Neuronal nitric oxide synthase: a substrate for SHP-1 involved in sst2 somatostatin receptor growth inhibitory signaling.

Somatostatin receptor sst2 is an inhibitory G protein-coupled receptor, which inhibits normal and tumor cell growth by a mechanism involving the tyrosine phosphatase SHP-1. We reported previously that SHP-1 associates transiently with and is activated by sst2 and is a critical component for sst2 growth inhibitory signaling. Here, we demonstrate that in Chinese hamster ovary cells expressing sst2, SHP-1 is associated at the basal level with the neuronal nitric oxide synthase (nNOS). Following sst2 activation by the somatostatin analog RC-160, SHP-1 rapidly recruits nNOS tyrosine dephosphorylates and activates it. The resulting NO activates guanylate cyclase and inhibits cell proliferation. Coexpression of a catalytically inactive SHP-1 mutant with sst2 blocks RC-160-induced nNOS dephosphorylation and activation, as well as guanylate cyclase activation. In mouse pancreatic acini, RC-160 treatment reduces nNOS tyrosine phosphorylation accompanied by an increase of its activity. By opposition, in acini from viable motheaten (mev/mev) mice, which express a markedly inactive SHP-1, RC-160 has no effect on nNOS activity. Finally, expression of a dominant-negative form of nNOS prevents both RC-160-induced p27 up-regulation and cell proliferation inhibition. We therefore identified nNOS as a novel SHP-1 substrate critical for sst2-induced cell-growth arrest.

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs.

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.

Inhibition of growth and metastatic progression of pancreatic carcinoma in hamster after somatostatin receptor subtype 2 (sst2) gene expression and administration of cytotoxic somatostatin analog AN-238.

The sst2 somatostatin receptor mediates the antiproliferative effects of somatostatin analogs. The present study demonstrates that stable expression of sst2 in the hamster pancreatic cancer cells PC-1 and PC-1.0 activates an autocrine negative loop leading to an in vitro inhibition of cell proliferation. In vivo studies conducted in Syrian golden hamsters after orthotopic implantation of PC-1.0 cells showed that both tumor growth and metastatic progression of allografts containing 100% of sst2-expressing cells were significantly inhibited for up to 20 days after implantation, as compared with control allografts that did not express sst2. A local antitumor bystander effect was observed after induction of mixed tumors containing a 1:3 ratio of sst2-expressing cells to control cells. Tumor volume and incidence of metastases of mixed tumors were significantly reduced at day 13 post implantation. This effect decreased with time as at day 20, growth of mixed tumors was similar to that of control tumors. After administration of the cytotoxic somatostatin conjugate AN-238 on day 13, antitumor bystander effect observed in mixed tumors was significantly extended to day 20. We also observed that in vitro invasiveness of sst2-expressing PC-1.0 cells was significantly reduced. Tyrosine dephosphorylation of E-cadherin may participate in restoring the E-cadherin function, reducing in turn pancreatic cancer cell motility and invasiveness. This dephosphorylation depends on the tyrosine phosphatase src homology 2-containing tyrosine phosphatase 1 (SHP-1) positively coupled to sst2 receptor. The inhibitory effect of sst2 gene expression on pancreatic cancer growth and invasion combined with chemotherapy with targeted cytotoxic somatostatin analog administration provides a rationale for a therapeutic approach to gene therapy based on in vivo sst2 gene transfer.

The activation of neuronal NO synthase is mediated by G-protein betagamma subunit and the tyrosine phosphatase SHP-2.

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L-NAME and alpha-guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK-induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP-2 mutant and the betaARK1 (495-689) sequestrant peptide, indicating the requirement of activated SHP-2 and G-betagamma subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP-2 activity) revealed that in response to CCK-treatment, SHP-2 associated to G-beta1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.

sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1.

Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1 cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27(Kip1). Consequently, a decrease of p27(Kip1)-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21(Waf1/Cip1). When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27(Kip1) and prevented modulation by insulin and RC-160 of p27(Kip1) expression, p27(Kip1)-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27(Kip1) induced by a mitogen, and this effect did not occur in acini from viable motheaten (mev/mev) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27(Kip1) and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27(Kip1) and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27(Kip1).

Gene therapy for pancreatic carcinoma: local and distant antitumor effects after somatostatin receptor sst2 gene transfer.

Human pancreatic adenocarcinomas lose the ability to express sst2, the somatostatin receptor, which mediates the antiproliferative effect of somatostatin. Reintroducing sst2 into human pancreatic cancer cells by stable expression evokes an autocrine negative feedback loop leading to a constitutive activation of the sst2 gene and an inhibition of cell proliferation and tumorigenicity. In vivo studies have been conducted in athymic mice to investigate the antitumor bystander effects resulting from the transfer of the sst2 gene into human pancreatic cancer cell line BxPC-3. In mixing experiments, a local bystander effect was observed: mixed tumors containing a ratio of sst2-expressing cells to control cells of 25:75, 50:50, and 75:25 grew with a time delay of 31, 44, and 50 days, respectively, when compared with control tumors derived from control cells. Tumors containing 100% sst2-expressing cells remained quiescent for up to 80 days. A significant increase in apoptosis and a decrease in the Ki67 index were detected in mixed and sst2 tumor when compared with control tumors. In combined experiments, mice were separately xenografted with control cells on one flank and with sst2-expressing cells on the other flank. A distant antitumor effect was induced: growth of control tumors was delayed by 33 days, the Ki67 index decreased significantly, and apoptosis increased when compared with control tumors that grew alone. The distant bystander effect may be explained in part by a significant increase in serum somatostatin-like immunoreactivity levels resulting from the autocrine feedback loop produced by sst2-expressing cells and inducing an upregulation of the type 1 somatostatin receptor, sst1, which also mediates the antiproliferative effect of somatostatin. In conclusion, the local and distant antitumor bystander effects obtained in this experimental model suggest that sst2 gene transfer may represent a new therapy for pancreatic cancer.

sst2 somatostatin receptor mediates negative regulation of insulin receptor signaling through the tyrosine phosphatase SHP-1.

We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulin-induced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.

Expression of somatostatin receptor SST4 in human placenta and absence of octreotide effect on human placental growth hormone concentration during pregnancy.

In pregnancy, the human placenta GH acts as a growth-promoting hormone and appears to be the main stimulator of insulin-like growth factor I (IGF-I) secretion. In a woman with a TSH-secreting macroadenoma, successful treatment with the somatostatin analog octreotide was conducted during the first month and the second half of pregnancy without side-effects on placental and fetal development. As observed in normal pregnancy, both serum placental GH and IGF-I levels increased throughout pregnancy and dropped sharply after delivery. In placental membranes from both treated and healthy untreated patients, we demonstrated the presence of high affinity binding sites for somatostatin-14 (Kd, 4.6 and 5.3 nmol/L; binding capacity, 1.53 and 1.35 pmol/mg protein, respectively). These receptors displayed low affinity for octreotide (IC50, 1.2-2 mumol/L), suggesting the presence of SST1 and/or SST4 receptors. We found that messenger ribonucleic acids of these two subtypes were expressed in both human placental tissue and purified human cytotrophoblast cells. Finally, the SST1-selective analog, des-AA1,2,5[D-Trp8,IAmp9]S-14 had low affinity for placental somatostatin receptors. These results argue in favor of the presence of the SST4 subtype in human placenta. At the doses administered, octreotide did not bind to placental somatostatin receptors. Our results may explain the absence of changes in both human placental GH and IGF-I concentrations that we observed during octreotide treatment.

The tyrosine phosphatase SHP-1 associates with the sst2 somatostatin receptor and is an essential component of sst2-mediated inhibitory growth signaling.

Activation of the somatostatin receptor sst2, a member of the Gi protein-coupled receptor family, results in the stimulation of a protein-tyrosine phosphatase activity involved in the sst2-mediated growth inhibitory signal. Here, we report that SHP-1, a cytoplasmic protein-tyrosine phosphatase containing two Src homology 2 domains constitutively associated with sst2 as evidence by coprecipitation of SHP-1 protein with sst2, in Chinese hamster ovary cells coexpressing sst2 and SHP-1. Activation of sst2 by somatostatin resulted in a rapid dissociation of SHP-1 from sst2 accompanied by an increase of SHP-1 activity. SHP-1 was phosphorylated on tyrosine in control cells and somatostatin induced a rapid and transient dephosphorylation on tyrosine residues of the enzyme. Stimulation of SHP-1 activity by somatostatin was abolished by pertussis toxin pretreatment of cells. Gialpha3 was specifically immunoprecipitated by anti-sst2 and anti-SHP-1 antibodies, and somatostatin induced a rapid dissociation of Gialpha3 from sst2, suggesting that Gialpha3 may be involved in the sst2.SHP-1 complexes. Finally, somatostatin inhibited the proliferation of cells coexpressing sst2 and SHP-1, and this effect was suppressed in cells coexpressing sst2 and the catalytic inactive SHP-1 (C453S mutant). Our data identify SHP-1 as the tyrosine phosphatase associated with sst2 and demonstrate that this enzyme may be an initial key transducer of the antimitogenic signaling mediated by sst2.

Characterization of the antiproliferative signal mediated by the somatostatin receptor subtype sst5.

We investigated cell proliferation modulated by cholecystokinin (CCK) and somatostatin analogue RC-160 in CHO cells bearing endogenous CCKA receptors and stably transfected by human subtype sst5 somatostatin receptor. CCK stimulated cell proliferation of CHO cells. This effect was suppressed by inhibitor of the soluble guanylate cyclase, LY 83583, the inhibitor of the cGMP dependent kinases, KT 5823, and the inhibitor of mitogen-activated protein (MAP) kinase kinase, PD 98059. CCK treatment induced an increase of intracellular cGMP concentrations, but concomitant addition of LY 83583 virtually suppressed this increase. CCK also activated both phosphorylation and activity of p42-MAP kinase; these effects were inhibited by KT 5823. All the effects of CCK depended on a pertussis toxin-dependent G protein. Somatostatin analogue RC-160 inhibited CCK-induced stimulation of cell proliferation but it did not potentiate the suppressive effect of the inhibitors LY 83583 and KT 5823. RC-160 inhibited both CCK-induced intracellular cGMP formation as well as activation of p42-MAP kinase phosphorylation and activity. This inhibitory effect was observed at doses of RC-160 similar to those necessary to occupy the sst5 recombinant receptor and to inhibit CCK-induced cell proliferation. We conclude that, in CHO cells, the proliferation and the MAP kinase signaling cascade depend on a cGMP-dependent pathway. These effects are positively regulated by CCK and negatively influenced by RC-160, interacting through CCKA and sst5 receptors, respectively. These studies provide a characterization of the antiproliferative signal mediated by sst5 receptor.

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells.

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.

Molecular mechanisms of antiproliferative effect of somatostatin: involvement of a tyrosine phosphatase.

A protein of 66 kd immunoreactive to anti-tyrosine phosphatase (PTP1C) antibodies coeluted with, and so may be associated with, somatostatin receptors (ssts) from rat pancreatic membranes. Also, anti-PTP1C antibodies immunoprecipitated functional ssts from pancreatic membranes, suggesting a PTP1C protein can associate with ssts at the membrane level. Somatostatin analog RC 160 had good affinity for sst2,3 and sst5 (IC50 = 0.2, 0.1, and 21 nmol/L) and low affinity for sst1 and sst4 (IC50 = 200 and 620 nmol/L), and induced rapid dose-dependent stimulation of PTP activity (maximal at 1 nmol/L and half maximal at 5 pmol/L) in NIH3T3 and CHO cells expressing sst2, with similar results for sst1, but no stimulation with sst3,4 or sst5. Treatment of cells expressing sst2 with RC 160 for 24 hours inhibited serum- or growth factor-induced cell proliferation dose-dependently (maximal at 1 nmol/L, half maximal at 6 to 53 pmol/L RC 160). In cells expressing sst1, weak inhibition of fibroblast growth factor 2-induced NIH3T3 cell proliferation was provoked by somatostatin analogs (> 10 nmol/L). The good correlation between inhibition of somatostatin binding, stimulation of PTP activity, and inhibition of cell proliferation implicates a PTP in growth inhibition mediated by sst2 and sst1.

Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells.

The somatostatin receptor subtype sst2 mediates both activation of a tyrosine phosphatase activity and inhibition of cell proliferation induced by somatostatin analogues. In the absence of exogenous ligand, expression of sst2 in NIH 3T3 cells resulted in inhibition of cell growth. Polymerase chain reaction coupled to reverse transcription demonstrated that expression of sst2 in NIH 3T3 cells stimulated the expression of preprosomatostatin mRNA accompanied by a production of immunoreactive somatostatin-like peptide which corresponded predominantly to somatostatin 14. Moreover anti-somatostatin antibodies suppressed sst2-promoted inhibition of cell proliferation. Inhibition of cell proliferation associated with increased secretion of somatostatin-like immunoreactivity was also observed after expression of sst2 in human pancreatic tumor cells BxPC3 devoid of endogenous receptors. In addition, expression of sst2 in NIH 3T3 cells was associated with constitutive activation of tyrosine phosphatase PTP1C that resulted from enhanced expression of the protein. Blocking of PTP1C tyrosine phosphatase activity with orthovanadate or that of PTP1C protein with antisense PTP1C oligonucleotides decreased the sst2-induced inhibition of cell proliferation. These results, taken together, show that expression of sst2 in NIH 3T3 cells generated a negative autocrine loop by stimulating sst2 ligand production and amplifying PTP1C sst2-transducer. Sst2/ligand may function as a determinant factor involved in the negative growth control of cells.

Regulation of the src homology 2 domain-containing protein tyrosine phosphatase PTP1C by glucocorticoids in rat pancreatic AR42J cells.

The effect of glucocorticoids, known to induce inhibition of growth and differentiation of pancreatic cells, has been examined on the tyrosine phosphatase containing two src homology 2 domains, PTP1C, in rat pancreatic cancer AR42J cells. Immunoblotting analysis revealed that PTP1C protein was present in AR42J cells as two PTP1C species of 66 and 31 kilodaltons (kDa), the 31-kDa species representing a proteolytic product of the larger form. Dexamethasone increased the level of the two PTP1C species by 2 to 3 times. Nearly 80% of the PTP1C molecules were found in the particulate fraction in control cells and dexamethasone did not change the distribution of PTP1C. The increase of PTP1C protein was also detected by immunohistochemical analysis. Dexamethasone increased the tyrosine phosphatase activity of immunoprecipitated PTP1C. In addition, dexamethasone raised the level of expression of PTP1C messenger RNA in a time- and dose-dependent manner in relation with its effect on cell growth and differentiation. This effect was selective, the messenger RNA levels of the other tyrosine phosphatase containing two src homology 2 domains (SH2), PTP1D, and that of the cytosolic PTP1 being not affected. This is the first report of glucocorticoid increase of PTP1C expression, suggesting that PTP1C may be involved in the glucocorticoid-mediated pancreatic cell differentiation.

Somatostatin receptor-effector system in rat pancreatic acinar membranes after subtotal enterectomy.

In the present study we found that exocrine pancreatic hyperplasia observed after proximal small bowel resection is accompanied by an increase in pancreatic somatostatin (SS) content at 1 mo and an increase in the number of SS receptors at 2 wk and 1 mo after intestinal surgery. At 6 mo after small bowel resection SS content and SS receptors had returned to control values. However, the original increase in SS receptor number was accompanied by a decrease in the ability of SS to inhibit forskolin-stimulated adenylyl cyclase (AC) activity. In addition, the ability of 5'-guanylylimidodiphosphate (a nonhydrolyzable GTP analogue) to inhibit SS receptor binding was decreased in pancreatic acinar membranes from enterectomized rats at 2 wk and 1 mo after jejunoileal resection. These data suggest that there is an abnormality in the integrity of SS receptor binding site-G protein interactions and would explain the decreased inactivation of AC by SS at 2 wk and 1 mo after proximal small bowel resection.

Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms.

Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.

A tyrosine phosphatase is associated with the somatostatin receptor.

Regulation of tyrosine phosphorylation is thought to be an essential step in signal transduction mechanisms that mediate cellular responses. In pancreatic tumour cells we demonstrated that somatostatin analogues inhibited cell proliferation and stimulated a membrane protein tyrosine phosphatase (PTP) activity at concentrations at which they bind to the somatostatin receptor. To elucidate the role of PTP in the signal transduction pathway activated by somatostatin receptors we first studied the interaction of PTP with the somatostatin receptor at the membrane. We purified somatostatin receptors by immunoaffinity from pancreatic membranes that strongly expressed the type 2 somatostatin receptor sstr2. We identified the receptor as an 87 kDa protein. We demonstrated that a PTP activity co-purified with somatostatin receptors. The PTP was identified as a 66 kDa protein immunoreactive to antibodies against SHPTP1. These antibodies immunoprecipitated somatostatin receptors either occupied or unoccupied by ligand indicating that SHPTP1 is associated with somatostatin receptors. We then expressed sstr2A in monkey kidney COS-7 cells and mouse NIH/3T3 fibroblasts and demonstrated that somatostatin analogues (RC 160, octreotide and BIM 23014) which exhibited high affinity for sstr2 stimulated a PTP activity and inhibited cell proliferation in proportion to their affinities for sstr2. Under the same conditions these analogues have no effect on the growth of cells expressing sstr1. All these results suggest that a PTP related to SHPTP1 is associated with somatostatin receptors and may be involved in the negative growth signal promoted by sstr2.

Co-purification of a protein tyrosine phosphatase with activated somatostatin receptors from rat pancreatic acinar membranes.

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Up-regulation of somatostatin receptors by epidermal growth factor and gastrin in pancreatic cancer cells.

Interactions between growth factor receptor systems may be important in the regulation of cell growth. The proliferation of pancreatic tumor AR42J cells has been shown to be stimulated by Epidermal growth factor (EGF) and gastrin and inhibited by somatostatin. To analyze the interaction between these different peptides, we explored the influence of EGF and gastrin on the somatostatin receptors. Treatment of AR42J cells with 10 nM EGF or gastrin for 24 hr increased specific binding of [125I] Tyr3SMS to 131 and 147% of that in control cells, respectively. The effect of peptides on [125I]Tyr3SMS binding was time- and dose-dependent, with half-maximal effect at 0.2 +/- 0.03 nM EGF and 0.3 +/- 0.15 nM gastrin. Scatchard plots revealed an increase in somatostatin receptor number of 27 and 80% after 48 hr of treatment with EGF and gastrin, respectively, without any change in receptor affinity. The increase in somatostatin receptor density was accompanied by the enhancement of biological responses to somatostatin. In cells pretreated with EGF or gastrin, the potency of somatostatin for inhibiting vasoactive intestinal peptide-stimulated cAMP content was increased 2-fold as that of somatostatin analog, SMS, for inhibiting cell proliferation. Furthermore, the efficiency of SMS as antiproliferative agent was greatly increased. Vasoactive intestinal peptide or forskolin did not modify [125I]Tyr3SMS binding of control or treated cells. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not affect [125I]Tyr3SMS binding. On the other hand, cycloheximide completely blocked the increase in [125I]Tyr3SMS binding induced by EGF and gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of tyrosine phosphatase and inhibition of cell proliferation by somatostatin analogues: mediation by human somatostatin receptor subtypes SSTR1 and SSTR2.

The effects of somatostatin analogues RC-160 and SMS-201-995 on tyrosine phosphatase and cell proliferation were investigated in COS-7 and NIH 3T3 cells expressing human somatostatin receptor subtype 1 or 2 (SSTR1 or SSTR2). Binding experiments were performed on membranes from COS-7 cells expressing human SSTR1 or SSTR2 using 125I-labeled [Tyr11]S-14 or [Tyr3]SMS-201-995, respectively. The somatostatin analogues RC-160 and SMS-201-995 exhibited low affinity for SSTR1 (IC50 of 0.43 and 1.5 microM, respectively) and high affinity for SSTR2 (IC50 of 0.27 and 0.19 nM). Addition of these analogues to cells expressing either SSTR1 or SSTR2 did not result in an inhibition of adenylate cyclase activity. In SSTR2-expressing cells, both analogues induced a rapid stimulation of a tyrosine phosphatase activity (EC50: RC-160, 2 pM; SMS-201-995, 6 pM) and an inhibition of serum-stimulated proliferation (EC50: RC-160, 6.3 pM; SMS-201-995, 12 pM). In SSTR1-expressing cells, only RC-160 induced stimulation of a tyrosine phosphatase activity. Both analogues caused an inhibition of cell proliferation at a concentration higher than 10 nM in accordance with their affinities for the SSTR1 receptor subtype. A good correlation between the affinities of RC-160 and SMS-201-995 for each receptor subtype and their potencies to inhibit cell proliferation suggests the involvement of these receptors in cell growth regulation. Tyrosine phosphatase was stimulated by both these analogues in SSTR2 and by RC-160 in SSTR1 at affinities similar to their ability to inhibit growth and bind to receptors, implicating tyrosine phosphatase as a transducer of the growth inhibition signal. We also found that mRNAs of receptor subtypes were variably expressed in different pancreatic and colon cancer cell lines, indicating the necessity of a precise analysis of receptor subtypes in target tissues before therapy with analogues.