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Effects of Hypothalamic Neuropeptides on Extracellular Signal-Regulated Kinase (ERK1 and ERK2) Cascade in Human Tumoral Pituitary Cells

Institute of Endocrine Sciences (A.L., M.F., S.C., E.B., P.B.-P., A.S.), University of Milan, Ospedale Maggiore Instituto di Ricovero e Cura a Carattere Scientifico, and Department of Neurosurgery (M.L.), Istituto Scientifico San Raffaele, Milan 20122, Italy

Address all correspondence and requests for reprints to: Anna Spada, M.D., Institute of Endocrine Sciences, Padiglione Granelli, Via Francesco Sforza, 35, 20122 Milan, Italy. E-mail: anna.spada{at}unimi.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The G protein-coupled receptor (GPCR) activation has been demonstrated to affect the ERK1/2 cascade in different cell lines. We investigated the effects of hypothalamic neuropeptides acting via GPCR on this pathway in GH-secreting (GH-oma) and nonsecreting (NFPA) pituitary adenomas. GHRH increased ERK1/2 activity (236 ± 80%) in both gsp- and gsp+ GH-omas, this effect being almost completely abolished by protein kinase C (PKC) blockade. Both GnRH and pituitary adenylate-activating peptide caused a similar PKC-dependent activation of ERK1/2 in most NFPA. Increasing cAMP by forskolin caused a protein kinase A-dependent increase of ERK activity (287 ± 37%) in GH-omas and had no effect in NFPA. ERK cascade blockade in GH-omas did not affect basal and GHRH-stimulated GH release, whereas it totally prevented the 3-fold increase in cyclin D1 protein expression induced by GHRH. In conclusion, this study demonstrated that in pituitary adenomas the activation of GPCR by neurohormones caused a PKC-dependent activation of ERK1/2 cascade that, at least in GH-omas, resulted to be involved in cyclin D1 induction by GHRH. Moreover, a stimulatory effect of the protein kinase A-dependent pathway on ERK1/2 cascade occurred selectively in GH-omas, probably contributing to the mitogenic potential of the cAMP pathway in this cell type.

	Introduction

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PITUITARY CELL FUNCTIONS are under the control of the hypothalamic neuropeptides, which are known to interact with specific receptors belonging to the superfamily of G protein-coupled receptors (GPCRs). The activation of these receptors induces the generation of intracellular cascades. In particular, TRH and GnRH receptors signal through the calcium-calmodulin pathway, whereas the cAMP-dependent pathway is activated by GHRH and CRH (1, 2). The MAPK are serine/threonine-specific protein kinases and include the extracellular signal-regulated kinases ERK1 and ERK2, the c-Jun NH2-terminal kinases, and the p38-MAPKs (3, 4). Indeed, phosphorylated ERK1/2 participate in the regulation of cell growth through phosphorylation of transcription factors, such as Elk1, and induction of proteins involved in the cell cycle, such as cyclins and cell cycle-related proteins (5, 6). Although in the past MAPK pathway was thought to represent the intracellular cascade specifically activated by growth factors, it is now well established that several GPCRs are able to stimulate ERK1/2 by different mechanisms (7, 8, 9).

Among the GPCRs expressed in the pituitary, recent studies showed that receptors operating through the calcium-calmodulin pathway, such as TRH and GnRH receptors, may activate ERK1/2 cascade by triggering protein kinase C (PKC) activation (10, 11, 12). By contrast, although several studies suggest that cAMP represents a mitogenic signal in selected cell types (13, 14), the possibility that cAMP-dependent pathway might cross-talk and activate ERK pathway in the pituitary has been so far poorly investigated. Moreover, to date, no information on the ability of GPCRs to modulate ERK1/2 activity in human adenomatous pituitary cells is available, as the data present in the literature concern receptors expressed in the rat pituitary or cell lines (14, 15, 16, 17).

The aim of the present study was to investigate the effect of the activation of either calcium-calmodulin- or cAMP-dependent pathways on ERK1/2 cascade in human pituitary tumors and to evaluate the possible impact of these events on hormone secretion and cyclin D1 protein expression.

	Materials and Methods

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Pituitary tissue samples and cell cultures

The study included eight GH-secreting (GH-omas) and eight nonfunctioning pituitary (NFPA) adenomas surgically removed by the transsphenoidal route. Small adenoma fragments were fixed for light and electron microscopy, to check the adenomatous nature of the material, as previously described (18). Part of the tissue was quickly frozen for Gs gene analysis. Tissues from all tumors were placed in sterile medium for cell culture. Local ethical approval was obtained for all studies.

Cells, enzymatically dispersed as previously described (19), were plated at the density of 1 x10⁶ cells/ml in six-well plastic cluster dishes and cultured in DMEM supplemented with 10% fetal calf serum and antibiotics in an atmosphere of 95% air-5% CO₂ in a humidified incubator. For the determination of ERK1/2 phosphorylation and activity, cell monolayers were incubated with different agents for 10 min at 37 C after 24 h of serum starvation. Incubation was stopped by placing the cells on ice, the medium was removed, and cells were treated with 500 µl ice-cold lysis buffer in the presence of protease and phosphatase inhibitors, as previously described (20). For in vitro GH secretion, dispersed cells were plated in 24-well plastic cluster dishes at a density of 2 x 10⁵ cells/ml and after 24 h of serum starvation incubated with and without the MAP kinase kinase (MEK) inhibitor PD98059 (50 µM) and GHRH (10 nM) for 30 min in triplicate. At the end of incubation, medium was removed and stored at -20 C for determination of GH, as previously described (19).

Analysis of mutations in Gs gene

Genomic DNA was obtained from tissue homogenates by acid guanidine thiocyanate-phenol-chloroform extraction, as previously described (21). Sequence analyses of Gs genes were directly performed by the dideoxy-nucleotide method and by automatic techniques (ABI Prism 310, Perkin-Elmer, Norwalk, CT) on PCR products. The hotspots of Gs gene were amplified using intronic oligonucleotide primers, as previously described (21). Three of eight GH-omas included in our study were gsp+, whereas all NFPA were negative for mutations of Gs gene.

Determination of ERK1/2 phosphorylation and activity

Briefly, active ERK was selectively immunoprecipitated from cell lysates with an equal amount of protein (as detected by bicinchoninic protein assay) by using an immobilized anti-phospho-p44/42 ERK monoclonal antibody (Thr202 and Tyr204), and the resulting immunoprecipitates were then incubated with a Elk-1 fusion protein (2 µg) in the presence of ATP (200 µM) and kinase buffer for 30 min at 37 C, which allow immunoprecipitated active MAPK to phosphorylate Elk-1, as previously described (20). Phosphorylation of Elk-1 at Ser383 was measured by Western blotting using an anti-phospho-Elk-1 (Ser383) antibody. For the determination of phosphorylated ERK1/2 levels, Western blot analysis was performed using an anti-phospho-p44/42 ERK polyclonal antibody diluted 1:1000 and detected by chemiluminescent method. The densitometric readings of the resulting bands were evaluated using a GS-670 imaging densitometer (Bio-Rad Laboratories, Inc., Hercules, CA). Experiments were repeated at least twice.

Determination of cyclin D1 protein

After 24 h of serum starvation, cell monolayers were incubated with different agents for 8 h at 37 C. Incubation was stopped by placing the cells on ice, the medium removed and cells were treated with 500 µl ice-cold lysis buffer in the presence of protease and phosphatase inhibitors. For the determination of cyclin D1, Western blot analysis was performed after immunoprecipitation of equal amount of cell lysates (as detected by bicinchoninic protein assay) with a specific monoclonal antibody, detected by chemiluminescent method and the resulting bands evaluated by imaging densitometer. Experiments were repeated at least twice.

Materials

Phospho-p42/44 ERK (Thr 202 and Tyr 204) monoclonal antibody, phospho-p42/44 ERK (Thr 202 and Tyr 204) polyclonal antibody, phospho-Elk-1 (Ser 383) antibody the phototope-horseradish perozidase enhanced chemiluminescence kit (LumiGLO) were obtained from New England Biolabs, Inc. (Beverly, MA). Cyclin D1 monoclonal antibody was from Novocastra (Newcastle, UK). PD98059 (2'-amino-3'-methoxyflavone), calphostin C, H89, DMEM, collagenase, 3-isobutyl-1-methylxanthine, GHRH, GnRH, pituitary adenylate activating peptide (PACAP), and forskolin were obtained from Sigma Chemical (St. Louis, MO).

Statistical analysis

The results are expressed as the mean ± SD. A paired or unpaired two-tailed Student’s t test or ANOVA for multiple comparisons with Bonferroni’s multiple comparison test was used to detect the significance between two series of data where appropriate. P value less than 0.05 was accepted as statistically significant.

	Results

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ERK1/2 activity in adenomatous somatotrophs

The ERK1/2 activity was determined by evaluating the phosphorylation of Elk-1 at Ser383 that represents the major phosphorylation site for ERK1/2 required for Elk-1-dependent transcriptional activity. Basal ERK1/2 activity in cells from tumors carrying or not gsp, the oncogene constitutively activating Gs, was similar [OD 0.56 ± 0.01 in gsp+ and 0.41 ± 0.15 in gsp-; P = not significant (NS)]. Cell exposure to 10 nM GHRH for 10 min resulted in a significant ERK1/2 activation, the percent stimulation ranging from 110- 590 (236 ± 80%; Fig. 1). The pattern of ERK1/2 activation correlated well with the levels of ERK1/2 phosphorylation (data not shown). This effect required PKC activation because pretreatment with the specific inhibitor calphostin C (1 µM for 30 min), as well as PKC down-regulation by prolonged incubation with the PKC activator phorbol 12-myristate 13-acetate, almost completely abolished GHRH-induced ERK1/2 stimulation (Fig. 1 and data not shown). Conversely, blockade of cAMP-dependent protein kinase A (PKA) by the inhibitor H89 (20 µM for 1 h), did not significantly reduce GHRH-induced ERK1/2 activity, indicating a poor, if any, involvement of the cAMP pathway in this effect (Fig. 1). Consistent with a marginal involvement of the cAMP pathway, no difference in GHRH induced ERK1/2 activation between gsp+ and gsp- tumors was observed (210 vs. 247% of stimulation).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of GHRH on ERK1/2 activity in human adenomatous somatotrophs. Lysates from cells incubated or not with GHRH 10 nM for 10 min were immunoprecipitated with anti-phospho-p44/42 ERK monoclonal antibody, incubated with an Elk-1 fusion protein and kinase buffer to induce the phosphorylation of Elk-1, which was measured by Western blotting using an anti-phospho-Elk-1 (Ser383) antibody (see Materials and Methods). A, Representative immunoblotting showing the increase of ERK1/2 activity induced by GHRH and the effects of PKC inhibitor calphostin C (1 µM for 30 min) and PKA inhibitor H89 (20 µM for 1 h) on ERK1/2 activity. B, Immunoblots were measured by an imaging densitometer and the values expressed in arbitrary units. Data represent the percent stimulation (mean ± SD) of ERK1/2 activity over basal values (control), which are arbitrarily defined as 100% (GH-secreting adenomas, n = 8). *, P < 0.05.

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of forskolin on ERK1/2 in pituitary adenomas. A, Representative immunoblotting showing the modification of ERK1/2 activity induced by forskolin (1 mM) in cells from one GH-oma and from one (NFPA). B, Immunoblots were measured by an imaging densitometer and the values expressed in arbitrary units. Data represent the percent variation (mean ± SD) of ERK1/2 activity over basal values induced by forskolin in three gsp- GH-secreting adenomas and in four NFPA (for experimental details see legend to Fig. 1). Basal levels (control) are arbitrarily defined as 100%. *, P < 0.05.

In cells obtained from 6 of 8 NFPA studied, 10 nM GnRH caused a significant increase in ERK1/2 activity, the percent stimulation ranging from 70–323% (181.7 ± 106%; Fig. 3). Moreover, in these adenomas, which are known to express specific PACAP receptors (22), PACAP caused a significant ERK1/2 activation (105 ± 40.0% of stimulation; Fig. 3). ERK1/2 activation by both GnRH and PACAP was almost completely abolished by calphostin C and not affected by H89 (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Effect of GnRH (10 nM) and PACAP (10 nM) on ERK1/2 activity in cells from NFPA. Immunoblots were measured by an imaging densitometer and the values expressed in arbitrary units. Data represent the percent modification (mean ± SD) of ERK1/2 activity over basal values that are arbitrarily defined as 100% (NFPA, n = 6; for experimental details, see legend to Fig. 1). *, P < 0.001; , P < 0.05.

Effect of MAPK blockade on GH release and cyclin D1 levels in adenomatous somatotrophs

To evaluate the possible role of the ERK1/2 cascade elicited by GHRH on hormone secretion, we measured basal and GHRH-stimulated GH release from cultured somatotroph cells obtained from 3 gsp- GH-omas in the presence or absence of PD98059, a compound specifically inhibiting the upstream kinases MEK1 and MEK2, which in turn phosphorylate ERK1/2. In these cells, basal GH levels were significantly stimulated by 10 nM GHRH (120 ± 9% stimulation). MEK1 and MEK2 blockade by PD98059 (50 µM) had no effect on both basal and GHRH-stimulated GH release (Fig. 4). Different results were obtained by evaluating cyclin D1 expression in these tumors. In fact, the incubation with 1 nM GHRH for 8 h induced a significant increase in cyclin D1 levels (210 ± 35% stimulation) that was totally abrogated by preincubating the cells with the inhibitor PD98059 (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of ERK1/2 on in vitro GH release. In cells obtained from three gsp- tumors GHRH (10 nM) caused a significant increase of GH release that was not affected by preincubating cultured cells for 1 h with PD98059 (50 µM), a MEK1/2 inhibitor. Values given are the means ± SD of GH determinations, carried out in duplicate, on three incubation media. *, P < 0.05 vs. GH release in basal conditions.

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of GHRH on cyclinD1 expression in GH-secreting adenomas. A, Representative immunoblotting showing the modification of cyclinD1 levels induced by GHRH (10 nM for 8 h). The stimulatory effect of GHRH was completely abolished by MEK1/2 blockade with PD98059 (50 µM). B, Immunoblots were measured by an imaging densitometer and the values expressed in arbitrary units. Data represent the percent modification (mean ± SD) of cyclin D1 levels over basal values (control) in three GH-omas. Basal levels are arbitrarily expressed as 100%. *, P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In pituitary tumors, ERK1/2 activation was also induced by peptides that classically operate through the cAMP-dependent pathway. Indeed, both GHRH and PACAP, at nanomolar concentrations, caused a significant increase in ERK1/2 phosphorylation both in GH-omas and NFPA. This effect was marginally dependent from the activation of the cAMP dependent pathway because the specific PKA blocker H89 caused a slight, not significant, reduction of GHRH-induced ERK1/2 activation in GH-omas and did not modify the PACAP action in NFPA. Consistent with a poor, if any, involvement of cAMP, GHRH stimulated ERK1/2 activity also in gsp+ tumors, despite the poor effectiveness of this peptide in increasing cAMP levels in these tumors, due to the constitutive activation of the mutant Gs (25, 26). The stimulation of ERK1/2 activity induced by GHRH and PACAP required PKC activation because it was prevented by PKC blockade or down-regulation. The ability of GHRH and PACAP receptors to signal through PKC may be due to the fact that both agents belong to the glucagon/secretin superfamily that has been demonstrated to activate receptors coupled with multiple G proteins, particularly Gs and Gq (27). Therefore, in addition to cAMP accumulation and PKA activation via Gs, these receptors may induce calcium rise and PKC activation via Gq. Alternatively, PKC activation by GHRH and PACAP might be triggered by calcium rise due to channel opening directly by Gs or secondary to cAMP accumulation. Although PKC was found to be the main mediator of ERK1/2 activation induced by hypothalamic neurohormones, an additional effect of cAMP on this cascade was documented in pituitary tumors. Indeed, in GH-omas the increase in cAMP accumulation caused by forskolin, an agent known to directly activate adenylyl cyclase, caused a ERK1/2 activation that was PKA dependent. Therefore, consistent with the view that somatotrophs are a selected cell type in which cAMP pathway is a mitogenic signal (13, 28), in these cells cAMP was able to activate ERK1/2 via PKA. Although it is not clear why forskolin activated ERK1/2 via PKA and GHRH activated ERK1/2 via PKC, it is tempting to speculate that the inability of forskolin to affect PKC may account for this phenomenon.

Interestingly, in cells obtained from NFPA that are mainly constituted by cells of the gonadotroph lineage, forskolin was unable to significantly modify ERK1/2 activity. Consistent with the absent action of forskolin on ERK1/2 in these cell types, PACAP-induced ERK1/2 activation in NFPA was fully PKA independent. Therefore, consistent with the notion that cross-signaling between different pathways is a cell-specific event that largely depends on the signaling molecules available for activation in the specific cell (8, 9, 13), cAMP differently affected ERK1/2 activity depending on the cell type within the pituitary. Admittedly, there are no data indicating that the different effects on ERK1/2 cascade induced by PKA in GH-omas and NFPA may also occur in the normal corresponding cell type.

The impact of ERK1/2 activation was irrelevant for GH release from cultured GH-omas in basal condition, as well as in response to GHRH, because the prevention of its activation by a specific inhibitor did not affect hormone secretion. Different results were obtained by investigating the expression of cyclin D1, a key regulator of G₁ phase progression in mammalian cells. In agreement with previous studies demonstrating the expression of cyclin D1 in pituitary tumors, particularly in the aggressive ones (29), cyclin D1 protein was detectable in the GH-omas investigated. Interestingly, GHRH caused a significant increase in cyclin D1 protein expression, which was abolished by preventing ERK1/2 activation, in agreement with the notion that cyclin D1 is a downstream target of ERK pathway (30). Although the induction of cyclin expression by GPCR activation has not been so far investigated in the pituitary, previous studies indicated that this event occur in endocrine tissues. In particular, it has been reported that TSH induced D-type cyclin expression and G₁ phase progression in thyroid FTRL5 cells (31, 32). Therefore, in tumoral somatotrophs GHRH acted as a specific growth factor, able to trigger up-regulation of cyclin D1 by activating ERK1/2 cascade. These data indicate that, although PKA activation has been reported to account for most GHRH biological activities, the mitogenic action of GHRH may be at least in part mediated by PKC activation that in turn activated ERK cascade.

In conclusion, this study first demonstrated that the activation of GPCR by specific neurohormones caused a PKC-dependent activation of ERK1/2 cascade that may be involved, at least in GH-omas, in cyclin D1 induction. Moreover, the study provided evidence for an additional cross-signaling between PKA and ERK1/2 pathways that resulted on different effects on GH-omas and NFPA.

	Footnotes

 
This work was supported in part by Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) Grant 2001068427 and Ricerca Corrente funds of Ospedale Maggiore Instituto di Ricovero e Cura a Carattere Scientifico (IRCCS) and Istituto Auxologico Italiano IRCCS.

Abbreviations: GH-oma, GH-Secreting pituitary adenoma; GPCR, G protein-coupled receptor; MEK, MAP kinase kinase; NFPA, nonsecreting pituitary adenomas; NS, not significant; PACAP, pituitary adenylate-activating peptide; PKA, protein kinase A; PKC, protein kinase C.

Received August 1, 2002.

Accepted December 20, 2002.

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				R. C. Fowkes, M. Desclozeaux, M. V. Patel, S. J. B. Aylwin, P. King, H. A. Ingraham, and J. M. Burrin Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone {alpha}-Subunit Gene in Gonadotropes Mol. Endocrinol., November 1, 2003; 17(11): 2177 - 2188. [Abstract] [Full Text] [PDF]

				A. Lania, G. Mantovani, and A. Spada Genetics of Pituitary Tumors: Focus on G-Protein Mutations Experimental Biology and Medicine, October 1, 2003; 228(9): 1004 - 1017. [Abstract] [Full Text] [PDF]

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