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J. Biol. Chem., Vol. 281, Issue 28, 19156-19171, July 14, 2006
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1
From the
INSERM U531, IFR31, Centre Hospitalier Universitaire Rangueil, 31432 Toulouse Cedex 4, France and the Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
Received for publication, March 3, 2006 , and in revised form, April 14, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nitric oxide (NO) regulates a number of physiological processes, including smooth muscle contractility, platelet reactivity, neurotransmission, and the cytotoxic activity of immune cells. Three isoforms of nitric-oxide synthase (NOS) that generate NO from L-arginine have been characterized and named according to the cell type or conditions under which they were first identified. Both endothelial NOS and neuronal NOS (nNOS) are constitutively expressed, whereas expression of inducible NOS requires transcriptional activation. Because of its ubiquitous nature, inappropriate release of NO has been linked to a number of pathological conditions. In recent years, a growing body of evidence has depicted NO as a modulator of cell proliferation and/or survival of several normal and tumor systems such as vascular and airway smooth muscle cells, myoblasts, glomerular mesangial cells, pancreatic tumor cells, aortic adventitial fibroblasts, and neuronal cells (14–38). However, signaling pathways involved in NO-mediated control of cell proliferation are still poorly understood and debated. We identified nNOS as a novel critical partner for somatostatin receptor SST2-induced cell growth arrest (39). Following SST2 activation by the somatostatin analog RC-160, protein-tyrosine phosphatase SHP-1 rapidly recruits nNOS to induce its dephosphorylation at tyrosyl residues and subsequent activation. The resulting NO production activates guanylate cyclase and inhibits cell proliferation of CHO-DG44 cells stably expressing the SST2 receptor (39). In addition, somatostatin has been recently demonstrated to modulate NO production through SST1, SST2, and SST3 (40). However, participation of NO in the SST5-mediated signaling pathway is not known.
We demonstrated previously that, in CHO-K1 cells, cell proliferation is stimulated through an nNOS/NO/cGMP-dependent pathway elicited by cholecystokinin following protein-tyrosine phosphatase SHP-2 activation of nNOS by dephosphorylation at tyrosyl residues (41). Parallel work from the laboratory demonstrated that treatment of CHO-K1 cells stably expressing the SST5 receptor with the somatostatin analog RC-160 results in a marked reduction of cell proliferation induced by cGMP and cGMP-dependent protein kinase (11). The present study was conducted to investigate whether somatostatin can act antagonistically in regulating nNOS activity following binding to the SST5 receptor subtype. Molecular mechanisms involved in such regulation and subsequent inhibition of cell proliferation were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Minimal essential medium (-MEM), Fungizone, streptomycin, penicillin, trypsin, and fetal calf serum (FCS) were purchased from Invitrogen. [
-33P]ATP (3000 Ci/mmol) was purchased from Isotopchim (Ganagobie, France). AG-50W-X8 resin (sodium form) was from Bio-Rad. L-[14C]Arginine and the ECL immunodetection system were from Amersham Biosciences. Hybond ECL nitrocellulose membrane was from VWR International S.A.S. (Fontenay-sous-Bois, France). CHAPS was from Serva (Heidelberg, Germany). Sodium nitroprusside, leupeptin, tetrahydro-L-biopterin (BH4), 
-NADPH, FMN, FAD, N
-nitro-L-arginine, calmodulin, pertussis toxin, soybean trypsin inhibitor, PP-2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), dithiothreitol, enolase, L-citrulline, L-arginine, protein A-Sepharose beads, NaF, and Geneticin (G418) were from Sigma. FuGENE 6 was from Roche Applied Science. Antibodies—Monoclonal anti-human nNOS antibody (catalog no. 610308) was from BD Transduction Laboratories. Polyclonal anti-nNOS (R-20) and anti-p60src (SRC2, catalog no. sc-18) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal anti-phosphotyrosine antibody (4G10) was from Euromedex (Souffelweyersheim, France). Polyclonal antibody against SST5 was generated in rabbits immunized with peptide EPRPDR, corresponding to amino acids 334–339 of the human SST5 C-terminal end (42).
Cell Lines—Human pancreatic carcinoid BON cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, (1 x 105 units/liter penicillin, 0.5 mg/liter Fungizone, and 2 mmol/liter L-glutamine. CHO-K1 cells were cultured in -MEM containing 5% FCS. The human SST5 cDNA, subcloned into the pcDNA1/AMP vector (Invitrogen) (3), was stably cotransfected into CHO cells with the pSV2neo vector (Clontech) using Lipofectin reagent (Invitrogen). Stable transfectants were selected in -MEM containing Geneticin at 600 µg/ml. Geneticin-resistant clones expressing the SST5 somatostatin receptor (CHO/SST5 cells) were selected by somatostatin binding using [125I-Tyr11]somatostatin-14 as the tracer (43). Two clones were selected for subsequent experiments. After selection, cells were cultured in -MEM containing 5% FCS and 400 µg/ml Geneticin. The pSV2neo vector-alone stable transfectants were used as control clones. All cultures were tested mycoplasma-free.
Plasmids and Cell Transfection—Rat nNOS cDNA (kindly provided by Dr. M. Marletta, Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI) was subcloned into the pCMV5 vector (kindly provided by P. Rouet, INSERM U586, Toulouse, France) to make pCMV5/nNOS, in which nNOS expression is under the control of the cytomegalovirus promoter. A catalytically inactive p60src mutant (K295R) (44) subcloned into the pSGT vector (pSGT/
p60src) was a generous gift from Dr. S. Roche (Centre de Recherches de Biochimie Macromoléculaire, CNRS, Montpellier, France). The primers used for site-directed mutagenesis of nNOS p60src SH2 domain-binding sites (Y604F) were as follows: 5'-aac tct cga ttc aac atc ctc gag gaa gta gc-3' and 5'-gct act tcc tcg agg atg ttg aat cga gag tt-3'. Mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Roche Applied Science) following the manufacturer's instructions.
For transient transfection, CHO/SST5 cells were grown for 8hin -MEM containing 5% FCS. After the -MEM-containing serum was removed, cells were transfected in low serum-containing medium for 16 h with the pCMV5/nNOS and/or pSGT/p60src vector at the concentrations indicated using FuGENE 6 (3 µl/µg of DNA) as described previously (41). Cells transfected with the pCMV5 vector were used as a control. Under these conditions of transient transfection, the percentage of cells transfected at 24 h was 48 ± 4 (mean ± S.E., n = 3) as quantified by green fluorescent protein expression (driven by the cytomegalovirus promoter) using a VisioLab 2000 image analysis system (Biocom, Paris, France) (41).
nNOS Activity—CHO/SST5 cells were plated in 60-mm diameter dishes at 50 x 103 cells/ml in -MEM containing 5% FCS (5 ml/dish) before transient transfection, serum starvation, and treatment with RC-160 with or without other agents tested at the times and concentrations indicated. nNOS activity was measured as described previously (39, 41). Briefly, cells were homogenized using a Dounce homogenizer (60 strokes at 4 °C) in 50 mM Tris buffer (pH 7.4) supplemented with 1 mM EDTA, 10% glycerol, 20 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.01% soybean trypsin inhibitor, and 0.1% CHAPS. 25 µg of cellular proteins were incubated for 15 min at 37 °C in 50 mM Tris buffer (pH 7.4) containing 50 µM L-[14C]arginine (150,000 cpm, specific activity of 58.7 Ci/mmol), 10 mM -NADPH, 1 mM dithiothreitol, 4 µM FMN, 4 µM FAD, 10 µM BH4, 2 µg of calmodulin, and 1 mM CaCl2 in a final volume of 200 µl. The reaction was terminated by addition of 500 µl of Dowex AG-50W-X8 (sodium form) pre-equilibrated in 50 mM (v/v) Hepes (pH 5.5) overnight at 4 °C. The mixture was gently agitated for 15 min at room temperature and centrifuged at 5000 x g for 5 min at 4 °C. Radioactivity in the supernatant was measured by liquid scintillation counting.
BON cells were plated for 24 h in 8-chamber slides at 50 x 103 cells/chamber in DMEM containing 10% FCS (0.25 ml/chamber). Following overnight serum starvation, cells were washed twice with serum-free medium before treatment for 30 min with 10 nM RC-160. Real-time cell-associated NOS activity was monitored using an NOS fluorometric detection system (Sigma) with a Zeiss LSM510 confocal microscope at the times indicated following the manufacturer's instructions.
Cell Growth Assay—CHO/SST5 cells were cultured in -MEM containing 5% FCS and plated in 35-mm diameter dishes at 50 x 103 cells/ml (2 ml/dish). After an overnight attachment phase and/or transient transfection, the cell medium was changed to serum-free -MEM with or without different agents tested at the times and concentrations indicated. Cell growth was measured after 24 h of culture by cell counting using a Model ZM Coulter counter as described previously (6, 11). For cell growth assays, treatment with exogenous L-arginine was carried out in L-arginine-free -MEM. For viability assays, BON cells were plated for 24 h in flat-bottomed 96-well plates at 10 x 103 cells/well in DMEM containing 10% FCS (0.1 ml/well). Following overnight serum starvation, cells were treated in triplicate for 24 h with 100 µl of 10% FCS-containing medium in the presence or absence of RC-160 (10 nM) or the nNOS inhibitor -guanidinoglutaric acid (0.1 µM). Cell viability was measured at 490 nm using the CellTiter 96 AQueous One Solution cell proliferation assay (Promega Corp.) following the manufacturer's instructions.
p60src Kinase Activity Assay—CHO/SST5 cells were plated in 100-mm diameter dishes at 75 x 103 cells/ml (10 ml/dish) in -MEM containing 5% FCS. After an 18-h period of serum deprivation and/or transient transfection, cells were or were not treated with RC-160 at the times and concentrations indicated. CHO/SST5 cells were then washed twice with 20 mM Tris buffer (pH 7.4) containing 150 mM NaCl and 0.5 mM orthovanadate (buffer A). p60src activity was quantitated as described previously (45) with some minor modifications. Briefly, cells were scrapped and solubilized at 4 °C for 15 min in buffer A containing 1% Nonidet P-40, 10 mM NaF, 2.5 mM EDTA, 20 µM leupeptin, and 1% aprotinin. Supernatant-containing soluble proteins were obtained by centrifugation at 13,000 x g for 10 min at 4 °C. Soluble proteins (250 µg) were incubated for 1.5 h at 4 °C without agitation with 0.5 µg of anti-p60src kinase antibody or preimmune serum prebound to protein A-Sepharose beads prewashed with buffer A as a control. The beads were then washed once with buffer A and once with 20 mM Hepes (pH 7.4) containing 10 mM MnCl2 and 1.5 mM orthovanadate (buffer B). The reaction was initiated by addition of 20 µl of buffer B containing 50 µM ATP, 5 µCi of [-33P] ATP, and 4 µg of heat-denatured enolase. The reaction was carried out for 15 min at 30 °C and terminated by addition of 30 µl of protein loading buffer containing 3% SDS and -mercaptoethanol. Samples were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were air-dried and exposed on a PhosphorImager screen for 16 h. Phosphorylated enolase was visualized using a PhosphorImager (GE Healthcare) and quantified by image analysis using ImageQuant software (GE Healthcare).
Immunoprecipitation and Immunoblotting—CHO/SST5 cells were plated in 100-mm diameter dishes at 100 x 103 cells/ml (10 ml/dish) for 8 h in -MEM containing 5% FCS before transient transfection and serum starvation. BON cells were plated in 100-mm diameter dishes at 150 x 103 cells/ml (10 ml/dish) for 16 h in DMEM containing 10% FCS. Cells were or were not treated with RC-160 with or without other agents tested at the times and concentrations indicated. Cells were then washed twice with phosphate-buffered saline (PBS) and scrapped on ice with 50 mM Tris buffer (pH 7.4) containing 140 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride (buffer C) in the presence of 1.5% CHAPS, 0.05% soybean trypsin inhibitor, and 0.5 mM sodium orthovanadate. The mixture was gently agitated for 30 min at 4 °C and thereafter centrifuged at 13,000 x g for 20 min at 4 °C. Soluble proteins (500 µg to 1 mg) were incubated overnight at 4 °C with a 1:100 dilution of polyclonal anti-nNOS, monoclonal anti-phosphotyrosine, polyclonal anti-p60src kinase, or polyclonal anti-SST5 antibody prebound to protein A-Sepharose beads prewashed with buffer C in the presence of 0.1% CHAPS, 0.01% soybean trypsin inhibitor, and 0.3% bovine serum albumin (buffer D). Immunoprecipitation with preimmune serum was used as a control. Following incubation, beads were washed twice with buffer D and resuspended in 35 µl of protein loading buffer containing 3% SDS prior to electrophoresis. For immunoblotting, solubilized (50 µg) or immunoprecipitated proteins were resolved on 7.5 or 10% SDS-polyacrylamide gels before transfer to nitrocellulose membranes. After blocking at room temperature for 2 h in PBS (pH 7.4), 0.05% Tween 20, and 5% dry milk, blots were immunoprobed overnight at 4 °C with monoclonal anti-nNOS (1:1000), polyclonal anti-nNOS (1:500), monoclonal anti-phosphotyrosine (1:1000), polyclonal anti-human SST5 (1:500), or anti-p60src kinase (1:500) antibody diluted in PBS (pH 7.4), 0.05% Tween 20, and 1% dry milk. The corresponding horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Perbio Science, Brebières, France) diluted 1:20,000 in PBS (pH 7.4), 0.05% Tween 20, and 1% dry milk were added, and blots were incubated for 1 h at room temperature. Each antibody incubation was followed by extensive washing with PBS (pH 7.4) and 0.05% Tween 20. Immunoreactive proteins were visualized using a SuperSignal West Pico chemiluminescent substrate system (Perbio Science) and by image analysis using ImageQuant software. To visualize nNOS dimerization, low temperature SDS-PAGE was performed as described (41). Briefly, cells were solubilized for 30 min at 4 °C in 50 mM Tris buffer (pH 7.4) containing 140 mM NaCl, 5 mM MgCl2, 0.5 mM orthovanadate, 0.05% soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 1% CHAPS and then centrifuged at 13,000 x g for 20 min at 4 °C. After addition of protein loading buffer containing 3% SDS under nonreducing conditions, 100 µg of solubilized proteins were resolved by discontinuous gradient 5–15% SDS-PAGE performed at a constant current of 30 mA. Gels and buffers were equilibrated at 4 °C prior to electrophoresis, which was performed below 15 °C. Immunoblotting was performed using monoclonal antihuman nNOS antibody as described previously (41).
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In Vitro Assays for Recombinant nNOS Dimerization and Phosphorylation by Recombinant p60src—Low temperature-PAGE using recombinant nNOS was conducted as described previously (46) with some modifications. Briefly, 500 ng of recombinant rat nNOS (purity >95%, specific activity of 858 units/mg of protein; Sigma) were incubated in 50 mM Hepes (pH 7.4) containing 1 µM BH4 for 10 min at 37 °C in a final volume of 10 µl. To induce nNOS homodimerization, BH4 was raised to 100 µM. Samples were subsequently incubated with 2 units of human purified active p60src (Calbiochem) diluted in 50 mM Hepes (pH 7.5) containing 0.1 mM EDTA, 0.015% Tween 20, 0.1 mg/ml bovine serum albumin, and 0.2% -mercaptoethanol as described previously (47). p60src activity was initiated by addition of 10 µl of ATP mixture (0.15 mM ATP and 30 mM MgCl2 diluted in 50 mM Hepes (pH 7.5) containing 0.1 mM EDTA and 0.015% Tween 20) and further incubation at 30 °C for 30 min. Incubations were terminated by addition of 40 µl of Laemmli buffer containing 0.125 M Tris-HCl (pH 6.8), 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 20% (w/v) glycerol, and 0.02% (w/v) bromphenol blue, followed by storage on ice. As a monomerization control, samples were heat-denatured at 95 °C for 5 min. To visualize nNOS dimerization, samples were subjected to low temperature SDS-PAGE, followed by Western blotting for nNOS as described. To visualize nNOS phosphorylation, samples were first subjected to immunoprecipitation using anti-phosphotyrosine antibody before low temperature SDS-PAGE, followed by Western blotting for nNOS as described.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Next, we investigated whether activated SST5 inhibits nNOS/NO-induced cell proliferation. CHO/SST5 cells were treated for 24 h with NO donors in the presence of the somatostatin analog RC-160. As shown in Fig. 1C, treatment of CHO/SST5 cells with 10 nM RC-160 strongly antagonized sodium nitroprusside (–68 ± 1.5%)-, L-arginine (–60 ± 4.5%)-, and nNOS (–95 ± 2%)-induced cell proliferation. Taken together, these results demonstrated that 1) cell proliferation of CHO/SST5 cells was induced by endogenous and exogenous NO donors and that 2) treatment of CHO/SST5 cells with RC-160 strongly inhibited nNOS/NO-induced cell proliferation.
nNOS Activity Is Inhibited following SST5 Somatostatin Receptor Activation—We then investigated whether activated SST5 inhibits nNOS activity in CHO/SST5 cells. CHO/SST5 cells were transiently transfected with nNOS DNA before treatment with 10 nM RC-160. As shown in Fig. 2A, SST5 somatostatin receptor activation resulted in a time-dependent inhibition of nNOS-induced NO production, with a maximal effect observed after 30 s of treatment with RC-160 (–74 ± 15%). Because the nNOS enzyme is known to be active as a dimer (48), nNOS homodimerization status was monitored in CHO/SST5 cells by low temperature SDS-PAGE. As shown in Fig. 2B, the proportion of the inactive monomeric form of transfected nNOS was increased by 82 ± 6% 30 s following addition of 10 nM RC-160 and was stable up to 15 min of treatment. Taken together, these results suggest that RC-160 inhibits nNOS activity and prevents dimerization of the enzyme.
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SST5 Associates with and Activates p60src Kinase—We first tested whether p60src kinase interacts with the SST5 receptor by immunoblotting anti-SST5 immunoprecipitates with anti-p60src antibody. As shown in Fig. 5A, p60src kinase was detected within anti-SST5 immunoprecipitates in resting cells, whereas preimmune antiserum was ineffective in immunoprecipitating protein complexes. Exposure of CHO/SST5 cells to RC-160 increased the amount of p60src kinase immunoprecipitated with SST5 in a time-dependent fashion to a maximal within 15–30 s following treatment (6.2 ± 0.2-fold over basal levels). This latter result was further confirmed by the increase of SST5 in anti-p60src immunoprecipitates from RC-160-treated CHO/SST5 cells (Fig. 5C).
To test whether the activated SST5 receptor regulates p60src activity, CHO/SST5 cells were treated with RC-160, and cell lysates were immunoprecipitated with anti-p60src antibody. p60src activity was quantified by Src kinase-specific substrate enolase phosphorylation. As shown in Fig. 5D, p60src activity was very low to undetectable at the basal level in CHO/SST5 cells. Treatment with RC-160 strongly induced p60src activity in a time-dependent fashion to a maximal 30 s following treatment (70.8 ± 5-fold over basal levels) (Fig. 5E). The kinetics of p60src activation correlated well with RC-160-induced SST5-p60src complex formation. Taken together, these data demonstrated that SST5 associated with p60src in resting cells. RC-160 treatment promoted a rapid increase in SST5-p60src complexes and the subsequent activation of p60src.
nNOS Is a New p60src Kinase Substrate—We next examined whether p60src kinase interacts with nNOS in CHO/SST5 cells transiently transfected with nNOS by immunoblotting anti-nNOS immunoprecipitates with anti-p60src antibody. As shown in Fig. 6A, p60src kinase was weakly detected within anti-nNOS immunoprecipitates in resting cells. However, exposure of CHO/SST5 cells to 10 nM RC-160 increased the amount of p60src kinase immunoprecipitated with nNOS in a time-dependent fashion. This effect was maximal from 15 s (7.1 ± 0.3-fold over basal levels) up to 60 s (7.9 ± 0.4-fold over basal levels) of treatment with the somatostatin analog. The kinetics of p60src association with nNOS correlated well with RC-160-induced SST5-p60src complex formation and dephosphorylation of nNOS at tyrosyl residues.
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We next devised an nNOS mutant in which Tyr604 was replaced with phenylalanine (pCMV5/nNOS(Y604F)). CHO/SST5 cells were transiently transfected with wild-type nNOS or nNOS(Y604F). Successful expression of both proteins in CHO/SST5 cells was verified by Western blotting (Fig. 7B). We next measured the ability of the nNOS mutant to interact with p60src and SST5 by immunoprecipitation. Both wild-type and mutant nNOS were faintly detected within anti-p60src and anti-SST5 immunoprecipitates in resting cells (Fig. 7, C and D, respectively). As expected, exposure of CHO/SST5 cells to 10 nM RC-160 strongly increased the amount of nNOS and SST5 immunoprecipitated with wild-type p60src (Fig. 7, C and D, respectively). However, mutation of Tyr604 strongly inhibited RC-160-induced nNOS binding to p60src and SST5 (Fig. 7, C and D, respectively). Finally, RC-160 failed to inhibit mutant nNOS-induced elevation of intracellular NO (Fig. 7E). Taken together, these results demonstrated that Tyr604 mediated nNOS binding to p60src and that such an interaction was critical to SST5-mediated inhibition of nNOS activity.
The SST5-mediated Anti-proliferative Action Initiated by p60src-dependent nNOS Phosphorylation and Inactivation Is Effective in Pancreatic Tumor Cells—Because somatostatin and somatostatin analogs are well characterized inhibitors of gastroenteropancreatic neuroendocrine tumor cell secretion and growth in vivo and in vitro (55–59), we investigated whether SST5-mediated inhibition of nNOS activity following p60src recruitment might account for inhibition of pancreatic neuroendocrine tumor-derived cell proliferation. BON cells, isolated from human pancreatic carcinoid (60), endogenously expressed the SST5 receptor, p60src, and nNOS, but not endothelial NOS, as ascertained by reverse transcription-PCR and Western blotting (data not shown). We examined whether SST5 interacts with p60src in BON cells by immunoblotting anti-SST5 immunoprecipitates with anti-p60src antibody. As shown in Fig. 8A, p60src was detected in resting cells within anti-SST5 immunoprecipitates, whereas preimmune antiserum was ineffective in immunoprecipitating protein complexes (data not shown). Exposure of BON cells to RC-160 treatment increased the amount of p60src immunoprecipitated with SST5 1 min following RC-160 treatment (Fig. 8A). Next, we examined whether SST5 interacts with nNOS in BON cells by immunoblotting anti-nNOS immunoprecipitates with anti-SST5 antibody. As shown in Fig. 8B, SST5 was detected in resting cells within anti-nNOS immunoprecipitates, whereas preimmune antiserum was ineffective in immunoprecipitating protein complexes (data not shown). Exposure of BON cells to RC-160 treatment increased the amount of the SST5 receptor immunoprecipitated with nNOS 1 min following RC-160 treatment (Fig. 8B). We therefore explored whether RC-160 could regulate the level of nNOS tyrosine phosphorylation following SST5 activation in BON cells. As shown in Fig. 8B, nNOS was weakly phosphorylated at tyrosyl residues in resting cells. Treating BON cells for 1 min with 10 nM RC-160 strongly increased nNOS tyrosine phosphorylation. Taken together, these data demonstrated that SST5 associated with nNOS and p60src in resting BON cells. RC-160 treatment promoted a rapid increase in SST5-nNOS and SST5-p60src complexes associated with the phosphorylation of nNOS at tyrosyl residues.
We next investigated whether activated SST5 inhibits nNOS activity in BON cells. BON cells were incubated for 30 min with 10 nM RC-160. Real-time NO production was measured in live BON cells. As shown in Fig. 8C, SST5 somatostatin receptor activation strongly and irreversibly antagonized nNOS-induced NO production in BON cells compared with control cells. Treating BON cells with either RC-160 (10 nM) or the nNOS inhibitor -guanidinoglutaric acid (0.1 µM) drastically inhibited cell proliferation (Fig. 8D). Interestingly, no synergistic inhibitory effect on cell proliferation was measured when RC-160 and -guanidinoglutaric acid were concomitantly administrated to BON cells. Taken together, these results demonstrated that the endogenous SST5 receptor might interact with p60src upon RC-160 stimulation of human gastroenteropancreatic neuroendocrine tumor cells to inhibit nNOS activity and subsequent cell proliferation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thus, the role of NO in the SST5-promoted growth inhibition signal was further investigated. Treating SST5-bearing CHO cells with the somatostatin analog RC-160 strongly reduced cell proliferation induced by NO donors, probably through the inhibition of cGMP-induced MAPK activity as we reported previously (11). Furthermore, RC-160 treatment drastically inhibited cell proliferation following nNOS expression. These data suggest that somatostatin acting through the SST5 receptor could inhibit NO production originating from nNOS. This possibility was confirmed by the study of nNOS activity in CHO/SST5 cells following treatment with RC-160. We observed that RC-160 induced a rapid inhibition of nNOS activity and strongly inhibited the formation of active nNOS dimers in favor of the accumulation of inactive nNOS monomers. We demonstrated that nNOS was constitutively associated with SST5 in CHO/SST5 cells and that SST5-nNOS complex formation was up-regulated after somatostatin binding to SST5.
GPCRs that have been shown to positively regulate nNOS activity include -adrenoreceptors and glutamate, bradykinin, endothelin-1, purinergic, opioid, cannabinoid, serotonin, and cholecystokinin receptors (41, 61, 62). To our knowledge, the capacity of SST5 to bind and inhibit nNOS is the unique demonstration of negative coupling between a GPCR and the nNOS isoform. Interestingly, we reported the positive coupling of the SST2 somatostatin receptor and nNOS in the SST2-mediated anti-proliferative effect (39). The activated SST2 receptor simulates the tyrosine phosphatase SHP-1, which results in nNOS tyrosyl dephosphorylation and activation. Conversely, somatostatin was found to be a powerful inhibitor of tumor angiogenesis through SST3-mediated inhibition of both endothelial NOS and MAPK (63). In addition, a recent study linked SST2 activation to inducible NOS-derived production inhibition (64). Such bimodal regulation of NO production by different receptor subtypes has been observed following angiotensin (AT) II treatment. Through its AT1 receptor, AT II stimulates the long-term increase in several membrane components of NADPH oxidase and indirectly inhibits NO production, leading to endothelium dysfunction. Conversely, the AT2 receptor counterbalances the deleterious effect of AT II-induced AT1 receptor stimulation through bradykinin and direct NOS stimulation (for review, see Ref. 65). Recently, receptor subtype-selective inhibition of long-term NO production by somatostatin was reported (40).
This study provides the first demonstration that somatostatin receptor subtypes can directly act antagonistically in regulating nNOS activity. Such opposite regulation of NOS may prove to be one of the mechanisms by which a seemingly ubiquitous hormone such as somatostatin with widespread receptor distribution can achieve functional selectivity in different tissues and physiological states.
The activity of the constitutive NOS isoforms such as nNOS is critically controlled by calcium, which promotes binding of calmodulin to the enzymes and subsequent activation. We demonstrated previously that RC-160 inhibits cholecystokinin-induced mobilization of intracellular calcium in CHO/SST5 cells (6). However, this effect is not implicated either in cholecystokinin-induced nNOS activation or in the anti-proliferative effect mediated by the SST5 somatostatin receptor in these cells (6, 11). Variations in the level of phosphorylation have been recently suggested to alter conformation, activity, and/or protein coupling of nNOS. In this context, phosphorylation of nNOS at Ser847 by calcium/calmodulin-dependent protein kinase II reduces nNOS activity, whereas dephosphorylation of the same residue by protein phosphatase-1 and protein phosphatase-2A is correlated with an increase in nNOS activity (66, 67). Accordingly, we described an original mode of activation of nNOS by dephosphorylation of tyrosyl residues (39, 41). In the present work, we observed that RC-160 treatment of SST5-expressing cells resulted in transient phosphorylation of nNOS at tyrosyl residues and consequent nNOS inhibition. The kinetics of phosphorylation were in accordance with RC-160-induced SST5-nNOS complex formation and the RC-160 inhibitory effect on nNOS activity. These results confirmed the critical role of tyrosyl phosphorylation in controlling nNOS activity.
Like other GPCRs, the SST5 somatostatin receptor does not harbor intrinsic tyrosine kinase activity. Recruitment and activation of Src family kinases are known to play a role in GPCR-mediated signaling pathways (51, 68–73). Src has been recently described to be positively coupled to the SST1 and SST2 somatostatin receptor subtypes (10, 74–76). The data presented herein clearly demonstrate that p60src is involved in SST5-mediated inhibition of nNOS activity and cell proliferation. We first demonstrated that transfecting CHO/SST5 cells with the catalytically inactive p60src mutant abrogated the RC-160 anti-proliferative effect. We next demonstrated that p60src specifically interacted with the SST5 somatostatin receptor and that the somatostatin analog RC-160 strongly induced p60src activation. The participation of p60src in SST5 receptor-mediated activation of the N-methyl-D-aspartate receptor in hippocampal noradrenergic nerve endings has been reported recently (77). The present study provided, to our knowledge, the first demonstration of the activation of p60src by the SST5 somatostatin receptor. Several mechanisms have been shown to mediate p60src activation by GPCRs (50, 52, 78). Recently, we (10) and others (79, 80) demonstrated that -arrestin, which activates p60src by a G protein -subunit-dependent mechanism (for review, see Ref. 71), can be recruited by activated SST2 receptors. We obtained preliminary results suggesting that expression of a G protein -subunit-sequestering agent might inhibit RC-160-induced binding of p60src to SST5 and subsequent p60src activation. Thus, one possibility is that, in SST5 signaling, G protein -subunits are critical for p60src recruitment and subsequent activation.
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1-syntrophin (84), and the protein chaperone CAPON (85). Other proteins such as the protein inhibitor of NOS PIN and the dynein light chain dissociate the active NOS homodimer (86). We demonstrated previously that protein-tyrosine phosphatases SHP-1 and SHP-2 can associate with nNOS (39, 41). Formation of nNOS-Hsp90 heterocomplexes resulting in enhanced NO formation has also been reported (87). The interaction of nNOS with caveolin-3 in skeletal muscle has been described (88). The present study provides, to our knowledge, the first demonstration of the direct interaction between the proteintyrosine kinase p60src and the nNOS isoform. Somatostatin and somatostatin analogs are well characterized inhibitors of pancreatic neuroendocrine tumor cell secretion and growth in vivo and in vivo (55–59). Thus, we investigated whether the mechanism we proposed was effective in tumor cells. We demonstrated that SST5 associated with nNOS and p60src in resting BON cells (derived from human pancreatic carcinoid). RC-160 treatment promoted a rapid increase in SST5-nNOS and SST5-p60src complexes associated with the phosphorylation of nNOS at tyrosyl residues, leading to nNOS inactivation and subsequent inhibition of cell proliferation. The SST5 somatostatin receptor subtype is expressed in various tissues, including the central and peripheral nervous systems and the gastrointestinal tract, and in inflammatory and immune cells. In addition, tumors originating from brain, pancreas, prostate, lung, kidney, breast, and neuroendocrine tissues express SST5, with preferential expression in growth hormonesecreting pituitary adenomas (89). nNOS has been detected in different tissues, including brain, skeletal muscle, intestine, and pancreas, where SST5 expression occurs (90, 91). It would therefore be of interest to investigate whether control of NO production by SST5 mediates other cellular functions in addition to control of cell proliferation.
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FOOTNOTES |
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1 To whom correspondence should be addressed: INSERM U531, IFR31, Inst. Louis Bugnard, CHU Rangueil, Batîment L3, BP 84225, 31432 Toulouse Cedex 4, France. Tel.: 5-6132-2404; Fax: 5-6132-2403; E-mail: pierre.cordelier{at}toulouse.inserm.fr.
2 The abbreviations used are: CHO, Chinese hamster ovary; MAPK, mitogen-activated protein kinase; NOS, nitric-oxide synthase; nNOS, neuronal nitricoxide synthase; -MEM, -minimal essential medium; FCS, fetal calf serum; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BH4, tetrahydro-L-biopterin; DMEM, Dulbecco's modified Eagle's medium; SH2, Src homology 2; PBS, phosphate-buffered saline; GST, glutathione S-transferase; GPCR, G protein-coupled receptor; AT, angiotensin.
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