Somatostatin Acts by Inhibiting the Cyclic 3',5'-Adenosine Monophosphate (cAMP)/Protein Kinase A Pathway, cAMP Response Element-Binding Protein (CREB) Phosphorylation, and CREB Transcription Potency
John J. Tentler,
John R. Hadcock and
Arthur Gutierrez-Hartmann
Departments of Medicine and of Biochemistry, Biophysics,and
Genetics, Program in Molecular Biology, Colorado Cancer
Center, University of Colorado Health Sciences Center (J.J.T.,
A.G.-H.), Denver, Colorado 80262,
Wyeth Ayerst Research
(J.R.H.), Princeton, New Jersey 08543
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ABSTRACT
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Somatostatin (SRIF) was discovered as an inhibitor
of GH secretion from pituitary somatotroph cells. SRIF analogs are very
effective agents used to treat neuroendocrine tumors and are now being
used with increasing frequency in clinical trials to treat more
aggressive malignancies. However, the cellular components mediating
SRIF signal transduction remain largely unknown. We have stably
overexpressed the SRIF type 2 receptor (SST2) in
GH4 rat somatomammotroph cells, establishing a
physiologically relevant model system. In this model, the SRIF analog,
BIM23014, inhibited forskolin-induced cAMP accumulation, protein kinase
A activation, cAMP response element-binding protein phosphorylation,
and Pit-1/GHF-1 promoter activation in an okadaic acid-insensitive
manner. Pertussis toxin inhibited the effects of BIM23014, documenting
that SST2 signaling was coupled to Gi.
Moreover, the inhibitory effects of BIM23014 were reversed by
overexpression of protein kinase A catalytic subunit, indicating that
SRIF does not act via serine/threonine phosphatases, but, rather, by
lowering protein kinase A activity. These data define the components of
the SRIF/SST2 receptor signaling pathway and provide important
mechanistic insights into how SRIF controls neuroendocrine tumors. As
SRIF analogs are effective antitumor agents, and many other related
compounds are in development, the knowledge gained here will further
our understanding of their mechanism of action in other malignancies as
well.
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INTRODUCTION
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The neuropeptide somatostatin (SRIF) has potent inhibitory effects
on cellular proliferation in a wide variety of tissues. Therefore,
several long lasting SRIF analogs, such as SMS 201995 (octreotide),
MK 678 (seglitide), and BIM23014 (somatuline), have been developed as
anticancer agents (1). Because SRIF was discovered as the key
physiological inhibitor of GH secretion in pituitary somatotroph cells,
octreotide was first approved for use in treating GH-secreting
pituitary somatotroph tumors resulting in acromegaly (2, 3). Octreotide
and other stable SRIF analogs cause a significant reduction of both GH
secretion and pituitary tumor size in acromegaly. More recently, SRIF
analogs have shown significant utility in the treatment of other
endocrine and nonendocrine tumors, where it can inhibit tumor
progression and in some cases result in tumor shrinkage (4, 5).
Critical to SRIF action is its binding to specific plasma membrane
receptors (SSTx, where x is the receptor subtype number). Five distinct
SST receptor subtypes (SSTs 15) have been characterized and shown to
be members of the seven transmembrane-spanning,
Gi-protein-coupled superfamily of receptors
(6, 7, 8). All five SSTs have been detected in a variety of human tumors.
However, the SST2 subtype is the most frequently expressed, providing a
potential explanation for the significant effects of SST2-selective
agents, such as octreotide, on many different types of malignancies (4, 5, 9). Elucidation of downstream effectors in a native cellular
environment has been hampered by the very low expression of SST protein
in most cell types. To circumvent this problem, investigators have
overexpressed individual SST subtypes in heterologous cells, such as
Chinese hamster ovary (CHO) and HEK 293, and reported that SSTs can
couple to multiple cellular effector systems, including adenylyl
cyclase, Ca2+ and K+ channels,
phospholipase A, serine/threonine phosphatases, and tyrosine
phosphatases (6, 7, 8, 9, 10). One SST subtype may be linked to more than one
effector system, and the actual pattern of SST-effector coupling
appears to be cell or tissue specific, most likely based on which
Gi proteins and effector systems are present. Although
these fibroblast expression studies have provided important insights
into the SRIF signaling pathway, these cells are not physiological
targets of SRIF and thus may lack critical cellular SRIF signaling
component(s). In this report, we describe the use of a novel,
physiologically relevant, GH4 pituitary cell line that
stably overexpresses the long isoform of the type 2 SRIF receptor
(SST2). This model system was characterized and used to begin to
identify the signaling components and gene targets of the SRIF pathway
mediated by SST2. We find that SRIF inhibits the cAMP/protein kinase A
(PKA)/cAMP response element-binding protein (CREB) pathway, lowers CREB
transcription potency, and decreases GHF-1/Pit-1 promoter activity in a
pertussis toxin-sensitive, but okadaic acid (OA)-insensitive, manner.
Moreover, we map the effect of SRIF to the PKA step. As the cAMP/PKA
pathway is important for pituitary somatotroph ontogeny, proliferation,
and cellular function, we conclude that SRIF acts by targeting its
inhibitory effects to signaling components critical to somatotroph
function.
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RESULTS
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Development and Characterization of SST2 Expressing Cell Lines
To identify the physiologically relevant signaling components by
which SRIF controls cell function, we developed an experimental model
system by stably transfecting GH4C1 rat
pituitary somatomammotroph tumor cells with a plasmid encoding for a
full-length rat SST2A, the long SST2 isoform, and the predominant
pituitary SRIF receptor (hereafter termed SST2). Control cells were
transfected with an empty vector (CMV). Several clones were isolated
and, along with wild-type nontransfected controls, were subjected to
saturation binding analysis using [125I]SRIF-14 to
determine levels of SST2 protein. The results of the saturation binding
analysis are given in Fig. 1
and expressed as picomoles
per mg protein. [125I]SRIF-14 binding to membranes
prepared from all GH4C1 cells was saturable and
of high affinity at all levels of SST2 expression. The nontransfected
wild-type control and six transfected clones expressing different
levels of SST2 were then cultured in either the absence or presence of
the long acting SRIF analog, BIM23014, and the levels of SST2 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA)
expression were determined by Northern blot analysis (Fig. 1
). Although
endogenous SST2 mRNA levels could not be detected in the wild-type
(WT), the CMV control, or the R2/4 cell lines after a 36-h exposure,
[125I]SRIF-14 binding analysis revealed that the controls
each expressed about 0.4 pmol SST2/mg protein and that the R2/4 clone
expressed approximately a 5-fold higher level of SST2 (2.2 pmol/mg
protein). Detection of the endogenous SST2 mRNA in the control cells
required a 14-day exposure, revealing two bands of 2.3 and 2.6 kb (data
not shown), consistent with the size of endogenous SST2 mRNA (11). By
contrast, the R2/25, R2/34, R2/20, and R2/21 clones resulted in a
detectable signal of about 1.9 kb, consistent with the size of the
transfected SST2 complementary DNA (cDNA), and clones R2/20 and R2/21
produced a relatively strong SST2 mRNA signal. Again, the mRNA signals
in these clones generally agreed with the levels of SST2 protein
detected by saturation binding analysis, with the intermediate clones
expressing 15 and 25 pmol/mg protein (40- and 60-fold), and the high
expressing clones producing 40 pmol/mg protein (100-fold). The
effects of BIM23014 on endogenous SST2 mRNA expression in WT and CMV
cells are negligible (data not shown), as are its effects on exogenous
SST2 mRNA expression, except for clone R2/34. It is unlikely that
BIM23014 affects the CMV promoter activity controlling SST2 expression,
because several of the clonal lines are not affected; it is more likely
an integration site-specific effect in the R2/34 clone. The GAPDH band
(Fig. 1
, bottom panel) reveals equal loading of RNA and
showed that the ratio of exogenous/endogenous SST2 mRNA expression was
at least 100-fold, consistent with the data from Scatchard analysis.
Together, the Scatchard and Northern data verify the scarcity of
endogenous SST2 mRNA and protein and the abundant levels of the
transfected SST2. The high expressing R2/20 clone was used in all of
the following studies.

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Figure 1. Northern Blot Analysis of SST2 mRNA in Control and
SST2-Stable GH4C1 Cell Lines
Clonal cell lines (R2/4, R2/25, R2/34, R2/20, and R2/21) were subjected
to saturation binding analysis and found to express varying amounts of
SST2 receptor protein (picomoles per mg). WT and CMV represent control
nontransfected and pRC/CMV only-transfected wild-type GH4
cells, respectively. Cells were exposed to PBS (-) or 10
nM BIM 23014 (Bachem, Torrance, CA; +) in DMEM plus 10%
FBS for 36 h, replenishing the analog at 6-h intervals. Total RNA
(20 µg) from each clonal cell line was electrophoresed on a 1.4%
agarose-formaldehyde gel and transferred to a nitrocellulose membrane;
the membrane probed with a radiolabeled SST2 cDNA hybridization probe
at high stringency and exposed to film for 36 h (top
panel). The same blot was reprobed with a labeled cDNA for
GAPDH (bottom panel) to assure equal loading of RNA. A
representative autoradiograph is presented.
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Effects of BIM23014 on the 3',5'-cAMP/PKA/CREB Pathway in R2/20
Pituitary Cells
The cAMP/PKA/CREB signaling pathway is critical for somatotroph
ontogeny, proliferation, and cell-specific gene expression (12, 13, 14).
Also, 3050% of human somatotroph tumors contain activating
Gs mutations, resulting in constitutively elevated cAMP
levels and constitutively phosphorylated and activated CREB
transcription factor (15, 16, 17). As SRIF analogs inhibit somatotroph
tumors, we sought to determine whether the SRIF analog BIM23014
inhibited any of the components of the cAMP signaling cascade. The
R2/20 clone expressed lower basal levels of cAMP compared with control
GH4 cells, and they displayed a 5-fold lower
ED50 for SRIF-14-mediated reduction of forskolin
(FSK)-stimulated cAMP levels (data not shown). Using a serine 133
phospho-specific CREB antibody (17), we showed that
FSK-stimulated CREB phosphorylation (Fig. 2A
) by about
8.5-fold (Fig. 2B
). Pretreatment with increasing doses of BIM23014
reversed the FSK-induced CREB phosphorylation, with an IC50
of approximately 7 nM (Fig. 2
, A and B). BIM23014 (10
nM) alone had no effect (Fig. 2A
, lane 2, and Fig. 2B
), and
the BIM23014-mediated inhibition of the FSK response was blocked by
pretreatment with pertussis toxin (Fig. 2A
, lanes 12 and 13, and Fig. 2B
), verifying that the effect of BIM23014 was mediated by
Gi, as predicted (6, 7, 8, 10). Also, BIM23014 had minimal
effects on FSK-mediated CREB phosphorylation in GH4 WT
cells (data not shown), indicating that the effects of BIM23014 were
not through an endogenously expressed SST receptor and substantiating
the idea that SST2 overexpression amplifies the effects of BIM23014. As
CREB phosphorylation at serine 133 directly correlates with its
activity as a transcription factor (17), these data suggest that
BIM23014 inhibits CREB transcription potency.

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Figure 2. Effect of BIM23014 on FSK-Stimulated
Phosphorylation of CREB
A, R2/20 cells were cultured in 1% FBS for 6 h, and then treated
with the indicated doses of BIM23014 for 10 min, followed by treatment
with FSK (1 µM). After 10 min, the cells were lysed by
the addition of Laemmlis sample buffer. Some cells were pretreated
with pertussis toxin (PTX; 100 ng/ml) for 24 h before and during
the experimental protocol noted above. Equal amounts (50 µg) of whole
cell extracts were resolved by SDS-PAGE, followed by Western blotting,
and probed with an antibody specific for
[Ser133]phospho-CREB (top panel). The blot
was subsequently stripped and reprobed with an antibody to both
phospho- and dephospho-CREB (bottom panel). Bands were
detected by enhanced chemiluminescence (Amersham). B, Quantification of
the Western blot shown in A. Phosphorylation was quantitated by
scanning laser densitometry of the autoradiograph (Molecular Dynamics,
Sunnyvale, CA), and values were corrected for levels of CREB in each
sample.
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Effect of BIM23014 on Transcription of CREB-Dependent Genes
To determine the functional significance of the BIM23014-mediated
attenuation of FSK-induced CREB phosphorylation, we tested the response
of the CREB-dependent, pituitary-specific POU homeodomain transcription
factor promoter, Pit-1/GHF-1, to FSK with or without BIM23014 (Fig. 3B
). The Pit-1/GHF-1 promoter is a particularly relevant
target, because it has been shown that the Pit-1 protein is one of the
factors that controls somatotroph cellular proliferation (18). FSK
treatment resulted in a 13-fold activation of a -200Pit-1/CAT reporter
construct (set at 100%) transiently transfected into the R2/20
pituitary cells (Fig. 3B
) (12). Moreover, the dose response of the
BIM23014-mediated inhibition of FSK-induced Pit-1 promoter activation
(Fig. 3B
) closely paralleled the inhibition of CREB phosphorylation
(Fig. 2
), with an IC50 of approximately 15 nM.
Similarly, the inhibitory effect of BIM23014 on the Pit-1 promoter was
reversed by pretreatment with pertussis toxin (Fig. 3B
). The brief
period of BIM23014 treatment (6 h) was insufficient to mediate
inhibition of Pit-1 mRNA levels (data not shown). However, as Pit-1
protein autoregulates its own promoter, we used a separate
pituitary-specific, CREB-dependent, Pit-1-independent promoter, the
-1760
-subunit (
-SU) glycoprotein promoter (19), to insure that
the effects were due to inhibition of CREB alone (Fig. 3C
). FSK
stimulated
-SU promoter activity 22-fold, and 10 nM
BIM23014 reduced this activation by 52%, mimicking the effects on the
Pit-1 promoter (Fig. 3B
). Finally, BIM23014 had no effect on the CMV
promoter used as an internal control to drive ß-galactosidase, thus
showing that the BIM23014 effect was specific for cAMP-responsive
promoters. These results corroborate that the BIM23014-mediated effects
on CREB phosphorylation status govern its potency as a transcription
factor.

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Figure 3. Dose Effects of BIM23014 on -200 Rat GHF-1/Pit-1
Promoter Activity
A, Schematic map of the GHF-1/Pit-1 proximal promoter region. Binding
sites for CREB (CRE1 and CRE2) and for Pit-1 (Pit-1B1) are indicated.
B, R2/20 cells were cultured in complete DMEM and transfected by
electroporation with 9 µg Pit-1-CAT and 3 µg CMV ß-galactosidase
(ß-gal), as previously described (40). After transfection, cells were
cultured in DMEM plus 1% FBS for 18 h and then treated with FSK
(1 µM) and the indicated doses of BIM23014 for 6 h.
Some cells were pretreated with pertussis toxin (PTX; 100 ng/ml) for
24 h before and during the transfection protocol. The amounts of
extracts used to determine CAT activity were normalized according to
the level of ß-galactosidase. The results shown are expressed as a
percentage of the value with FSK alone (100% = 13-fold). C, Effects of
BIM23014 on FSK-activated human -SU. R2/20 cells were cultured in
complete medium and transfected by electroporation with 3 µg
-SU-luciferase and 3 µg CMV-ß-gal. After transfection, cells
were cultured in DMEM plus 1% FBS. FSK (1 µM) and the
indicated doses of BIM23014 were added 18 h after transfection,
and cells were collected 6 h later. Results are corrected for
ß-galactosidase activity and expressed as fold promoter activation.
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Effects of OA on SRIF Action
It has been proposed that activation of serine/threonine
phosphatases may mediate some of the effects of SRIF, as the Ser/Thr
phosphatase inhibitor OA can reverse the stimulatory effects of SRIF on
large conductance Ca2+- and voltage-activated
K+ (BK) channel activity (20). Serine/threonine
phosphatases 1 (PP1) and 2A (PP2A) have been shown to dephosphorylate
CREB in vivo and thus attentuate its transcription activity
(21, 22, 23). To investigate the roles of PP1 and PP2A in the
BIM23014-mediated reduction in CREB phosphorylation, we treated cells
with or without OA, a specific inhibitor of PP1 and PP2A, at a dose (25
nM) that will inhibit both of these phosphatases (22, 23).
Western analysis (Fig. 4A
), using the CREB
phospho-specific antibody, and quantification of these data (Fig. 4B
)
show that OA, either alone or in combination with FSK, slightly
stimulates CREB phosphorylation, yet OA fails to reverse the
BIM23014-mediated inhibition of CREB phosphorylation. To corroborate
these results in a functional manner, we tested the ability of 25
nM OA to reverse the inhibitory effects of BIM23014 on the
-SU promoter (Fig. 4C
). Again, OA alone activated the
-SU
promoter and enhanced the effects of FSK, verifying that OA is
functional. However, OA failed to reverse the inhibitory effects of
BIM23014
-SU promoter activity. We found that the effects of OA are
specific. All transfections were internally controlled with pCMV
ß-galactosidase, and this cAMP-unresponsive promoter is unaffected by
OA, with or without BIM23014.
PKA Catalytic Subunit Expression Reverses Inhibitory Effects of
BIM23014 on
-SU Promoter
The biochemical and functional data presented above indicate that
SRIF does not act via PP1 or PP2A to inhibit CREB phosphorylation and
activity. Therefore, we postulated that BIM23014 may act by lowering
PKA activity. To determine whether BIM23014 is acting upstream or
downstream of PKA, an expression vector encoding the ß-catalytic
subunit of PKA was cotransfected with the
-SU reporter gene, and the
effects of BIM23014 on PKA promoter activation were assessed
(Fig. 5
). These results show that PKAß, in the absence
of BIM23014, activated the
-SU promoter 18-fold, consistent with the
FSK results shown in Fig. 3C
. However, addition of increasing amounts
of BIM23014, from 0.1100 nM, had no statistically
significant effect on the PKA response. These data are in striking
contrast to the inhibitory effects of BIM23014 on FSK activation of
this same promoter (Fig. 3C
). More importantly, we have mapped the site
of SRIF action between adenylate cyclase and PKA, with the data clearly
implicating PKA as a key target. Finally, these data independently
corroborate that phosphatases are unlikely to be involved in SRIF
action.

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Figure 5. Overexpression of PKA Catalytic Subunit Blocks
Effects of BIM23014
R2/20 cells were cotransfected by electroporation with and without (+
or -) 9 µg of an expression vector encoding an RSV-driven PKA
catalytic subunit (RSV PKAß) plus 3 µg -SU-luciferase and 3 µg
CMV ß-galactosidase. The indicated doses of BIM23014 were added
18 h after transfection, and cells were collected 6 h later.
Results are corrected for ß-galactosidase activity and expressed as
fold promoter activation.
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DISCUSSION
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SRIF and its analogs have found clinical utility as potent
antisecretory and antiproliferative agents in tumors derived from a
wide variety of tissues. Despite this widespread use of SRIF analogs,
little is known regarding the functional cellular components that
mediate SRIF signal transduction. In this paper, we report the
development and characterization of a novel SST2-overexpressing model
system in a physiologically relevant cell line, GH4
pituitary cells. Using this system, we demonstrated that the SRIF
analog, BIM23014, acts via a Gi-coupled process
to inhibit the cAMP pathway, acting at or upstream of PKA and resulting
in decreased CREB phosphorylation and transcription potency in an
OA-insensitive manner. As cAMP/PKA, CREB, and Pit-1/GHF-1 exert
important growth regulatory effects on somatotroph cells (12, 13, 14, 15, 16), we
have linked the inhibitory effects of SRIF to specific cellular
constituents that are critical for neuroendocrine cellular function and
proliferation (24, 25).
Trophic hormone activation of the cAMP/PKA pathway has been shown to be
critical for the maintenance of cell-specific function and cell
proliferation in many neuroendocrine cell types (24, 25). Underscoring
this idea has been the identification of several naturally occurring
mutations that result in the constitutive activation of receptors for
trophic hormones, such as TSH, LH, and PTH/PTH-related peptide
(26, 27, 28, 29), and the demonstration that these receptor mutations cause
elevations in cAMP and hyperfunction of the respective endocrine end
organ, leading to endocrine tumor formation. Conversely, inactivating
mutations of trophic hormone receptors have also been identified, and
patients harboring these mutations display a phenotype consistent with
the loss of endocrine end organ cell function (30). These trophic
hormone receptors typically act by coupling to heterotrimeric
Gs
proteins, and activating mutations of
Gs
have also been identified and denoted the
gsp oncogene, because they result in endocrine end-organ
hyperfunction and tumor formation (15, 16, 24, 26, 31). Taken together,
these findings corroborate the importance of the cAMP/PKA pathway in
endocrine cells. Indeed, this pathway has been shown to be critical for
normal pituitary somatotroph cell ontogeny, maintenance of cell
function, and cellular proliferation (18, 32). For example, an
inactivating mutation of the GRF receptor results in a lack of the
normal expansion of somatotroph cells during development and leads to
the little mouse phenotype (33). Similarly, targeted
transgene expression of a cholera toxin to somatotroph cells leads to a
persistently activated Gs
, increased cAMP, and
somatotroph hyperplasia and tumorigenesis (13), whereas targeted
expression of a dominant negative CREB leads to somatotroph hypoplasia
(14). Finally, many GH-secreting somatotroph adenomas from acromegalic
patients arise from expression of the constitutively active
gsp oncogene, resulting in activated adenylyl cyclase,
elevated cAMP levels, persistently phosphorylated CREB, and
up-regulated Pit-1 promoter activity (13, 14, 15, 16, 17, 34). Of note, these
patients are particularly responsive to the SRIF analog octreotide
(35). Thus, our results showing that SRIF acts via SST2 to inhibit the
cAMP/PKA/CREB pathway and Pit-1 promoter activity provide important
insights into the cellular mechanisms by which SRIF analogs effectively
control tumors derived from cAMP-regulated endocrine cells.
Having determined that BIM23014 inhibits FSK-induced CREB
phosphorylation, we sought to identify the mechanism of this action. In
theory, the inhibitory effects of BIM23014 on CREB phosphorylation
could be mediated by either increased phosphatase activity, decreased
PKA activity, or a combination of these two possibilities. The results
of our studies indicate that in our system, BIM23104 is not activating
phosphatases, as it is capable of attenuating CREB phosphorylation and
transcription potency in the presence of OA, a specific inhibitor of
Ser/Thr phosphatases PP1 and PP2A. The observation that addition of OA
alone results in increased basal CREB phosphorylation and
-SU
promoter activity (Fig. 4
) indicates that OA is entering the cells and
exerting its expected effect on CREB phosphorylation, by modulating
either PP1 and/or PP2A (21, 22). By contrast, our results showing that
overexpression of a PKA catalytic subunit can reverse the inhibitory
effects of BIM23014 on
-SU promoter (Fig. 5
) indicate that the
effect of SRIF maps upstream or at the level of PKA. Furthermore, this
finding also supports the idea that phosphatases are not the key
regulatory effectors, as it would be expected that SRIF stimulation of
phosphatase catalytic activity should diminish the effects of
transfected PKA. In this regard, the effects of SRIF on somatotrophs
mimic the tonic inhibitory effects of dopamine on lactotrophs, by which
dopamine agonists also act via a Gi-coupled D2
receptor to lower cAMP and diminish Pit-1 promoter activity (36, 37).
Similarly, these dopamine agonists inhibit pituitary lactotroph cell
function and shrink pituitary lactotroph adenomas (38). Together, these
data indicate a convergence of mechanisms for the clinical
antiproliferative effects of SRIF analogs on somatotrophs and of
dopamine analogs on lactotrophs, indicating that the cAMP/PKA pathway
is critical to both of these pituitary cell types. Therefore, the
mechanism of SRIF action in somatotrophs that we show here may be
generalizable to other tumors that use cAMP as a key regulator of
cellular growth or to maintain cell function (24, 25).
Although these data clearly implicate the cAMP/PKA pathway as a
critical target of SRIF, the possibility remains that SRIF may inhibit
other, OA-resistant signaling pathways as well. For example, SRIF might
alter calcium-dependent pathways (such as calcineurin/calmodulin
kinase, phosphoinositol-3 kinase, or protein kinase C) or tyrosine
kinase/MAP kinase pathways. All of these signaling constituents have
been shown to play important roles in somatolactotroph cell function
(39, 40, 41, 42, 43), and thus, SRIF inhibition of one or more of these signaling
cascades would result in a pleiotropic and more effective inhibition of
pituitary cell functions. Previous reports have shown that SRIF analogs
inhibit the inositol phospholipid/calcium pathway in rat pancreas (44)
and clonal hamster ß-cells (45). Also, SRIF analogs have been show to
stimulate p66 PTP1C tyrosine phosphatase activity in MCF-7 breast
cancer cells (45, 46), AR42J rat pancreatic acinar cells (47), and
COS, CHO, and NIH3T3 cells stably expressing the SST2 receptor subtype
(48), each in a vanadate-sensitive manner. However, the SRIF analog,
RC-160, inhibited cellular proliferation of CHO cells overexpressing
the SST5 receptor subtype in a vanadate-insensitive manner, indicating
differential effector coupling to the distinct SST subtypes (48). These
results raise the possibility that SRIF/SST2-activated PTP1C tyrosine
phosphatase may dephosphorylate and inactivate membrane receptor and
cytoplasmic tyrosine kinases required for cell growth (46, 47, 48, 49). In the
studies reported here, we have not examined whether BIM23014 results in
activation of tyrosine phosphatase activity, because we have focused on
the cAMP/PKA pathway. Therefore, the possibility remains that BIM23014
might induce tyrosine phosphatase activity via the overexpressed SST2
in the GH4 pituitary model system described here.
Finally, and of particular significance for the future discovery of
clinically applicable agents, the SSTR2-overexpressing GH4
somatolactotroph model that we have developed can be used to identify
and characterize SST2 subtype-selective agonists and antagonists, using
the biochemical and functional assays that we have reported here. In
this regard, this system provides an important and biologically
pertinent model that will allow us to further elucidate the molecular
mechanisms of SRIF actions that are selective for the SST2 receptor
subtype. These approaches should begin to decipher the
structure-function code by which specific SST receptor subtypes target
distinct and overlapping downstream effectors.
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MATERIALS AND METHODS
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The long isoform of the SST2 SRIF receptor (SST2A) was used for
these studies. The SST2 cDNA was isolated by the PCR using specific
primers and GH4C1 RNA. Both strands of the SST2
cDNA were sequenced using an Applied Biosystems automated DNA sequencer
(Foster City, CA). The full-length rat SST2 SRIF receptor was subcloned
into the expression vector, pRC/CMV (Invitrogen, San Diego, CA), and
stable transfections of GH4C1 cells were
accomplished using a calcium phosphate transfection kit (Stratagene, La
Jolla, CA). Six individual clones were selected using 500 µg/ml G-418
and were amplified and maintained in 100 µg/ml G-418. Saturation
binding was performed on membranes of WT and transfected
GH4C1 cells with 02500 pmol
[125I]SRIF-14. Nonspecific binding for each point (counts
per min bound in the presence of 5 µM cold SRIF-14)
ranged from 1040%. Experiments for each cell line were performed in
triplicate. Transient transfection experiments of
GH4C1 rat pituitary cell lines were performed
as described previously, using pCMV-ß-galactosidase as an internal
control for transfection efficiency (40, 50). The -200 Pit-1 promoter
and the human glycoprotein
-SU promoter plasmid constructs have been
described previously (12, 19). Cells were harvested, and reporter
enzyme activities were assayed as previously described (40, 50).
Normalized data are depicted as the mean of several experiments ±
SEM.
Cell extracts for Western blotting were prepared from 80% confluent
60-mm dishes. Cells were harvested with Laemmli-SDS sample buffer and
cell scraping. Cell extracts were boiled for 5 min and sheared through
a 22-gauge needle. Samples were resolved on 12% SDS-polyacrylamide
gels and transferred to nitrocellulose as described previously (40).
Filters were blocked in 5% nonfat milk and 0.2% Tween-20, probed with
antibodies to phospho-CREB (provided by Dr. M. Montminy) and CREB
(provided by Dr. D. Klemm), and developed using enhanced
chemiluminescence (Amersham Corp., Arlington Heights, IL) according to
the manufacturers protocols.
Total RNA was prepared from the indicated clones with the RNA Stat 60
Kit (Tel-Test, Inc., Friendswood, TX). Samples (20 µg total RNA) were
electrophoresed on a 1.4% agarose-formaldehyde gel and transferred to
a nylon-reinforced nitrocellulose membrane. A
[
-32P]CTP-labeled hybridization probe was prepared
from the full-length SST2 cDNA template by random primer synthesis.
After high stringency hybridization, the membrane was washed three
times with 0.1 x saline sodium citrate (SSC) and 0.1% SDS at 65
C for 30 min. The same blot was reprobed with a labeled cDNA for GAPDH
to assure equal loading of RNA.
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ACKNOWLEDGMENTS
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We thank M. C. Eppler for providing cell lines, M. Montminy for
providing antibody to phospho-CREB, M. Karin for the -200 GHF-1
promoter construct, and K. N. Farrow, A. P. Bradford, P. Zeitler, S. E.
Diamond, and A. James for critical comments on the manuscript.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Arthur Guitierrez-Hartmann, Departments of Medicine and of Biochemistry, Biophysics, and Genetics, Program in Molecular Biology, Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262.
This work was supported by NIH Grants DK-46868 and DK-37667, and NIH
SBIR Subcontract N44-DK-22214 (to A.G.-H.). Additionally, partial
support was provided by the Lucille P. Markey Charitable Trust and the
University of Colorado Cancer Center Core Grant CA-46934.
Received for publication December 11, 1996.
Revision received January 31, 1997.
Accepted for publication March 17, 1997.
 |
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