C-Terminal Region of Human Somatostatin Receptor 5 Is Required for Induction of Rb and G1 Cell Cycle Arrest
Kamal Sharma,
Yogesh C. Patel and
Coimbatore B. Srikant
Fraser Laboratories Departments of Medicine (K.S., Y.C.P.),
Neurology (Y.C.P.), and Neurosurgery (Y.C.P.) McGill University
and Royal Victoria Hospital Montreal, Quebec, Canada, H3A 1A1
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ABSTRACT
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Ligand-activated somatostatin receptors (SSTRs)
initiate cytotoxic or cytostatic antiproliferative signals. We have
previously shown that cytotoxicity leading to apoptosis was signaled
solely via human (h) SSTR subtype 3, whereas the other four hSSTR
subtypes initiated a cytostatic response that led to growth inhibition.
In the present study we characterized the antiproliferative signaling
mediated by hSSTR subtypes 1, 2, 4, and 5 in CHO-K1 cells. We report
here that cytostatic signaling via these subtypes results in induction
of the retinoblastoma protein Rb and G1
cell cycle arrest. Immunoblot analysis revealed an increase in
hypophosphorylated form of Rb in agonist-treated cells. The relative
efficacy of these receptors to initiate cytostatic signaling was
hSSTR5>hSSTR2>hSSTR4
hSSTR1. Cytostatic signaling via hSSTR5
also induced a marginal increase in cyclin-dependent kinase inhibitor
p21. hSSTR5-initiated cytostatic signaling was G protein dependent and
protein tyrosine phosphatase (PTP) mediated. Octreotide treatment
induced a translocation of cytosolic PTP to the membrane, whereas it
did not stimulate PTP activity when added directly to the cell
membranes. C-tail truncation mutants of hSSTR5 displayed progressive
loss of antiproliferative signaling proportional to the length of
deletion, as reflected by the marked decrease in the effects of
octreotide on membrane translocation of cytosolic PTP, and induction of
Rb and G1 arrest. These data demonstrate
that the C-terminal domain of hSSTR5 is required for cytostatic
signaling that is PTP dependent and leads to induction of
hypophosphorylated Rb and G1 arrest.
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INTRODUCTION
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The antiproliferative actions of somatostatin (SST) signaled via
cell surface SST receptors (SSTRs) regulate cellular protein
phosphorylation and elicit cytostatic (growth arrest) and cytotoxic
(apoptosis) responses in tumor cells. For instance, SST treatment
causes apoptosis in MCF-7 and AtT-20 cells, whereas it induces cell
cycle arrest in GH3 cells (1, 2, 3, 4, 5). Such discrepant findings
may be due to the existence of five distinct SSTR subtypes and their
differential expression in these tumor cells (6, 7, 8, 9, 10). We have reported
that human (h) SSTR3 is the only subtype that is capable of
cytotoxic signaling: upon ligand activation, cells transfected
with hSSTR3 respond with induction of wild-type (wt) tumor suppressor
protein p53, the proapoptotic protein Bax, and an acidic endonuclease
and intracellular acidification and undergo apoptosis (11, 12). The
antiproliferative action of SST is also signaled via SSTRs 2, 4, and 5.
However, neither apoptosis nor changes in any of the above parameters
were seen in cell lines stably expressing these subtypes (11, 13).
While SST was shown to inhibit cell growth via hSSTRs 2, 4, and 5, such
a conclusion was based on measurement of thymidine incorporation or
cell number at a single time point during SST treatment (14, 15, 16, 17). Thus,
the mechanism underlying the antiproliferative signaling mediated by
the SSTR subtypes incapable of triggering apoptosis remained unknown.
Since these SSTR subtypes do not initiate apoptotic signals, it
appeared likely that they may transduce cytostatic signals leading to
cell cycle arrest.
Cytostatic events leading to G1 cell cycle arrest are
associated with the induction of two proteins Rb (retinoblastoma
tumor suppressor protein) and p21 (cyclin-dependent
kinase inhibitor, also called Waf-1/Cip1) (18). Rb is a phosphorylated
protein: it remains hyperphosphorylated (ppRb) in S and
G2/M phases and becomes hypophosphorylated (pRb) in
G1. pRb negatively regulates the G1/S
transition and promotes accumulation of cells in the G1
phase (18, 19). Rapid phosphorylation of Rb occurs before entry of
cells into S phase. While Rb functions independently of p53, p21
mediates p53-dependent G1 arrest (18, 20). Nevertheless,
overexpression of p21 can induce G1 arrest in the absence
of p53 induction (21). To determine whether the antiproliferative
signaling via these hSSTRs causes cell cycle arrest and to identify the
molecular mediators involved in this process, we evaluated the effect
of SST in CHO-K1 cells expressing hSSTRs 1, 2, 4, and 5 on cell cycle
progression and induction of Rb and p21. We report here that
SST-induced G1 cell cycle arrest in these cells is due
mainly to the induction of Rb. Maximal effect was exerted via hSSTR5
followed by hSSTR 2, 4, and 1. In hSSTR5-expressing cells, a major
portion of SST-induced Rb was hypophosphorylated. SST-induced
G1 arrest and induction of Rb were pertussis toxin
sensitive, G protein dependent, and protein tyrosine phosphatase (PTP)
dependent. In octreotide (OCT)-treated cells there was a redistribution
of PTP activity from the cytosol to the membrane. Mutational analysis
of the C tail of this receptor revealed that the C tail of the receptor
is essential for PTP-dependent cytostatic signaling.
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RESULTS
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We first compared the effect of SST agonists [OCT (hSSTRs 2, 3,
and 5) or D-Trp8 SST-14 (hSSTRs 1 and 4)] on
cell cycle parameters in CHO-K1 cells expressing individual hSSTRs.
Cells were incubated for 24 h in the absence or presence of
agonists at a maximal stimulatory concentration of 100 nM.
(11). Cell cycle analysis revealed that cells expressing hSSTRs 1, 2,
4, and 5 responded with a decrease in the rate of proliferation. This
was evident from the agonist-induced increase in cells in
G1 and a decrease in S (Fig. 1
). The greatest cytostatic response was
elicited through hSSTR 5 followed by hSSTR2>hSSTR4
hSSTR1. The
effect of agonist treatment on cell cycle parameters is shown in Fig. 2
. In addition to the changes in
G1 and S phases, a relative increase of cell number in
G2/M was also seen. The absence of oligonucleosomal DNA
fragmentation, even after treatment for 48 h, indicated that
SST-induced cell cycle arrest via hSSTR5 did not lead to apoptosis
(data not shown).

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Figure 1. Effect of Peptide Treatment on Cell Cycle
Parameters in CHO-K1 Cells Expressing hSSTR15
Representative plots showing the phase distribution of cells incubated
for 24 h in the absence (top panel) or presence
(bottom panel) of 100 nM OCT (hSSTRs 2, 3,
and 5) or D-Trp8 SST-14 (hSSTRs 1 and 4). Cellular DNA was
stained with PI and analyzed by flow cytometry. An increase in
G1 peak can be seen in cells expressing four of the five
hSSTR subtypes (hSSTR5>hSSTR2>hSSTR4 hSSTR1). This contrasts with
the decrease in G1 peak and the appearance of a hypodiploid
peak in the region A0 after peptide treatment in cells
expressing hSSTR3.
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Figure 2. Effect of Agonist Treatment on Cell Cycle
Parameters in CHO-K1 Cells Expressing Individual hSSTR Subtypes
The distribution of cells in G0/G1 (top
panel), S (middle panel), and G2/M
(bottom panel) were quantitated by analysis of
PI-stained cells by flow cytometry. The increase in the number of cells
G0/G1 was accompanied by a decrease in cell
number in S phase. A small increase in G2/M was also
evident in agonist-treated cells (mean ± SE, n =
3). *, P < 0.005; **, P <
0.05.
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Inhibition of cell cycle progression signaled via hSSTR 5 was
associated with induction of Rb. The increase in intensity of
fluorescence of immunolabeled Rb counterstained with fluorescein
isothiocyanate (FITC)-conjugated second antibody was seen in all
phases of the cell cycle after OCT treatment. Dual label analysis of
fluorescence emissions of propidium iodide (PI) and immunostained Rb
revealed that the majority of cells were in
G0/G1 phase (Fig. 3A
). Immunoblot analysis of cell extracts
revealed that the level of Rb was low in untreated cells and was
present mainly as ppRb. An OCT-induced increase in Rb was reflected in
both hyper- and hypophosphorylated (ppRb and pRb) forms detectable by
their differential electrophoretic mobility (Fig. 3B
). A marked
enlargement of the nuclei in hSSTR5-expressing cells was observed in
OCT-treated cells (Fig. 3C
), a typical characteristic of G1
arrested cells (22). OCT-induced increase in Rb was time dependent and
was detectable by 4 h (2.7 ± 0.9 fold) and was maximal at
24 h (8.1 ± 0.8 fold) (Fig. 4A
). Induction of Rb preceded the onset
of G1 arrest since an increase in G1/S ratio,
which is an index of inhibition of cell proliferation, was detectable
only by 8 h (Fig. 4B
). The ability of OCT to induce Rb during
24 h incubation was dose dependent and occurred over the
concentration range 10100 nM (Fig. 5A
). Immunoblot analysis revealed a
dose-dependent increase in both ppRb and pRb in OCT-treated cells (Fig. 5B
).

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Figure 3. hSSTR5-Mediated Antiproliferative Signaling in
CHO-K1 Cells Causes G1 Cell Cycle Arrest Associated with Induction of
Rb
A, Flow cytometric analysis of hSSTR5 expressing cells incubated in the
absence and presence of 100 nM OCT for 24 h.
Scattergram represents dual label plot of FITC fluorescence of
immunostained Rb measured on a log scale against PI fluorescence
measured on a linear scale. In addition, PI fluorescence depicting the
cell cycle distribution is shown on the top, and the
FITC fluorescence of immunostained Rb is shown on the
right of the scattergram. An OCT-induced increase in Rb
occurred in all phases of the cell cycle and was associated with an
increase in the number of cells in G1. Data are
representative of three separate experiments. B, Western blot analysis
of Rb in CHO-K1 cells. In addition to the total increase in Rb in
OCT-treated cells, a significant portion was in the hypophosphorylated
form (pRb) that could be distinguished from the hyperphosphorylated
form (ppRb) on the basis of differential electrophoretic mobility. C,
Nuclear morphology of PI-stained cells revealed nuclear enlargement, a
feature that is characteristic of G1 arrested cells.
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Figure 4. Time Dependency of hSSTR5-Mediated Induction of Rb
and Inhibition of Cell Cycle Progression
After incubation in the presence of 100 nM OCT for the
indicated time, Rb and DNA were quantitated in the same cell
populations by dual label flow cytometry (mean ± SE,
n = 3; *, P < 0.05; **, P
<0.005). A, Rb was quantitated by flow cytometry after immunolabeling.
Values represent as percent change in fluorescence intensity measured
on a log scale and compared with that in untreated cells taken as 100%
(mean ± SE, n = 3). B, Inhibition of cell
proliferation by OCT is reflected in the increase in the ratio of cells
in G1 and S phases is evident by 8 h and was maximal
at 24 h.
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Figure 5. Dose-Dependent Induction of Rb by OCT via
hSSTR5
A, Rb was measured by flow cytometry after immunolabeling in cells
incubated with the indicated concentrations of the peptide for 24
h. Values represent percent change in fluorescence intensity measured
on a log scale and compared with that in untreated cells taken as 100%
(mean ± SE, n = 3). B, Immunoblot demonstrating
that OCT-induced augmentation in Rb is associated with a dose-dependent
increase in hypophosphorylated form of Rb (pRb).
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OCT-treated hSSTR5 cells also displayed an increase in p21 (3 ±
0.6 fold over basal level) and was of much smaller magnitude compared
with that of Rb. OCT-induced increase in Rb and p21 was abolished by
pertussis toxin pretreatment (Fig. 6
).
Sodium orthovanadate, an inhibitor of PTP, also abrogated the inductive
effect of OCT on Rb and p21Waf1 in these cells. To confirm
that PTP activity is involved in the cytostatic signaling via hSSTR5,
we measured PTP activity in extracts of cells before and after
incubation with SST. In OCT-treated cells there was a 40% increase in
membrane-associated PTP while the cytosolic enzyme activity decreased
by 20% (Fig. 7
). By contrast, when added
to the membrane fractions at the time of enzyme assay, OCT failed to
stimulate PTP activity (data not shown). While the maximal induction of
Rb was hSSTR5 mediated, three other subtypes were also found to
initiate cytostatic signals leading to Rb induction (Fig. 8
). The rank order potency of these SSTRs
for signaling the increase in Rb was hSSTR5>2 >4>1, the same as that
observed for triggering G1 arrest (Figs. 1
and 2
). By
contrast, no increase in Rb occurred in hSSTR3 expressing cells, in
agreement with our previously reported finding that OCT does not induce
G1 arrest via this subtype (11, 12).

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Figure 6. hSSTR5-Mediated Cytostatic Signaling Is Pertussis
Toxin Sensitive, G Protein Mediated, and PTP Dependent
OCT-treated cells displayed an increase in p21 in addition to Rb.
Fluorescence intensity of antibody-labeled Rb and p21 was quantitated
by flow cytometry after immunolabeling. Values represent percent change
in fluorescence intensity in OCT-treated cells measured on a log scale
and compared with that in untreated cells taken as 100%. The increase
in p21 in cells incubated with 100 nM OCT was less than
that of Rb (3.0 ± 0.8 vs. 8.1 ± 0.8 fold,
respectively). hSSTR5-signaled induction of these proteins was
abolished by pretreatment of the cells with 100 ng pertussis toxin for
18 h before incubation with the peptide. Na orthovanadate (10
mg/ml) present during the incubation with the peptide also inhibited
the action of OCT (mean ± SE, n = 3, *,
P < 0.005; **, P < 0.05).
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Figure 7. Effect of OCT on PTP Activity in CHO-K1 Cells
Expressing hSSTR5
PTP activity was measured in membrane and cytosolic fractions prepared
from cells incubated in the absence or presence of 100 nM
OCT for 24 h. The enzyme activity was measured using pNPP as the
substrate (mean ± SE, n = 3). By contrast, OCT
did not stimulate PTP activity of the membrane fractions when added at
the time of enzyme assay (not shown).
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Figure 8. hSSTR Subtype Selectivity for Induction of Rb
Maximum induction of Rb was seen in cells expressing hSSTR5, followed
by hSSTR2>hSSTR4>hSSTR1. The fluorescence intensity of immunostained
Rb was measured in cells incubated with 100 nM OCT (hSSTRs
2, 3, and 5) or D-Trp8 SST-14 (hSSTRs 1 and 4)
for 24 h. Induction of Rb by agonists was quantitated by flow
cytometry after immunolabeling. Values represent percent change in
fluorescence intensity measured on a log scale and compared with that
in untreated cells taken as 100% (mean ± SE, n
= 3; *, P < 0.05; **, P <
0.005).
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The C tail of several G protein-coupled receptors has been implicated
in G protein interaction and effector coupling (23, 24). To evaluate
the importance of the C tail of hSSTR5 in cytostatic signaling, we
investigated the effect of mutant hSSTR5 receptors with progressive
truncation of the C tail (Fig. 9
). These
mutants have been previously reported to display binding
characteristics and G protein coupling comparable to wild-type hSSTR5
(25). Progressive truncation of the C tail of hSSTR5 was associated
with an impaired ability of OCT to signal activation of Rb and induce
G1. Compared with the wild-type receptor, which triggered
8.1 ± 0.8 fold increase in Rb in response to OCT, the
347
mutant displayed only a 6.1 ± 0.4 fold increase in Rb (Fig. 10
). The
338,
328, and
318
mutants displayed more marked loss in the ability to activate Rb in
response to OCT (3.0 ± 0.9 fold for
338, 2.1 ± 0.7 fold
for
328, and 1.3 ± 0.2 fold for
318). To determine whether
progressive loss of ability to induce Rb parallels the decrease in
membrane-associated PTP, we compared PTP activity in cytosolic and
membrane fractions in cells incubated in the absence and presence of
OCT. In contrast to the more than 2-fold increase induced by OCT
pretreatment in cells expressing hSSTR5, only
25% increase occurred
with the mutant
347, and no change was seen with the shorter hSSTR5
mutants (Fig. 11
). Interestingly, the
basal membrane-associated PTP activity was higher in untreated cells
expressing each of the mutant receptors compared with wild-type
hSSTR5.

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Figure 9. Topographical Arrangement of Primary Amino Acid
Sequence of hSSTR5 Showing the N-glycosylation Sites (CHO), S/T
Phosphorylation Sites (), and Palmitoylation Site ( )
C tail truncation mutants of this receptor were generated by inserting
stop codons at sites indicated by solid lines.
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Figure 10. Effect of C Tail Deletion Mutations on
hSSTR5-Initiated Rb Induction
Induction of Rb by agonists was quantitated by flow cytometry after
immunolabeling. Values represent percent change in fluorescence
intensity measured on a log scale and compared with that in untreated
cells taken as 100% (mean ± SE, n = 3, *,
P < 0.01; **, P < 0.0001).
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Figure 11. Effect of C Tail Truncation on hSSTR5-Signaled
Change in Cellular Distribution of PTP Activity in CHO-K1 Cells
PTP activity was measured using pNPP as the substrate in membrane and
cytosolic fractions of cells incubated for 2 h in the absence and
presence of 100 nM OCT (mean ± SE, n
= 3; *, P < 0.0001; **, P <
0.005).
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DISCUSSION
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The present study establishes that SSTR-mediated antiproliferative
signaling elicits subtype-selective cytostatic effect via hSSTRs 1, 2,
4, and 5. In CHO-K1 cells expressing each of these four hSSTR subtypes,
there was decreased proliferation due, in part, to a G1
cell cycle arrest associated with an increase in Rb. The extent of Rb
induction and inhibition of cell cycle progression was the greatest in
CHO-K1 cells expressing hSSTR5, followed by hSSTR2>hSSTR4
hSSTR1. A
significant proportion of Rb induced via hSSTR5 was present in a
hypophosphorylated form as evident from its greater electrophoretic
mobility. We show that OCT treatment caused nuclear enlargement in
hSSTR5-expressing cells, a feature that is characteristic of cells in
G1 arrest (22). Additionally, hSSTR5-mediated cytostatic
signaling did not lead to apoptosis. While the cytostatic action
exerted through hSSTR5 by OCT was dose- and time dependent, such an
effect occurred with a relatively slow time course and could be seen
only at concentrations greater than 10 nM. This is in
contrast to the greater sensitivity of hSSTR3-mediated induction of
wild-type p53, which was clearly discernible within minutes and could
be elicited at less than 10 nM concentration of OCT
(11).
Pretreatment of cells with PTx abolished the induction of Rb, p21, and
G1 arrest, indicating that the cytostatic signaling by
hSSTRs 1, 2, 4, and 5 is G protein dependent. Likewise, our finding
that orthovanadate abolishes the effects of SST suggests a mediatory
role for PTP in the cytostatic signaling initiated via hSSTRs 1, 2, and
4 as well. PTP-mediated antimitogenic effect of SST has previously been
reported to be signaled through hSSTR1, mouse and human SSTR2, human
and mouse SSTR3, and rat SSTR4 (11, 15, 16, 26, 27, 28). By contrast, rat
SSTR5-initiated antiproliferative signaling was found to be PTP
independent (14, 17). The present findings suggest that the
antiproliferative signaling via hSSTR 5 leading to growth inhibition is
also PTP dependent and contradicts the reported inability of the rat
homolog of SSTR5 to regulate PTP (14). Such a difference between the
rat and human SSTR5 receptors is surprising given the high degree of
C-terminal sequence identity between the two receptors. It remains to
be seen whether structural differences in other regions of rat and
human SSTR5 contribute to their divergent behavior.
The mechanism involved in SST induction of hypophosphorylated Rb
remains to be elucidated. The concomitant, albeit smaller, induction of
p21 raises the possibility that it may inhibit cyclin-dependent
kinase-mediated phosphorylation of Rb that is required for the cells to
exit G1. Alternatively, SST may activate phosphatase(s)
that may dephosphorylate hyperphosphorylated Rb. Evidence for the
existence of such a phosphatase comes from studies using anticancer
drugs that promote p53-independent G1 arrest in the absence
of p21 induction (29). It remains to be tested whether hSSTR5- mediated
increase in hypophosphorylated Rb is due to activation of Rb
phosphatase alone or in conjunction with p21-mediated inhibition of Rb
phosphorylation. Another possibility is that SST may inhibit
Ca2+/calmodulin-mediated hyperphosphorylation of Rb (30, 31). Cross-talk between SST-induced PTP and mitogenic signaling
pathways involving mitogen-activated protein (MAP) kinase may also
contribute to the regulation of serine phosphorylation in Rb as well as
cell cycle progression. It has been shown that cell cycle progression
due to induction of cyclin-dependent kinase and phosphorylation of Rb
can occur after MAP kinase activation (32). It is likely that
inhibition of MAP kinase activity by SST may be an additional factor
involved in its cytostatic signaling. SSTR regulation of MAP kinase
activation is complex and involves inhibition by SSTR2 and SSTR5,
stimulation through SSTR4, or a transient increase followed by
subsequent decrease elicited by (murine) SSTR3 (33).
Gß
-subunit-mediated activation of Ras is implicated in
the induction of MAP kinase (34, 35). On the other hand, PTP-dependent
regulation of serine/threonine phosphorylation inactivates Raf-1, which
functions downstream of Ras in the mitogenic signaling cascade (27, 36, 37, 38, 39). Thus, regulation of MAP kinase cascade by SST may occur at two
levels: activation of Ras by ß
-subunits of G protein and tyrosine
phosphorylation-dependent inactivation of MAP kinase. Thus, it is
plausible that activation of different
phosphorylation/dephosphorylation mechanisms by SST elicited in a
receptor subtype-specific or cell-specific manner may exert dual
effects on cell growth and proliferation (40, 41). A direct correlation
between subtype-selective change(s) in MAP kinase and cell cycle arrest
or apoptosis remains to be established.
Of the five hSSTRs, only hSSTR3 induces cytotoxicity, the other four
subtypes being cytostatic (Refs. 11, 13 and the present study). We
have previously reported that the
347,
338,
328, and
318
hSSTR5 mutants show progressive loss of the ability to inhibit
forskolin-stimulated cAMP and variable impairment of agonist-dependent
desensitization and internalization responses (25). This suggests a
multifunctional role of the C tail of hSSTR5 in mediating effector
coupling, desensitization, and internalization (25). Here we have
extended an analysis of these mutants to their ability to regulate
membrane-associated tyrosine phosphatase activity and to determine
whether decreased potency to recruit cytosolic PTP to the membrane may
account for their inability to initiate cytostatic signaling. After OCT
treatment, only a 25% increase in PTP activity in the membrane
fraction was seen in cells expressing hSSTR5
347, in contrast to the
100% increase detected in cells expressing the wild-type receptor.
Membrane-associated PTP activity did not increase in response to OCT in
cells expressing
338,
328, and
318 mutants. Surprisingly,
however, the enzyme activity was 2035% higher in the membrane
fraction of these cells under basal conditions than in
hSSTR5-expressing cells. We do not know the reason for this, but this
observation raises the intriguing possibility that, in the absence of
ligand activation, the C tail of the wild-type receptor may inhibit the
association of PTP with the membrane. Such a phenomenon has not
previously been described. However, chronic association of the tyrosine
phosphatase SHP-1 to the killer cell- inhibitory receptor in natural
killer cells has been reported to tonically inhibit the function of the
receptor in the inactivated state; dissociation of SHP-1 upon receptor
ligation restores its function (42). Despite its inability to recruit
cytosolic PTP to the membrane in cells expressing these mutants, OCT
was still capable of inducing Rb, albeit with progressively less
efficiency paralleling the length of C tail deletion. While this raises
the possibility that OCT may be able to elicit cytostatic signaling
through the PTP already present at the membrane, alternate,
PTP-independent mechanisms may also contribute to hSSTR5-initiated
antiproliferative signaling. For instance, hSSTR5 can decrease
intracellular Ca2+, thereby inhibiting cell growth (14, 43). While the nature of SST-induced, Ca2+-sensitive growth
inhibition was not established in these studies, it may invoke
hypophosphorylation of Rb. Indeed,
Ca2+/calmodulin-dependent Rb hyperphosphorylation occurs
during cell proliferation (30, 31).
In summary, the present findings demonstrate that SST peptides exert a
cytostatic action via SSTR 1, 2, 4, and 5. Such subtype-specific
cytostatic signals target Rb and p21, leading to G1
cell cycle arrest (hSSTR5>hSSTR2 >hSSTR4
hSSTR1). These effects are
pertussis toxin- and G protein dependent and are PTP mediated. The
marked decrease in the ability of C tail mutants of hSSTR5 to induce Rb
and G1 cell cycle arrest suggests that the C-terminal
domain of hSSTR5 is involved in cytostatic antiproliferative
signaling.
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MATERIALS AND METHODS
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Materials
The SST analog SMS 201995 (octreotide, OCT) was obtained from
Sandoz Pharmaceutical Co. (Basel, Switzerland). (PI) was purchased from
Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies
against p21 (C-19) and Rb (C-15) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). FITC-conjugated goat antimouse and
antirabbit IgG antibodies were supplied by Zymed Laboratories (San
Francisco, CA). All other reagents were obtained from local commercial
sources and were of analytical quality.
CHO-K1 Cells Stably Expressing hSSTR15 and Mutant
hSSTR5
Genomic fragments of hSSTR 2, 3, and 5 or cDNA clones for hSSTR
1 and 2A containing the entire coding sequences were subcloned into the
polylinker region of the mammalian expression vector pRc/CMV
(Invitrogen, San Diego, CA). Mutant hSSTR5 receptors with progressive
truncation of the C tail (
347,
338,
328, and
318) were
created by introducing stop codons at positions 347, 338, 328, and 318
of a cassette cDNA construct using the PCR overlap extension technique
(25); the mutant cDNAs were cloned into the mammalian expression vector
PTEJ8 (25). Wild-type hSSTRs and the mutant hSSTR5 receptors were
stably transfected in CHO-K1 cells maintained under G418 selection (11, 44, 45). The binding characteristics of the different hSSTRs and the
hSSTR5 C tail deletion mutants are compared in Tables 1
and 2
.
These values were estimated from saturation binding analysis using
[125I-LTT]SST-28 as the radioligand as previously
described (25, 44, 45). Cells were grown in T75 flasks in Hams F-12
medium containing 5% FCS (Life Technologies, Grand Island, NY) and 400
U/ml G-418 and cultured for 35 days at 37 C in a humidified
atmosphere with 5% CO2. When the cells had reached
6070% confluency, medium was replaced with fresh medium containing
100 nM of either OCT (hSSTRs 2, 3, and 5) or
D-Trp8 SST-14 (hSSTR 1 and 4 subtypes to which
OCT does not bind) (11, 44). To investigate the G protein dependency of
hSSTR5-mediated cytostatic signaling, cells were preincubated for
18 h with pertussis toxin. To determine whether such action was
PTP mediated, the effect of OCT was compared in the absence and
presence of the tyrosine phosphatase inhibitor Na orthovanadate. The
pertussis toxin and Na orthovanadate were used at optimal
concentrations of 100 ng/ml and 10 mg/ml, as determined in earlier
studies (11, 46). After 24 h incubation, the cells were washed in
PBS, scraped, and fixed sequentially in 1% paraformaldehyde and 70%
ethanol. Cellular DNA was labeled with the intercalating dye PI (50
mg/ml) in PBS and incubated at 37 C for 5 min in the presence of RNAse
A (50 mg/ml). Rb and p21 were immunolabeled with their respective
antibodies, followed by counterstaining with FITC-conjugated secondary
antibodies as previously described (11).
Flow Cytometry
Flow cytometry was carried out in an EPICS 750 series flow
cytometer (Coulter Electronics, Hialeah, FL). Fluorescence was excited
by a 5-watt argon laser generating light at 351363 nm. PI emission
was detected through a 610-nm long pass filter, and FITC fluorescence
was detected with a 560-nm short pass dichroic filter. At least 10,000
gated events were recorded for each sample, and the data were analyzed
by Winlist software (Verity Software House).
Analysis of Nuclear Morphology
Aliquots of cells stained with PI for analysis by flow cytometry
were cytospun onto microscope slides, mounted using Immunomount
(Shandon, Pittsburgh, PA), viewed, and photographed through a Reichert
Polyvar 2 fluorescence microscope (original magnification
x 400).
Western Blot Analysis
Cells were lysed in Tris-HCl buffer (100 mM, pH 7.2)
containing 300 mM NaCl, 2% Nonidet P-40, 20% glycerol, 2
mM ZnCl2, 10 mg/ml pepstatin, and 0.2
mM pefabloc (Boehringer Mannheim, Canada). Protein
measurement was performed using the Bio-Rad protein assay kit (Bio-Rad
Laboratories, Hercules, CA). Aliquots (30 µg) were electrophoresed in
10% SDS-polyacrylamide gel in running buffer (50 mM
Tris-HCl, 60 mM boric acid, 1 mM EDTA, 0.1%
SDS) and transferred onto Protran plus membranes electrophoretically in
a buffer containing 25 mM Tris, 192 mM glycine,
and 15% methanol. Blots were probed with anti-RB antibody (Pharmingen,
San Diego, CA) and visualized with alkaline phosphatase conjugate
detection kit (Bio-Rad). Molecular size was determined using 10 kDa
protein ladder (Life Technologies) and staining with Ponceau S
(46).
Measurement of PTP Activity
Phosphatase activity in whole-cell extracts or membrane and
cytosolic fractions was determined using pNPP as the substrate
as described previously (47).
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. J. Cai for technical assistance with cell culture,
Dr. N. Hukovic for binding studies with hSSTR5 mutants, and Ms. S.
Schiller and Dr. Halwani for assistance with flow cytometry.
 |
FOOTNOTES
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Address requests for reprints to: Dr. C. B. Srikant, M3.15, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada, H3A 1A1. E-mail: mdcs{at}musica.mcgill.ca
This work was supported by grants from the Medical Research Council of
Canada (MT 12603 and MT 10411) and the US Department of Defense. K.S.
is a recipient of a studentship award of the Fonds de la Recherche en
Sante du Quebec.
Received for publication May 8, 1998.
Revision received September 22, 1998.
Accepted for publication September 28, 1998.
 |
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