| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
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
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: fscadigo{at}usc.es
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 different 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. 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). ß-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 Raf-MEK-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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 µg), T3 (0.4 µg),
transferrin (10 mg), glucagon (10 ng), epidermal growth factor
(0.1 µg), and fibroblast growth factor (0.2 µg).
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 ß-glycerophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM dithiothreitol, 400 µM phenylmethylsulfonylfluoride, 2 µM 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 (CHO-K1
strain), as previously described (20). Briefly, these cells were
selected with 400 µg/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 µg expression plasmid DNA were used per plate and adjusted to 26 µg 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-µl volume
of the corresponding assay buffer containing 100 µM
[-32P]ATP (1–5000 cpm/pmol) and 10 µg MBP
as substrate for the MAP kinases. The assay time was 20 min at 30 C.
Reactions were stopped by the addition of 6 x 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, 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.
|
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.
|
, 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. 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.
|
, 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.
|
, 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.
|
dimers
of the G protein (22, 25, 26, 27). A way to assess the involvement
of ß-subunits in a signaling pathway is to transiently express the
-subunit of transducin (Gt) that can bind
ß-subunits and thus block ß-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
ß-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 ß
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.
|
, 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.
|
, 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.
|
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 ß-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 PI3K activity is
involved in GHRH receptor-MAP kinase signaling.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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, 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
ß-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 ß 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 ß-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 ß-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 ß-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 |
|---|
|
Footnotes |
|---|
Received January 3, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain
of Gs and stimulate adenylyl cyclase in human
pituitary tumours. Nature 340:692–696[CrossRef][Medline]
-dependent activation and a
Gs-cAMP-mediated inhibition. J Biol Chem 270:25259–25265-subunits activate
mitogen-activated protein kinase via a novel protein kinase C-dependent
mechanism. J Biol Chem 271:1266–1269-mediated mitogen-activated protein kinase signaling
pathway. J Biol Chem 271:12133–12136 subunits. Nature 356:159–161[CrossRef][Medline]
subunits. Nature 369:418–420[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  K. Bedard, J. Strecko, K. Theriault, J. Bedard, C. Veyrat-Durebex, and P. Gaudreau Effects of a high-glucose environment on the pituitary growth hormone-releasing hormone receptor: type 1 diabetes compared with in vitro glucotoxicity Am J Physiol Endocrinol Metab, April 1, 2008; 294(4): E740 - E751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Hohla, A. V. Schally, K. Szepeshazi, J. L. Varga, S. Buchholz, F. Koster, E. Heinrich, G. Halmos, F. G. Rick, C. Kannadka, et al. Synergistic inhibition of growth of lung carcinomas by antagonists of growth hormone-releasing hormone in combination with docetaxel PNAS, September 26, 2006; 103(39): 14513 - 14518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Siriwardana, A. Bradford, D. Coy, and P. Zeitler Autocrine/Paracrine Regulation of Breast Cancer Cell Proliferation by Growth Hormone Releasing Hormone via Ras, Raf, and Mitogen-Activated Protein Kinase Mol. Endocrinol., September 1, 2006; 20(9): 2010 - 2019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Klausen, T. Tsuchiya, J. P. Chang, and H. R. Habibi PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1625 - R1633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Alba and R. Salvatori Naturally-occurring missense mutations in the human growth hormone-releasing hormone receptor alter ligand binding J. Endocrinol., September 1, 2005; 186(3): 515 - 521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Luque, R. D. Kineman, S. Park, X.-D. Peng, F. Gracia-Navarro, J. P. Castano, and M. M. Malagon Homologous and Heterologous Regulation of Pituitary Receptors for Ghrelin and Growth Hormone-Releasing Hormone Endocrinology, July 1, 2004; 145(7): 3182 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lania, M. Filopanti, S. Corbetta, M. Losa, E. Ballare, P. Beck-Peccoz, and A. Spada Effects of Hypothalamic Neuropeptides on Extracellular Signal-Regulated Kinase (ERK1 and ERK2) Cascade in Human Tumoral Pituitary Cells J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1692 - 1696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Mayo, L. J. Miller, D. Bataille, S. Dalle, B. Goke, B. Thorens, and D. J. Drucker International Union of Pharmacology. XXXV. The Glucagon Receptor Family Pharmacol. Rev., March 1, 2003; 55(1): 167 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. AMIEUX and G. S. MCKNIGHT The Essential Role of RI{alpha} in the Maintenance of Regulated PKA Activity Ann. N.Y. Acad. Sci., June 1, 2002; 968(1): 75 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Lee, W. R. Duan, T. Kotlar, and J. L. Jameson Restoration of Growth Hormone-Releasing Hormone (GHRH) Responsiveness in Pituitary GH3 Cells by Adenovirus-Directed Expression of the Human GHRH Receptor Endocrinology, January 1, 2001; 142(1): 414 - 420. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
