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(r-PTP) Is Responsible for the Somatostatin Inhibition of PC Cl3 Thyroid Cell Proliferation
Pharmacology and Neuroscience (T.F., S.A., S.T., A.C., A.M., A.P., A.B., G.S.), National Institute for Cancer Research (IST) and Advanced Biotechnology Center (CBA) Genova 16132, Italy; Department of Biomedical Sciences (T.F.), Section of Pharmacology, University G. D’Annunzio of Chieti, Chieti 66013, Italy; Department of Oncology Biology and Genetics (S.A., S.T., A.C., A.M., A.P., A.B., G.S.), Section of Pharmacology, University of Genova 16132, Italy; Department of Clinical and Experimental Medicine (R.I., F.T., A.F.) University of Catanzaro, Catanzaro 88100, Italy
Address all correspondence and requests for reprints to: Professor Gennaro Schettini, Neuroscience and Pharmacology, Advanced Biotechnology Center (CBA), Largo R. Benzi, 10, 16132 Genova, Italy. E-mail: schettini{at}cba.unige.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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whose expression was abolished in the PC mos cells. To
directly prove the involvement of r-PTP in somatostatin’s effect,
we stably transfected this phosphatase in PC mos cells. This new cell
line (PC mos/PTP) recovered somatostatin’s ability to inhibit cell
proliferation, showing dose-dependence and time course similar to those
observed in PC Cl3 cells. Conversely, the transfection of a
catalytically inactive mutant of r-PTP did not restore the
antiproliferative effects of somatostatin. PC mos/PTP cells showed a
high basal phosphotyrosine phosphatase activity which, similarly
to PC Cl3 cells, was further increased after somatostatin treatment.
The specificity of the role of r-PTP in somatostatin receptor signal
transduction was demonstrated by measuring its specific activity after
somatostatin treatment in an immunocomplex assay. Somatostatin highly
increased r-PTP activity in PCCl3 and PC mos/PTP (+300%,
P < 0.01) but not in PCmos cells. Conversely, no
differences in somatostatin-stimulated SHP-2 activity, (
+50%,
P < 0.05), were observed among all the cell lines.
The activation of r-PTP by somatostatin caused, acting downstream of
MAPK kinase, an inhibition of insulin-induced ERK1/2 activation
with the subsequent blockade of the phosphorylation, ubiquitination,
and proteasome degradation of the cyclin-dependent kinase inhibitor
p27kip1. Ultimately, high levels of p27kip1
lead to cell proliferation arrest. In conclusion, somatostatin
inhibition of PC Cl3 cell proliferation requires the activation of
r-PTP which, through the inhibition of MAPK activity, causes the
stabilization of the cell cycle inhibitor p27kip1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The effects of SST are mediated by a family of five different G protein-coupled receptors (named SSTR1 through 5), which are variably expressed in both normal tissues and tumors (3), often showing the presence of multiple subtypes in the same cell.
A role for this peptide as endogenous regulator of cell proliferation in a variety of epithelial (4, 5, 6, 7) and endocrine tissues has also been proposed (8). In recent years, the molecular mechanisms involved in this activity have begun to be identified, and both direct and indirect antiproliferative effects of SST have been observed. SST indirectly controls cell growth in vivo through its inhibitory effects on the release of growth-promoting hormones (9) or through a recently identified antiangiogenic mechanism involving regulation of proliferation and migration of endothelial cells as well as monocytes (10). Moreover, a direct antiproliferative activity in both normal and tumoral cells has also been reported (2, 9). This effect is mainly cytostatic (5) although it was reported that the activation of SSTR3 is able to induce apoptosis (11).
The modulation of phosphotyrosine phosphatase (PTP) activity has been proposed as being one of the main intracellular pathways responsible for the inhibition of cell growth by SST (5, 12, 13, 14, 15). A SST-dependent increase of PTP activity has been shown to induce dephosphorylation of the epidermal growth factor receptor, inhibiting the proliferative activity of epidermal growth factor (13, 16). Thus, hormonally-regulated PTPs were proposed to play a key role in the control of cell proliferation. SST, as well as other hypothalamic hormones (17, 18, 19, 20), was suggested to be an important endogenous modulator of the activity of this class of enzymes. The PTP activity stimulated by SST is associated with the plasma membrane and shares biochemical features with the phosphatases SHP1 and SHP2. SHP1 and SHP2 belong to a family of cytosolic PTPs that contains motifs called src homology 2 (SH2) domains, involved in protein-protein interaction via their association with specific phosphotyrosine residues (21). It has been reported that SHP proteins are rapidly recruited to the plasma membrane upon SST treatment of breast cancer cells (22). Moreover, SHP PTPs can be immunoprecipitated in a complex containing SST (23) or SST receptors (24). Finally, overexpression of an interfering mutant of SHP2 abolished SST-stimulated membrane-associated PTP activity (25). More recently, it has been reported that the antiproliferative activity mediated by SSTR1, transfected in CHO-K1 cells, was mediated by a SHP2-dependent regulation of MAPK activity, and that c-src could be the substrate for the PTP activity regulated by SST (26). Although all this evidence supports a role of SHPs in SST cell proliferation control, other PTPs also seem to be involved in the effects of SST. While SHP1 or SHP2 activation by SST occurs after a very short latency time (1–5 min), which is followed by a rapid return to basal levels (24, 26), a long lasting PTP activity was also observed in some cell types after SST treatment, with statistically significant increased PTP activity even after 2 h of stimulation (5, 15, 27). This activity was detected in membrane preparations of the responsive cells, and it was thought to be dependent on the activation of a still-unidentified receptor-like PTP.
We have recently reported that the SST-dependent cytostatic effects in
the normal thyroid cell line PC Cl3 were mediated by the activation of
a long-lasting membrane PTP activity (5). In an attempt to
identify the molecular correlate for this activity, we evaluated the
effects of SST on both PTP activity and cell proliferation in subclones
of these cells, transfected with different oncogenes, as a model of
malignant transformation (28). By expressing the E1A
and/or middle T oncogenes in these cells, we obtained different cell
lines representing different degrees of malignant transformation. In
particular, wild-type (w.t.) PC Cl3 cells retain most
of the typical markers of thyroid differentiation in vitro,
including dependence on TSH for proliferation and function,
thyroglobulin synthesis and secretion, and the ability to trap iodide
from the medium. In contrast, the cell lines obtained with stable
expression of the E1A and/or middle T oncogenes gradually lost these
differentiative markers, became independent of TSH for proliferation,
and acquired the capability to generate tumors when injected in nude
mice (28, 29, 30, 31). In all the oncogene-transformed cell lines,
SST was unable to inhibit cell proliferation and to stimulate PTP
activity (5, 32). The expression of a newly cloned rat PTP
(32), named r-PTP on the basis of its homology with the
human DEP-1/HPTP gene (33), correlated with the lack of
SST efficacy. We therefore proposed that this PTP may be responsible
for the antiproliferative activity of SST. R-PTP is expressed
ubiquitously, but is mainly found in the brain, liver, and spleen. The
predicted protein contains a unique intracellular catalytic domain, a
short transmembrane domain, and an extracellular region containing
eight fibronectin type III-like repeats (33). The
expression of the r-PTP gene is induced by TSH in normal thyroid
cells and correlates with their differentiation state; r-PTP is
down-regulated by transformation induced by several oncogenes as well
as in malignant human thyroid tumors (33, 34).
The aim of this paper was to establish a role for r-PTP in the
antiproliferative activity of SST. We therefore compared the effects
induced by SST in the w.t. PC Cl3 cells, which express
native r-PTP, with those obtained in three new subclones of these
cells: 1) PC mos cells (PC Cl3 cells infected with MPSV carrying the
v-mos oncogene) in which the loss of most of thyroid
differentiation markers and the acquisition of autonomous growth and
high tumorigenicity in nude mice correlates with the lack of the
expression of r-PTP (28); 2) PC mos/PTP (PC mos
cells in which r-PTP has been stably retransfected)
(34); and 3) PC mos/PTP[C/S] (PC mos cells in which a
catalytically inactive mutant r-PTP has been stably
transfected) (34).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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shows Northern blot
analysis of the expression of the mRNA for r-PTP, in the PC Cl3, PC
mos, and PC mos/PTP. R-PTP was expressed in the
w.t. cells, and its expression was lost after cell
transformation by stable expression of the v-mos oncogene.
In the PCmos/PTP the cDNA for the phosphatase is transfected in the
PC mos cells and the expression of r-PTP was restored. Equal RNA
loading was demonstrated by evaluation of the GAPDH mRNA content (Fig. 1A). Similar results were observed in Western blotting experiments
using an 
–DEP-1 antibody (the human form of r-PTP) (Fig. 1B).
Interestingly, the expression of other PTPs involved in the SST signal
transduction, such as SHP2 (26), was not affected in the
PC mos cells (Fig. 1B).
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cell lines were studied by means of both DNA synthesis and cell number
analysis, using the [3H]thymidine incorporation
and 3-(4, 5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide
(MTT) assays, respectively.
Table 1 shows the time course (up
to 3 d) of the proliferative effects of TSH, insulin, and a
combination of the two factors, analyzed using the MTT test, as cell
number index. In this preliminary experiment, cells were serum- and
growth factor-starved for 24 h and then treated as indicated. In
the absence of growth factors, the w.t. PC Cl3 cells showed
very little proliferative capability and after 4 d (one of
starvation and three of the experiment) clear signs of cell death were
apparent. As previously reported (5), the treatment with
TSH alone had little effect on the mitogenic activity. Conversely,
insulin was a very powerful mitogen, and the combination treatment with
TSH and insulin resulted in a synergistic stimulation of cell growth.
After 3 d of treatment a diminution in cell proliferation,
measured by the MTT test, was observed in the insulin and
insulin+TSH-stimulated cells. This effect is probably due to contact
inhibition as the cells had reached confluency at that time point. A
different proliferation pattern was observed in the PC mos cell line.
In these cells, similar to the other transformed PC Cl3 subclones
(32), up to 3 d of serum starvation did not arrest
the proliferative activity. Conversely, these transformed cells showed
an autonomous pattern of proliferation, since stimulation with the
mitogen (insulin and insulin+TSH) did not significantly increase the
proliferation rate. In the PC mos/PTP cells an intermediate pattern
of response was observed. Although less pronounced than in PC mos
cells, a proliferative activity, even after 72 h of starvation,
was observed. However, the reexpression of r-PTP restored the
responsiveness to the growth factors (insulin and insulin+TSH), which
showed a synergistic activity on the proliferation of these cells
(Table 1).
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). The expression of r-PTP in the PC
mos/PTP cells completely restored SST inhibition of cell
proliferation (Fig. 2A).
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). A similar pattern of expression was also observed in
the PC mos cells transfected with the cDNA encoding for r-PTP (Fig. 2B).
We then performed time course and dose-response experiments to compare
the characteristics of SST cell proliferation inhibition in both the
w.t. cells (which express the endogenous r-PTP) and the
transformed cells transfected with r-PTP cDNA. The data reported in
Figs. 3 and 4, while confirming the
absence of responses in the PC mos cells,
show that the pharmacological profile of the SST’s antiproliferative
effects in the PC Cl3 and PC mos/PTP cells was almost identical,
having superimposable time course and dose-response curves. Moreover,
to demonstrate that the recovery of the responsiveness to SST in PC
mos/PTP was specifically due to r-PTP and not to the transfection
procedure and clonal selection, we analyzed the effect of SST on
insulin+TSH-stimulated cell growth in PC mos cells transfected with a
catalytically inactive mutant of r-PTP (PC mos/PTP[C/S]). This
cell line showed growth characteristics identical to the PC mos cells
(only a 12% increase in proliferative activity after insulin+TSH
stimulation); SST (0.01–10 µM) did not
affect the proliferation of these cells (Fig. 4).
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cells (Fig. 5), indicating that the signal
transduction activated by this peptide was dependent on the activation
of a G protein that appears to belong to the
Gi/G0 family. Again, no
effects were detected in the PC mos cells (Fig. 5). These data show
that the antiproliferative effects of SST in these thyroid cell lines
were correlated with the expression of r-PTP. To more directly
demonstrate the involvement of a PTP in SST activity, we treated the
cells with vanadate, a selective inhibitor of all the PTPs. As shown in
Fig. 6, vanadate (25 µM)
treatment increased basal [3H]thymidine
incorporation in PC Cl3 cells, confirming that a basal PTP activity was
present that could maintain a low rate of cell proliferation.
Pretreatment with the PTP inhibitor completely reverted the inhibitory
activity of SST on both insulin- and insulin+TSH-stimulated conditions.
In the transformed PC mos cells, in which r-PTP was completely
down-regulated, vanadate did not modify the basal activity, suggesting
the loss of cell proliferation control by a PTP, likely r-PTP. In
addition, no effects of SST or vanadate were observed in
TSH+insulin-stimulated conditions. In PC mos/PTP cells, vanadate
pretreatment increased basal [3H]thymidine
uptake, confirming that the lack of effects observed in the PC mos
cells was due to the lack of expression of r-PTP. Similarly,
vanadate completely reverted the inhibitory activity of SST in
stimulated conditions, showing a pattern of response similar to that
observed in the w.t. PC Cl3 cells (Fig. 6). Using the
synthetic substrate p-nitrophenylphosphate (pNPP), we
directly measured the SST-inducible total membrane PTP activity in the
different cell lines (Fig. 7). The
expression of the oncogene v-mos in the PC Cl3, which caused
a down-regulation of r-PTP gene expression, significantly reduced
the total membrane PTP activity (-50%), together with the
disappearance of the SST-dependent (1 µM,
1 h of treatment) increase in PTP activity observed in the PC Cl3
cells. In the PC mos/PTP cells, basal PTP activity increased to
levels similar or even higher than those observed in the PC Cl3 cells.
More importantly, the expression of r-PTP in the PC mos cells
totally restored the stimulation of a membrane PTP activity by SST
(Fig. 7). The PC mos/PTP[C/S] cells, which express the
catalytically inactive mutant of r-PTP, showed the same pattern of
response as the PC mos cells (low basal level and lack of response to
SST) (Fig. 7). Moreover, we directly evaluated the specific r-PTP
activity in these cell lines after SST treatment (1
µM). As a control we evaluated the SST
modulation of another PTP, SHP2, endogenously expressed in all the cell
lines used in this study (Fig. 1B), which was previously described to
be activated by SST (26). Because different kinetics of
activation between these two PTPs were reported (5, 26),
we analyzed SST effects after both 10 min and 1 h of treatment.
The experiments were performed in an immunocomplex assay. After
insulin+TSH and insulin+TSH+SST treatments, cells were lysed and
immunoprecipitated with SHP2 and PTP antibodies, and PTP
activity, measured as hydrolysis of pNPP, was assayed in the
immunocomplexes. An aliquot of the immunoprecipitate was analyzed in
Western blot to normalize the level of r-PTP and SHP2 proteins in
the pNPP hydrolysis assay. As shown in Fig. 8C, the level of SHP2 immunoprecipitated
was similar in all the cell lines and for all the treatments. As
expected, no r-PTP was immunoprecipitated in PC mos cells, although
the level of protein detected in the other cell lines was comparable
for all the treatments (Fig. 8C). The PTP activity experiments
showed that r-PTP activity was increased after only 10 min of SST
treatment (+300%) in both PC Cl3 and PC mos/PTP cell lines. If
compared with the untreated or insulin+TSH-treated cells, the activity
was still significantly higher after 1 h of stimulation
(+150%)(Fig. 8A). As expected, no r-PTP specific activity was
detected in the PC mos cell line (Fig. 8A). Conversely, 10 min of SST
treatment significantly increased SHP2 activity to a similar level in
all the cell lines ( +50%), but after 1 h of SST treatment,
SHP2 activity returned to the basal level (Fig. 8B).
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was able to control cell
proliferation through a stabilization of the cyclin-dependent kinase
(CDK) inhibitor p27kip1 (34). In the
different cell lines, we evaluated whether the SST treatment was able
to increase the amount of nuclear p27kip1 and
whether this effect correlated with the activation of r-PTP. For
this purpose we analyzed by immunofluorescence confocal microscopy the
nuclear presence of p27kip1 using a specific
polyclonal antibody. In
G0/G1 synchronized
w.t. PC Cl3 (24 h of serum and growth factor deprivation) a
clear nuclear expression of p27kip1 was observed
(Fig. 9a). The nuclear staining was
greatly reduced after TSH+insulin treatment (Fig. 9b). SST powerfully
inhibited the degradation of p27kip1 in
stimulated conditions, suggesting that the cytostatic signals, mediated
by the SST-sensitive PTP, have p27kip1 as a
nuclear effector (Fig. 9c). In PC mos cells
p27kip1 levels were extremely low after 24 h
of starvation and did not change after insulin+TSH or insulin+TSH +SST
treatments (Fig. 9, d, e, and f). The expression of r-PTP in the PC
mos cells restored both the basal levels of
p27kip1 and the sensitivity to SST. Indeed, the
mitogenic stimuli (TSH+insulin) significantly reduced the number of
p27kip1 positive cells (>80% of the cells did
not show any immunostaining for p27kip1), while
cotreatment with SST reversed this effect, showing more than 70% of
positive cells (Fig. 9, g, h, and i). Similar results were obtained in
Western blot experiments using a p27kip1
selective antibody (Fig. 10). In PC Cl3
and PC mos/PTP cells, basal p27kip1 expression
was significantly reduced by the mitogenic insulin+TSH stimulation,
while SST treatment prevented this effect. Interestingly, the
pretreatment with the MAPK kinase (MEK) and proteasome
inhibitors, PD98059 and ZIE [Ot-Bu]-A-leucinal (PSI), respectively,
per se ineffective (data not shown), inhibited the reduction
in p27kip1 cell content induced by insulin+TSH
stimulation (Fig. 10), suggesting that the decrease in the
concentration of this CDK inhibitor requires the activation of ERK1/2
which through the phosphorylation of p27kip1
induces its ubiquitination and proteasome degradation, as recently
reported (36). Moreover, this result indicates that SST
negatively interferes with this pathway. In PC mos cells, the basal or
insulin+TSH levels of p27kip1 were extremely low
and were not affected by SST treatment. Conversely, both PD98059 and
PSI significantly increased the p27kip1 content
in both basal (data not shown) or insulin+TSH-stimulated
conditions, indicating that ERK1/2 and proteasome activation were
downstream to the v-mos effects. The relevance of
p27kip1 expression for the growth
factor-dependent proliferation of all the cell lines was also
demonstrated evaluating the effects of the PD98059 and PSI compounds on
the insulin+TSH-stimulated cell growth. In these experimental
conditions both basal (in the PC mos cells) and insulin+TSH-stimulated
(in PC Cl3 and PC mos/PTP cells) proliferation was completely
blocked (data not shown).
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activity induced
by SST, we evaluated the phosphorylation state (activation) of
different components of the insulin-dependent proliferative pathway:
the insulin receptor-ß (IRß) subunit, MEK and ERK1/2. To
evaluate the effects of SST on the insulin-dependent IRß tyrosine
phosphorylation, serum-starved PC Cl3, PC mos, and PC mos/PTP cells
were treated for 5 min with 100 nM insulin in the presence
or absence of 1 µM SST and then immunoprecipitated with
-IRß antibody and evaluated with -phosphotyrosine antibody in
Western blot (Fig. 11), or vice versa
(data not shown). Insulin treatment induced a significant
phosphorylation of its receptor, and SST significantly inhibited this
effect in all the cell lines. MEK activation was tested in all the cell
lines in basal and insulin+TSH- or insulin+TSH+SST-stimulated
conditions (Fig. 12). In PC Cl3 cells
insulin+TSH increased MEK phosphorylation while SST completely blocked
this effect. Conversely, in PC mos and PC mos/PTP MEK,
phosphorylation was already elevated in basal conditions, probably due
to the expression of mos, of which MEK is a direct substrate. However,
in both cell lines, neither insulin+TSH nor insulin+TSH+SST treatments
modified this effect.
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cells), insulin+TSH treatment (100 nM and
10 nM, respectively) significantly increased ERK1/2
phosphorylation, and SST completely reverted this effect. In PC mos
cells basal ERK1/2 phosphorylation was significantly elevated, and
neither insulin+TSH nor insulin+TSH+SST treatments were able to modify
it (Fig. 12).
Interestingly, other signal transduction systems were not recovered
after r-PTP expression in the PC mos cells. Indeed, similarly to the
transformed cells, in the PC mos/PTP the treatment with TSH did not
induce an increase in intracellular calcium concentration
([Ca++]i), as observed in the w.t. PC Cl3 (Fig. 13).
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antiproliferative activity of SST was studied analyzing the capability
of TGFß1 to control the proliferative activity in the different cell
lines we studied. The data in Fig. 14
show that, irrespectively to the expression of r-PTP, TGFß1
induced a significant inhibition of
[3H]thymidine uptake, in all the cell lines
studied. Moreover, the dose-responses of TGFß1 observed in the
w.t. PC Cl3, PC mos, and PC mos/PTP cell lines were
almost superimposable, showing that the expression of r-PTP is not
necessary for the antiproliferative activity of TGFß1 and confirming
that this PTP may represent a specific effector in the signal
transduction of SSTRs, to control cell proliferation.
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DISCUSSION |
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, in the antiproliferative activity of SST
in the PC Cl3 clonal thyroid cell line.
PC Cl3 cells are dependent on insulin and TSH for their proliferation,
and the activation of SSTRs mediates the generation of cytostatic
signals (5). In these cells, the antiproliferative
activity of SST is dependent on the activation of a membrane-associated
PTP through a PTX-sensitive GTP-binding protein. In the past, the
involvement of PTPs in the antiproliferative effects of SST and other
endogenous compounds, such as dopamine, angiotensin II, and LHRH, was
well characterized (16, 17, 18, 19). More recently, in an effort
to identify at molecular level the PTP(s) involved in the
antiproliferative activity of SST, many studies identified the
SH2-containing PTPs as effectors of different SSTRs. However these
PTPs, namely SHP1 and SHP2, cannot account for all the PTP activities
induced by SST. Indeed, while the activation of SHP1 and SHP2 occurs
very rapidly (0.5–3 min) and is rapidly inactivated (10 min)
(24, 26), in different experimental models a SST-inducible
long lasting PTP activity (still elevated after 1 h of treatment)
was reported (13, 15, 27). Also in PC Cl3 cells we
previously reported (5, 32), and confirmed in this study,
a stimulatory effect on a membrane PTP activity after 1 h of SST
treatment.
The main goal of our work was to identify, at molecular level, a
possible candidate for this PTP activity. For this purpose we used a PC
Cl3 subclone that was stably infected with the plasmid MPSV carrying
the oncogene v-mos (PC mos). These cells acquire a
transformed phenotype and loose the thyroid differentiation markers.
Like other transformed subclones of PC Cl3 cells (stably expressing the
oncogenes E1A and/or middle T) (32), PC mos cells show an
autonomous pattern of proliferation, probably due to the constitutive
activation of the ERK1/2 pathway that occurs by the direct MEK
phosphorylation induced by the active mos oncogene. Moreover, they are
insensitive to the antiproliferative effects of SST. These growth
characteristics correlate with the loss of the expression of the
receptor-like r-PTP, while other PTPs, such as PTPµ
(32) or SHP2 (present paper) are unaffected. r-PTP is
expressed in most of the normal rat tissues, and the reduction of its
expression was observed in most oncogene-transformed thyroid cell lines
(33). Interestingly, a down-regulation of the expression
of the human homolog of r-PTP, DEP-1/HPTP, was also observed in
human malignant thyroid tumors, suggesting that this PTP may represent
an important regulator of thyroid cell differentiation and
transformation, behaving as an oncosuppressor gene (34).
Indeed, the stable expression of r-PTP in the transformed PC mos
cells was sufficient to cause a partial recovery of thyroid
differentiation markers and a reduction of the growth potential of the
transformed cells. In particular, the mechanisms controlling the
proliferation, such as contact inhibition, were restored in these cells
through an increase in the steady-state level of the cyclin-dependent
kinase inhibitor p27kip1 (Ref. 34
and present paper). This effect was specific for r-PTP since the
transfection of another receptor-like PTP, PTP
, did not cause the
reversion of the transformed phenotype (34). In this paper
we evaluated the involvement of r-PTP in the antiproliferative
signals induced by SST. Indeed, while in the w.t. PC Cl3
cells SST seems to be one of the major inhibitors of the proliferative
pathways, this control is completely lost in the transformed PC mos
cells. The lack of responsiveness to SST in PC mos cells is not due to
an alteration in the mRNA expression of SSTR, which is not changed in
the different cell lines, but seems due to the down-regulation of
r-PTP. Indeed, in the PC mos/PTP cells a complete recovery of the
SST control of cell proliferation was observed. Conversely, the
transfection of a catalytically inactive mutant of r-PTP in the PC
mos cells did not cause any recovery of the antiproliferative effects
of SST. In PC mos/PTP cells, the expression of r-PTP restored the
PTP tonic activity that controls basal cell proliferation, as
demonstrated by the capability of vanadate to increase basal
proliferative activity that correlated with a reduction in the basal
proliferation rate, features also observed in the w.t. PC Cl3 cells.
More importantly, the expression of r-PTP was absolutely necessary
for the SSTergic PTP-mediated control of cell proliferation. Indeed,
the transfection of r-PTP in the PC mos cells restored the
responsiveness to SST with pharmacological characteristics
superimposable to that observed in the w.t. cells. SST treatment was
able to induce a membrane PTP activity only in the r-PTP-expressing
cell line. Immunocomplex assays showed that this activity was mainly
ascribable to r-PTP, the activity of which was increased by 300%.
However, the specific activity of SHP2, another SSTR-regulated PTP
(26), was also increased although to a much lower level
(150% of the basal) irrespective of the modulation of cell
proliferation induced by SST. This observation suggests that, at least
in these cells, SHP2 activity is not sufficient, to inhibit cell
growth. Conversely, the activation of r-PTP was absolutely
necessary for such an effect.
Interestingly, although PC mos/PTP cells show a higher proliferation
rate compared with the w.t. PC Cl3 cells, the dose-response
curves, time course, and G protein dependence of the antiproliferative
effects of SST were similar in the two cell lines, indicating that the
exogenous expression of this PTP did not alter the normal regulation of
its activity and final physiological effects. The role of
r-PTP in the control of cell proliferation seems to be selective for
the signal transduction activated by the SSTR, since no alterations in
the antiproliferative effects of TGFß1 were observed in the
transformed cells, regardless of the expression of r-PTP. Similarly
the transfection of r-PTP did not cause, in the PC mos cells, the
recovery of other transduction signals lost in the transformed cells,
such as the mobilization of intracellular Ca++
observed in the PC Cl3 cells, which was probably involved in biological
functions other than cell proliferation (i.e. hormonal
secretion). These data demonstrate that r-PTP is mainly
responsible for the control of the antiproliferative signals in these
thyroid cell line and that its activation is selectively regulated by
SST receptors.
The inhibition of ERK1/2 phosphorylation, with the subsequent
ubiquitination and proteasome-dependent proteolytic degradation of the
CDK-inhibitor p27kip1, was recently reported to
represent an important mechanism of cell cycle arrest in normal and
tumoral cells (36). Moreover, r-PTP was shown to
represent a possible regulator of p27kip1
expression (34). Here we report that the activation of
r-PTP by SST is able to revert the activation of the proteolytic
degradation of p27kip1 induced by the treatment
with TSH and insulin, an effect dependent on the blockade of the
insulin-stimulated ERK1/2 activation. Next, we tried to identify
possible substrates dephosphorylated by r-PTP, after SST
stimulation. We found that in all the cell lines (including the PC mos
cells that do not express r-PTP), SST caused dephosphorylation of
the insulin-phosphorylated IRß, thus indicating that in these cells
SST inhibition of IR activation is not mediated by r-PTP.
Interestingly, the recombinant human homolog of r-PTP, DEP-1, was
reported to be unable to dephosphorylate in vitro a
tyrosine-phosphorylated IRß (37). Conversely, it was
reported that SSTR2 activation caused, in CHO cells, dephosphorylation
of IRß through the activation of SHP1 (38). Since in our
cell system SST causes, in all the cell lines, the activation of the
related PTP SHP2, we can speculate that also in thyroid cells the
dephosphorylation of the IRß may be mediated by SST through SHP2.
Thus, r-PTP specific target seems to be downstream from IRß in the
signal transduction cascade. We evaluated MEK and ERK1/2
phosphorylation after SST-induced PTP activation. We found that the
activation of both kinases was inhibited by SST in PC Cl3 cells, and
that MEK phosphorylation was not affected in PC mos and PC mos/PTP.
Since in these cell lines the oncogene v-mos, which is able to directly
phosphorylate MEK, is constitutively expressed, the lack of effects of
SST in the PC mos/PTP cells clearly suggests that the activity of
r-PTP should be downstream from MEK. Indeed, we found that,
similar to w.t. PC Cl3 cells, in PC mos/PTP, SST treatment
caused a significant reduction of the insulin-dependent ERK1/2
phosphorylation. Although we do not have evidence of a direct
interaction, we propose that r-PTP activation leads to the
inhibition of ERK activity, either through a direct dephosphorylation
or through the regulation of still unidentified intermediate proteins.
Interestingly, other PTPs, namely PTP-SL and striatum enriched
phosphatase, were previously reported to directly interact and
dephosphorylate ERK1/2 (39). Thus, we propose that, at
least in PC Cl3 cells, SST is able to exert a cytostatic activity
through the control of p27kip1 expression,
mediated by the activation of r-PTP that, acting directly or
indirectly, downstream of MEK, interferes with the insulin-dependent
ERK1/2 activation. The establishment of a role for the control of
p27kip1 expression in the SST cytostatic effects
is in agreement with previous observations showing, in different cell
lines, a SST-dependent blockade of the growth factor-induced
p27kip1 degradation, although through different
mechanisms. In the FRTL-5 thyroid cell line, the cytostatic effects of
SST were mediated by the inhibition of the down-regulation of
p27kip1 induced by the TSH-dependent PKA and PI3K
activation, via an interference with cAMP generation (40, 41). Conversely, in CHO-k1 cells overexpressing SSTR2, SST was
reported to induce a stabilization of p27kip1
levels in a PTP-dependent manner involving SHP1 (42).
In our experimental model SST is able to block the
insulin-induced degradation of p27kip1 by
preventing the activation of the ERK1/2 pathway, via the activation of
r-PTP (Fig. 15).
|
, which mediates the antiproliferative signals activated by
SSTR, including the stabilization of the CDK inhibitor
p27kip1, acting directly as an inhibitor of
growth factor-mediated ERK1/2 activation. This observation shows, for
the first time, the capability of G protein-coupled receptors to
regulate the activity of receptor-like PTPs to control cell
proliferation. The intracellular pathway leading to the activation of
r-PTP by SST is still to be determined, and we cannot exclude the
involvement of other cytosolic PTPs such as SHP-1/2, as reported
in other cell systems. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies
For r-PTP detection, we used antibodies raised against the
intracellular region of r-PTP expressed as a recombinant protein
fused to GST, and affinity purified (34), used in Western
blot experiments at the dilution of 1:500. Other antibodies used were:
p27kip1 (clone 57) (Transduction Laboratories, Inc., Lexington, KY), p44/42 and phospho-p44/p42
MAPK, MEK and phospho-MEK (New England Biolabs, Inc.,
Beverly, MA), SHP2 (C18) (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) phosphotyrosine P-Tyr-100 (Cell Signaling Technology,
Beverly, MA), all used at the dilution of 1:1,000; IRß (clone 46)
(Transduction Laboratories, Inc.) was used at the dilution
of 1:250.
Cell Culture
PC Cl3 and PC mos/PTP cell lines were
grown in Ham’s F12 medium, Coon’s modification (Sigma)
supplemented with 5% FCS (ICN) and a mixture of growth factors
(TSH, 10 nM; hydrocortisone, 10 nM; insulin,
100 nM; transferrin, 5 µg/ml; glycyl-hystidyl-lysine,20
µg/ml), as previously reported (5). Since SST (5
nM) that was present in the original culture medium
reduced the proliferation rate of these cells, it was removed from the
growth factor mixture (5).
PC mos and PC mos/PTP[C/S] cells were grown in the same medium
without the growth factors.
PTX (180 ng/ml) treatment was performed in serum-free medium for 18 h before the experimental treatments, as previously reported (43).
[3H]Thymidine Incorporation Assay
DNA synthesis activity was measured by means of the
[3H]thymidine uptake assay, as previously
reported (17). Briefly, cells were plated at the density
of 5 x 105 in 24-well plates. After 24
h the cells were serum- and growth factor-starved for 48 h.
Subsequently, cells were treated with the test substances for 16
h, and in the last 4 h cells were pulsed with 1 µCi/ml of
[3H]thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL). At the end of the incubation
time, cells were trypsinized (15 min at 37 C), extracted in 10%
trichloroacetic acid (TCA), and filtered under vacuum through fiber
glass filters (GF/A; Whatman, Clifton, NJ). The filters
were then sequentially washed, under vacuum, with 10% and
5% TCA and 95% ethanol. The TCA-insoluble fraction was counted in a
scintillation counter.
MTT Assay
Mitochondrial function, as an index of cell viability, was
evaluated by measuring the levels of mitochondrial dehydrogenase
activity using reduction of MTT as the substrate. Its cleavage to a
purple formazan product by dehydrogenase was quantified
spectrophotometrically measuring the absorbance at 570 nm
(44).
Immunoprecipitation
Total cell lysates (250 µg) were used in immunoprecipitation
experiments. The proteins were incubated with the appropriate
antibodies (1 µg/1 mg of proteins) for 2 h at 4 C, in RIPA
buffer and then with protein A sepharose for an additional hour. After
three washes with RIPA buffer, the immunocomplexes were analyzed in
Western blot. For the PTP immunocomplex assay, the precipitation was
done with IgG-coupled magnetic beads (Dynabeads), since the protein A
caused, per se, hydrolysis of p-nitrophenylphosphate
(pNPP).
PTP Assay
Cells, plated at 50% confluence in 10-cm Petri dishes, were
preincubated with the test substances for 1 h in FCS-free medium
at 37 C in a CO2 incubator. Then the cells were
washed with PBS and mechanically scraped in a buffer containing 0.32
M sucrose, 10 mM Tris, pH 7.5, 5 mM
EGTA, and 1 mM EDTA, and the membranes were isolated as
previously reported (43). Nuclei were removed by
centrifugation at 2,000 x g at 4 C for 10 min.
Membrane fraction was isolated by a further centrifugation at
15,000 x g at 4 C for 60 min, resuspended in a buffer
containing 250 mM HEPES, pH 7.2, 140
mM NaCl, 1% NP40, and PMSF and leupeptin as
protease inhibitors, and assayed for protein content using the method
of Bradford (45) using BSA as a standard and the
Bio-Rad Laboratories, Inc. (Hercules, CA) reagent. Twenty
micrograms of control or treated membranes were used in the PTP assay.
PTP assay was performed using the synthetic substrate pNPP in a
spectrophotometric assay. pNPP is a general phosphatase substrate that
in the presence of inhibitors of Ser/Thr phosphatases is specific for
PTP (13, 17). Membranes were preincubated for 5 min at 30
C in 80 µl volume containing 20 µl of a 5x reaction buffer [250
mM HEPES, pH 7.2, 50 mM
dithiothreitol, 25 mM EDTA, 500
nM microcystin-leucine-arginine (Alamone
Laboratories, Jerusalem, Israel), as a Ser/Thr phosphatase
inhibitor]. The reaction was started by adding 20 µl of 50
mM pNPP, carried out for 30 min at 30 C and
stopped by adding 900 µl of 0.2 N NaOH. The
absorbance of the sample, directly proportional to the amount of
dephosphorylated substrate, was measured at 410 nm (46).
The extinction coefficient for pNPP, at this wavelength is 1.78 x
104
M-1cm-1
(46). In the immunocomplex specific PTP assay, all the
immunoprecipitated proteins (see above) with the specific -PTP
antibodies were assayed.
mRNA Analysis
The expression of specific mRNAs was evaluated by means of
RT-PCR technique for the SST receptor subtypes and by means of the
Northern blot technique for r-PTP, as described below.
RNA Isolation and RT-PCR
Total RNA was isolated using the acidic phenol technique
(47). RT-PCR was performed as previously reported
(32). Briefly, 10 µg of total RNA were treated for 45
min with RNAse-free DNAse at 37 C to remove genomic DNA contamination,
phenol/chloroform was extracted and ethanol was precipitated. RT
reaction was performed using oligo-dT(16) primer
and the AMV RT (Amersham Pharmacia Biotech), for 40 min at
42 C. PCR reaction was performed on 10 ng of cDNA as follows: (final
volume 50 µl) 5 min of denaturation at 94 C followed by 40 cycles of
1 min at 94 C, 1 min at 60 C, and 1 min at 72 C, followed by 7 min at
72 C, using the Taq DNA polymerase (2.5 U/reaction)
(Roche Molecular Biochemicals, Indianapolis, IN).
Amplified DNA fragments were then visualized on agarose gel
electrophoresis. The primers used were the following: SSTR1: 5'-sense
primer corresponded to the amino acids 86–91 and 3'-antisense primer
corresponded to the amino acids 211–217 of SSTR1 sequence; SSTR2:
5'-sense primer corresponded to the amino acids 71–77 and 3'-antisense
primer corresponded to the amino acids 195–181 of SSTR2 sequence;
SSTR3: 5'-sense primer corresponded to the amino acids 189–196 and
3'-antisense primer corresponded to the amino acids 304–311 of SSTR3
sequence; SSTR4: 5'-sense primer corresponded to the amino acids
284–291 and 3'-antisense primer corresponded to the amino acids
367–374 of SSTR4 sequence; SSTR5: 5'-sense primer corresponded to the
amino acids 270–276 and 3'-antisense primer corresponded to the amino
acids 354–362 of SSTR5 sequence; expected lengths for the amplified
products were the followings: SSTR1 = 395 bp, SSTR2 = 392 bp,
SSTR3 = 370 bp; SSTR4 = 270 bp, SSTR5 = 275 bp.
Northern Blot
Northern blot and hybridization procedures were performed
according to standard procedures (48). A mouse GAPDH probe
was used to ascertain the equal RNA loading.
Immunofluorescence
Indirect immunofluorescence was performed on the different cell
lines, plated on glass coverslips. Cells were fixed in paraformaldehyde
(4% in PBS) for 15 min, washed three times with PBS, and permeabilized
with 0.1% Triton X-100, for 5 min. After washing in PBS, cells were
treated with 0.1 M glycine and incubated in PBS containing
0.2% gelatin. Cells were stained with anti-p27 (Transduction Laboratories, Inc.) antibody in PBS-0.2% gelatin for 1 h,
washed three times in PBS, and incubated with antirabbit fluorescein
isothiocyanate conjugates for 20 min. Finally after washing in PBS,
coverslips were mounted in Moviol (Calbiochem, La Jolla,
CA) and analyzed on a confocal microscope MRC 1024ES (Bio-Rad Laboratories, Inc.).
[Ca++]i Measurement
Cells were plated on 25-mm glass coverslips and transferred to a
35-mm Petri dish. After 24 h cells were serum starved for an
additional 24 h. On the day of the experiment cells were washed
for 10 min with a balanced salt solution (HEPES 10 mM, pH
7.4; NaCl, 150 mM; KCl, 5.5 mM;
CaCl2, 1.5 mM;
MgSO4, 1.2 mM; glucose, 10
mM). Then cells were loaded with Fura-2 penta-acetoxymethyl
ester (4 µM) (Calbiochem) for 20 min at room
temperature. For fluorescence measurements, coverslips were mounted on
a coverslip-chamber, and fura-2 fluorescence was imaged with an
inverted diaphot microscope (Nikon, Melville, NY) using a
Nikon 40X/1.3 NA Fluor DL objected lens. Fluorescence
(ratio 340/380) was then evaluated and converted in
[Ca++]i using the
Quanticell apparatus (Visitech, London, UK), as previously reported
(49). Calibration of fluorescence signals was performed as
described (49). The values of the
[Ca++]i were calculated
using Quanticell software, according to the equation of Grynkiewicz
et al. (50).
Statistical Analysis
Experiments were performed in quadruplicate and repeated at
least three times. Statistical analysis was performed by means of
one-way ANOVA. A P value less than or equal to 0.05 was
considered statistically significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: [Ca++]i, Intracellular Ca++ concentration; CDK, cyclin-dependent kinase; IRß, insulin receptor-ß; MEK, MAPK kinase; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5, diphenyl tetrazolium bromide; pNPP, p-nitrophenylphosphate; PSI, ZIE [Ot-Bu]-A-leucinal; PTP, phosphotyrosine phosphatase; PTX, pertussis toxin; SST, somatostatin; SSTR, a family of five different G protein-coupled receptors; TCA, trichloroacetic acid; w.t., wild-type.
Received for publication April 10, 2001. Accepted for publication June 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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suppresses the neoplastic phenotype of
retrovirally transformed thyroid cells through the stabilization of the
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