The Activation of the Phosphotyrosine Phosphatase {eta} (r-PTP{eta}) Is Responsible for the Somatostatin Inhibition of PC Cl3 Thyroid Cell Proliferation

Tullio Florio, Sara Arena, Stefano Thellung, Rodolfo Iuliano, Alessandro Corsaro, Alessandro Massa, Alessandra Pattarozzi, Adriana Bajetto, Francesco Trapasso, Alfredo Fusco and Gennaro Schettini

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The aim of this study was the characterization of the intracellular effectors of the antiproliferative activity of somatostatin in PC Cl3 thyroid cells. Somatostatin inhibited PC Cl3 cell proliferation through the activation of a membrane phosphotyrosine phosphatase. Conversely, PC Cl3 cells stably expressing the v-mos oncogene (PC mos) were completely insensitive to the somatostatin antiproliferative effects since somatostatin was unable to stimulate a phosphotyrosine phosphatase activity. In PC mos cells basal phosphotyrosine phosphatase activity was also reduced, suggesting that the expression of a specific phosphotyrosine phosphatase was impaired in these transformed cells. We suggested that this phosphotyrosine phosphatase could be r-PTP{eta} whose expression was abolished in the PC mos cells. To directly prove the involvement of r-PTP{eta} in somatostatin’s effect, we stably transfected this phosphatase in PC mos cells. This new cell line (PC mos/PTP{eta}) 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{eta} did not restore the antiproliferative effects of somatostatin. PC mos/PTP{eta} 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{eta} in somatostatin receptor signal transduction was demonstrated by measuring its specific activity after somatostatin treatment in an immunocomplex assay. Somatostatin highly increased r-PTP{eta} activity in PCCl3 and PC mos/PTP{eta} (+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{eta} 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{eta} which, through the inhibition of MAPK activity, causes the stabilization of the cell cycle inhibitor p27kip1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SOMATOSTATIN (SST) IS a highly expressed peptide hormone involved in a wide range of biological activities, including inhibition of pituitary and gastrointestinal hormone secretion and modulation of neurotransmitter release in the brain (1, 2).

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{eta} on the basis of its homology with the human DEP-1/HPTP{eta} 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{eta} 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{eta} gene is induced by TSH in normal thyroid cells and correlates with their differentiation state; r-PTP{eta} 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{eta} 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{eta}, 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{eta} (28); 2) PC mos/PTP{eta} (PC mos cells in which r-PTP{eta} has been stably retransfected) (34); and 3) PC mos/PTP{eta}[C/S] (PC mos cells in which a catalytically inactive mutant r-PTP{eta} has been stably transfected) (34).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Figure 1AGo shows Northern blot analysis of the expression of the mRNA for r-PTP{eta}, in the PC Cl3, PC mos, and PC mos/PTP{eta}. R-PTP{eta} 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{eta} the cDNA for the phosphatase is transfected in the PC mos cells and the expression of r-PTP{eta} was restored. Equal RNA loading was demonstrated by evaluation of the GAPDH mRNA content (Fig. 1AGo). Similar results were observed in Western blotting experiments using an {alpha}–DEP-1 antibody (the human form of r-PTP{eta}) (Fig. 1BGo). 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. 1BGo).



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Figure 1. Identification of mRNA and Protein Content of r-PTP{eta} in the Different Cell Lines

A, Expression of mRNA for r-PTP{eta} in PCCL3 (lane 1), PC mos (lane 2), and PC mos/PTP{eta} (lane 3) (upper panel) by means of Northern blot analysis. Equal loading of RNA was confirmed after rehybridization of the filter with a GAPDH probe (lower panel). B, Protein expression of r-PTP{eta} in PCCL3 (lane 1), PC mos (lane 2), and PC mos/PTP{eta} (lane 3) (upper panel) and SHP2 (lower panel).

 
The proliferative characteristics of PC Cl3, PC mos, and PC mos/PTP{eta} 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 1Go 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{eta} 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{eta} restored the responsiveness to the growth factors (insulin and insulin+TSH), which showed a synergistic activity on the proliferation of these cells (Table 1Go).


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Table 1. Time Course of the Proliferative Effects of TSH, Insulin, and TSH + Insulin in the PC Cl3, PC mos, and PC mos/PTP{eta} Cell Lines, Evaluated Using the MTT Test

 
The effects of SST on both insulin- and insulin+TSH-stimulated cell proliferation were also studied using the [3H]thymidine incorporation assay. In PC Cl3 cells, in agreement with previous data (5, 32), both proliferative stimuli were suppressed by SST treatment while no SST effects were observed in the v-mos transformed cells (Fig. 2aGo). The expression of r-PTP{eta} in the PC mos/PTP{eta} cells completely restored SST inhibition of cell proliferation (Fig. 2AGo).



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Figure 2. Somatostatin Effects on Cell Proliferation

A, Effect of somatostatin (SST) (1 µM) on insulin (100 nM)- and insulin (100 nM)+TSH (10 nM)-stimulated cell proliferation evaluated by means of the [3H]thymidine incorporation assay after 24 h of treatment. Somatostatin inhibition of proliferation, observed in PC Cl3 stimulated with the two growth factors, is abolished in v-mos-transformed cells while in the PC mos/PTP{eta} cells the antiproliferative effect of somatostatin is completely recovered. Data are expressed as percentage of basal [3H]thymidine incorporation activity. Basal values were (counts per min/50,000 cells): PC Cl3 = 367 ± 23; PC mos = 7,893 ± 289; PC mos/PTP{eta} = 4,245 ± 394. {square}{square} = P < 0.01 vs. respective basal values; ** = P < 0.01 vs. respective insulin- or insulin+TSH-stimulated values. B, Expression of SST receptor subtypes in PC Cl3, PC mos, and PC mos/PTP{eta} evaluated by means of RT-PCR. The pattern of expression of the receptors was not changed by v-mos or r-PTP{eta} expression. The number of the lanes corresponds to the SST receptor subtype; "A" represents amplification of a ß-actin cDNA fragment, used as internal control.

 
The lack of responsiveness to SST in the PC mos cells was not due to the absence of the expression of the SSTRs since all the cloned SSTRs (SSTR1–5) were expressed in the PC mos cells as well as in the PC Cl3 cells (Fig. 2BGo). A similar pattern of expression was also observed in the PC mos cells transfected with the cDNA encoding for r-PTP{eta} (Fig. 2BGo).

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{eta}) and the transformed cells transfected with r-PTP{eta} cDNA. The data reported in Figs. 3Go and 4Go, 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{eta} 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{eta} was specifically due to r-PTP{eta} 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{eta} (PC mos/PTP{eta}[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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Figure 3. Time Course of the Antiproliferative Effects of SST on PC Cl3, PC mos, and PC mos/PTP{eta} Cell Growth Induced by Insulin+TSH, Evaluated Using the MTT Test

The expression of the mos oncogene caused an abolishment of the sensitivity to SST, while the transfection of r-PTP{eta} favored the restoration of the inhibitory response to SST. Data are expressed as percentage of the absorbance values measured at time 0. **, P < 0.01 vs. respective TSH+insulin values.

 


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Figure 4. Dose-Response (10 nM-10 µM) of SST on Insulin+TSH-Stimulated DNA Synthesis in PC Cl3, PC mos, PC mos/PTP{eta}, and PC mos/PTP{eta} [C/S] Cell Lines

Data are expressed as percentage of basal [3H]thymidine incorporation activity (dotted line). Basal values (counts per min/50,000 cells) were: PC Cl3 = 617 ± 13; PC mos = 8,593 ± 392; PC mos/PTP{eta} = 4,511 ± 409; PC mos/PTP{eta} [C/S] = 7,665 ± 523. *, P < 0.05; and **, P < 0.01 vs. respective TSH+insulin values.

 
Almost all the physiological effects mediated by SSTR are dependent on the activation of a pertussis toxin (PTX)-sensitive GTP-binding protein (for review see ref. 35). Therefore, we also tested the role of G proteins in the antiproliferative activity of SST in the different cell lines. PTX pretreatment (180 ng/ml, 18 h) completely reverted the inhibitory effects of SST in PC Cl3 and PC mos/PTP{eta} cells (Fig. 5Go), 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. 5Go). These data show that the antiproliferative effects of SST in these thyroid cell lines were correlated with the expression of r-PTP{eta}. 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. 6Go, 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{eta} was completely down-regulated, vanadate did not modify the basal activity, suggesting the loss of cell proliferation control by a PTP, likely r-PTP{eta}. In addition, no effects of SST or vanadate were observed in TSH+insulin-stimulated conditions. In PC mos/PTP{eta} 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{eta}. 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. 6Go). Using the synthetic substrate p-nitrophenylphosphate (pNPP), we directly measured the SST-inducible total membrane PTP activity in the different cell lines (Fig. 7Go). The expression of the oncogene v-mos in the PC Cl3, which caused a down-regulation of r-PTP{eta} 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{eta} 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{eta} in the PC mos cells totally restored the stimulation of a membrane PTP activity by SST (Fig. 7Go). The PC mos/PTP{eta}[C/S] cells, which express the catalytically inactive mutant of r-PTP{eta}, showed the same pattern of response as the PC mos cells (low basal level and lack of response to SST) (Fig. 7Go). Moreover, we directly evaluated the specific r-PTP{eta} 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. 1BGo), 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 {alpha}SHP2 and {alpha}PTP{eta} 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{eta} and SHP2 proteins in the pNPP hydrolysis assay. As shown in Fig. 8CGo, the level of SHP2 immunoprecipitated was similar in all the cell lines and for all the treatments. As expected, no r-PTP{eta} was immunoprecipitated in PC mos cells, although the level of protein detected in the other cell lines was comparable for all the treatments (Fig. 8CGo). The PTP activity experiments showed that r-PTP{eta} activity was increased after only 10 min of SST treatment (+300%) in both PC Cl3 and PC mos/PTP{eta} 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. 8AGo). As expected, no r-PTP{eta} specific activity was detected in the PC mos cell line (Fig. 8AGo). 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. 8BGo).



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Figure 5. Involvement of PTX-Sensitive G Protein on the Activity of SST in PCCL3, PC mos, and PCmos/PTP{eta}

Cells were pretreated with the PTX (180 ng/ml) for 18 h and then treated for 24 h with TSH and insulin in the presence or absence of SST. Data are expressed as percentage of TSH+insulin-stimulated conditions. Basal values were (counts per min/50,000 cells): PC Cl3 = 524 ± 43; PC mos = 8,913 ± 779; PC mos/PTP{eta} = 5,655 ± 416. **, P < 0.01 vs. respective TSH+insulin values.

 


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Figure 6. Effect of Vanadate (25 µM) Pretreatment on SST Inhibition of PC Cl3 (upper panel), PC mos (middle panel) and PC mos/PTP{eta} (lower panel) Cell Proliferation

Data are expressed as percentage of basal [3H]thymidine incorporation activity. °, P < 0.05; and °°, P < 0.01 vs. respective basal values; ##, P < 0.01 vs. respective insulin value; ++, P < 0.01 vs. respective insulin+TSH values.

 


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Figure 7. Total PTP Activity in Membrane Preparations Derived from PC Cl3, PC mos, PC mos/PTP{eta}, and PC mos/PTP{eta}[C/S] cells in the Presence or Absence of SST (1 µM, 1 h), Measured Evaluating The Hydrolysis of the Synthetic Substrate pNPP, in a Spectrophotometric Assay

**, P < 0.01 vs. respective basal values; °, P < 0.05 vs. PC Cl3 basal value.

 


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Figure 8. Somatostatin-Specific Activation of r-PTP{eta} and SHP2

A, r-PTP{eta} specific activity in PC Cl3, PC mos, and PC mos/PTP{eta} after 10 min or 1 h of treatment with vehicle or insulin (100 nM)+TSH (10 nM) in the presence or absence of SST (1 µM). After the treatment 250 µg of total cell lysate were immunoprecipitated with {alpha}-r-PTP{eta} antibody, and the immunocomplexes, isolated using protein A-coated magnetic beads (Dynabeads), were tested in the PTP assay. The hydrolysis of pNPP, used as substrate, was detected in a spectrophotometric assay (O.D. 410 nm). B, SHP2 specific activity in PC Cl3, PC mos, and PC mos/PTP{eta} in basal conditions or after 10 min or 1 h of stimulation with insulin (100 nM)+TSH (10 nM) in the presence or absence of SST (1 µM). After the treatment, 250 µg of total cell lysate were immunoprecipitated with {alpha}-SHP2 antibody, and the immunocomplexes, isolated using protein A-coated magnetic beads (Dynabeads), were tested in the PTP assay. The hydrolysis of pNPP, used as substrate, was detected in a spectrophotometric assay (O.D. 410 nm). C, Western blot (using {alpha}-r-PTP{eta} or {alpha}-SHP2 antibodies) on an aliquot of the immunoprecipitated proteins, to compare the amount of specific proteins used in the PTP assay. *, P < 0.05 and ** = P < 0.01 vs. respective basal values.

 
It was previously reported that r-PTP{eta} 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{eta}. 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. 9aGo). The nuclear staining was greatly reduced after TSH+insulin treatment (Fig. 9bGo). 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. 9cGo). 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. 9Go, d, e, and f). The expression of r-PTP{eta} 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. 9Go, g, h, and i). Similar results were obtained in Western blot experiments using a p27kip1 selective antibody (Fig. 10Go). In PC Cl3 and PC mos/PTP{eta} 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. 10Go), 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{eta} cells) proliferation was completely blocked (data not shown).



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Figure 9. Detection of Nuclear Presence of p27kip1 by Immunofluorescence Using a Polyclonal Antibody in PC Cl3 (a, b, c), PC mos (d, e, f), and PC mos/PTP{eta} (g, h, i)

Panel a, Immunoreactivity for p27kip1 in PC Cl3 cells after a 24-h starvation. p27kip1 nuclear expression is greatly reduced after treatment with insulin+TSH (panel b), while treatment with SST restored basal levels of p27kip1 (panel c). In G0/G1 synchronyzed PC mos cells p27kip1 was not detectable in the nucleus (/panel d) as it was not after the treatment with the growth factors (panel e) and after the addition of somatostatin (panel f). The expression of r-PTP{eta} is responsible for the nuclear induction of p27kip1 in 24-h starved PC mos/PTP{eta} cells (panel g). p27kip1 positive cells are less than 20% after growth factor stimulation (panel h), while a significant immunostaining for p27kip1 was observed when treated with SST (panel i).

 


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Figure 10. Expression of p27kip1, in the Different Cell Lines, Measured by Western Blot Analysis

1, Untreated (control) cells; 2, insulin (100 nM)+TSH (10 nM) treatment for 4 h; 3, insulin+TSH+SST (1 µM) treatment for 4 h; 4, insulin+TSH+PD98059 (10 µM) treatment for 4 h; 5, insulin+TSH+PSI (10 µM) treatment for 4 h.

 
To identify the possible level of action of r-PTP{eta} 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{eta} cells were treated for 5 min with 100 nM insulin in the presence or absence of 1 µM SST and then immunoprecipitated with {alpha}-IRß antibody and evaluated with {alpha}-phosphotyrosine antibody in Western blot (Fig. 11Go), 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. 12Go). In PC Cl3 cells insulin+TSH increased MEK phosphorylation while SST completely blocked this effect. Conversely, in PC mos and PC mos/PTP{eta} 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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Figure 11. Reversal by SST of the Insulin-Dependent Tyrosine Phosphorylation of the IRß-Subunit in PC Cl3, PC mos, and PC mos/PTP{eta} Cell Lines, Measured in Western Blot after Immunoprecipitation, as Indicated

C, Untreated (control) cells; I, insulin (100 nM); I+S, insulin + SST (1 µM).

 


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Figure 12. Somatostatin Regulation of MEK and ERK1/2 Activities

A, Effect of SST treatment on TSH+insulin-dependent MEK (left panels) and ERK1/2 (right panels) phosphorylation in PC Cl3, PC mos, and PC mos/PTP{eta} cell lines. B, Normalization of the results obtained with the phospho-specific antibodies, evaluating total MEK and ERK1/2 expression in the different cell lines. C, Untreated (control) cells; TI, TSH (10 nM)+insulin (100 nM) treatment for 10 min.; TIS, TSH+insulin+SST (1 µM) treatment for 10 min.

 
As far as ERK1/2 activation is concerned, in PC Cl3 cells (as well as in PC mos/PTP{eta} 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. 12Go).

Interestingly, other signal transduction systems were not recovered after r-PTP{eta} expression in the PC mos cells. Indeed, similarly to the transformed cells, in the PC mos/PTP{eta} the treatment with TSH did not induce an increase in intracellular calcium concentration ([Ca++]i), as observed in the w.t. PC Cl3 (Fig. 13Go).



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Figure 13. Microfluorimetric Analysis of the Increase in [Ca++]i Elicited by TSH, using the Fluorescent Probe Fura 2

In the graph are reported the mean values of at least 10 cells, individually analyzed. SE was less than 5% for all the values. The expression of the mos oncogene completely abolished the TSH-induced calcium increase, and the coexpression of r-PTP{eta} did not modify this effect.

 
Moreover, the specificity of the coupling of r-PTP{eta} to the 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. 14Go show that, irrespectively to the expression of r-PTP{eta}, 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{eta} cell lines were almost superimposable, showing that the expression of r-PTP{eta} 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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Figure 14. Effect of Different TGFß1 Concentrations (1, 5, and 10 nM) on the DNA Synthesis Induced by Insulin+TSH Stimulation, Evaluated by Means of [3H]Thymidine Uptake Assay

Data are expressed as percentage of the TSH+insulin stimulation of DNA synthesis. *, P < 0.05; and **, P < 0.01 vs. respective TSH+insulin values.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we have characterized the role of the receptor-like PTP, r-PTP{eta}, 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{eta}, while other PTPs, such as PTPµ (32) or SHP2 (present paper) are unaffected. r-PTP{eta} 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{eta}, DEP-1/HPTP{eta}, 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{eta} 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{eta} since the transfection of another receptor-like PTP, PTP{gamma}, did not cause the reversion of the transformed phenotype (34). In this paper we evaluated the involvement of r-PTP{eta} 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{eta}. Indeed, in the PC mos/PTP{eta} cells a complete recovery of the SST control of cell proliferation was observed. Conversely, the transfection of a catalytically inactive mutant of r-PTP{eta} in the PC mos cells did not cause any recovery of the antiproliferative effects of SST. In PC mos/PTP{eta} cells, the expression of r-PTP{eta} 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{eta} was absolutely necessary for the SSTergic PTP-mediated control of cell proliferation. Indeed, the transfection of r-PTP{eta} 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{eta}-expressing cell line. Immunocomplex assays showed that this activity was mainly ascribable to r-PTP{eta}, 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{eta} was absolutely necessary for such an effect.

Interestingly, although PC mos/PTP{eta} 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{eta} 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{eta}. Similarly the transfection of r-PTP{eta} 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{eta} 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{eta} was shown to represent a possible regulator of p27kip1 expression (34). Here we report that the activation of r-PTP{eta} 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{eta}, after SST stimulation. We found that in all the cell lines (including the PC mos cells that do not express r-PTP{eta}), 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{eta}. Interestingly, the recombinant human homolog of r-PTP{eta}, 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{eta} 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{eta}. 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{eta} cells clearly suggests that the activity of r-PTP{eta} should be downstream from MEK. Indeed, we found that, similar to w.t. PC Cl3 cells, in PC mos/PTP{eta}, 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{eta} 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{eta} 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{eta} (Fig. 15Go).



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Figure 15. Diagrammatic Representation of the Intracellular Mechanisms by Which SST, via the Activation of r-PTP{eta} or SHP2, Is Able to Control the Proliferation of PC Cl3 Cells

The exact mechanism linking the SST receptor to the PTPs, involving a PTX-sensitive G protein, is still to be identified. Although the r-PTP{eta} substrate has not yet been identified (the possible pathways are reported as dotted arrows), the activation of this PTP by SST resulted in a dephosphorylation of ERK1/2, also in conditions of constitutive activation of MEK (due to the activity of v-mos). We propose that in the w.t. PC Cl3 cells, SST inhibits the insulin-dependent activation of the ERK1/2 cascade, acting both at level of the insulin receptor (likely via SHP2) and via r-PTP{eta} causing a dephosphorylation of ERK1/2. The blockade of ERK cascade results in an inhibition of the degradation of the CDK inhibitor p27kip1 and cell cycle arrest. The red arrows indicate the substrates of the activity of the signal transduction inhibitors used in this paper.

 
In conclusion, in this paper we demonstrate that in the PC Cl3 thyroid cell line, SST is able to activate the receptor-like PTP r-PTP{eta}, 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{eta} 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
SST, PD98059, ZIE [Ot-Bu]-A-leucinal (PSI), and TGFß1were purchased from Calbiochem (Lucerne, Switzerland), and vanadate was obtained from ICN Biochemicals, Inc. (Cleveland, OH); all other reagents were from Sigma (Milano, Italy) unless otherwise specified.

Antibodies
For r-PTP{eta} detection, we used antibodies raised against the intracellular region of r-PTP{eta} 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{eta} 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{eta}[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 {alpha}-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{eta}, 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.


    ACKNOWLEDGMENTS
 
We would like to thank D. Noonan for scientific discussion.


    FOOTNOTES
 
This work was supported by grants from Consiglio Nazionale delle Ricerche 98.03031.Ct04 and 99.02482.Ct04 (to T.F. and Italian Association for Cancer Research (AIRC, 1999) and contract QRTL-1999–00908 by the European Union (to G.S.)

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.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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