha} overexpression and gsp oncogene induce a sustained activation of the MAPK ERK1/2
In the parental GH4C1 pituitary cells, we reported previously that activation of the VIP/pituitary adenylate cyclase-activating polypeptide type 2 receptor, coupled to the cAMP pathway, activated the MAPK ERK1/2 (9). We therefore investigated the ability of the gsp oncogene and WtGs{alpha} overexpression to activate ERK1/2. Induction of both transgenes increased the phosphorylated ERK1/2 levels, whereas total ERK1/2 remained constant (Fig. 4Go, A and B). The maximum activation of ERK1/2 observed after 2 d of induction was sustained throughout the induction and did not differ significantly between the two clones (Fig. 4CGo).


Figure 4
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  		FIG. 4. Overexpression of the WtGs{alpha} and expression of the gsp oncogene induce ERK1/2 activation. Inducible GH4C1 cell lines were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. Serum-starved GH4C1 cell lines were incubated 30 min in Ham’s F10 20-mM HEPES medium. Cell lysates (40 µg) were resolved on a denaturing 10% polyacrylamide gel before being transferred onto polyvinylidene fluoride membrane. Phospho-ERK1/2 (P-ERK1/2) was detected using a phospho-specific polyclonal antibody, and total ERK1/2 was recognized by a specific polyclonal antibody. Representative immunoblots of ERK expressed in the WtGs{alpha} cell line (A) and the R201CGs{alpha} cell line (B) are shown. Mean values of phosphorylated ERK2 (means ± SEM) in the WtGs{alpha} cell line ({square}) and the R201CGs{alpha} cell line ({blacksquare}) from three independent experiments are given. Phospho-ERK2 level of noninduced cells exposed 2 min to 0.1 µM VIP was increased 5.4 ± 0.9-fold (n = 11) and 9.1 ± 2.1-fold (n = 7), respectively, in the R201C cell line and the Wt {alpha}s cell line.

 
WtGs{alpha} overexpression and gsp oncogene increase hPRL and hGH promoter activities via a MAPK ERK1/2-dependent process
We then investigated whether gsp oncogene and WtGs{alpha} overexpression stimulate hPRL and hGH transcription. Both the hPRL proximal promoter (–164, –1) and the hGH proximal promoter (–493 bp), which contain cAMP response element (CRE) sequences [respectively, in the sequence A (–115, –85) (36) and the two core CREs at (–187/–183), (–99/–95) (37)] were regulated by 0.1 µM VIP in the noninduced clonal cell lines (Fig. 5AGo). VIP-activated hPRL and hGH promoters were inhibited in the presence of 10 µM U0126, the pharmacological inhibitor of ERK1/2 activation (Fig. 5BGo). The activity of hPRL and hGH promoters was also increased by 1 nM epidermal growth factor treatment (Fig. 5AGo). Induction of the gsp oncogene rapidly and stably increased the luciferase activity in the clonal cell line transiently transfected with the hPRL proximal promoter luciferase reporter construct. Luciferase activity was similarly increased during WtGs{alpha} overexpression (Fig. 6AGo). Gsp oncogene and WtGs{alpha} overexpression also increased the hGH proximal promoter activity but to a lesser extent (1.5-fold) (Fig. 6BGo).


Figure 5
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  		FIG. 5. hPRL and hGH promoters are regulated in the clonal cell lines. Inducible Wt (open bars) and R201C (hatched bars) GH4C1 cell lines were grown in vitro in the presence (noninduced cells) of 20 ng/ml doxycycline. Three days after plating, GH4C1 cell lines were cotransfected with 100 ng/well PRL-164Luc plasmid and 4 ng/well phRL-TK (hPRL) or 100 ng/well Pa3-Ghp-Luc and 4 ng/well phRL-TK (hGH). At 48 h after the transfection process, serum-starved cells were incubated for an additional 6 h in the absence or presence of 0.1 µM VIP or 1 nM epidermal growth factor (A) and in the absence or presence of 0.1 µM VIP and in the absence or presence of 10 µM U0126 (B). Cells were then lysed, and luciferase activity was measured using the dual luciferase reporter assay system. Each assay was performed in triplicate, and a representative experiment of at least three independent experiments is given. C, Control.

 

Figure 6
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  		FIG. 6. Overexpression of the WtGs{alpha} and expression of the gsp oncogene increase hPRL and hGH promoter activities. Inducible Wt ({square}, {circ}) and R201C ({blacksquare}, bullet) GH4C1 cell lines were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. Three days after the last passage, GH4C1 cell lines were cotransfected with 100 ng/well PRL-164Luc plasmid and 4 ng/well phRL-TK (A) or 100 ng/well Pa3-Ghp-Luc and 4 ng/well phRL-TK (B). At 48 h after the transfection process, serum-starved cells were incubated for an additional 6 h in the absence ({square}, {blacksquare}) or presence ({circ}, bullet) of 10 µM U0126. Cells were then lysed, and luciferase activity was measured using the dual luciferase reporter assay system. Each assay was performed in triplicate, and a representative experiment of at least three independent experiments is given.

 
In the present study, we established that both the gsp oncogene and WtGs{alpha} overexpression induce a sustained activation of the MAPK ERK1/2. The potential involvement of ERK1/2 in the regulation of hPRL and hGH transcription by Gs{alpha} was investigated using U0126. The results presented in Fig. 6Go, A and B, show that both the hPRL and hGH promoter activation induced by the gsp oncogene and WtGs{alpha} overexpression decreased in the presence of 10 µM U0126, which suggests that activated ERK1/2 may be involved in the up-regulation of the hormone transcription process exerted by wild-type and mutated Gs{alpha}.

WtGs{alpha} overexpression and gsp oncogene increase endogenous PRL synthesis and release
All acromegalic patients suffer from excessively high GH secretion levels. Forty-two percent of these patients also have elevated serum PRL levels (38). Although GH4C1 cells have retained the capacity to secrete both GH and PRL, they produced higher basal PRL than GH levels (39). In the present study, we therefore used PRL synthesis and release as an index to endogenous hormone regulation. The PRL mRNA levels determined by performing Northern blot analysis were stably increased during WtGs{alpha} overexpression and expression of the gsp oncogene (Fig. 7AGo). In addition, induction of both transgenes was correlated with a similarly sustained increase in the PRL release rates (Fig. 7BGo).


Figure 7
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  		FIG. 7. Overexpression of the WtGs{alpha} and expression of the gsp oncogene increase endogenous PRL mRNA and release. Inducible GH4C1 cell lines were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. Serum-starved cells were incubated 6 h in Ham’s F10 20-mM HEPES medium containing 10–5 M Bacitracin. A, PRL mRNA levels in the Wt cell line ({square}) and the R201C cell line ({blacksquare}) were analyzed by performing Northern blotting. Each assay was performed in triplicate, and a representative experiment is given. B, PRL release into the culture medium of Wt cell line ({square}) and R201C cell line ({blacksquare}) were analyzed by RIA. Each assay was performed in quadruplicate, and data from a representative experiment are given.

 
Effects of WtGs{alpha} overexpression and gsp oncogene on GH4C1 cell line proliferation
Expression of the gsp oncogene has been found to confer growth advantages on NIH3T3, FRTL5 thyroid cells, and pituitary GH3 cells, whereas patients with gsp+ tumors do not show any differences in their tumor growth and recurrence rates compared with patients with gsp– tumors (40). Based on these findings, we examined the effects of induced WtGs{alpha} overexpression and gsp oncogene expression on DNA synthesis, cell number, and cell cycle progression in both cell lines. During induction of the gsp oncogene, both [3H]thymidine incorporation, which is not modified by U0126 treatment, and cell number were slightly reduced (Fig. 8Go, B and C). Moreover, FACS analysis showed a decrease in the fraction of cells in the G2/M phase after several days of induction (Fig. 9BGo). WtGs{alpha} overexpression induced a small increase in [3H]thymidine incorporation, which is not affected by U0126 treatment (Fig. 8AGo), associated with a small increase in cell number (Fig. 8CGo) observed only after long-term induction of WtGs{alpha}. After several days of induction of WtGs{alpha}, FACS analysis showed a small decrease in the fraction of cells in the G2/M phase compensated by a small increase in the fraction of cells in the S phase (Fig. 9AGo). FACS analysis did not show a sub-G1 DNA peak, characteristic of cell undergoing apoptosis, in either induced WtGs{alpha} or R201C cell lines, whereas 5 µM staurosporine for 16 h induced 5.65 ± 0.2% (n = 4) of cells in the sub-G1 phase (data not shown).


Figure 8
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  		FIG. 8. Effects of overexpression of the WtGs{alpha} and expression of the gsp oncogene on [3H]thymidine incorporation and cell number. Inducible GH4C1 cell lines were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. Incorporation of [3H]thymidine (0.5 µCi) was performed for 4 h into the Wt cell line in the absence ({square}) or presence ({circ}) of 10 µM U0126 (A) or into the R201C cell line in the absence ({blacksquare}) or presence (bullet) of 10 µM U0126 (B). Each assay was performed in triplicate, and the results of a representative experiment of at least three independent experiments are given. C, Cells from the Wt cell line ({square}) and R201C cell line ({blacksquare}) were counted with a hemocytometer. Values (means ± SEM) of six independent experiments obtained at different passages are given. *, P < 0.05 and **, P < 0.01 compared with the respective control values (noninduced cells) using Student’s t test.

 

Figure 9
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  		FIG. 9. Effects of overexpression of the WtGs{alpha} and expression of the gsp oncogene on cell cycle progression. Inducible Wt cell line (A) and R201C cell line (B) were grown in vitro in the presence (noninduced cells) or absence (induced cells) of 20 ng/ml doxycycline for the times indicated. Cells were stained with propidium iodide and analyzed for DNA content by flow cytometry as described in Materials and Methods. Percentage of cells in the S phase (open bars) and in the G2/M phase (hatched bars) are represented in A and B. Each assay was performed in triplicate, and the results of a representative experiment of two similar experiments are given. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 compared with the respective control values (noninduced cells) using Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The relative frequency of occurrence of the gsp oncogene (18) and the high levels of Gs{alpha} expression (23) often detected in GH-secreting adenomas, as well as the similarities between the clinical phenotypes of patients with tumors, whether or not they carry the gsp oncogene, prompted us to explore the contribution of Gs{alpha} overexpression compared with those of gsp oncogene expression to pituitary cell regulation. Using GH4C1 cell lines, with conditional Gs{alpha} overexpression and gsp oncogene expression, it was established here for the first time that a weak level of Gs{alpha} overexpression can recapitulate the hormone dysregulation, observed in the presence of the gsp oncogene. Moreover, the present findings provide some evidence that Gs{alpha} overexpression and gsp oncogene both induced a sustained activation of ERK1/2, which seems to be involved in the hyperactivation of hPRL and hGH transcription.

We established doxycycline-dependent Gs{alpha}-expressing cell lines derived from the well differentiated rat pituitary GH4C1 cells. This system allowed us to minimize the secondary changes taking place during the selection procedure and thus to examine the early effects of the oncoprotein and those of WtGs{alpha} overexpression. We selected various clones with undetectable levels of transgene expression in the presence of doxycycline, showing functional membrane-targeted transgene expression after doxycycline withdrawal. A similar conditional pattern of expression was obtained previously with the dopamine D3 receptor in the GH3 pituitary cell line (41) and recently with the oncogenic B-Raf kinase in thyroid PPC3 cells (42). Surprisingly, the levels of expression of the gsp oncogene were found to be systematically lower (2.5- to 4-fold) than those of the WtGs{alpha} transgene expression in all of the clones screened. Moreover, an increase in total Gs{alpha} expression (2.5-fold) was observed only in GH4C1 clonal cell lines expressing the WtGs{alpha} transgene. This suggests that the degradation process is faster and/or that a subcellular redistribution of the gsp oncogene occurs (43).

Induction of the gsp oncogene initiates a considerable increase in the activity of the AC, associated with an increase in the intracellular cAMP level. Although the AC activity is closely related to the level of expression of the oncogene, the cAMP level rapidly decreases, suggesting the possible involvement of phosphodiesterases, as found previously to occur in gsp+ GH-secreting adenomas (20, 21). A weak but long-lasting activation of the AC is also observed in response to overexpression of Gs{alpha}. The associated increase in the intracellular cAMP level is rapidly and progressively decreased, also suggesting a potential contribution of the phosphodiesterases. Induction of a 2- to 3-fold membrane Gs{alpha} overexpression therefore has an impact on the activation of the AC pathway in pituitary cells, as observed previously in HEK 293 cells (44).

Despite the great difference in the activation of the AC pathway, induction of both transgenes was found to initiate a similar sustained 2- to 3-fold activation of the ERK1/2 cascade. It has been reported previously that ERK1/2 is one of the main MAPK pathways regulated by various GPCRs or tyrosine kinase receptors in pituitary somatolactotroph cell lines (9, 10, 45, 46). In addition, ERK1/2 was activated in GH-secreting adenoma cell cultures by increasing the cAMP levels with forskolin and by applying PKC-dependent GHRH treatment (47). The present results show for the first time that WtGs{alpha} overexpression or mutated Gs{alpha} are able to activate ERK1/2 without any need for an activated receptor in pituitary cells. Additional experiments are now required to determine the molecular mechanisms underlying the temporal relationships between the increase in cAMP levels and the sustained ERK activation, such as cAMP/protein kinase A-dependent specific recruitment of the monomeric GTPases Ras and Rap1 exchange factors (48) or direct activation of the tyrosine kinase Src by Gs{alpha}, as suggested by in vitro experiments (49).

In line with results obtained previously when performing transient and stable transfection of the R201C and Q227L oncogenic forms of Gs{alpha} (26, 28, 50), the present data show that induction of the R201C gsp oncogene initiates the activation of endogenous PRL transcription and release as well as that of hPRL and hGH proximal promoters. In addition, induction of a 2- to 3-fold WtGs{alpha} overexpression was found to have similar effects on hormone regulation. In a previous study, we reported that the ERK1/2 cascade and the upstream monomeric Ras and Rap1 GTPases play a pivotal role in rat PRL gene regulation (11). Here we provide evidence that ERK1/2 activation is involved in the chronic hyperactivation of hPRL and hGH genes, which suggests that sustained ERK1/2 activation may contribute, at least partly, to the dysregulation of the hormone promoters. The CRE-binding protein (CREB), which is involved in pituitary hormone regulation, may be one of the ERK1/2-targeted transcription factors. In fact, although the CREB mRNA levels are higher in gsp+ adenomas (51), high Ser133 CREB phosphorylation has been detected in both gsp+ and gsp– GH-secreting adenomas (52).

Studies on several transgenic mice models have suggested that cAMP provides important signals for somatotroph cell proliferation (38). In addition, stable expression of the Q227L gsp oncogene slightly increases the growth rate in the GH3 cell line (28). It is noteworthy that GH4C1, like the GH3 cell line, has a faster growth rate than normal pituitary cells and pituitary adenomas. However, in the present study, induction of Gs{alpha} overexpression and R201C gsp oncogene expression was not found to clearly initiate cell growth. A modest increase in the number of cells was observed only after long-lasting induction of the WtGs{alpha}. On the contrary, induction of the gsp oncogene seemed to slightly reduced [3H]thymidine incorporation, cell number, and the fraction of cells in the G2/M phase, which suggested a slowing down of proliferation. It has also been established that the overexpression of functional Gs{alpha} protein observed in thyroid adenomas is correlated with neither AC activity nor the proliferation rate of these tumors (53).

In conclusion, results obtained here in inducible GH4C1 cell lines are consistent with previous clinical observations done on patients carrying gsp+ or gsp– tumors or on GH-secreting tumors in culture (19, 22, 40, 54, 55). In addition, the present study provides evidence that the induction of WtGs{alpha} overexpression and gsp oncogene expression have a primary effect on hormone regulation, whereas this might not suffice to enhance cell growth. Secondary genetic and/or epigenetic alterations may be required to promote cell proliferation. Furthermore, our results favor the idea that the control of the expression level of the heterotrimeric Gs protein is critical to maintain hormone secretion at a physiological level. The pharmacological treatments based on somatostatinergic analogs used at present to treat somatotroph adenomas, efficacious on only 50% of the patients, seem to be mainly indicated for patients bearing gsp+ adenomas (22). In view of the functional similarities observed in these two cell lines (overexpressing WtGs{alpha} and expressing R201CGs{alpha}), downstream of the AC pathway, additional studies are now required to elucidate the molecular mechanisms underlying signaling cascade alterations, which should lead to the development of novel therapeutic approaches for treating GH-secreting adenomas of all kinds.


   Acknowledgments
 
We express our gratitude to Drs. H. Bujard, L. Journot, C. Berlot, J. Martial, N. L. Eberhart, and A. Parlow for the gift of the various plasmids and reagents described in Materials and Methods. We acknowledge the expert assistance of Dr. Jose Boucraut (Centre National de la Recherche Scientifique, Marseille) for the FACS analysis.


   Footnotes
 
First Published Online March 15, 2007

1 D.R. and K.M. contributed equally to this work. Back

Abbreviations: AC, Adenylyl cyclase; CRE, cAMP response element; CREB, CRE-binding protein; FACS, fluorescence-activated cell sorting; GPCR, G protein-coupled receptor; gsp, oncogenic Gs protein; gsp–, gsp-negative; gsp+, gsp-positive; GTPase, guanosine triphosphatase; h, human; PRL, prolactin; tet, tetracycline; tTA, tet-dependent transactivator; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylthio) butadiene; VIP, vasoactive intestinal polypeptide; WtGs{alpha}, wild-type Gs{alpha} subunit.

This research was supported by the Centre National pour la Recherche Scientifique and by grants from Ligue Contre le Cancer, Association pour le Développement des Recherches Biologiques et Médicales, and Fondation pour la Recherche Médicale and by a Provence Alpes Côte d’Azur regional grant.

The authors have nothing to disclose.

Received September 18, 2006.

Accepted for publication February 28, 2007.


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