Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells

Molecular Brain Research 87 (2001) 12–21 www.elsevier.com / locate / bres

Research report

Influence of the somatostatin receptor sst2 on growth factor signal cascades in human glioma cells Janka Held-Feindt*, Frauke Forstreuter, Thomas Pufe, Rolf Mentlein Department of Anatomy, University of Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany Accepted 12 September 2000

Abstract The somatostatin receptor subtype sst2A is highly expressed, non-mutated and functionally active in gliomas. After stimulation of cultivated human U343 glioma cells with somatostatin, octreotide (sst2-, sst3- and sst5-selective peptide agonist) or the sst2-selective non-peptide agonist L-054,522 multiple signal transduction pathways are induced: elevated cAMP levels are reduced, protein tyrosine phosphatases (especially SHP2) are activated and mitogen-activated protein kinases are inhibited. Stimulation of the phosphatases resulted in dephosphorylation of activated receptors for EGF and PDGF (epidermal and platelet-derived growth factor), and as a consequence the mitogen-activated protein kinases ERK 1 and 2 (p42 / p44) were de-phosphorylated in co-stimulation experiments. Furthermore, somatostatin or sst2-selective agonists reduced EGF-stimulated expression of the AP-1 complex (c-jun / c-jun) on the transcriptional and translational level. These experiments show that the interaction of stimulatory and inhibitory receptors are important mechanisms for the regulation of signal cascades and gene expression.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neuro-oncology Keywords: Somatostatin receptor; Glioma; Glial tumor; Octreotide; Phosphorylation; Epidermal growth factor receptor

1. Introduction Receptors for the neuro / endocrine peptide somatostatin (SS) are expressed — beside on physiological target cells — on a variety of tumors including glioblastomas, meningiomas, neuroblastomas, pituitary adenomas, carcinoids, pheochromocytomas, renal, pancreatic, prostate, breast and hematopoietic carcinomas [28, for review]. In normal and malignant target cells SS often induces antisecretory and antiproliferative responses [24, for review] that are mediated by five SS receptor subtypes (sst1–5, for sst2 two Abbreviations: AP-1, activator protein-1; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellularsignal related kinase; MAPK, mitogen-activated protein kinases; PDGF, platelet-derived growth factor; PTP, protein tyrosine phosphatase; SS, somatostatin or somatotropin release-inhibiting factor; RT–PCR, reverse transcriptase–polymerase chain reaction; sst, somatostatin receptor *Corresponding author. Tel.: 149-431-880-2466; fax: 149-431-8801557. E-mail address: [email protected] (J. Held-Feindt).

splice variants sst2A and sst2B exist). SS receptors are G protein-coupled seven transmembrane domain receptors which induce several and mostly inhibitory signal transduction pathways that depend on the sst subtype and the target cell involved [21]. In solid and cultivated gliomas of different WHO grades and in glioma cell lines the SS receptor subtype sst2A, sometimes in combination with other sst subtypes, is highly expressed [6], is non-mutated and functionally active [13]. Stimulation of sst2 in glioma cells in vitro inhibits the expression of distinct genes under basal, but especially under growth factor-stimulated conditions, e.g., of the vascular endothelial cell growth factor (VEGF) (Eichler, Forstreuter, Held-Feindt and Mentlein, submitted for publication), reduces elevated cyclic AMP levels [6], but has no long-lasting effects on basal or stimulated glioma cell proliferation [6]. Since metabolically stable SS derivatives inhibit the growth of human glioma cells transplanted into nude mice in vivo [25], an important effect of SS appears to be the reduction of glioma growth factors and their actions.

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00225-4

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To understand the inhibitory influence of SS / sst2-selective agonists on basal and especially growth factor-induced gene expression we investigated therefore the signal transduction of sst2 in human glioma cells and its interaction with that of epidermal growth factor (EGF). As found for sst2 also EGF receptors (EGFR) are expressed in high densities in all gliomas. For our investigations we used the human glioma cell line U343, and initially verified the high expression of sst2 and absence of other sst-subtypes. Then, we stimulated these glioma cells with SS, the sst2- (sst3- and sst5-) selective peptide agonist octreotide (SMS 201-995), or the sst 2-selective non-peptide agonist L-054,522 alone or in combination with EGF and other growth factors, and investigated the effects on the autophosphorylation of the growth factor receptors, as well as on the subsequent activation of mitogen-activated protein kinases (MAPK) and the activator protein-1 (AP-1) transcription factor complex. We further analyzed the effects of SS- / sst2selective agonists on protein tyrosine phosphatases (PTPs). We could show that stimulation of sst2 in glioma cells activated the PTP SHP2 (also termed PTP1D or SHPTP2), dephosphorylated stimulated EGF and PDGF receptors, reduced the phosphorylation of EGF-stimulated MAPK extracellular-signal related kinases ERK 1 and ERK 2 as well as the EGF-induced c-jun expression and the formation of the AP-1 transcription complex in a time-dependent sequence.

2. Materials and methods

2.1. Peptides, agonists and inhibitors Synthetic SS1–14 was purchased from Bachem, Bubendorf, Switzerland. SS agonists were generous gifts from Novartis Pharma, Basel, Switzerland (octreotide5 SMS201–995) and Dr Arthur A. Patchett, Merck Research Laboratories, Rahway NJ, USA (L-054,522). Recombinant growth factors (human sequences) were obtained from Pepro Tech, Rocky Hill, NJ, USA (EGF) or Biomol, Hamburg, Germany (PDGF-BB). All peptides and peptide agonists were dissolved in bidistilled water to yield stock solutions of 1 mM or 0.1 mg / ml. The MAPK kinase inhibitors PD 98059 (29-amino-39-methoxy-flavone) and U0126 were purchased from Alexis, San Diego, CA, USA, (PD 98059) or Promega, Madison, USA (U0126) and dissolved at 1 mM in anhydrous dimethylsulfoxide (DMSO). Tetradecanoyl phorbol acetate (TPA) and forskolin were purchased from Biomol and freshly dissolved as 10 mM stock solutions in DMSO.

2.2. Cell culture The human glioma cell line U343 was obtained from Deutsches Krebsforschungszentrum (Heidelberg, Ger-

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many) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal calf serum [6]. For experimental studies subcultures from 5 until 15 were used. Purity of the cultures was checked routinely by immunostaining for the cell type specific markers glial fibrillary acidic protein (astrocytes / glioma cells; antibody from Boehringer, Mannheim, Germany) and CD68 (contaminating microglial cells and macrophages, compare [7]). Moreover, contaminations by Mycoplasma were checked by staining with bisbenzimide (Merck, Darmstadt, Germany).

2.3. DNA–polymerase chain reaction ( DNA–PCR) and reverse transcriptase–PCR ( RT–PCR) DNA was isolated from surgical human gliomas (25 mg fresh weight each) with the QIAamp Tissue Kit (Qiagen, Hilden, Germany). Cell cultures (10 6 ) were lysed with guanidinium thiocyanate, RNA was isolated by CsCl density centrifugation, and for destroying genomic DNA total RNA was digested with DNase I for 20 min at 258C. DNA–PCR was performed with 200–300 ng DNA, 5 ml 103 PCR buffer (Amersham Pharmacia Biotech, Uppsala, Sweden), 1 ml 10 mM desoxynucleotide triphosphates, 10–20 pmol sst1–5 specific primer pairs [6] and 1–4 ml 25 mM MgCl 2 in a final volume of 50 ml at 548C (sst 3, 4) or 568C (sst 1, 2, 5) annealing temperatures. For preparing a cDNA-stock solution for RT–PCR experiments, 10 mg of total RNA was subjected to a DNase I digestion by adding 3 ml DNase incubation buffer (Boehringer) and 30 U of RNase-free DNase I (Boehringer) in a final volume of 30 ml for 20 min at 258C. After destroying DNase at 658C for 15 min, 3 ml (60 pmol) oligo (dT) 15 primer (Pharmacia) were added, the sample was incubated for another 5 min at 658C, and quickly chilled on ice. Reverse transcription was performed after addition of 12 ml 53 reverse transcription buffer (Gibco-BRL, Eggenstein, Germany), 6 ml 100 mM dithiothreitol, 6 ml 10 mM desoxynucleotide triphosphates and 3 ml superscript RNase H 2 reverse transcriptase (Gibco BRL) for 90 min at 378C. For amplification 4 ml cDNA were incubated with 5 ml 103 PCR buffer (Promega), 10 pmol sst1–5 PCR sense / antisense primers [6], 3–7 ml 25 mM MgCl 2 , 1 ml 10 mM dNTP and 0.5 ml (2.5 U) Taq DNA polymerase (Promega) in a final volume of 50 ml at 548C (sst3, sst4) or 568C (sst1, 2, 5) annealing temperature.

2.4. Stimulation of glioma cells 2310 6 U343 glioma cells were grown for 3 days in serum-supplemented DMEM, washed and equilibrated overnight in 378C-thermostatted 0.05% bovine serum albumin (BSA)-supplemented DMEM–HEPES without glutamine (3320 min), stimulated next day in the same medium with or without SS (10 nM) / -agonists (1 nM octreotide or L-054,522) alone or in combination with

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EGF or PDGF-BB (20 ng / ml each). After different times at 378C, the incubation medium was withdrawn, and cells were lysed immediately as described below. Aliquots of the lysate were used for determination protein content by a modification of the Coomassie Blue-binding method for membrane bound proteins [6].

2.5. Analysis of tyrosine phosphorylation Stimulated cells were quickly rinsed twice with thermostatted 0.14 M NaCl 10 mM HEPES buffer, pH 7.4, lysed with ice-cold, hypotonic 5 mM HEPES buffer, pH 7.4, supplemented with 1 mM Na 3 VO 4 , and scraped off from the dishes by a rubber policeman. The lysate was sonicated (20 s), and after addition of 1.4 M NaCl 200 mM HEPES, pH 7.4, (100 ml per 1 ml lysate) nuclei and debri removed by centrifugation (10 min at 8003g at 48C). Centrifugation of the supernatant (60 min 48,0003g at 48C) yielded cytosol and membrane fractions, the later were suspended in 50 ml 10 mM HEPES 1 mM Na 3 VO 4 , pH 7.4. For detection of activated MAPK, cells were rinsed once in cold phosphate-buffered saline (PBS), lysed with Tritonlysis-buffer (50 mM Tris–HCl, pH 7.8, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 2 mM Na 3 VO 4 ) and scraped off by a rubber policeman. The lysates were mixed vigorously (vortex mixer) and clarified in an Eppendorf centrifuge (15 min, 14,0003g, 48C). After protein determination from an aliquot, samples with equal amounts were boiled in 50–200 ml SDS–PAGE sample buffer, separated by SDS–PAGE (7% gel for growth factor receptors, 10% for MAPK), transferred onto a polyvinylidene difluoride (PVDF) membrane that was blocked with 5% BSA overnight or for 1 h. The blots were incubated in 1% BSA either with a horseradish peroxidase labeled anti-phosphotyrosine antibody (1:1000, mouse monoclonal; ECL phosphorylation detection system from Amersham, UK), or anti-phosphorylated EGFR (1:500, mouse monoclonal; Transduction Laboratories E12120, Lexington, KY, USA), or anti-phosphorylated extracellular-signal related kinases ERK 1 / 2 (1:200, mouse monoclonal reacting with Tyr-204 phosphorylated ERK 1 / 2; Santa Cruz sc-7383, CA, USA), or anti-phosphorylated c-Jun NH 2 -terminal kinase (JNK, 1:500, mouse monoclonal IgG 1 , Santa Cruz sc-6254), or anti-phosphorylated p38 (1:500, rabbit polyclonal, New England Biolabs, Beverly, MA, USA) later incubation (after washings) with horseradish peroxidase-labeled anti-mouse or anti-rabbit IgG (1:30,000; DAKO, Glostrup, Denmark) and visualized by enhanced chemiluminescence (ECL system; Amersham). In some experiments the blots were stripped by washing with methanol (18 h 48C) and re-probed with antibodies to EGFR (1:250; Santa Cruz sc-03-G), or PDGF-a receptor (1:200; Santa Cruz sc-432), or ERK 2 (1:200; Santa Cruz sc-1647).

2.6. Detection and activity of protein tyrosine phosphatases 2.6.1. Detection of phosphatases Presence of the PTPs SHP1 and SHP2 was proved by Western blot experiments. U343 cells were homogenized (see above), and samples were separated by SDS–PAGE (10%). Blots were blocked overnight at 48C in 1% BSA, incubated at 378C for 1 h with anti-SHP1 (1: 250; Transduction Laboratories P17320, Lexington, KY, USA) or anti-SHP2 (1:1000; Transduction Laboratories P54420) followed (after washings) by horseradish peroxidase labeled anti-mouse IgG (1:30,000, rabbit; DAKO P026002, Glostrup, Denmark), and bound antibodies were visualized by enhanced chemiluminescence (ECL system; Amersham). 2.6.2. Activity Stimulated cells were lysed under gentle shaking in ice-cold 0.5% Nonidet P-40, 10% glycerol in 50 mM HEPES buffer, pH 7.4, containing 10 mg / ml aprotinin and 10 mg / ml leupeptin (lysis buffer) for 30 min at 48C, and the lysate was centrifuged for 15 min at 14,0003g and 48C. Aliquots of the supernatant were retained for determination of protein content, and the rest was subjected to immunoprecipitation with 10 ml mouse monoclonal anti-SHP2 or anti-SHP1 (3 h; Transduction Laboratories P54420 or P17320) followed by 20 ml protein G agarose (pre-washed with 0.14 M NaCl 10 mM HEPES, pH 7.4, 18 h; Santa Cruz). The immunoprecipitates were sedimented (3 min, 14,0003g at 48C) and washed with lysis and phosphatase buffer (twice each). PTP activities of the immunoprecipitates were assayed with 0.2 mM of the phosphorylated synthetic peptide DADEpYLIPQQG (Sigma) derived from the EGFR (residues 1014–1024) in 50 mM NaCl in 25 mM imidazole, pH 7.0, containing 2.5 mM EDTA and 5 mM dithiothreitol (phosphatase buffer) in a final volume of 50 ml. Liberated phosphate was determined as the Malachite Green / ammonium molybdate complex using a commercial kit (Sigma PTP-101). 2.7. Detection of transcription factors 2.7.1. Electrophoretic mobility shift assays ( EMSA) Stimulated cells were lysed in 500 ml ice-cold buffer 1 (10 mM HEPES, pH 7.9, containing 10 mM KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride and 10 mg / ml aprotinin) and scraped off from the dishes by a rubber policeman. After incubation of the lysate for 1 h at 48C (last 5 min under gentle shaking) and a centrifugation step for 1 min at 1,3003g at 48C, the nuclear pellet was dissolved in 50 ml buffer 2 (same contents as buffer 1, but 10 mM KCl replaced by 0.4 M NaCl), incubated at 48C under gentle shaking for 15 min, and centrifuged for 8 min at 14,0003g and 48C. Aliquots

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of the supernatant were retained for determination of protein content. Five micrograms of the isolated nuclear proteins were incubated with 1 ml 32 P–AP-1 oligonucleotide probe (30,000–60,000 cpm / ml) and 2 ml 53gel shift binding buffer (Promega) for 30 min at 48C. For supershift experiments 2 ml c-jun /AP-1 antibody (Santa Cruz sc-44G) were added. Samples were incubated for 60 min at room temperature, for another 30 min at 48C and separated on a non-denaturing 4% polyacrylamide gel at 208C and 140 V for 1.5 h. Oligonucleotide labeling was performed with the Gel Shift Assay Core System (Promega), and labeled probes were purified with Superdex G-25 MicroSpinE columns (Pharmacia).

2.7.2. Western blot experiments Five micrograms of the isolated nuclear proteins were boiled in 30 ml SDS–PAGE sample buffer, separated by SDS–PAGE (10%), and transferred onto a PVDF-membrane that was blocked with 5% casein overnight at 48C. The blots were incubated either with anti-c-jun /AP-1 (1:400 in blocking reagent, Santa Cruz sc-44-G) or anti-cfos (1:100, Santa Cruz sc-7202) overnight at 48C. After different washing steps blots were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:500, Santa Cruz sc-2004) and visualized by enhanced chemiluminescence (ECL system; Amersham). 2.8. Proliferation assays 1310 5 U343 glioma cells were grown for 3 days in serum-supplemented DMEM and stimulated in the same medium with or without SS (10 nM) or U0126 (10 mM) alone or in combination with EGF (20 ng / ml) for another 2 days. After this, 0.25 mCi [methyl-19,29-H 3 ]thymidine (130 Ci / mmol; Amersham) was added for 5 h. Radioactivity incorporated into DNA was measured after washing cells with phosphate-buffered saline (PBS, two-fold), fixing with methanol (5 min), washing with PBS, fixing the DNA with 10% trichloroacetic acid (5 min) and washing twice with PBS. The DNA was dissolved in 1 ml 0.3 M NaOH (15 min), the solution was neutralized with 1 ml 0.3 M HCl, 10 ml scintillation liquid added, and radioactivity measured in a b-counter. All measurements were done in quadruplicate.

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Fig. 1. U343 glioma cells express the somatostatin receptor subtype 2 (sst2). Total RNA of U343 cells was isolated by CsCl density gradient centrifugation and RT–PCR was performed with subtype specific primer pairs. Only the sst2 could be detected in glioma cells as a clearly prominent PCR product with 377 bp (lane 3).

gical samples from gliomas all five known somatostatin receptor subtypes could be detected.

3.2. Activation of the sst2 induces the dephosphorylation of the activated EGF and PDGF receptors When the cells were stimulated with 20 ng / ml EGF, lysed and analyzed by Western blots using an anti-phosphotyrosine antibody, a 170 kDa protein corresponding in molecular mass to the non-truncated, human EGFR [3] was clearly detectable after 5 min and 30 min incubation time (Fig. 2). After longer stimulation times the autophosphorylation of the activated EGFR decreased (120 min up to 160 min). Neither a constitutive activation of the EGFR, nor a phosphorylation of EGFR by stimulation with SS alone could be observed. In co-incubation experiments with EGF and 1 nM octreotide (or 10 nM SS or 1 nM L-054,522, not shown) the phosphorylation was suppressed after 5 min and 30 min incubation time. This inhibition of the autophosphorylation of the EGFR by SS could also be observed in Western blots when using antibodies directed against the phosphorylated EGFR. In a similar experiment, octreotide inhibited the phos-

3. Results

3.1. Human U343 glioma cells express the sst2 Initially, we analyzed the expression of the five known sst subtypes in the human U343 glioma cell line used for our subsequent studies. By RT–PCR we could show that U343 cells expressed only the sst2 (377 bp product, Fig. 1), whereas in DNA–PCR control experiments with sur-

Fig. 2. The phosphorylation of the EGF receptor (EGFR) is inhibited by octreotide (SMS). Human U343 glioma cells were stimulated with EGF (20 ng / ml) in the absence or presence of octreotide (1 nM) for 5, 30 or 120 min, lysed, membranes were isolated and analysed for Tyr-phosphorylation by SDS–PAGE (7%) followed by Western blotting with anti-phosphotyrosine, see Materials and methods. EGF induced the phosphorylation of a 170 kDa protein corresponding to the EGFR after 5 and 30 min that vanished after 120 min. Octreotide suppressed this phosphorylation after 5 min and 30 min incubation time.

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Fig. 3. The phosphorylation of the PDGF receptor is inhibited by increasing concentrations of octreotide (SMS). Human U343 glioma cells were stimulated with PDGF-BB (20 ng / ml) in the absence or presence of different concentrations of octreotide for 45 min, lysed, membranes were isolated and analyzed for Tyr-phosphorylation by SDS–PAGE (7%) followed by Western blotting with anti-phosphotyrosine, see Materials and methods. Non-stimulated cells (lane 1) showed no Tyr-phosphorylated proteins. PDGF induced the phosphorylation of a 180 kDa protein corresponding to the PDGF receptor and — with less intensity — a few others, their phosphorylation was suppressed dose-dependently by octreotide.

phorylation of the PDGF receptor, a 180 kDa protein, dose-dependently (Fig. 3). No activation of the PDGF receptor by autocrine stimulation could be detected under the conditions used. Beside the 180 kDa protein, other minor ones were phosphorylated after 45 min stimulation with 10 ng / ml PDGF-BB, this could also be suppressed by octreotide. In conclusion, stimulation of the sst2 inhibits the autophosphorylation or dephosphorylates the activated receptors for EGF and PDGF.

3.3. Stimulation of the sst2 activates the protein tyrosine phosphatase SHP2 The most probable explanation for the observed dephosphorylation of activated growth factor receptors by SS or sst2-selective agonists is the activation of PTPs after sst2-stimulation. In Western blot experiments of homogenates from human U343 glioma cells we detected the phosphatase SHP2 and traces of the phosphatase SHP1 (not shown). The activity of SHP2 was induced more than 10-fold after stimulation of the glioma cells with L054,522 (Fig. 4). PTP activity was raised already 1 min after stimulation and the elevation of SHP2 lasted at least 30 min. Activation of SHP1 could not be measured within the sensitivity of the immunoprecipitation method followed by a phosphatase assay with a synthetic Tyr-phosphorylated peptide derived from the EGFR. Since the SHP2 contains two SH2 domains binding to Tyr-phosphorylated growth factor receptors, we conclude that activated EGF and PDGF receptors are dephosphorylated after stimulation of the sst2 receptor by induction of PTP activity.

Fig. 4. The protein tyrosine phosphatase SHP2 is activated by the sst2-agonist L-054,522. Human U343 glioma cells were stimulated with 1 nM L-054,522 for 0–30 min, lysed, SHP2 was immunoprecipitated and the protein tyrosine phosphatase activity measured as described under Materials and methods. The sst2-agonist stimulated SHP2 for at least 30 min with a maximum at 10 min.

3.4. Stimulation of the sst2 dephosphorylates MAPK Stimulation of growth factor receptors induces via adapter and Ras proteins the phosphorylation and activation of different MAPK. To evaluate whether the inhibitory effect of SS / -derivatives on the phosphorylation of growth factor receptors could also be observed in the downstream signal transduction cascade, we stimulated U343 cells with EGF and sst2-agonists alone or in combination and measured the phosphorylation of the MAPK ERK 1 and 2 (p44 and p42), c-Jun NH 2 -terminal kinase (JNK) and p38. In accordance with the activation of the receptor, EGF induced the phosphorylation of ERK 1 / 2 strongly after 5 min and 20 min and to a lesser extent up to 160 min (Fig. 5). The phosphorylation process could be inhibited by MAPK kinase inhibitors PD 98059 or U0126 (not shown). As expected, SS (or the sst2 agonist L-054,522) reduced this phosphorylation after 5 min and 20 min of co-stimulation. Thereby, equal amounts of proteins were used in Western blot experiments as shown by incubating the membranes with an antibody against non-activated MAPK. EGF, SS or their combinations had no detectable effects on the phosphorylation of JNK and p38.

3.5. Stimulation of sst2 inhibits the activation of the AP-1 complex Activation of the early intermediate genes c-fos and c-jun was investigated by Western blot experiments. After 20 min incubation time, no stimulatory effect on c-fos / cjun expression by EGF or SS could be detected (Fig. 6). In contrast, after longer incubation times (160 min) EGF

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Fig. 5. EGF induces the phosphorylation of the MAPK ERK 1 and ERK 2 which is suppressed by co-incubation with somatostatin (SS). Human U343 glioma cells were stimulated with EGF (20 ng / ml) and SS (10 nM) alone or with combination for different times, lysed and analyzed for Tyr-204 phosphorylated ERK 1 (p44) and ERK 2 (p42) by Western blotting using a specific antibody (left site). EGF induced the phosphorylation of ERK 1 / 2 strongly after 5 min and 20 min and to a lesser extend up to 160 min. Co-stimulation with SS reduced phosphorylation of ERK 1 and ERK 2 after 5 min and 20 min. Equal amounts of proteins were used in Western blot experiments, this was proved by incubating membranes with an antibody against non-activated MAPK (right-hand side).

Fig. 6. Stimulation of sst2 inhibits c-jun transcription and the activation of the AP-1 complex. U343 glioma cells were stimulated with EGF (20 ng / ml) and SS (10 nM) alone or in combination. Nuclear proteins were isolated and separated by SDS–PAGE (10%) followed by Western blotting with anti-c-fos or anti-c-jun (see Materials and methods). After 20 min incubation time no stimulatory effect on c-fos / c-jun expression by EGF or SS could be detected (left side, first and second line). In contrast, after longer incubation times (160 min) EGF could activate c-jun expression whereas SS reduced this (right side, first line). Up to 160 min c-fos expression was not influenced by EGF or SS (right side, second line). A corresponding result was detected in electrophoretic mobility shift assays for the AP-1 complex (third line). After 160 min a prominent effect of EGF on AP-1 activation was inhibited by co-stimulation with SS.

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could activate c-jun expression and SS was able to suppress this activation. In some experiments (e.g., one shown in Fig. 6) SS alone could slightly activate c-jun expression. Up to 160 min c-fos expression was neither influenced by EGF nor by SS. Corresponding results were obtained using electrophoretic mobility shift assays (EMSA) for the AP-1 complex (Fig. 6). After 160 min a prominent effect of EGF on AP-1 activation was inhibited by co-stimulation with SS. Similarly, AP-1 activation by EGF alone could be reduced up to 75% by application of MAPK kinase inhibitor U0126 (not shown). Specificity of AP-1 signal could be verified by addition of an antibody directed to c-jun /AP-1 that resulted in a supershift of the visible band. These results show that stimulation of sst2 in glioma cells influences the signal transduction cascade of growth factors up to the level of transcription factors. Stimulation of U343 cells by TPA (50 nM) or forskolin (50 mM) activated NFkB or CREB transcription factors after 20 min up to 160 min stimulation, but an inhibitory effect of SS in co-stimulation experiments could not be detected (not shown).

3.6. Stimulation of the sst2 slightly influences proliferation of glioma cells Proliferation of U343 glioma cells was measured by H-thymidine incorporation experiments. In comparison to control cells ( 3 H-thymidine incorporation: 100%) EGF was able to stimulate proliferation up to 268%. Additional application of the MAPK kinase inhibitor U0126 reduced EGF-induced stimulation to 200% whereas SS was able to diminish EGF-induced proliferation only to 246% in comparison to control cells. These experiments show that SS has only an slight effect on growth factor-induced proliferation in U343 glioma cells (not shown). 3

shown to reduce elevated levels of intracellular cAMP [6] and to activate phospholipase A 2 [24]. To prove the inhibitory actions of SS in glioma cells — especially in interaction with growth factor signal cascades and gene expression — we investigated the signal transduction pathways of the sst2 in connection with the EGFRinduced signal cascade. In previous experiments we could show that the activation of the sst2 receptor by the specific SS-analogue octreotide inhibited the adenylyl cyclase when stimulated by forskolin [6]. Now we could detect that stimulation of sst2 in glioma cells also induced the potency of a PTP capable to dephosphorylate the activated (autophosphorylated) single transmembrane growth factor receptors for EGF and PDGF (Figs. 2, 3, 7). In malignant gliomas, EGFR are frequently overexpressed due to gene amplification, partly in an extracellularly truncated form (DEGFR) that is constitutively tyrosine phosphorylated independent of ligand binding [15,26,30]. Since DEGFR and ligand-activated EGFR increase cellular proliferation and reduce apoptosis by activation of the Shc-Grb2-Ras pathway, they are considered as oncogenes for gliomas. We could now show that activation of sst2 reduced the tyrosine phosphorylation of the EGFR. The PTP activity associated with sst stimulation has been attributed to the src homology (SH2) domain containing cytosolic PTPs whose members include SHP1 and SHP-2. SHP1 is expressed mainly in hematopoetic cells, whereas SHP2 occurs more widely in many different cell types including CHO cells [23]. A 66-kDa PTP identified as SHP1 co-purifies with membrane from AR42J pancreatic cells that are enriched in sst2 [36]. Based on this findings SHP1 has been postulated as the PTP responsible for sst2-mediated inhibitory growth signaling. In contrast to this, we could detect an activation of the SHP2 in U343

4. Discussion Gliomas are characterized by a strong overexpression of receptors for the inhibitory peptide SS, especially the subtype sst2 that is found only in trace amounts in nonmalignant astrocytes [6,16,20,29]. Therefore, receptor scintigraphy with labeled octreotide is a clinical tool for the localization and diagnosis of these tumors [17,20]. Stimulation of the sst2 by SS or derivatives is suspected to induce inhibitory actions in receptor-positive tumors. Via G i - or G o -proteins sst subtypes may transduce multiple intracellular signals [9]. Activated sst have been shown (partly only in transfected cells) to inhibit adenylyl cyclase (all subtypes) and Ca 21 -channels (sst2), or to activate K 1 -channels (sst2) and phospholipase A 2 (sst4). The five human sst-subtypes also stimulate PTPs through pertussis toxin-sensitive pathways [2,8] and modulate MAPK [24]. In non-malignant astrocytes, SS has been

Fig. 7. Schematic drawing of the interaction between the signal transduction of SS and EGF in human glioma cells, see text.

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glioma cells within 3–30 min (with an activation maximum after about 10 min) incubation time using the sst2specific analogue L-054,522 (Figs. 4 and 7). These findings are in accordance with studies of Reardon et al. [27] who showed by transfection of a catalytically inactive SHP-2 mutant form that SHP2 is the PTP activated upon sst2-, sst3- and sst4-stimulation in membranes from rastransformed NIH3T3 fibroblasts. As a further step in growth factor activated signal cascade processes an activation of the MAPK ERK 1 and 2 (p42 / p44) are frequent. In our studies we could demonstrate an activation of p42 / p44 after EGF stimulation within 5–20 min (Figs. 5 and 7) and this activation could be inhibited by MAPK kinase inhibitors PD 98059 or U0126. MAPKs are phosphorylated and activated on tyrosine and threonine sites by MAPK kinases (MKK) and up to now 12 MAPKs and seven MKKs are known [11]. MAPKs in turn phosphorylate numerous cellular proteins on both, serine and tyrosine residues, including transcription factors such as p62 TCF or Elk-1 that play an important role in the transcriptional regulation of c-fos through a specific region of its promoter known as the serum response element [14,31]. The influence of SS on MAPKs appear to be complex and cell type specific. On the one hand SS activates MAPK in transfected CHO-K1 cells via sst1 and sst4 [1,10], and on the other hand three of the sst subtypes inhibit the MAPK signaling cascade: sst2 in SY-5Y neuroblastoma cells, sst3 in NIH3T3 fibroblasts and mouse insulinoma cells, and sst5 in transfected CHO-K1 cells [4,5,35]. In U343 glioma cells we could now show that SS inhibits the EGF-activated MAPKs ERK 1 and ERK 2 after 5–20 min stimulation time. This inhibition could result from the activation of SHP2 by SS followed subsequent direct dephosphorylation of p42 / p44 or by terminating the EGF signal cascade through dephosphorylation of the activated EGFR by SHP2 (Fig. 7). In MiaPaCa human pancreatic cancer cells a sst-sensitive PTP was shown to dephosphorylate and inactivate the EGFR kinase and to antagonize additional tyrosine kinases such as the insulin and the IGF-1 receptor tyrosine kinases [19]. Acting through a putative sst2 in SY-5Y neuroblastoma cells, SS was shown to inhibit MAPK activities and cell proliferation induced by receptor tyrosine kinases. This effect was blocked by orthovanadate, suggesting dephosphorylation-dependent inactivation of MAPK through sst activation of PTP [4]. Activated MAPKs can phosphorylate transcription factors (e.g., ATF-2, c-Myc, Chop, Elk-1, Max, ETS-1, SAP1), other kinases (MAPKAP kinase, p90 rsk S6 kinase), upstream regulators (EGFR, son of sevenless (SOS) Ras exchange factor), and other regulatory enzymes such as phospholipase A 2 [34]. Especially through activation of Elk-1 the c-fos promoter can be influenced, and ERK 1 / 2 were shown to induce c-jun expression and phosphorylation, indicating cross talk between ERK 1 / 2 and JNK / SAPK pathways in the regulation of c-jun activity [18].

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Moreover, although p38 does not directly phosphorylate c-jun, it contributes to enhanced AP-1 activation that up-regulate c-jun and c-fos promoter activities [22]. In our studies we could now show by Western blot experiments that EGF was able to activate c-jun after 160 min incubation time on the translational level (Figs. 6 and 7), but had no influence on c-fos protein amount. This activation was probably mediated by ERK 1 / 2, because p38 or JNK activation by EGF could be not detected in U343 glioma cells. The products of these early-response genes are known to form heterodimeric (c-jun / c-fos) or homodimeric (c-jun / c-jun) transcription factor complexes termed activator protein-1 (AP-1) that bind to the consensus sequence TGAC(G)TCA [12]. In accordance with this the increased translation of c-jun, an activation of the AP-1 complex could be detected after 160 min incubation with EGF. Both, the EGF-induced induction of the c-jun protein as well as AP-1 activation could be inhibited by SS in U343 glioma cells (Figs. 6 and 7). The slight induction of c-jun by SS alone observed in part of the Western blot experiments cannot be readily explained. So far, an induction of MAPK has been only observed with the sst4 [1]. Under normal quiescent, basal conditions, the low levels of nuclear c-jun possess a phosphorylation pattern that inhibits DNA-binding ability and hence prevents transcriptional activation. Exposure to ligands results in activation of phosphatases that rapidly dephosphorylate two inhibitory C-terminal sites near the DNA-binding domain of c-jun, but also leads to activation of kinases that activate c-jun by phosphorylation on different residues. Thus, the equilibrium between various nuclear kinases and phosphatases appears to be a major locus of control over the transactivating ability of c-jun and other early-response genes. Possibly, SS influences this equilibrium in U343 glioma cells by activation of SHP2 or by interaction with other steps of different signal cascades. Previously, the effect of SS on transcription factors in other cell types has been rarely investigated. In isolated gastric parietal cells and the GH 3 pituitary cell line SS inhibited c-fos expression and the AP-1 binding [32] which could be abolished by phosphatase inhibitors (sodium orthovanadate and okadaic acid) and pertussis toxin [33]. In conclusion, in human glioma cells SS interferes with growth factor-activated signal transduction cascades and finally reduces the activation of transcription factors. Analysis of further steps of the signal transduction cascade and gene expression will show whether the interaction of sst2 with EGFR can reduce the malignancy of gliomas. In consideration of the only slight antiproliferative effect of somatostatin on EGF-induced proliferation of U343 glioma cells other functions of somatostatin have to be taken into account. One possibility for this could be the 50–60% inhibition the EGF-induced secretion of the vascular endothelial growth factor (VEGF) in the human glioma cell line U343 by SS (Eichler, Forstreuter, Held-Feindt and

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Mentlein, submitted). A similar inhibition was also reached by the MAPK-inhibitors PD98059 and UO126 which points to comparable intracellular targets. VEGF is the pivotal factor for angiogensis of gliomas which is required for supply of nutrients and tumor growth. Such influences may explain why SS / -analogues inhibits the growth of glioblastoma cell lines after transplantation into nude mice [25].

Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 415) and the ‘Forschungs¨ Integrative Neurowissenschaften’ at the Unizentrum fur versity of Kiel. We thank Martina Burmester and Marita Krumbholz for their excellent technical assistance and Alexander Arlt for introducing us into the EMSA technique. We are indebted to Novartis Pharma, Basel (Switzerland) and Dr Arthur A. Patchett, Merck Research Laboratories, Rahway, NJ (USA) for gifts of SS agonists.

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