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INTRODUCTION |
Investigations of the growth effects of somatostatin on both
normal and neoplastic human tissues suggest that it has a complex mechanism of action, inducing a direct antiproliferative response in a
variety of cell types (1-3) in addition to reducing the circulatory
levels of mitogenic hormones and growth factors (4, 5). Somatostatin
transduces its direct action by stimulating G protein-coupled
receptors, named sst1-5 (6), although little is known as
to the identity of the receptor types mediating its antiproliferative
functions in tissues, and information has been largely restricted to
studies utilizing partially selective receptor analogs (7). Numerous
reports have demonstrated the expression of a high density of
somatostatin receptors on a variety of human cancer cells (4, 8). The
antiproliferative action of either somatostatin or its more
metabolically stable analog octreotide, however, does not correlate
with this expression, having inhibitory actions on pancreatic (9) and
breast tumors (1) but eliciting no effect on the growth of small cell
lung (10) and colon tumors (11). Growth-promoting effects of
somatostatin have also been described in vitro on human
pancreatic carcinoid (12) and epidermoid carcinoma cells (13), whereas
in rat mesangial cells, somatostatin stimulates proliferation in the
absence of serum but inhibits the growth of proliferating cells
(14).
As part of a study to resolve some of the apparent conflicting actions
of somatostatin on cell proliferation and to identify the receptor
types involved, clonal lines have been established expressing the
individual human recombinant forms in Chinese hamster ovary
(CHO)1 cells. Using a well
characterized in vitro model, changes in cellular
proliferation on the application of test substances have been assessed
by determining regeneration into denuded areas of a previously
confluent monolayer as well as by direct cell counting after a 24-h
recovery period (15). Both the activated human recombinant
sst2 (16) and sst5 receptors (7) in this model have been shown to have no effect on basal proliferation in the absence
of exogenously added mitogenic agents, but they can inhibit that
induced by a submaximal concentration of basic fibroblast growth factor
(bFGF). This is in agreement with earlier studies demonstrating a
reduced cell count after the application of the somatostatin analog
RC-160 to complete media in cell lines expressing either the mouse
sst2 or the sst5 receptor type (17). By
contrast, preliminary studies employing the regeneration model have
shown somatostatin to stimulate basal proliferation in cells expressing the human recombinant sst4 receptor in the absence of other
mitogenic agents (18).
The existence of different somatostatin receptors suggests that their
functional responses may be mediated by a variety of effector systems.
Although this aspect is not yet fully clarified, so far all five human
receptors have been shown to be functionally coupled to inhibition of
adenylate cyclase (19) and mediate, albeit to different degrees, the
stimulation of phospholipase C with subsequent calcium mobilization
(20). However, it is difficult to explain the specificity and diversity
of the responses to somatostatin through the apparently similar
modulation and subsequent actions of a small number of second
messengers. The mechanism involved in transmission of the
antiproliferative effect of the sst2 receptor has been
attributed to the activation of a protein tyrosine phosphatase, SHP-1
(21), and it has been suggested that such an activity may counteract
the growth-promoting properties of receptors containing an intrinsic
tyrosine kinase domain (22).
Activation of the mitogen-activated protein (MAP) kinase cascade has
been demonstrated after stimulation of sst4 receptors (23),
which is an important transduction mechanism utilized by many growth
factor receptors, although increasing evidence suggests that the
outcome of the receptor's signal depends both on the duration of
extracellular signal-regulated kinase (ERK) activation and cell context
(24, 25). A critical aspect of cell phenotype will be which
ERK-responsive transcription factors are present. A number of
transcription factors have been identified as targets for MAP kinase,
which enters the nucleus after phosphorylation by MAP/ERK kinase (MEK)
(26). The Ras/ERK pathway provides a common route by which signals from
different growth-inducing receptors converge at a major regulatory
element of the promoters of the c-fos and other coregulated
genes, the serum response element. The major targets for the pathway in
the c-fos promoter are the ternary complex factor family
members, Elk1 and SAP1, whose activity is regulated by phosphorylation
of a cluster of Ser/Thr-Pro motifs (Ser383 and
Ser389 of Elk1) at their COOH termini (27).
Another potential substrate for MAP kinase, belonging to a very
different family of transcription factors, is signal transducer and
activator of transcription 3 (STAT3). The STAT proteins are involved in
the transcriptional attenuation of many cytokine- and growth
factor-inducible genes, and, prior to receptor activation, they appear
to be cytoplasmic (28). They at least associate transiently with their
cognate receptors during activation; after phosphorylation at a
conserved tyrosine, which allows dimerization through reciprocal
phosphotyrosine-SH2 domain interactions, they translocate to the
nucleus and bind the cis-inducible element found in the
promoter of the c-fos gene. STAT activation by receptors lacking intrinsic kinase activity, including G protein-coupled receptors (29), involves specific members of the Janus kinase (JAK)
family (30). However, STAT3 is also phosphorylated by a
serine/threonine kinase thought to be MAP kinase which enhances its
transcriptional activity (31).
The purpose of this study was to investigate the transduction mechanism
mediating the proliferative action of somatostatin through stimulation
of the human sst4 receptor and to compare the effects with
those induced by the somatostatin analog L-362,855, which displays full
agonist activity at this receptor type (32). In particular, the
kinetics of the phosphorylation of ERK1 and ERK2 were monitored
throughout the initial period of proliferation and compared with
responses induced by bFGF. In addition, correlation between any late
phase MAP kinase activity and induced phosphorylation of Elk1 or STAT3
was also determined.
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EXPERIMENTAL PROCEDURES |
Materials--
Chinese hamster ovary (CHO-K1) cells were
obtained from The European Collection of Animal Cell Cultures (ECACC).
LipofectAMINE, Geneticin (G418 sulfate, specific activity 500 µg/ml),
and reagents for culturing cells were obtained from Life Technologies.
Thermanox coverslips, manufactured by Nunc, were purchased through Life Technologies, otherwise Costar tissue culture plastic ware was used.
bFGF and monoclonal antibodies to
-actin were obtained from Sigma.
Somatostatin and L-362,855 were synthesized by the GlaxoWellcome
Chemistry Unit, Department of Chemistry, University of Cambridge,
U. K. PD 98059 (2'-amino-3'-methoxyflavone) and Bordetella
pertussis toxin were from Calbiochem. Antibodies to ERK1 (C-16)
and ERK2 (C-14) were obtained from Santa Cruz Biotechnology; that
specific to the phosphoforms (at both Thr202 and
Tyr204) was supplied by New England Biolabs.
Phosphospecific STAT3 antibodies recognizing either the serine
(Ser727) or the tyrosine phosphorylation sites
(Tyr705) and that to the phosphorylated form of Elk1
(Ser383) were also obtained from New England Biolabs.
Antibodies to these transcription factors but whose specificity is
independent of the phosphorylation state were supplied by Santa Cruz Biotechnology.
Stable Expression of Human Somatostatin sst4
Receptors in CHO Cells--
The cDNA encoding the human
sst4 receptor was subcloned into the mammalian expression
vector pCIN4 harboring a neomycin-resistant gene as a selection marker.
CHO-K1 cells were cultured in Dulbecco's modified Eagle's
medium/Ham's F-12 medium (1:1) containing 10% fetal calf serum and 1 mM Glutamax I and transfected in the absence of serum with
10 µg of sst4-pCIN4 using a cationic liposome
formulation-mediated transfer (LipofectAMINE). Selection was performed
in the presence of complete medium containing 1 mg/ml G418 sulfate, and
clonal cell lines expressing the cDNA were isolated by single cell
cloning. Receptor expression was assessed by binding of
125I-Tyr11-somatostatin. The transfected cell
line expressing the human recombinant sst4 receptor
(CHOsst4) used in the current study gave IC50
values for somatostatin binding of 0.33 ± 0.06 nM
with the corresponding Bmax value being
2.07 ± 0.43 pmol/mg of membrane protein (n = 3).
All cultures were routinely maintained in their appropriate growth
medium at 37 °C in humidified air containing 5% carbon dioxide and
passaged when 95% confluence was reached.
Partial Denudation of Confluent Cell Monolayers and Assessment of
Proliferation--
Cells were grown to confluence in complete media on
Thermanox coverslips. Proliferation was assessed by determining the
regeneration of multiple denuded areas (400 µm wide), created by
dragging a Perspex comb across the surface of the coverslip, according
to the method described previously (7). The Perspex comb was designed so that 50% of the confluent monolayer was removed by the partial denudation process, leaving parallel strips of cells. Importantly, for
CHO-K1 cells, it has been demonstrated previously that repopulation of
the denuded areas in the presence of bFGF for 24 h is associated with a concomitant increase in cell number, abolished by protein synthesis inhibitors and independent of motorgenic processes (7, 15).
After partial denudation, coverslips were washed twice in
phosphate-buffered saline and placed in a fresh well containing drug or
vehicle in appropriate media but in the absence of fetal calf serum.
Regeneration of the cell monolayer was terminated after 24 h by
rapid washing in ice-cold phosphate-buffered saline followed by fixing
in absolute ethanol. Regeneration of the denuded areas was quantified
using a Leica Q500 MC image analysis system. Image analysis has
revealed that regeneration occurs by an outgrowth of CHO-K1 cells along
the perimeter of the denuded areas with an insignificant number of
cells becoming detached from the coverslip at any time point during the
investigative period (15). For each coverslip, four fields of view
selected at random were analyzed, and data are expressed as a
percentage of the denuded area recovered. All results were calculated
from a minimum of three experiments with four replicates per test
group; values are expressed as the arithmetic mean ± S.E.
Statistical analysis was by analysis of variance followed by Tukey's
test, taking p < 0.05 as the level of significance
(SigmaStat® version 2.0). The EC50 values were
calculated by interpolation from the concentration-effect curves
plotted and are defined as the concentration of drug which produced
half its maximal possible response.
Determination of Change in the Phosphorylation Status of ERK1,
ERK2, STAT3, and Elk1--
To analyze changes in the phosphorylation
status of ERK1 and ERK2 and the transcription factors STAT3 and Elk1 at
various stages during the regenerative processes after partial
denudation, whole cell protein extract was combined from eight
coverslips for each treatment group. Immediately before partial
denudation, cells forming the confluent monolayers of
CHOsst4 cells were in either the G0 or early
G1 phase of the cell cycle. Producing multiple denuded
areas on a single coverslip dramatically increases the number of cells
recruited into the regenerative process and amplifies the resultant
biochemical signals. For endothelial cells, it has been shown that
after denudation, at least 10 cells adjacent to the denuded edge
respond to this process. Subsequent analysis of changes induced in the
phosphorylation state of effector proteins involved in basal or growth
factor-stimulated regeneration for CHO-K1 cells will thus reflect those
of a large synchronized, proliferating cell population as well as from
a small, contact-inhibited subpopulation localized in the central
regions of the confluent strips. Application of bFGF to nondenuded,
confluent CHO-K1 monolayers has very little effect on the
phosphorylation status of ERK1 or ERK2, a weak transient increase in
phosphorylation between 10 and 20 min after
application.2
Regeneration was terminated by washing the CHOsst4 cell
monolayers in ice-cold phosphate-buffered saline before applying
SDS-polyacrylamide gel sample buffer (50 µl of 3 × strength) to
each test well (1 × sample buffer: 4% SDS, 5% glycerol, 60 mM Tris, pH 6.8, and 0.01% bromphenol blue) under reducing
conditions (50 mM 2-mercaptoethanol). After solubilization
of cellular protein by rapid mixing, the well contents were transferred
to a separate tube and combined with two further washings of the well
with deionized water (50 µl). Samples were vortexed, centrifuged at
10,000 × g for 2 min, and heated at 95 °C for 5 min. Total cell protein for each of the extracts was measured by
microBCA (Pierce), and equivalent amounts of protein were resolved
electrophoretically on 10% polyacrylamide gels.
After electrophoretic transfer onto nitrocellulose (0.22 µm) using a
semidry blotter, the membrane was washed briefly in Tris-buffered saline (TBS: 50 mM Tris, pH 7.5, 250 mM NaCl)
and saturated overnight in TBS supplemented with 0.1% Tween 20 and 5%
dried milk. For detection with the antibodies to ERK1 and ERK2, the
membranes were incubated with a 1:2,000 dilution (1:1 mix of ERK1 and
2) or 1:1,000 dilution of the anti-phospho-ERK antibody. Antibodies to
the phosphospecific forms of the transcription factors were used at a
1:500 dilution, whereas those to STAT3 and Elk1 were at a 1:1,000
dilution. When used, the antibody to
-actin was at a 1:5,000
dilution. All primary incubations were for 1 h at 22 °C in TBS
containing 0.1% Tween 20 (TBST) followed by washing five times for 10 min each in TBST. Membranes were incubated for 1 h at 22 °C
with a 1:5,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody in TBST containing 5% dried
milk. Excess antibody was removed by washing as above, and immunocomplexes were visualized using enhanced chemiluminescence (ECL)
detection, according to the manufacturer's instructions (Amersham
Pharmacia Biotech). The Western blots shown are representative of at
least three separate experiments, and each panel is taken from a single immunoblot.
 |
RESULTS |
Effect of Somatostatin and L-362,855 on the Proliferation of
Chinese Hamster Ovary Cells Recombinantly Expressing Human
sst4 Receptors--
Using the in vitro model to
determine changes in proliferation of CHOsst4 cells, the
basal rate of regeneration in incomplete media, 24 h after partial
denudation of a previously confluent monolayer, was 7.1 ± 0.4%.
Application of somatostatin caused a
concentration-dependent increase in the proliferation of
these cells (Fig. 1) with its potency,
expressed as an EC50 value, estimated at 19.9 ± 0.3 nM. The synthetic peptide, L-362,855, showed some agonist
activity, producing concentration-dependent increases in
the regeneration of CHOsst4 cells with an EC50
of 63.1 ± 0.8 nM, but the maximal response (10.0 ± 0.1%) was greatly suppressed compared with that induced by
somatostatin (24.1 ± 0.2%) and was only just significantly
different from basal values (p = 0.045) (Fig. 1). The
concentration-effect curve for bFGF reached a maximal response similar
to that produced by somatostatin (Fig. 1) but was at least 300-fold
more potent (EC50 0.05 ± 0.004 nM). In
all further experiments, submaximal concentrations of bFGF and
somatostatin were used at 10 ng/ml (0.6 nM) and 100 nM, respectively, which gave approximately equivalent
amounts of regeneration (80% of the maxima). Two concentrations of
L-362,855 were used: L-362,8551 at 100 nM and
L-362,8552 at 1 µM.

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Fig. 1.
Concentration-effect curves for bFGF ( ),
somatostatin ( ) and L-362,855 ( ) on the proliferation of CHO-K1
cells expressing human recombinant sst4 receptors.
Values are expressed as the mean percentage regeneration 24 h
after partial denudation of a previously confluent monolayer, against
log molar concentrations of drug (n = 3). The
vertical bars represent S.E.; where no error bar is shown,
the S.E. lies within the symbol.
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Effect of Pertussis Toxin Pretreatment or MEK Inhibition on
Proliferation--
Pretreatment for 20 h with pertussis toxin
(100 ng/ml) had no significant effect on either basal proliferation of
CHOsst4 cells (6.9 ± 0.2%) or that induced by bFGF
(10 ng/ml) treatment (22.5 ± 0.2% and 22.7 ± 0.4% with or
without pertussis toxin, respectively). However, the increased
proliferation evoked by application of somatostatin (100 nM) was abolished after pretreatment with the toxin to
values not significantly different from basal (7.6 ± 0.5% and
21.6 ± 0.5% with or without toxin treatment, respectively). The
increased proliferation of CHOsst4 cells in the presence of L-362,8552 (1 µM) also decreased after
pertussis toxin pretreatment, again to values not significantly
different from basal (6.8 ± 0.6% and 9.3 ± 0.2% with and
without pertussis toxin, respectively).
The inhibitor of MEK1, PD 98059 (2 µM), had no
significant effect on the basal proliferation of CHOsst4
cells (7.3 ± 0.4%) but abolished that elicited by the
application of somatostatin (100 nM) from 21.2 ± 0.6% to 7.1 ± 0.6%. Proliferation induced by bFGF (10 ng/ml)
was only partially blocked by PD 98059 treatment (from 21.9 ± 0.4% to 15.1 ± 0.7%). The small proliferative effect of
L-362,8552 (1 µM) was also blocked by
coincubation with PD 98059 (7.3 ± 0.7% and 9.2 ± 0.3%
with and without PD 98059, respectively).
Changes in the Phosphorylation Status of ERK1 and
ERK2--
Activation of MAP kinase during the initial processes
involved in the proliferative response of CHOsst4 cells was
assessed by monitoring changes in the phosphorylation status of ERK1
and ERK2. A time course of the immunoreactivity detected with the anti-phospho-ERK1 and ERK2 antibody by Western analysis of whole cell
extracts of CHOsst4 cells over the initial 4 h of
regeneration is shown in Fig. 2. During
this period, there was no detectable change in the expression of ERK1
or ERK2 protein in CHOsst4 cells (Fig. 2A), and
this level was not affected by any drug treatment (data not shown).

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Fig. 2.
Changes in the phosphorylation status of MAP
kinase during initial events in the induced proliferation of
CHOsst4 cells as determined by Western analysis. Whole
cell extracts were prepared from confluent monolayers before denudation
( ) immediately after denudation ( ), and after recovery in
incomplete media (BASAL), somatostatin (SRIF; 100 nM), L-362,8551 (100 nM), or bFGF
(10 ng/ml) for the times shown (in minutes). Panel A shows
the immunoreactivity obtained with anti-ERK1 and anti-ERK2 antibodies;
panels B and C are after detection with the
antibody that recognizes the dually phosphorylated forms of ERK (at
Thr202 and Tyr204). The time course of the
phosphorylation changes induced by the various treatments are shown in
panel B, with a comparative lane at the end of each
immunoblot indicating the level of phosphorylation after incubation in
the presence of somatostatin (SRIF) or incomplete media
(CON) for 4 h. Panel C shows a direct
comparison of the phosphorylation induced by the various drug
treatments at 10 and 20 min or at 2 and 4 h after denudation. It
should be noted that the concentration of L-362,855
(L362) used in panel C was at 1 µM instead of 100 nM as in panel
B.
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Proliferation under basal conditions produced little apparent change in
the phosphorylation of ERK1 and ERK2 during the initial 10 min compared
with levels detected in confluent monolayers prior to denudation (Fig.
2B). However, this apparent "steady state" of
phosphorylation decreased over the next 20 min to almost undetectable levels by 30 min (Fig. 2B). Proliferation in the presence of
somatostatin (100 nM), L-362,8551 (100 nM), or bFGF (10 ng/ml) produced a marked increase in the phosphorylated forms of both ERK1 and ERK2 compared with predenudation levels (Fig. 2B). The increase in the phosphorylation
induced by L-362,8551 reached a peak after recovery for 20 min but diminished to undetectable levels by 1 h. In contrast,
somatostatin treatment evoked a maximal response at 10 min, and
although this level subsequently declined, that observed at 4 h
postdenudation was substantially increased over basal (Fig.
2B). The phosphorylation of ERK1 and ERK2 in the presence of
bFGF appeared biphasic in that a transient increase occurred at 10 min
with a second peak following at 4 h postdenudation, although a
persistent increase in the immunoreactivity over basal levels was
observed throughout the entire time course (Fig. 2, B and
C).
The intensity of the immunoreactivity detected with the
anti-phospho-ERK1 and ERK2 antibody was strongest after somatostatin (100 nM) treatment for 10 min or bFGF (10 ng/ml) treatment
for 4 h (Fig. 2C). The level of phosphorylation after
regeneration in somatostatin (100 nM) or a high
concentration of L-362,8552 (1 µM) for 20 min
was comparable (Fig. 2C). The level of phosphorylation observed after a 2- or 4-h incubation period with
L-362,8552 was considerably lower for both ERK1 and ERK2
compared with that induced by somatostatin and comparable to that
detected for samples allowed to recover for these same times under
basal conditions (Fig. 2C).
Changes Induced in the Serine Phosphorylation Levels of STAT3 and
Elk1--
Activation of the transcription factors STAT3 and Elk1 is
associated with phosphorylation on serine residues Ser727
(33) and Ser383 (27), respectively. A time course of the
immunoreactivity detected with anti-STAT3 or anti-Elk1 antibodies
showed that the expression of these transcription factors remained
unchanged during the early basal regenerative processes of partially
denuded CHOsst4 cell monolayers (Fig.
3A). The expression of STAT3
and Elk1 was also unaffected by incubation with any of the drug
treatments during this initial 4-h period (data not shown).

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Fig. 3.
Changes in the phosphorylation status of
Ser727 of STAT3 and Ser383 of Elk1 during
initial processes in the induced proliferation of CHOsst4
cells. Whole cell extracts were prepared from confluent monolayers
immediately after denudation ( ) and after recovery in incomplete
media (BASAL), somatostatin (SRIF; 100 nM), L-362,8551 (100 nM), or bFGF
(10 ng/ml) for the times shown in min. Panel A shows
anti-STAT3 and anti-Elk1 antibody detection; the immunoblots in
panel B are after Western analysis using the
anti-phosphospecific antibodies for residues Ser727 (STAT3)
and Ser383 (Elk1).
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Under basal conditions, regeneration was associated with a small
increase in the serine phosphorylation of both STAT3 and Elk1 as
demonstrated by the enhanced immunoreactivity compared with samples
prepared immediately after denudation, together with a concomitant
electrophoretic mobility shift of the proteins (Fig. 3B).
Recovery in the presence of somatostatin (100 nM) increased the phosphorylation on Ser727 of STAT3, producing two
peaks: one at 20 min and a second at 4 h (Fig. 3B).
Somatostatin treatment, however, appeared to have no effect on the
basal level of phosphorylation of Elk1. Recovery in the presence of
L-362,8551 (100 nM) produced little change in
the intensity of the immunoreactivity detected by the
anti-phosphospecific antibodies to either transcription factor, and the
shift in mobility was comparable to that observed under basal
conditions over the same time interval (Fig. 3B). In
contrast, bFGF (10 ng/ml) induced a steady increase in the phosphorylation of both STAT3 and Elk1 throughout the first 4 h of
proliferative processes, producing maximal immunoreactivity and
electrophoretic mobility shifts at the longest recovery time examined
(Fig. 3B).
Increasing the concentration of L-362,8552 10-fold to 1 µM produced no further change in the level of
immunoreactivity detected with the anti-STAT3 phosphospecific antibody
compared with that obtained after recovery for 20 min or 2 h with
the lower dose (Fig. 4, A and
B). After the longer recovery time, the antibody appeared to
detect a doublet for all treatment groups, with a similar intensity of
staining for the band with the retarded mobility (Fig. 4B).
The phosphorylation induced by somatostatin (100 nM) or
bFGF (10 ng/ml) of the product with the greater mobility was similar at
2 h postdenudation and considerably increased over basal levels
(Fig. 4B). However, the intensity in staining of this latter
product appeared less than that of the control after recovery with
either concentration of L-362,855 (Fig. 4B). The increased
phosphorylation at Ser727 of STAT3 induced by somatostatin
(100 nM) or bFGF (10 ng/ml) after recovery for 4 h was
abolished by coapplication of PD 98059 (2 µM) (Fig.
4C). PD 98059 had no observable effect on the basal level of
serine phosphorylation of STAT3 (data not shown).

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Fig. 4.
Comparison of the changes induced in the
phosphorylation at Ser727 of STAT3 during initial events in
the proliferation of CHOsst4 cells. Analysis was made
of samples after recovery in the presence of incomplete media
(CON), somatostatin (SRIF; 100 nM),
L-362,855 (L361 at 100 nM and
L362 at 1 µM), or bFGF (10 ng/ml).
Panel A shows Western analysis using the phosphospecific
antibody (Ser727) of whole cell extracts prepared from
samples allowed to recover for 20 min; in panel B samples
left to recover for 2 h were analyzed by the anti-phosphospecific
antibodies to STAT3 (Ser727) as well as ERK1 and ERK2
(Thr202 and Tyr204). To illustrate consistency
in protein loading between the treatment groups in panel B,
detection of -actin was also made. Panel C shows the
effect of coincubation for 4 h with PD 98059 (PD; 2 µM) on the serine phosphorylation of STAT3 induced by
somatostatin (100 nM) or bFGF (10 ng/ml).
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Changes Induced in the Tyrosine Phosphorylation of
STAT3--
Tyrosine phosphorylation on residue Tyr705 of
STAT3 is required for dimerization of the transcription factor and
subsequent binding to DNA (34). During basal recovery over the initial
period of regeneration of CHOsst4 cells there was no change
in the expression of STAT3 (Fig.
5A), but a small and transient
increase in the immunoreactivity detected by the anti-phosphospecific
antibody was observed, producing a peak 5 min after denudation and
falling to levels below those detected in confluent monolayers by 30 min (Fig. 5B). Associated with this increased
phosphorylation was a concomitant splitting of the immunoreactivity
into discrete bands.

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Fig. 5.
Changes in the phosphorylation status of
Tyr705 of STAT3 during initial events in the induced
proliferation of CHOsst4 cells. Whole cell extracts
were prepared from confluent monolayers before denudation ( ),
immediately after denudation ( ), and after recovery in incomplete
media (BASAL), somatostatin (SRIF; 100 nM), L-362,8551 (100 nM), or bFGF
(10 ng/ml) for the times indicated (in minutes). Panel A
shows Western analysis using the anti-STAT3 antibody; panels
B and C represent the immunoreactivity obtained with
the antibody recognizing phosphorylated Tyr705. It should
be noted that the concentration of L-362,855
(L362) used in panel C was 1 µM instead of 100 nM as in panel
B.
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Regeneration in the presence of somatostatin (100 nM)
resulted in a marked and rapid increase in the tyrosine phosphorylation of STAT3, which was apparent after 5 min of regenerative processes and
appeared to be sustained throughout the first 4 h (Fig.
5B). After approximately 20 min of regeneration, multiple
products with discrete electrophoretic mobilities were detected by the anti-phosphospecific antibody, and the intensity of the staining shifted to those with retarded mobilities. L-362,8551 (100 nM) also induced rapid tyrosine phosphorylation of STAT3 which was sustained throughout the initial 20 min of recovery, but in
contrast to somatostatin-treated samples, the immunoreactivity subsequently diminished to predenudation levels by 1 h (Fig.
5B). The appearance of multiple products with distinct
electrophoretic mobilities, as observed for somatostatin-treated
samples, was also less evident during the longer recovery period in the
presence of L-362,8551 (100 nM). Treatment with
bFGF (10 ng/ml) produced a pattern of staining with the phosphospecific
antibody which was comparable to that obtained with somatostatin,
whereby sustained tyrosine phosphorylation of STAT3 was apparent with
multiple products being detected after regeneration for 30 min (Fig.
5B).
Increasing the concentration of L-362,8552 to 1 µM produced a pattern and intensity of immunoreactivity
similar to those observed after somatostatin (100 nM) or
bFGF (10 ng/ml) treatment for 5- and 20-min regeneration (Fig.
5C). At 30 min, the split into discrete products with a
concomitant shift in the immunoreactivity to those with retarded
mobilities was more apparent after bFGF or somatostatin treatment than
for L-362,8552. However, the 10-fold higher concentration
of L-362,8552 resulted in an intensity of staining
comparable to that produced by somatostatin (100 nM) after
30-min and 4-h regeneration (Fig. 5C). The separation of the
immunoreactivity detected with the anti-phosphotyrosine-specific STAT3
antibody into multiple products is most probably the result of
electrophoretic mobility shifts caused by the increased phosphorylation
at discrete sites within the protein. Although STAT3 is phosphorylated
on a single tyrosine residue (as detected by the antibody in Fig. 5,
B and C) there are alternative sites for the
addition of phosphate at serine/threonine residues. The separation into
multiple bands observed particularly after regeneration for 30 min and
longer in the presence of somatostatin or bFGF as shown in Fig.
5B is consistent with the concomitant increase in the
phosphorylation of the protein at Ser727 as shown in Fig.
3B.
Pretreatment of CHOsst4 cells for 20 h with pertussis
toxin (100 ng/ml) had no apparent effect on the intensity of
immunoreactivity or banding pattern after detection with the
anti-phosphotyrosine-specific STAT3 antibody, after recovery of
partially denuded monolayers in the presence of somatostatin (100 nM), L-362,8552 (1 µM), or bFGF
(10 ng/ml) for 10 min (Fig. 6).

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Fig. 6.
Effect of pretreatment for 20 h with
pertussis toxin (100 ng/ml) on the phosphorylation change induced at
Tyr705 of STAT3 during initial events in the induced
proliferation of CHOsst4 cells as determined by Western
analysis. Partially denuded monolayers were allowed to recover for
10 min in the presence of incomplete media (CON),
somatostatin (SRIF; 100 nM),
L-362,8551 (L361; 100 nM), or bFGF (10 ng/ml) which had been pretreated with
(PTX) or without pertussis toxin.
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Effect of Inhibiting MEK1 Activity on the Phosphorylation of ERK1
and ERK2 Induced by Somatostatin in Regenerating CHOsst4
Cells--
Coapplication of the MEK1 inhibitor PD 98059 (2 µM) (35) with somatostatin (100 nM) to
CHOsst4 cells immediately after denudation produced a
pattern and intensity of immunoreactivity with the anti-phosphospecific
ERK1 and ERK2 antibody similar to those produced by somatostatin
treatment alone over the initial 30 min of regenerative processes (Fig.
7). However, after 2 and 4 h of
regeneration, the level of ERK phosphorylation in samples treated with
PD 98059 and somatostatin was almost undetectable, similar to that
observed after incubation with L-362,8551 (100 nM) for 4 h and markedly reduced compared with samples
treated with somatostatin alone (Fig. 7). PD 98059 had no detectable
effect on basal levels of phosphorylation of ERK1 and ERK2 throughout the time course examined, and the immunoreactivity detected after regeneration with and without PD 98059 present for 10 min is shown in
Fig. 7.

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Fig. 7.
Effect of MEK1 inhibition on the
phosphorylation change induced in MAP kinase by somatostatin during
initial proliferative processes of CHOsst4 cells as
determined by Western analysis. Whole cell extracts were prepared
from confluent monolayers immediately after denudation ( ) and after
recovery in incomplete media (CON) or incomplete media
containing PD 98059 (PD; 2 µM) for 10 min as
well as somatostatin (SRIF; 100 nM) or
somatostatin in the presence of PD 98059 for the times indicated (in
minutes). For comparison, the level of immunoreactivity detected after
recovery in the presence of incomplete media or L-362,8551
(L361; 100 nM) for 4 h is also
shown.
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 |
DISCUSSION |
Despite numerous reports describing the ability of somatostatin to
regulate the growth of a variety of cell lines (1, 7, 12), little
information is available as to the identity of the receptor types
involved. An antiproliferative activity has been demonstrated in
recombinant systems for the sst2 and sst5
receptors (16, 17), and stimulation of cell growth in the absence of exogenously added mitogenic factors has been shown after
sst4 receptor activation (18). Thus, the biphasic and
sometimes contrasting nature of the growth responses observed on
application of somatostatin to cells expressing multiple somatostatin
receptors may be caused by the resultant effect of the activation of
those types with opposing proliferative functions. The aim of the
current investigation was to dissect the transduction mechanism through
which the human sst4 receptor mediates its proliferative
action so that a greater understanding of the functional interplay
between the individual somatostatin receptors can be made.
Alterations in the proliferative rate of transfected CHO-K1 cells after
24 h in the presence of test agents were assessed by determining
the regeneration of denuded areas of a previously confluent monolayer
(15). At various times throughout the initial stages of recovery,
analysis was made of any change induced in the phosphorylation status
of ERK1 and ERK2, in an attempt to correlate persistent or transient
activation of MAP kinase with the resultant proliferative effects
observed 24 h later. Somatostatin, its analog L-362,855, and bFGF,
all produced a rapid and marked increase in the phosphorylation state
of ERK1 and ERK2, which reached a peak between 10 and 20 min after
application. However, this rapid and potent phosphorylation did not
appear to be responsible for the induced proliferative effect of
somatostatin in CHOsst4 cells. Although somatostatin and
bFGF both increased cell growth, L-362,855 had little effect; and
perhaps more germane, coapplication of PD 98059 with somatostatin
abolished the somatostatin-induced proliferation but had no effect on
the phosphorylation changes evoked in ERK1 and ERK2 during the initial
hour of regeneration. The persistent increase in phosphorylation
observed after regeneration in the presence of somatostatin, however,
was abolished by PD 98059 treatment. It would thus seem that in CHO-K1
cells recombinantly expressing the sst4 receptor, it is the
prolonged activation of MAP kinase which is necessary for somatostatin
to promote cell growth. The functional role of the transient activity
of MAP kinase induced by activated sst4 and bFGF receptors
in CHO-K1 cells remains to be determined.
Recent reports using several cell types have suggested a role for
chronic MAP kinase activation in causing exit from the cell cycle
followed by reduced DNA synthesis and cellular differentiation (36,
37). These studies in particular have focused on the role of nerve
growth factor-induced growth arrest and inhibition of cell
cycle-dependent kinases. In both PC12 cells and primary cultures of hepatocytes, acute phasic activation (~30 min) of the MAP
kinase cascade was found to stimulate DNA synthesis, whereas chronic
activation (~5 h) in both cell types inhibited G1
progression/S phase entry. These data would appear to be in conflict
with the effects on proliferation observed in this study and the
associated duration of MAP kinase activity. However, several
publications have documented that both oncogenic Ras and Raf can
stimulate proliferation and suppress differentiation in SV40 virus
immortalized hepatocytes (38) in which potent and prolonged activation
of MAP kinase has been demonstrated. In addition, it has also been shown that in fibroblasts and other cell types, sustained activation of
MAP kinase is associated with proliferation and not differentiation (36); and depending on where it is expressed, the same receptor can
induce either response (39). Such observations thus indicate the
critical importance of cell phenotype in understanding signaling events
and predicting the functional outcome of a sustained or transient
activation of a particular transduction pathway.
One aspect that is fundamental to the resultant cellular effect induced
by the activation of the MAP kinase cascade will be which transcription
factors are present and if they are restricted in localization to the
nucleus. In every case examined thus far, sustained ERK activation is
associated with translocation of the kinases to the nucleus (39, 40),
whereas transient activation does not induce cytoplasmic nuclear
migration. Transient activation will therefore have very different
consequences for gene expression compared with that of sustained MAP
kinase activity. Nuclear accumulation of active ERK has been shown to
phosphorylate Elk1 in response to growth factors whose receptors
contain an intrinsic tyrosine kinase domain (27). Although MAP kinase
activation induced by bFGF in this study appeared biphasic, prolonged
phosphorylation of ERK1 and ERK2 was evident. Concomitant with the
marked increase in phosphorylation of MAP kinase after recovery in the
presence of the growth factor for 4 h was the enhanced serine
phosphorylation of both Elk1 and STAT3. In contrast, somatostatin had
no apparent effect on the phosphorylation at Ser383 of the
nuclear located Elk1 but increased that of STAT3 at Ser727.
The differential phosphorylation of Elk1 induced by somatostatin and
bFGF, despite both drugs producing a persistent activation of MAP
kinase, suggests that additional regulation of ERK-responsive transcription factors must be in place other than those enforced through compartmentalization restrictions. Even though Elk1 has been
shown to be a good substrate for ERK1 and ERK2 in vitro, there appears to be some selectivity for its requirement in the transduction of mitogenic responses induced through the different receptor types used in this study. The apparent lack of serine phosphorylation of either transcription factor on treatment with L-362,855 suggests that sustained activation of MAP kinase is critical
in CHO-K1 cells for the serine phosphorylation of STAT3 although it has
not yet been determined if this phosphorylation event takes place
within the nucleus.
STAT proteins are present in a latent form in the cytoplasm and become
phosphorylated on a single tyrosine, which is obligatory for STAT
activation (28, 34). Reports have shown this phosphorylation to occur
within minutes after ligand binding (28); this is consistent with the
rapid change in the immunoreactivity observed in this study using the
anti-phosphotyrosine-specific antibody to STAT3 after bFGF or
sst4 receptor activation. Rapid tyrosine phosphorylation of
STAT3 was observed in the regeneration model under basal conditions, although the increase was only transient in nature. A small but detectable increase in serine phosphorylation with associated electrophoretic mobility shifts was also observed for both STAT3 and
Elk1 under basal conditions, suggesting that these transcription factors may be involved in the basal regenerative processes of areas
denuded in confluent monolayers of CHO-K1 cells. It may be that
mitogenic factors are released from damaged cells at the denuded edge,
and/or signal transduction mechanisms are triggered by the disruption
of integrin clusters or intracellular zonular adheren sites. Regulation
of the cytoskeletal architecture is a requirement not only for cell
motility but also for growth, and it is possible that effectors
involved in the regulation of the actin cytoskeleton, such as Src, FAK,
and RhoGAP (41), can activate early response gene transcription in
CHO-K1 cells to promote cell proliferation under basal conditions.
An enhanced increase in the tyrosine phosphorylation of STAT3 over
basal levels was detected at all time points examined on application of
somatostatin, the higher concentration of L-362,855 as well as bFGF.
The rapidity of the induced phosphorylation suggests that this event is
placed upstream of the activation of MAP kinase and the serine
phosphorylation of STAT3. The lack of effect of pertussis toxin
treatment on the tyrosine phosphorylation also suggests that for the
sst4 receptor, activation of this pathway is independent of
the Gi/Go-mediated stimulation of MAP kinase. However, because the somatostatin-induced proliferation after activation of sst4 receptors can be inhibited by pertussis
toxin or PD 98059, the mechanism involved is presumably critically
dependent on serine phosphorylation of STAT3 because this was also
abolished by PD 98059 and pertussis toxin. The mechanism causing the
small proliferative response induced by L-362,855 is unlikely to be a
consequence of the activation of a receptor other than the
sst4 because L-362,855 is devoid of agonistic activity in
wild-type CHO-K1 cells (32).2 It is possible that L-362,855
does cause a small degree of serine phosphorylation of STAT3 but below
the level of detection by Western analysis using the
anti-phosphospecific antibody. Such a finding would be consistent with
both the partial agonist effect observed with L-362,855 in the
proliferation assay and the sensitivity of this effect to pertussis
toxin and PD 98059.
Tyrosine phosphorylation of STAT proteins has been demonstrated after
ligand activation of receptors with intrinsic tyrosine kinase activity
as well as the cytokine receptors that lack catalytic activity but to
which JAKs are noncovalently associated (42). Ligand-mediated
dimerization of either type of receptor results in reciprocal tyrosine
phosphorylation and consequent activation of the intrinsic or attached
kinase (43). The mechanism by which G protein-coupled receptors
activate the JAK/STAT pathway is less clear. In the case of the
angiotensin II receptor AT1, binding of JAK2 to the
receptor itself has been demonstrated (44). It has not been determined
here whether JAK activity was required for the tyrosine phosphorylation
of STAT3 either by activated bFGF or sst4 receptor types,
although STAT3 can be a substrate for other non-receptor tyrosine
kinases such as Src (45). However, what is clear is that tyrosine
phosphorylation is necessary for dimerization of the STAT3 proteins and
that serine phosphorylation can modulate binding to DNA and
transcriptional activity. The kinase responsible for the serine
phosphorylation of STATs is not known, but the phosphorylated residue
identified in the COOH terminus of STAT3 lies within a consensus site
for MAP kinase and is phosphorylated by this kinase in vitro
(31). The abolition of the serine phosphorylation induced by
somatostatin in STAT3 on application of the MEK1 inhibitor is
consistent with the concept that STAT3 is an ERK-responsive
transcription factor. Thus, although both phosphorylation events are
necessary for full transcriptional activity of STAT3, they are
controlled by distinct signaling pathways.
An unexpected and intriguing finding of this study came from the
comparison of the functional and transductional responses induced by
activation of the sst4 receptor type using different agonists. Because somatostatin and L-362,855 both caused a transient phosphorylation of ERK1 and ERK2 as well as a rapid and sustained tyrosine phosphorylation of STAT3, the lack of effect observed with
L-362,855 on serine phosphorylation of STAT3 together with its poor
proliferative action demonstrates agonist-selective receptor transduction. In view of the partial agonist effects observed with
L-362,855, these findings suggest that the receptor's mechanism mediating tyrosine phosphorylation of STAT3 is highly efficiently coupled in contrast to the more poorly coupled activation of MAP kinase
which results in serine phosphorylation of the transcription factor.
The phosphorylation changes in STAT3 are the result of somatostatin
stimulating distinct pathways with bifurcation at the level of its
receptor as suggested by the differential sensitivity of the
phosphorylation events to pertussis toxin.
There is an increasing number of examples in the literature where the
functional outcome in response to a mitogenic agent is determined not
only by the strength but also the duration of the stimulus, and small
differences in signal input can generate large differences in
transcriptional response. Even if two ligands activate the same STAT
protein, they may do so quantitatively differently for different
periods of time, thus contributing to different transcriptional
outcomes. Such quantitative variations can control physiological
decisions. In this study we have shown that CHO-K1 cells expressing the
recombinant sst4 receptor can be stimulated to proliferate
by the application of bFGF or somatostatin. This response is mediated
in the case of the sst4 receptor through a prolonged
activation of ERK1 and ERK2 which is dependent on the activity of
Gi/Go and is a common type of transduction
mechanism for many mitogenic G protein-coupled receptors (46).
Subsequent phosphorylation of Ser727 of STAT3 appears
dependent on this prolonged activation of MAP kinase and can be
activated differentially by agonist-selective stimulation of the
recombinant sst4 receptor. Agonist activation can, however,
promote tyrosine phosphorylation of the same transcription factor
possibly through a G protein-independent mechanism, in a functionally
cooperative but distinct signaling pathway.