From the INSERM U 151, Institut Louis Bugnard, CHU
Rangueil, F 31403 Toulouse Cedex, France, ¶ Department of
Medicine, Tulane University and Veterans Affairs Medical Center School
of Medicine, New Orleans, Louisiana 70112, and
Laboratoire
d'Immunologie, CHU Rangueil, F 31403 Toulouse Cedex, France
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ABSTRACT |
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Activation of the somatostatin receptor sst2
inhibits cell proliferation by a mechanism involving the stimulation of
the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory
events leading to sst2-mediated growth arrest are not known. Here, we
report that treatment of Chinese hamster ovary cells expressing sst2
with the somatostatin analogue, RC-160, led to G1
cell cycle arrest and inhibition of insulin-induced S-phase entry
through induction of the cyclin-dependent kinase inhibitor
p27Kip1. Consequently, a decrease of p27Kip1-cdk2
association, an inhibition of insulin-induced cyclin E-cdk2 kinase
activity, and an accumulation of hypophosphorylated retinoblastoma gene
product (Rb) were observed. However, RC-160 had no effect on the
p21Waf1/Cip1. When sst2 was coexpressed with a catalytically
inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1
induced entry into cell cycle and down-regulation of p27Kip1
and prevented modulation by insulin and RC-160 of p27Kip1
expression, p27Kip1-cdk2 association, cyclin E-cdk2 kinase
activity, and the phosphorylation state of Rb. In mouse pancreatic
acini, RC-160 reverted down-regulation of p27Kip1 induced by a
mitogen, and this effect did not occur in acini from viable motheaten
(mev/mev) mice
expressing a mutant SHP-1 with markedly deficient enzymes. These
findings provide the first evidence that sst2 induces cell cycle arrest
through the up-regulation of p27Kip1 and demonstrate that SHP-1
is required for maintaining high inhibitory levels of p27Kip1
and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27Kip1.
Somatostatin is a widely distributed inhibitory hormone that plays
an important role in several biological processes including neurotransmission, inhibition of exocrine and endocrine secretions, and
cell proliferation. The diverse biological effects of somatostatin are
mediated through a family of five somatostatin receptors (sst1-sst5) that belong to the family of G-protein-coupled receptors and that regulate diverse signal transduction pathways including adenylate cyclase, phospholipase C- The ability of somatostatin and its stable analogues to promote
inhibition of normal and tumor cell growth has been demonstrated in
various cell types including mammary, prostatic, gastric, pancreatic, colorectal, and small cell lung cancer cells (3, 4). However, the
mechanisms of cell growth arrest by somatostatin are still poorly
understood. Somatostatin analogues induce a
G0/G1 cell cycle arrest and thus prevent DNA
synthesis in GH3 rat pituitary tumor cells, whereas they induce a
transient G2/M blockade as well as apoptosis in MCF7 human
mammary tumor cells (5, 6). These tumor cells express multiple
somatostatin receptors and the question of whether different
somatostatin receptor(s) may be involved in eliciting these effects
still remains to be clarified. A specific role for sst3 in transducing
apoptosis through an induction of p53 and Bax has been reported (7).
Control of the cell cycle machinery by other receptors is an important
problem that remains to be addressed.
Our studies on the expression of somatostatin receptor subtypes in
heterologous systems led us to demonstrate that sst2 selectively mediates the antiproliferative effect of somatostatin analogues on
serum- or insulin-induced cell growth through the stimulation of a
protein-tyrosine phosphatase (8), which was recently identified as
SHP-1 (9, 10). SHP-1, a protein-tyrosine phosphatase with two SH2
domains, plays a role in terminating growth factor and cytokine signals
by dephosphorylating critical molecules (reviewed in Ref. 11). We
reported that SHP-1 is activated by somatostatin and participates in
the negative regulation of mitogenic insulin signaling as a result of
its association with and dephosphorylation of insulin receptor as well
as associated molecules (9, 10). However, the effect of sst2 as well as
the role of SHP-1 on cell cycle parameters remain unknown.
Cell cycle progression is dependent on the coordinated interaction,
posttranslational modification, and degradation of cyclins and their
catalytic partners, cyclin-dependent kinases
(cdks).1 Cyclins are
expressed at particular stages of the cell cycle and associate with
specific cdks to form active complexes that phosphorylate multiple
proteins and promote cell cycle progression. In mammalian cells,
progression through early to middle G1 phase of the cell
cycle is dependent on cdk4/and/or cdk6, which are activated by D-type
cyclins. Transition through middle G1 to S phase is
regulated by activation of cdk2 by cyclin E, cdk2, and cyclin A is
required for late G1 to S-phase progression and throughout S phase. One of the critical targets of cyclin-cdk complexes is the
retinoblastoma gene product (Rb). Rb acts as a transcriptional repressor. In its hypophosphorylated form, it binds to the E2F family of cell cycle transcription factors during G1 phase
and inhibits E2F activity. Rb is inactivated by cdk phosphorylation in
mid to late G1 phase of the cell cycle and dissociates from E2F, leading to activation of genes containing E2F sites and a progression from G1 to S phase (reviewed in Ref. 12).
Another level of regulation of cdk activity results from the action of
cdk inhibitors that bind cyclin-cdk complexes and either inhibit their
kinase activities or prevent their activation by cdk-activating kinase
(reviewed in Refs. 13 and 14). In mammalian cells, cdk inhibitors
comprise two classes of proteins, the Ink4 family including
p16Ink4a, p15 Ink4b, p18Ink4c, and
p19Ink4d, which specifically inhibit cyclin
D-dependent kinases, cdk4 and cdk6 (12), and the p21 family
including p21Cip1/Waf1, p27Kip1, and p57Kip2
(15-19), which can interact with many different cyclin-cdk complexes. Among them, p27Kip1 is a widely distributed cdk inhibitor that
has an important role regulating entry into and exit from the cell
cycle. p27Kip1 is abundantly expressed in normal quiescent
cells and is down-regulated by mitogens. The decrease in
p27Kip1 expression occurs through protein degradation via the
ubiquitin-proteasome pathway after p27Kip1 phosphorylation by
cyclin E-cdk2 complexes (20-22). Increased levels of p27Kip1
induced by transforming growth factor In this study, we investigated the potential effects of sst2 on cell
cycle progression and expression of cell cycle regulatory proteins in
CHO cells expressing sst2 (CHO/sst2). Activation of sst2 caused a
G1 cell cycle arrest in-phase accompanied by an increased
expression of cdk inhibitor p27Kip1, which resulted in an
increase of its association with cdk2 and a decrease in cdk2 activity
and led to dephosphorylation of protein Rb. The role of SHP-1 in
sst2-mediated regulatory mechanisms was investigated in CHO cells
coexpressing sst2 and a negative SHP-1 (C453S) mutant as well as in
acini isolated from viable motheaten mice expressing a mutant SHP-1
with markedly deficient enzyme activity. Our results provide evidence
that SHP-1 is a critical regulator of p27Kip1. This enzyme is
required for the maintenance of cell quiescence and is involved in the
sst2-mediated up-regulation of p27Kip1 leading to cell cycle arrest.
Materials--
Monoclonal anti-p27Kip1 and anti-Rb
antibodies, known to react with murine and human proteins, were
purchased from Transduction Laboratories and Pharmingen, respectively.
Monoclonal anti-human p21Waf1/Cip1 antibodies were from
Transduction Laboratories. Polyclonal anti-mouse p21Waf1/Cip1
and anti-cdk2 antibodies that react with human and murine proteins were
from Santa Cruz Biotechnology. Monoclonal anticyclin E antibodies react
with human and murine proteins and were from Calbiochem. RC-160 was
synthesized as described previously (28). [ DNA Transfection--
The 1.2-kilobase XbaI fragment
of mouse sst2A cDNA subcloned into pCMV6c vector was stably
co-transfected in CHO (DG44 variant) cells using Lipofectin reagent
with pSV2neo as described (kindly donated by Dr. G. I. Bell, Howard Hughes Medical Institute, University of Chicago and Dr. T. Reisine, University of Pennsylvania, School of Medicine, Philadelphia)
(8). Stable transfectants were selected in Cell Culture--
CHO-DG44 stably expressing sst2 (CHO/sst2) or
sst2 and SHP-1 (C453S) (CHO/sst2-SHP-1(C453S)) were cultured in Pancreatic Acini from Viable Motheaten
(mev/mev)
Mice--
C57BL6-mev/mev
mice were obtained by mating heterozygous
C57BL6-mev/+ mice (Jackson Laboratories, Bar
Harbor, ME) breeding pairs. Homozygous
mev/mev were screened by
reverse transcription-polymerase chain reaction as described (29).
Mev/mev were identifiable
by 10-15 days of age, because of the motheaten appearance of the skin.
Mev/mev or their
unaffected littermates were sacrificed at 3 weeks of age, and
pancreases were removed. Pancreatic acini were prepared using enzymatic
digestion of the pancreas with 1.5 mg/ml collagenase in an oxygenated
Krebs-Ringer buffer containing 0.2% bovine serum albumin and 0.01%
soybean trypsin inhibitor as described previously (buffer A) (30).
After washing with buffer A, acinar cells were incubated in the same
buffer in the presence or absence of peptides at 25 °C for indicated
times. Acini were then transferred to 0.3 M sucrose,
sedimented at 800 × g for 5 min at 4 °C, and
solubilized in lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM
Na4P2O7, 100 mM NaF, 2 mM sodium orthovanadate (pH 7.4)) (buffer B) containing 0.1% Triton X-100, 1 mM benzamidine, and 0.01% soybean
trypsin inhibitor at 4 °C for 30 min. Lysates were collected and
centrifuged at 13,000 × g for 10 min at 4 °C and
used for immunoprecipitation or immunoblotting.
Flow Cytometric Analysis--
Cells were harvested by trypsin
(0.5 mg/ml) and EDTA (0.02 mg/ml), washed twice with phosphate-buffered
saline (pH 7.4), and centrifuged at 800 × g for 5 min
at 4 °C. Cells were then incubated in the presence of 200 µl of
trypsin (30 mg/ml) for 10 min. 200 µl of RNase (0.1 mg/ml) and
trypsin inhibitors (0.5 mg/ml) were added for 10 min, and cells were
stained with 250 µl of propidium iodide solution (125 µg/ml) for at
least 15 min at 4 °C. Fluorescence of labeled cell nuclei was
measured by flow cytometry using a FACScan (Beckton Dickinson) with a
minimum of 10,000 events performed for each sample. Data were analyzed
using LYSIS II software.
Immunoprecipitation and Immunoblotting--
Cells were washed
with phosphate-buffered saline and then with buffer B. Cells were lysed
in 500 µl of buffer B containing 1% Triton X-100, 1 mM
phenylmethylsulfonylfluoride, 20 µg/ml aprotinin, 20 µM
leupeptin. After a 15-min incubation at 4 °C, the lysate was
collected and centrifuged at 13,000 × g for 10 min at
4 °C. Soluble proteins (300-500 µg) were incubated for 3 h
at 4 °C with specific antibodies or preimmune serum prebound to
Sepharose-protein A beads prewashed in buffer B. The beads were then
washed twice with buffer B and resuspended in sample buffer for immunoblotting.
For immunoblotting, solubilized proteins or immunoprecipitated proteins
(see above) were resolved on 7.5% or 12% SDS-polyacrylamide gels,
transferred to a nitrocellulose membrane, and immunoblotted with
specific antibodies as described previously (9). Immunoreactive proteins were visualized by the ECL immunodetection system and quantified by image analysis using a Biocom apparatus (Biocom, Paris, France).
Kinase Assay--
Immunoprecipitated proteins with anti-cdk2 or
anticyclin E antibodies were collected by centrifugation and washed
four times with buffer B and twice with 50 mM HEPES buffer
containing 1 mM dithiotreitol (pH 7.4). The beads were
suspended in 40 µl of kinase buffer containing 50 mM
HEPES (pH 7.5), 10 mM MgCl2, 1 mM
dithiothreitol, 4 µg of histone H1, 2.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM NaF, 50 µM ATP, and 10 µCi of [ Somatostatin Promotes G1 Cell Cycle Arrest and Blocks
Induction of the S Phase--
We previously reported that in CHO cells
expressing sst2, the addition of the somatostatin analogue, RC-160, for
24 h to the culture medium led to inhibition of the mitogenic
effect of insulin (10). To analyze whether RC-160-mediated inhibition
of cell proliferation reflects a stage-specific arrest of the cell
cycle, cells were rendered quiescent by serum deprivation and incubated with 100 nM insulin in the presence or absence of 1 nM RC-160 and then analyzed by flow cytometry. Cells grown
in the absence of fetal calf serum were taken as control values. The
treatment of cells with insulin increased the percentage of cells in
the S phase, which reached 27% at 6 h and increased up to 50% at
24 h of treatment (data not shown). The simultaneous treatment of cells with insulin and RC-160 for 6 h prevented cells from
entering into the S phase, RC-160 causing a decrease in the percentage of cells in the S phase (
G1 progression depends on an orderly and coordinated
expression of cyclins that bind to and activate cdks, the activity of which is negatively regulated by their association with a family of cdk
inhibitory proteins. Therefore we investigated whether sst2-mediated
cell cycle arrest is associated with a change in the expression of the
cdk inhibitors.
Somatostatin Analogue Induces a Rapid Accumulation of
p27Kip1--
We first examined the expression of the Kip/Cip
family cdk inhibitor p27Kip1, which has been demonstrated to be
involved in the regulation of cell cycle progression induced by various
antiproliferative stimuli that cause G1-phase arrest
(23-26). CHO/sst2 cells were treated for various times with insulin in
the presence or not of RC-160, and the level of p27Kip1 was
investigated by Western blot analysis. As observed in Fig. 2, p27Kip1 was expressed at high
level in growth-arrested control cells, and after 3 h of insulin
treatment, its expression decreased by 45% (p < 0.05), consistent with previous results reported for mitogenic signals
(26, 31, 32). The decrease of p27Kip1 level was transient, the
level of this cdk inhibitor being not significantly different from that
in control cells by 24 h. The addition of RC-160 resulted in a
4-fold increase (p < 0.02) in the level of
p27Kip1 during the first 3 h. Elevated levels of
p27Kip1 were found to return to control levels at 24 h of
treatment with RC-160. Treatment of cells with 1 µM
orthovanadate for 3 h suppressed the RC 160-induced increase of
p27Kip1, indicating that this effect is dependent on a tyrosine
phosphatase (Fig. 2).
The expression of the other member of the Kip/Cip family cdk
inhibitors, p21Waf1/Cip1, was also examined in CHO/sst2 cells.
In contrast to p27Kip1, p21Waf1/Cip1 was found to be
barely detectable in control cells (Fig.
3), as observed by others in quiescent
cells (33). As reported for mitogens in other cell systems (32, 34),
insulin induced an increase of its expression up to 24 h,
suggesting that elevated p21Waf1/Cip1 is not related to
insulin-mediated G1/S transition. However, the addition of
RC-160 did not significantly modify the insulin-induced expression of
p21Waf1/Cip1 irrespective of the time of treatment, suggesting
that this inhibitor is not involved in the somatostatin-mediated growth
arrest.
Somatostatin Analogue Induces Inhibition of cdk2 Kinase
Activity--
It has been shown in many cell types that among the
G1 cyclin-cdk complexes negatively regulated by
p27Kip1, the up-regulation of p27Kip1 in response to
growth inhibitory factors favors its association with cyclin E-cdk2,
resulting in kinase inhibition and contributing to cell growth arrest
(34). Therefore, we first tested whether somatostatin analogue-mediated
increases in the level of p27Kip1 should be reflected in a
change of the kinase activity of cdk2-associated complexes, as measured
by an in vitro assay on cdk2 immunoprecipitates using
histone H1 protein as a substrate. In comparison with the control
activity detected in resting CHO/sst2 cells, cdk2 kinase activity was
increased by about 2-fold after 3 h of treatment with insulin, as
revealed by the heavily phosphorylated histone H1 level. When RC-160
was added to the culture, the cdk2-dependent kinase
activity was inhibited by 80% during the first 3 h of culture (Fig. 4A). Similarly, RC-160
induced a decrease of about 80% insulin-induced increase of cyclin
E-cdk2 associated kinase activity (Fig. 4B). In addition,
the amount of p27Kip1 associated with cdk2 was decreased by
70% after treatment of cells for 3 h with insulin, whereas the
addition of RC-160 increased the level of the complexes by 87% (Fig.
4C). These results indicate that insulin and RC-160 could
contrarily modulate the level of cdk2-cyclin E complexes, free from the
constraining influence of p27Kip1 inhibitor and the resulting
kinase activity of the complexes.
We then analyzed the steady-state level of expression of cyclin E and
cdk proteins by immunoblotting. As shown in Fig. 4D, the
amount of cdk2 detected in control cells and in insulin-treated cells
was not significantly modified by RC-160 treatment. Anticyclin E
antibodies revealed that cyclin E was barely detectable in control cells, and treatment of cells for 3 h with insulin induced an increase of the level of cyclin E as observed for agents that promote
DNA synthesis (31). The addition of RC-160 resulted in a 50%
inhibition of cyclin E expression after 3 h of treatment.
Somatostatin Analogue Prevents pRb Phosphorylation--
One of the
targets of cdk includes pRb protein, as its hyperphosphorylated form
was proven to be a critical check point involved in regulating
progression through late G1 and into S phase. The extent of
pRb phosphorylation was first analyzed in soluble extracts of CHO/sst2
cells incubated in the presence of 100 nM insulin with or
without 1 nM RC-160 for various times. A specific antibody able to detect the active hypophosphorylated form of pRb (Fig. 5, lower band) as well as the
inactive slower migrating hyperphosphorylated form of the protein (Fig.
5, upper band) was used. In
serum-starved cells, pRb was found in its active hypophosphorylated
form in CHO/sst2 cells, and the inactive form of pRb became apparent
after 3 h of insulin stimulation and remained present 6 and
24 h after insulin treatment. During those periods, the cells
expressed fully hyperphosphorylated inactive pRb, in agreement with the
reported relation of the appearance of the slowly migrating
hyperphosphorylated form of pRb with the ability of mitogens to
subsequently induce DNA synthesis (31). Treatment of cells with RC-160
produced an accumulation of the fast-migrating hypophosphorylated form of pRb, which was evident at 3 h. Increased levels of the
hypophosphorylated form of pRb were no longer detectable by 6-24 h
after the addition of RC-160, indicating that the effect was
transient.
SHP1 Is Required for Somatostatin Analogue-mediated Inhibition of
S-phase Entry--
SHP-1 has been previously demonstrated to play a
role in the negative feedback of growth factor signaling and to be
required for early events in somatostatin-activated sst2 signaling
(9-11). However the role of SHP-1 in the regulation of cell cycle
machinery has not been delineated. To investigate whether SHP-1 is
critical for cell cycle arrest and p27Kip1 regulation, a
mutated SHP-1 cDNA in which the active cysteine at position 453 was
mutated to serine was generated and resulted in a catalytically
inactive enzyme. We stably co-transfected the cDNA coding for the
SHP-1 mutant and sst2 in CHO cells and selected the clones
(CHO/sst2-SHP-1(C453S)) that expressed sst2 receptors at a level
similar with that observed in CHO/sst2 cells (10). These clones
overexpressed the SHP-1 mutant protein approximately 4-fold as observed
by Western blotting (not shown).
Analysis of CHO/sst2-SHP-1(C453S) cells by flow cytometry revealed that
in the absence of serum, cells did not undergo G1 arrest as
observed with CHO/sst2 cells, and 22% of cells remained in S phase.
Furthermore, insulin and RC-160 did not modify the S phase, indicating
that expression of SHP-1 mutant promoted G1 progression of
cells and nullified the modulatory effect of insulin and RC-160 on cell
cycle progression (Fig. 6). These results
are in agreement with the previously observed increase in basal
proliferation and abrogation of regulatory effect of insulin as well as
RC-160 on cell proliferation in CHO/sst2-SHP-1(C453S) cells (10) and argue in favor of a role for SHP-1 in maintenance of cell
quiescence.
SHP-1 Is Required for Somatostatin Analogue-mediated Induction of
p27Kip1, Inhibition of cdk2 Activity, and pRb
Hypophosphorylation--
We then examined whether SHP-1 mutant
affected p27Kip1 protein levels. Western blotting analysis
demonstrated that in cells expressing mutant SHP-1, the basal level of
p27Kip1 decreased significantly as compared with control
CHO/sst2 cells (57 ± 5.6% of control) (Fig.
7A). Furthermore, the dominant
negative mutant SHP-1 was found to prevent insulin-mediated
down-regulation as well as RC-160-induced up-regulation of
p27Kip1 (Fig. 7B). These results strongly suggest
that SHP-1 might dephosphorylate some key substrate(s) in the insulin-
and sst2-mediated signaling pathway in order for p27Kip1
regulation to occur. We previously demonstrated that the
phosphotyrosine insulin receptor is an early substrate of
somatostatin-activated SHP-1 (11), suggesting that SHP-1 may exert at
least part of its effects on p27Kip1 expression by
dephosphorylating the insulin receptor.
These results prompted us to examine the effect of mutant SHP-1 on cdk2
and cyclin E-associated kinase activities. Expression of mutant SHP-1
prevented the effect of insulin and RC-160 on cdk2 as well as cyclin
E-associated kinase activity (Fig.
8A). In addition, the decrease
of p27Kip1 basal level observed in cells expressing mutant
SHP-1 was paralleled by a decrease in the association of
p27Kip1 with cdk2 (Fig. 8B). The amount of
p27Kip1 associated with cdk2 was decreased by 45% under basal
conditions, and insulin and RC-160 no longer had an effect on the
association p27Kip1-cdk2. These results provide evidence that
SHP-1 is involved in the retargeting of p27Kip1 to cyclin
E-cdk2 complexes and, in turn, in inhibition of the associated kinase
activity.
The effect of mutant SHP-1 on p27Kip1 and cdk2-associated
kinase activity suggests that mutant SHP-1 may affect the extent of pRb
phosphorylation. Immunoblotting of pRb showed that only the hyperphosphorylated inactive form of pRb was detected in
CHO/sst2-SHP-1(C453S) grown in serum-starved conditions and that mutant
SHP-1 prevented the mobility shift from hypo- to hyperphosphorylated
pRb as well as from hyper- to hypophosphorylated after growth in the
presence of insulin and RC-160, respectively (Fig. 8C).
To further examine the role of SHP-1 in p27Kip1 regulation,
experiments were performed using isolated pancreatic acini from viable motheaten (mev) mice. These animals express
negligible SHP-1 catalytic activity consequent to loss-of-function
mutation in the gene encoding SHP-1 (35). Pancreatic acini from normal
as well as mev mice expressed a high level of
sst2 receptors as revealed by anti-sst2
immunoblotting.2 However the
p27Kip1 protein level was decreased by about 50% in the
mev pancreatic acinar cells as compared with
normal acini (Fig. 9). We observed that
incubation of normal acini for 3 h with 1 nM epidermal
growth factor (EGF) at 25 °C induced a down-regulation of
p27Kip1. This effect was reversed by the addition of 1 nM RC-160, in agreement with the mitogenic effect of EGF
and the antiproliferative effect of somatostatin on pancreatic acini
(36). In contrast, when acini were isolated from
mev mice, EGF down-regulated p27Kip1 as
observed in control acini, but RC-160 had no more significant effect on
the level of p27Kip1 (Fig. 9).
Despite evidence for the role of somatostatin in negative growth
control in various cellular types, our understanding of the mechanisms
involved remains limited. Somatostatin analog, octreotide, inhibits
cell proliferation as a result of a blockade in
G0/G1 in GH3 rat pituitary tumor
cell, in contrast to a transient accumulation of cells in
G2/M and apoptosis in MCF-7 breast cancer cells and AtT-20
mouse pituitary tumor cells (5, 37). All these cell types express
multiple somatostatin receptor subtypes, and the involvement of each
receptor subtype in somatostatin response remains to be clarified. We
have previously demonstrated the role of sst2 in the
somatostatin-mediated inhibition of cell growth and the involvement of
SHP-1 in the transduction of the inhibitory growth signal (8-10, 38).
The present investigation was undertaken to further delineate the basis
of sst2-mediated control of cell cycle machinery. We have demonstrated
that in CHO cells expressing sst2, the inhibition of proliferation in
response to somatostatin analogue results from the suppression of cell
cycle progression and the arrest of cells in the
G0/G1 phase, which correlates with an increase
in expression of p27Kip1 but not p21Waf1/Cip1. This is
accompanied by an increase of association of p27Kip1with cdk2,
a concomitant inhibition of cyclin E-cdk2 activity and a consequent
decrease in the phosphorylation of pRb that precedes the inhibition of
entry into S phase. On the other hand, the data presented provide
strong support for the involvement of the tyrosine phosphatase SHP-1 in
maintaining cell quiescence as well as sst2-induced cell cycle arrest.
The expression of dominant negative SHP-1 is sufficient to induce
G1/S transition, allowing mitogen-independent cell
proliferation and abrogating the inhibitory effect of somatostatin. Consistent with its effect on cell cycle progression, expression of
dominant negative SHP-1 down-regulates p27Kip1, which results
in a decrease of association of p27Kip1, an increase of cyclin
E/cdk2 kinase activity, and a subsequent inactivation of the
growth-suppressive function of pRb protein, thus linking SHP-1 to
control of p27Kip1 expression.
The pivotal role of p27Kip1 in controlling cdk function and,
thus, cell cycle progression is well established. p27Kip1
mediates cell cycle arrest in response to various antimitogenic signals, including transforming growth factor p27Kip1 associates with G1-specific cyclin-cdk
complexes and inhibits their catalytic activities. Among them, cyclin
E-cdk2 p27Kip1 governs cdk2 activity, which is required for
G1 progression and contributes to the phosphorylation and
inactivation of the pRb protein. As reported for mitogenic factors, our
results demonstrate that after insulin treatment, the down-regulation
of p27Kip1 protein level is accompanied by an increase of
cyclin E- and cdk2-associated kinase activity, which is related to a
decrease of the amount of p27Kip1 associated with cdk2, thereby
providing conditions favorable for entry into S phase (21, 31). In
contrast, somatostatin analogue decreases the amount of p27Kip1
associated with cyclin E-cdk2 complexes, inactivates cyclin E-cdk2 complexes, and induces the disappearance of the hyperphosphorylated form of pRb protein. It has been reported that p27Kip1
inactivates cyclin E-cdk2-associated kinases (23), and conversely, inactivation of cyclin E-cdk2-associated kinases can lead to
accumulation of p27Kip1, because cyclin E-cdk2-associated
kinases phosphorylate p27Kip1 and induce its destruction by the
ubiquitin pathway (21). Somatostatin also decreases cyclin E protein
level. However, cyclin E overexpression does not prevent cell cycle
exit (44), and there is no significant relationship between cyclin E
expression and cyclin E-cdk2 activity (45). It is likely that the
negative regulation by somatostatin of the cdk2 activity could be one
of the consequences of the rise of p27Kip1, leading to an
increased interaction of p27Kip1 with cyclin E-cdk2 complexes
and, thus, resulting in an inhibition of their activity.
A critical finding of the present study is that unlike CHO cells
expressing sst2, which accumulate in G0/G1, CHO
cells coexpressing sst2 and a dominant negative mutant SHP-1 remain
distributed through the cell cycle after serum withdrawal, with a high
portion of cells being in the S phase. These cells express a
constitutively low level of p27Kip1; this in turn leads to an
activation of cyclin E-cdk2 complexes and accumulation of
hyperphosphorylated pRb. Furthermore, the expression of dominant
negative SHP-1 circumvents the requirement for insulin in
G1 cell cycle progression and is associated with a
resistance to the antiproliferative effect of somatostatin. Ours
results provide strong support for the hypothesis that SHP-1 is
required to revert mitogen-induced down-regulation of p27Kip1
and clearly demonstrate the importance of SHP-1 in sst2 signaling for
blockade of p27Kip1 down-regulation. This hypothesis is
strengthened by our results, obtained with acinar cells isolated from
mev mice that express a high level of sst2 and a
defective SHP-1 (35).2 As observed in CHO/sst2 cells
expressing a mutant SHP-1, acini from mev mice
express a low level of p27Kip1 that can be no longer
up-regulated by somatostatin, demonstrating the importance of SHP-1 in
the retention of high levels of p27Kip1 and its central role as
the downstream target of the sst2 signaling pathway leading to
up-regulation of p27Kip1. The functional role of SHP-1 in
pancreatic cells is not known, but the demonstration that SHP-1 is
necessary for regulation of p27Kip1 suggests that SHP-1 may be
important for the pancreatic cell development. It is notable that EGF
down-regulates p27Kip1 in acini from mev
mice, suggesting that SHP-1 is not the only negative regulatory protein-tyrosine phosphatase in growth factor signaling or that another
protein-tyrosine phosphatase can substitute for SHP-1 when it is not
functional. Most of the previous studies have focused on the role of
SHP-1 in response of quiescent cells to mitogenic stimulation. We and
others identified SHP-1 as a critical negative regulator of cytokine as
well as growth factor signaling; the recruitment of this enzyme to
activated membrane receptors causes dephosphorylation of the receptors
or/and of downstream signaling molecules (10, 46). The results
presented here extend these observations by demonstrating that SHP-1 is
necessary for negative regulation of cell cycle progression in the
G1 phase and is a key mediator of sst2 signaling pathway in
controlling high inhibitory levels of p27Kip1.
Regulation of p27Kip1 occurs by different mechanisms, including
transcriptional, post-transcriptional, or post-translational
mechanisms. Indeed, post-transcriptional mechanisms have been
implicated in the up-regulation of p27Kip1 protein induced by
antimitogen-transforming growth factor- In conclusion, this investigation shows that activation of sst2
promotes cell growth arrest through the ability of somatostatin to
maintain high levels of p27Kip1 and inactivate cyclin E-cdk2
complexes, thus leading to hypophosphorylation of pRb. Our findings
provide evidence that SHP-1 may be required for accumulation of
p27Kip1 and inhibition of cell cycle progression and indicate
that SHP-1 is a key mediator of sst2-induced p27Kip1
up-regulation and subsequent cell cycle arrest.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, phospholipase A2, guanylate
cyclase, ionic conductance channels, and tyrosine phosphatase (1,
2).
, contact inhibition, serum
deprivation, rapamycin, or staurosporine have been associated with a
G1 arrest (18, 23, 24). In contrast, an overexpression of
p27Kip1 antisense cDNA results in mitogen-independent
G1 progression, demonstrating the importance of
p27Kip1 in controlling cell cycle exit (25, 26). The
involvement of p27Kip1 in the negative regulation of cell
proliferation is related to its binding and subsequent inhibition of
the kinase activity of cdk2-cyclin complexes (17, 18, 23, 27).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
33P]ATP
(3,000 Ci/mmol) was purchased from Isotopchim (France), histone H1 was
from Sigma, and the enhanced chemiluminescence (ECL) immunodetection
system was from Amersham Pharmacia Biotech.
MEM (minimal essential
medium) containing geneticin at 600 µg/ml. Geneticin-resistant clones
expressing sst2 (CHO/sst2) were screened for somatostatin binding using
[125I-Tyr11] somatostatin as tracer as
described (8). The 2.1-kilobase HindIII/NotI
fragment of human SHP-1 cDNA (a gift of Dr. M. L. Thomas,
Howard Hughes Medical Institute, Washington University, St. Louis, MO)
was subcloned into the expression vector pcDNA I neo vector
(Invitrogen). The SHP-1 (C453S) mutant (a gift of Dr. C. Nahmias, ICGM,
Paris) was constructed as described (10). The mouse sst2 gene in the
pCMV6c vector was stably co-transfected in CHO cells using Lipofectin
reagent with the SHP-1 (C453S) mutant in pcDNA I neo. Stable
colonies obtained by selection with G418 (600 µg/ml) were screened
for somatostatin binding and the presence of SHP-1 as described
(9).
MEM
containing 10% fetal calf serum and G418 (200 µg/ml) as described
previously (8). After an overnight attachment phase, cells were
serum-starved in
MEM for 18 h without geneticin before peptide addition.
-33P]ATP
(1000-3000 Ci/mmol) and incubated for 30 min at 25 °C. Each sample
was mixed with 20 µl of 2× SDS sample buffer, heated for 5 min at
100 °C, and subjected to SDS-PAGE. The gel was fixed in 40%
methanol, 10% acetic acid and exposed to hyperfilm ECL.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
43%) and an accumulation of cells in the
G1 phase, which increased from 57% in the absence of
RC-160 to 72% (Fig. 1). For longer
treatment, RC-160 had no significant effect on the G1/S
transition (data not shown). We concluded that activation of sst2 by
ligand induces a G1 cell cycle arrest in CHO/sst2 cells.
Furthermore, treatment of cells with 1 µM orthovanadate suppressed the RC 160-induced decrease of number of cells in the S
phase as well as the increase of cells in the G1 phase,
indicating that a tyrosine phosphatase was required in the RC-160
effects (data not shown).
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Fig. 1.
Effect of insulin and RC-160 on cell cycle
distribution in CHO/sst2 cells. Subconfluent CHO/sst2 cells were
initially serum-starved for 18 h and subsequently treated with
MEM containing 0.1 µM insulin with or without 1 nM RC-160 for 6 h. Cell cycle distribution was
determined by flow cytometry analysis of DNA content in propidium
iodide-stained cells as described under "Experimental Procedures."
A, histogram representative of three independent
experiments. B, percentages of cells in
G0/G1, S, and G2/M phases are
presented as mean ±S.E. (statistical significance of insulin + RC-160
versus insulin, * p < 0.05; **
p < 0.01).
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Fig. 2.
Regulation of p27Kip1
protein by insulin and RC-160 in CHO/sst2 cells.
Serum-starved CHO/sst2 cells were incubated at 37 °C for the
indicated times with 0.1 µM insulin and with
(Ins+RC) or without (Ins) 1 nM RC-160
or with 0.1 µM insulin and 1 nM RC-160
supplemented with 1 µM orthovanadate (VO4) and
solubilized as described under "Experimental Procedures." Control
cells (Cont) were not treated. A, soluble
proteins were subjected to SDS-PAGE and immunoblotted (Blot)
with anti-p27Kip1antibodies. The arrow indicates the
position of p27Kip1. B, immunoblots were analyzed
densitometrically, and the data were plotted as the percentage of
control values obtained from cells incubated in serum-free MEM at
time 3, 6, and 24 h. Data from three separate experiments are
presented as the mean ±S.E.
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Fig. 3.
Effect of insulin and RC-160 on
p21Waf1/Cip1 expression in CHO/sst2 cells. A,
Serum-starved CHO/sst2 cells were incubated at 37 °C for indicated
times with 0.1 µM insulin and with (Ins+RC) or
without (Ins) 1 nM RC-160 or were not treated
(control (Cont)) and solubilized as described under
"Experimental Procedures." A, soluble proteins were
subjected to SDS-PAGE and immunoblotted (Blot) with
anti-p21Waf1/Cip1 antibodies. The arrow indicates
the position of p21Waf1/Cip1. B, immunoblots were
analyzed densitometrically, and the data were plotted as the percentage
of control values obtained from cells incubated in serum-free MEM at
time 3, 6, and 24 h. Data from three separate experiments are
presented as mean ±S.E.
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Fig. 4.
Effect of insulin and RC-160 on cyclin E- and
cdk2-associated kinase activity, the amount of p27Kip1
associated with cdk2, and the expression of cyclin E
and cdk2 protein in CHO/sst2 cells. Serum-starved CHO/sst2 cells
were incubated at 37 °C for 3 h with 0.1 µM
insulin with or without 1 nM RC-160 or were not treated and
then were solubilized. A and B, soluble proteins
were subjected to immunoprecipitation (Ip) with anti-cdk2
(A) or anticyclin E (B) antibodies. cdk2 kinase
activity was assayed in cdk2 or cyclin E immunoprecipitates using
histone H1 as substrate, followed by SDS-PAGE and autoradiography.
C, to detect the amount of p27Kip1 associated with
cdk2, soluble proteins were subjected to immunoprecipitation with
anti-p27Kip1 antibodies. Immunoprecipitates were resolved by
SDS-PAGE and analyzed by immunoblotting (blot) with
anti-cdk2 antibodies. The arrow indicate the position of
cdk2. D, soluble proteins were resolved by SDS-PAGE and
analyzed by immunoblotting (Blot) with anti-cdk2 or
anticyclin E antibodies. Arrows indicate the position of
cdk2 and cyclin E.
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Fig. 5.
Effect of insulin and RC-160 on pRb
phosphorylation in CHO/sst2 cells. CHO/sst2 cells were incubated
for indicated times with 0.1 µM insulin with or without 1 nM RC-160 or not treated and then solubilized. Soluble
proteins were subjected to SDS-PAGE and immunoblotted with anti-pRb
antibodies. The upper band represents the
hyperphosphorylated (pRbphos), whereas the lower band
(pRb) represents the hypophosphorylated form of protein
pRb.
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Fig. 6.
Effect of insulin and RC-160 on cell cycle
distribution in CHO/sst2-SHP1 (C453S) cells. Serum-starved
CHO/sst2-SHP1 (C453S) were treated for 6 h with 0.1 µM insulin and with or without 1 nM RC-160 or
were not treated (Control). Cell cycle distribution was
determined by flow cytometry analysis of DNA content in propidium
iodide-stained cells as described under "Experimental Procedures."
A, histogram representative of three independent
experiments. B, percentages of cells in
G0/G1, S, and G2/M phases are
presented as mean ±S.E.
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Fig. 7.
Expression of p27Kip1 in CHO/sst2 and
CHO/sst2-SHP1 (C453S) cells and regulation of its expression by insulin
and RC-160 in CHO/sst2-SHP1 cells (C453S). A, solubilized
proteins (µm/lane) from CHO/sst2 and CHO/sst2-SHP1 cells
(C453S) were subjected to SDS-PAGE and immunoblotted with
p27Kip1 antibodies. The arrow indicates the position
of p27Kip1. Serum-starved CHO/sst2-SHP1 (C453S) cells were
treated for indicated times with 0.1 µM insulin and with
or without 1 nM RC-160 or were not treated
(cont) and solubilized. A, soluble proteins were
subjected to SDS-PAGE and immunoblotted (Blot) with
anti-p27Kip1 antibodies. The arrow indicates the
position of p27Kip1. B, immunoblots were analyzed
densitometrically, and the data were plotted as percentage of control
values obtained from cells incubated in serum-free MEM at time 3, 6, and 24 h. Data from three separate experiments are presented as
mean ±S.E.
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Fig. 8.
Effect of insulin and RC-160 on cell cycle
proteins, cyclin E- and cdk2-associated kinase activity, and
p27Kip1-cdk2 association in CHO/sst2-SHP1
(C453S) cells. Serum-starved CHO/sst2-SHP1 (C453S) cells
were incubated at 37 °C for 3 h with 0.1 µM
insulin and with or without 1 nM RC-160 or were not treated
(control) and solubilized. A, soluble proteins were
subjected to immunoprecipitation (Ip) with anti-cdk2 or
anticyclin E antibodies. cdk2 kinase activity was assayed in cdk2 or
cyclin E immunoprecipitates using histone H1 as substrate, followed by
SDS-PAGE and autoradiography. B, to detect the amount of
p27Kip1 associated with cdk2, cells were subjected to
immunoprecipitation with anti-p27Kip1 antibodies.
Immunoprecipitates were resolved by SDS-PAGE and analyzed by
immunoblotting (Blot) with anti-cdk2 antibodies. The
arrow indicates the position of cdk2. Immunoblots were
analyzed densitometrically, and the data were plotted as percentages of
control values obtained from cells incubated in serum-free MEM. Data
from three separate experiments are presented as mean ±S.E.
C, soluble proteins were subjected to SDS-PAGE and
immunoblotted with anti-pRb antibodies to detect pRb. The upper
band represents the hyperphosphorylated (pRbphos), whereas
the lower band (pRb) represents the hypophosphorylated form
of protein pRb.
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Fig. 9.
Comparison of p27Kip1
expression in pancreatic acini from
C57BL6-mev/mev mice and their
normal littermates. A, pancreatic acinar cells were
isolated from control mice (control) or
mev/mev mice
(mev /
) and incubated in oxygenated Krebs-Ringer buffer
in the presence or not (control (cont)) of 1 nM
EGF with RC-160 (EGF+RC) or without 1 nM RC-160
(EGF) at 25 °C for 3 h. Acini were solubilized, and soluble
extracts were immunoblotted (Blot) using
anti-p27Kip1 antibodies. The arrow indicates the
position of p27Kip1. B, immunoblots were analyzed
densitometrically. Data were plotted as percentages of control values
obtained from acinar cells of normal mice incubated in Krebs-Ringer
buffer without peptide addition. Data from three separate experiments
are presented as mean ±S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, rapamycin, cAMP, cell-cell contact, and anti-epidermal growth factor antibody (18, 39-41). Overexpression of p27Kip1 leads to cell cycle arrest
(17) and inhibition of both normal and transformed human mammary
epithelial cell growth (42). Antisense inhibition of p27Kip1
expression can prevent quiescence upon withdrawal of growth factor (25,
26). In this study, insulin treatment of CHO/sst2 cells reduces the
level of p27Kip1, as expected for mitogenic signaling pathways.
In the presence of somatostatin analogue, p27Kip1 is rapidly
up-regulated, and this induction precedes somatostatin-induced G1 cell cycle arrest. Such a G1 blockade has
not yet been observed in cells expressing sst3, because it has been
reported that somatostatin induces a decrease of
G0/G1 and an increase in S phase via sst3 (7).
These results argue in favor of a receptor subtype selectivity for
somatostatin-mediated regulation of cell cycle progression. In contrast
to the ability of interferon
(43) and transforming growth
factor-
(34) to up-regulate both p27Kip1 and
p21Waf1/Cip1 cdk inhibitors, p21Waf1/Cip1 does not
appear to play a role in the sst2-mediated antimitogenic effect of
somatostatin. However, we cannot rule out the possibility that other
cdk inhibitors may be involved in this effect.
(47) and interferon
(48), whereas post-translational mechanisms involving desequestration
of p27Kip1 as a consequence of down-regulation of the
N-myc gene (49) or degradation of the protein (50) have been
proposed for the retinoic acid- and lovastatin-induced increases in
p27Kip1, respectively. It has been reported that the
mitogen-induced decrease in p27Kip1 expression occurs through
posttranslationally regulated protein degradation via the
ubiquitin-proteasome pathway (20). Recent data demonstrated the key
role for Ras signaling pathway in the down-regulation of
p27Kip1 and the involvement of RhoA in ubiquitin-mediated
p27Kip1 degradation (51). However, a regulation at a
transcriptional level can also occur, as shown in v-Src
oncoprotein-transformed cells, v-Src reducing the level of
p27Kip1 mRNA and preventing cellular quiescence (52). The
mechanisms involved in the SHP-1-induced up-regulation of
p27Kip1 remain to be elucidated. We previously demonstrated
that SHP-1 is associated with activated insulin receptor and is
involved in down-regulation of insulin signaling. In addition, upon
sst2 stimulation, somatostatin negatively regulates insulin signal transduction by controlling first the recruitment of SHP-1 to insulin
receptor and its activation and then causing a dephosphorylation and an
inactivation of insulin receptor and its substrates, thus leading to an
inhibition of the insulin downstream signaling (12). In agreement with
these results, activated SHP-1 may regulate the level of
p27Kip1 as a consequence of SHP-1-induced dephosphorylation of
insulin receptors and blockade of the insulin-induced catalytic cascade leading to down-regulation of p27Kip1. However, other
downstream effectors of somatostatin-activated SHP-1, different from
growth factor receptors and not yet identified, could also be involved
in the SHP-1-induced p27Kip1 regulation.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. G. I. Bell and Dr. T. Reisine for kindly providing CHO cells expressing sst2A receptors and to Dr. C. Nahmias for providing mutant SHP-1.
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FOOTNOTES |
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* This work was supported by Association pour la Recherche contre le Cancer Grant 9363, Conseil Régional Midi-Pyrénées Grant 2ACFH0113C, and Ligue Nationale contre le Cancer Grant (2578DB06D.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 33 5 61 32 2407; Fax: 33 5 61 32 2413; E-mail: susinich{at}rangueil.inserm.fr.
2 F. Lopez and G. Ferjoux, personal communication.
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ABBREVIATIONS |
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The abbreviations used are:
cdk, cyclin-dependent kinase;
Rb, retinoblastoma gene product;
pRb, phosphorylated Rb;
CHO, chinese hamster ovary;
MEM, minimal
essential medium;
PAGE, polyacrylamide gel electrophoresis;
EGF, epidermal growth factor.
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