Introduction

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G protein-coupled receptors (GPCRs) stimulate mitogenesis, in part, via extracellular signal-regulated kinases (ERKs), which are members of the mitogen-activated protein (MAP) kinase family. The mechanism of activation of the different MAP kinase signaling pathways by GPCRs is poorly understood, although it is becoming evident that many of the same transduction intermediates as those activated by the receptor tyrosine kinases are often used. For example, ERK1 and ERK2 are regulated by GPCRs through a Ras-dependent pathway by stimulating the recruitment of the guanine nucleotide exchange factor mSos (van Biesen et al., 1995) into a plasma membrane-associated signaling complex, where it activates Ras by catalyzing GTP for GDP exchange. This recruitment is the consequence of receptor-induced stimulation of tyrosine protein kinases such as Src (Dikic et al., 1996; Luttrell et al., 1996; Daub et al., 1997), which phosphorylate adapter proteins, including Shc (Luttrell et al., 1996) and Gab1 (Daub et al., 1997), followed by the Grb-2-mediated docking of mSos to the plasma membrane.

Considerable attention has also been focused recently on the functional outcome induced by the alternative kinase cascades that culminate in the activation of the MAP kinase family members, the stress-activated protein kinases (SAPKs) and p38 (Kyriakis and Avruch, 1996; Fanger et al., 1997). In contrast to ERKs that are stimulated almost universally by mitogens, a number of findings indicate that the activation of SAPKs and p38 can play a decisive role in the control of cell death (Verheij et al., 1996; Yang et al., 1997). Furthermore, deprivation of neurotrophic factors in PC-12 cells not only activates SAPKs and p38 but also leads to a dramatic inhibition of the ERK pathway (Xia et al., 1995; Berra et al., 1997). It has thus been suggested that the proliferative outcome of a cell may be dictated by a critical balance between the signaling pathways involving the various MAP kinase family members (Canman and Kastan, 1996).

In the present study, we have examined the abilities of human G protein-coupled somatostatin receptors to activate the various MAP kinase pathways and correlated any differential stimulation with the distinct effects on cell proliferation mediated by the individual receptor types. Opposing effects of somatostatin on the proliferative activity of a number of different cell types has been demonstrated, including inhibitory actions on prostatic (Brevini et al., 1993) and breast cell lines (Pagliacci et al., 1991), whereas growth-promoting effects of somatostatin have been described for human pancreatic carcinoid (Ishizuka et al., 1992), epidermoid carcinoma cells (Kamiya et al., 1993), and rat mesangial cells (Ruiz-Torres et al., 1993). However, little is known about the identity of the receptor types or the mechanisms involved in mediating the proliferative/antiproliferative functions of somatostatin in tissues.

We have previously shown that both activated human recombinant sst₂ and sst₅ receptors (Alderton et al., 1998) have no effect on basal proliferation, but can inhibit that induced by a submaximal concentration of basic fibroblast growth factor (bFGF). However, somatostatin can stimulate basal proliferation in cells expressing the human recombinant sst₄ receptor by a mechanism that is dependent on the sustained activation of ERK1 and ERK2, culminating in serine-phosphorylation of the transcription factor STAT3 (Sellers et al., 1999).

In addition to stimulating a survival/cell cycle progression pathway such as the ERK cascade, somatostatin may activate or inhibit apoptotic processes. Indeed, the well-documented antiproliferative effects of somatostatin may be the result of stimulating growth arrest rather than through a mechanism involving the direct inhibition of growth factor-activated transduction cascades. Although activation of p38 and SAPKs has been recently demonstrated for GPCRs (Yamauchi et al., 1997), the effect this activity has on regulating proliferative responses after stimulation by this receptor family is not well understood. Using a well-defined in vitro model (Sellers et al., 1999), we have examined the abilities of the human somatostatin sst₂, sst₃, and sst₄ receptor types to regulate cell proliferation by assessing changes in viable cell number, either in the presence or absence of bFGF. At intervals during the growth responses, the phosphorylation status of ERK1, ERK2, and p38 were determined to substantiate whether a correlation could be made with the resultant proliferative outcome and the activation of a particular kinase cascade. In addition, the induction of the cell cycle inhibitor p21^cip1 was assessed, as well as the involvement of the individual MAP kinase cascades in mediating its expression. Changes in the phosphorylation of the retinoblastoma protein (Rb) were also determined to substantiate if the levels of p21^cip1 were sufficient for cell cycle arrest.

	Experimental Procedures

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Materials. Chinese hamster ovary (CHO-K1) cells were obtained from The European Collection of Animal Cell Cultures (CAMR Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire, UK). Geneticin (G418 sulfate, specific activity 500 µg/ml) and reagents for culturing cells were obtained from Life Technologies, Inc. (Gaithersburg, MD) and plastic ware was from Costar (Cambridge, MA). Somatostatin and bFGF were obtained from Sigma (Poole, Dorset, UK). PD 98059 (2'-amino-3'-methoxyflavone), the pyridinyl imidazole compound PD 169316 [4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole], and Bordetella pertussis toxin were from Calbiochem (San Diego, CA). Antibodies to ERK1 (C-16) and ERK2 (C-14) were obtained from Santa Cruz Biochemicals (Santa Cruz, CA). Polyclonal antibodies specific for the dually phosphorylated and hence active forms of ERK1 and ERK2 (at Thr²⁰² and Tyr²⁰⁴) and phosphorylated p38 (Thr¹⁸⁰ and Tyr¹⁸² of , , and  isoforms) were obtained from New England Biolabs (Beverly, MA) together with an antibody to p38 with a specificity for the kinase independent of its phosphorylation state. Antibodies specific for the phosphorylated form of Rb and the transcription factor activating transcription factor 2 (ATF-2) were also obtained from New England Biolabs. A monoclonal antibody detecting p21^cip1 was supplied by Upstate Biotechnology (Lake Placid, NY). An antibody for STAT3 recognizing the phosphorylated form at a conserved tyrosine (Tyr⁷⁰⁵), which allows dimerization of the transcription factor through reciprocal phosphotyrosine-SH2 domain interactions (Darnell et al., 1994), was obtained from New England Biolabs.

Stable Expression of Human Somatostatin sst₂, sst₃, and sst₄ Receptors in Chinese Hamster Ovary Cells. The cDNA encoding each of the human receptors was subcloned into the mammalian expression vector pAlphaCA12 harboring a neomycin-resistant gene as a selection marker. CHO-K1 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) containing 10% (v/v) fetal calf serum and 1 mM Glutamax I and transfected in the absence of serum using a cationic liposome formulation-mediated transfer (LipofectAMINE; Life Technologies). Selection was performed in the presence of complete medium containing 1 mg/ml G418 sulfate and clonal cell lines expressing the cDNA were isolated by single cell cloning. Receptor expression was assessed by binding of ¹²⁵I-Tyr¹¹-somatostatin and the estimated B_max values for the clonal cell lines used were similar: 3.7 ± 0.5, 4.2 ± 1.2, and 3.3 ± 0.7 pmol/mg membrane protein for the sst₂ (CHOsst₂), sst₃ (CHOsst₃) and sst₄ (CHOsst₄) receptor-expressing cell lines, respectively (n = 3 for all data sets). No specific binding was detected in untransfected CHO-K1 cells. All cultures were routinely maintained in their appropriate growth medium at 37°C in humidified air containing 5% (v/v) carbon dioxide and passaged when 95% confluence was reached.

Partial Denudation of Confluent Cell Monolayers and Assessment of Change in Cell Number. To assess the effect of various treatments on cell number, the clonal cell lines were grown to confluence in complete media on Thermanox coverslips (Nunc, Naperville, CT). Multiple denuded areas (400 µm wide) were created by dragging a Perspex comb across the surface of the coverslip, according to the method described previously (Sellers, 1999). The Perspex comb was designed so that 50% of the confluent monolayer was removed by the partial denudation process, leaving parallel strips of cells. Repopulation of the denuded areas was investigated by placing the coverslip into a fresh well containing drug or vehicle in media without serum. Cells were harvested after incubation for 24 h by washing the coverslip in phosphate-buffered saline and adding 0.05% (w/v) trypsin/0.02% (w/v) EDTA solution for 2 to 5 min. The digestion process was terminated by adding complete media and the single cell suspension counted using a Coulter counter model Z1. Results were calculated from a minimum of three experiments with three replicates per test group and expressed as the arithmetic mean of the number of cells harvested from a single coverslip ± S.E.M. Statistical analysis was by Student's t test taking P < 0.05 as the level of significance.

Determination of the Phosphorylation Status of ERK1, ERK2, p38, STAT3, and ATF-2 or the Induction of p21^cip1. To analyze changes in the phosphorylation status of the MAP kinase family members or the transcription factors STAT3 and ATF-2 at various stages during the repopulation process, whole cell protein extract was combined from four coverslips for each treatment group. Immediately before partial denudation, cells forming the confluent monolayers will be in either G₀ or early G₁ of the cell cycle. Producing multiple denuded areas on a single coverslip dramatically increases the number of cells recruited into the growth responses and amplifies the resultant biochemical signals. Subsequent analysis of changes induced in the phosphorylation state of effector kinases involved in basal or growth factor-stimulated processes will thus reflect those of a large, synchronized cell population as well as from a small, contact-inhibited subpopulation localized to the central regions of the confluent strips.

Cell Cycle Analysis. The extent of Rb phosphorylation was measured directly on Western blots from 7.5% gels using specific antibodies supplied by New England Biolabs (at a 1:500 dilution).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effect of Somatostatin on Proliferation of Chinese Hamster Ovary Cells Recombinantly Expressing Human sst₂, sst₃, or sst₄ Receptors. The total number of CHO-K1 cells that remained on a single coverslip immediately after partial denudation of a confluent monolayer was 131 ± 4 × 10³, 140 ± 5 × 10³, and 138 ± 7 × 10³ for sst₂ (CHOsst₂), sst₃ (CHOsst₃), and sst₄ (CHOsst₄) receptor-expressing cell lines, respectively. After 24 h in the presence of incomplete media, this number had slightly increased for all recombinant lines (Fig. 1), with less than 0.7% of the cells detaching from the coverslip over the time course examined. Application of somatostatin (100 nM), immediately after denudation in the absence of other exogenously administered mitogenic factors, had no significant effect on the number of CHOsst₂ (Fig. 1A) or CHOsst₃ (Fig. 1B) cells counted 24 h later, compared with basal values. In contrast, somatostatin (100 nM) caused a significant increase in CHOsst₄ cell number (Fig. 1C), in agreement with previous observations (Sellers et al., 1999) and which was comparable with that induced by bFGF, using a concentration (10 ng/ml) that produced 80% of its maximal response.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effect of somatostatin on cell proliferation in the presence and absence of bFGF in CHO-K1 cells recombinantly expressing human sst2, sst3, or sst4 receptors. The mean number of CHOsst2 (A), CHOsst3 (B), and CHOsst4 (C) cells harvested from a single coverslip after incubation with incomplete media (basal; ), somatostatin (100 nM; SRIF; ), bFGF (10 ng/ml; ), or somatostatin and bFGF in combination (S+F; ) determined 24 h after application to partially denuded cell monolayers (n = 3). ***, significantly different from basal (P < 0.001); , significantly different from that incubated in the presence of bFGF alone (P < 0.001).

Effect of Somatostatin on bFGF-Induced Proliferation. In CHOsst₂ cells, the bFGF-induced (10 ng/ml) proliferative effect (Fig. 1A) was abolished by coapplication with somatostatin (100 nM) to values not significantly different from basal. However, somatostatin (100 nM) at the sst₃ receptor type had no significant effect on the increase in cell number induced by the growth factor (Fig. 1B). Paradoxically, activation of the sst₄ receptor abolished the bFGF-mediated proliferative effect using the same concentration of somatostatin (100 nM) that induced an increase in cell number in the absence of other mitogenic agents (Fig. 1C).

Effect of Pertussis Toxin Pretreatment on Proliferative Responses. Pretreatment with pertussis toxin (18 h at 100 ng/ml) had no significant effect on basal cell numbers determined 24 h after partial denudation of confluent monolayers of the recombinant cell lines (Table 1). CHOsst₂ and CHOsst₃ cell numbers after incubation with somatostatin (100 nM) were similarly unaffected by pertussis toxin, whereas the increased cell count mediated by sst₄ receptors was abolished by the toxin (Table 1). Pertussis toxin pretreatment had no effect on the proliferation induced by bFGF in all recombinant lines (Table 1) and stimulation of the sst₃ receptor in pertussis toxin-treated cells failed to significantly affect the bFGF-induced increase in cell number. However, the inhibition of bFGF-induced proliferation, mediated by somatostatin at the sst₂ and sst₄ receptor types, was abolished by pertussis toxin pretreatment (Table 1) to values not significantly different from those obtained after treatment with the growth factor alone.

Changes in the Phosphorylation Status of ERK1 and ERK2. Activation of MAP kinase family members during the initial repopulation events after partial denudation of confluent monolayers of the recombinant cell lines was assessed by monitoring changes in the kinase phosphorylation status using Western analysis. A time course of the immunoreactivity detected in whole cell extracts of the clonal lines with the anti-phosphospecific ERK1 and ERK2 antibody over the initial 4 h of basal repopulation and that in the presence of either bFGF (10 ng/ml) or somatostatin (100 nM) is shown in Fig. 2. During this period and irrespective of drug treatment, there was no detectable change in the expression of the ERK kinases examined for any of the recombinant lines, and the immunoreactivity obtained under basal conditions using phosphorylation state-independent pan antibodies is provided for the CHOsst₂ cells (Fig. 2A).

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Changes induced in the phosphorylation status of ERK1 and ERK2 during initial processes in the repopulation of partially denuded monolayers of the recombinant cell lines, as determined by Western analysis. Whole cell extracts were prepared from confluent monolayers immediately after denudation (T0) and after incubation with incomplete media (basal), bFGF (10 ng/ml), or somatostatin (100 nM) for the times shown in minutes. Consistency of protein loading was substantiated by determining the immunoreactivity of samples with phosphorylation state-independent anti-ERK antibodies as shown in A for CHOsst2 cells. Phosphorylation changes were demonstrated by detection with an antibody to ERK1 and ERK2 that recognizes only the dually phosphorylated and active forms (at Thr202 and Tyr204) in CHOsst2 (B-D), CHOsst3 (E), and CHOsst4 (F) cells.

Changes to the Phosphorylation Status of ERK1, ERK2, p38, and ATF-2 in the Presence of bFGF. Because ERK1 and ERK2 can be activated by either somatostatin or bFGF in all recombinant lines, we examined the effect on MAP kinase phosphorylation of the combined drug treatments, mimicking the conditions when somatostatin can exhibit an antiproliferative activity. Whole cell extracts from the recombinant cell lines were analyzed by Western blotting at both 20 and 120 min after denudation to determine the phosphorylation status during the transient and sustained phases of the kinase activity profiles. The expression of p38 was unaffected by any of the treatments in all recombinant cell lines and unchanged between the time points investigated and that detected immediately after denudation (data not shown). Phosphorylated p38 was only just detectable in confluent CHO-K1 cell monolayers and remained consistent in intensity after partial denudation and at all time points investigated throughout the basal repopulation processes (Fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Comparison of the changes induced by somatostatin and bFGF in the phosphorylation status of ERK1, ERK2, p38, and ATF-2 at various intervals during initial processes in the repopulation of partially denuded monolayers of CHOsst2, CHOsst3, and CHOsst4 cells. Analysis at 20 (A) and 120 (B) min after partial denudation of confluent monolayers was determined by Western detection using antibodies recognizing the active forms of ERK1, ERK2, and p38. Activation of p38 was assessed using an antibody specific for the doubly phosphorylated form at residues Thr180 and Tyr182 within the TGY sequence. Whole cell protein extracts were prepared from partially denuded monolayers incubated in the presence of incomplete media (CON), bFGF (10 ng/ml; FGF), somatostatin (100 nM; SRIF), or somatostatin in the presence of bFGF (S+F). Equivalent amounts of protein were analyzed for the three cell types and each panel has been taken from a single immunoblot. C, kinetic analysis of the change in p38 phosphorylation over a 4-h time course immediately after partial denudation (shown in minutes) in the presence of somatostatin (100 nM) for both CHOsst2 and CHOsst3 cells. D, changes in the phosphorylation of the transcription factor ATF-2 in CHOsst2, CHOsst3, and CHOsst4 cells, incubated for 60 min after partial denudation in the presence of incomplete media (CON), bFGF (10 ng/ml; FGF), somatostatin (100 nM; SRIF), somatostatin in the presence of bFGF (S+F), somatostatin in the presence of the p38 inhibitor PD 169316 (10 µM; -p38), or somatostatin in the presence of the MEK1 inhibitor PD 98059 (1-h preincubation at 40 µM; -MEK). Immunoreactivity was detected by Western analysis using the phosphospecific ATF-2 antibody, and each panel has been taken from a single immunoblot.

Effect of MEK1 or p38 Inhibition and Pertussis Toxin on the Induced Phosphorylation of ERK1, ERK2, and p38. The effect of pretreatment with the selective MEK1 inhibitor PD 98059 or pertussis toxin on somatostatin-induced phosphorylation of ERK1 and ERK2 either in the presence or absence of bFGF was examined. At 20 min after denudation, ERK activation in response to either somatostatin (100 nM) or bFGF (10 ng/ml) was partially reduced by PD 98059 (1-h pretreatment at 40 µM) in each of the recombinant cell lines (data not shown). By 60 min after denudation, the induced phosphorylation of ERK1 and ERK2 by either drug alone or in combination was abolished by PD 98059 pretreatment in all three cell lines (Fig. 4). The sustained activity of ERK detectable in CHOsst₂ and CHOsst₄ cell lines after 120 min in the presence of somatostatin or bFGF was also abolished by the MEK1 inhibitor, as was the enhanced phosphorylation observed after treatment with the drugs in combination (data not shown). Pretreatment with pertussis toxin (18 h at 100 ng/ml) had no effect on bFGF-induced phosphorylation of ERK1 and ERK2 at 20, 60 (Fig. 4), or 120 min after denudation in any of the recombinant cell lines. In contrast, ERK activation induced by somatostatin (100 nM) at all three receptor types was abolished by pretreatment with the toxin at the equivalent time points (Fig. 4). In addition, the additive effect of somatostatin and bFGF on the sustained ERK phosphorylation induced in CHOsst₂ (Fig. 4A) and CHOsst₄ (Fig. 4C) cells was also abolished by pertussis toxin, resulting in levels comparable with those obtained in the presence of bFGF alone. In contrast, the p38 inhibitor PD 169316 (10 µM) had no significant effect on either basal levels of ERK phosphorylation or on that induced by somatostatin for 60 min in any of the recombinant cell lines (Fig. 4). Treatment with the kinase inhibitors or pertussis toxin had no effect on the expression levels of ERK1 and ERK2 at the time points investigated (data not shown). Pertussis toxin pretreatment abolished the induced phosphorylation of p38 after incubation for 60 min with somatostatin in CHOsst₂ and CHOsst₄ cells (Fig. 4D). Pertussis toxin had no effect on the low levels of basal p38 phosphorylation (Fig. 4D) or on the expression level of the kinase in any of the recombinant lines (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of pertussis toxin, PD 98059, and PD 169316 on the phosphorylation status of ERK1, ERK2, and p38 induced by somatostatin. Confluent monolayers of CHOsst2 (A), CHOsst3 (B), and CHOsst4 (C) cells were treated with pertussis toxin (18 h at 100 ng/ml), PD 98059 (1 h at 40 µM), or PD 169316 (10 µM) immediately before partial denudation and in the presence of incomplete media (CON), bFGF (10 ng/ml; FGF), somatostatin (100 nM; SRIF), or somatostatin in the presence of bFGF (S+F) for 60 min after partial denudation. Detection was made by Western analysis of whole cell extract using phosphospecific ERK1 and ERK2 antibodies (A-C) or p38 antibodies (D).

Induction of p21^cip1 by Somatostatin and the Effect of the MEK1 Inhibitor or Pertussis Toxin. To determine whether activation of the individual somatostatin receptor types could regulate the accumulation of the cell cycle inhibitor p21^cip1, whole cell protein extract from each of the recombinant cell lines was analyzed, 24 h after partial denudation of confluent monolayers. Western analysis of equivalent protein loadings showed incubation in the combined presence of somatostatin (100 nM) and bFGF (10 ng/ml) to induce the accumulation of p21^cip1 in CHOsst₂ (Fig. 5A) and CHOsst₄ (Fig. 5C) cells but not in CHOsst₃ (Fig. 5B) cells. Treatment with somatostatin or bFGF alone had no effect on the basal expression level of p21^cip1 in any of the cell types at the time point investigated.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Induction of the cell cycle inhibitor p21cip1 after activation of human recombinant sst2 (A), sst3 (B), and sst4 (C) receptors and the effect of pertussis toxin and PD 98059 pretreatments. Immediately after denudation, cell monolayers were incubated in the presence of incomplete media (CON), bFGF (10 ng/ml; FGF), somatostatin (100 nM; SRIF), or somatostatin and bFGF (S+F) with and without an initial pretreatment of confluent monolayers with pertussis toxin (18 h at 100 ng/ml) or PD 98059 (1 h at 40 µM). Whole cell protein extracts were prepared 24 h after partial denudation and analyzed by an anti-p21cip1 antibody. Western detection was also made with an anti- actin antibody to demonstrate consistency of protein loading (data not shown).

Effect of the p38 Inhibitor on p21^cip1 Induction, Rb Phosphorylation, and Antiproliferative Activity of Somatostatin Receptors. The specific inhibitor of p38 kinase, PD 169316 (10 µM), had no significant effect on basal cell numbers obtained 24 h after partial denudation of CHOsst₂ or CHOsst₃ cells either in the presence or absence of somatostatin (100 nM) (Table 1). In CHOsst₄ cells, the increase in proliferation elicited by somatostatin (100 nM) was unaffected by coincubation with PD 169316 and basal values were similarly unaffected (Table 1). The p38 inhibitor also had no significant effect on the increase in cell number induced by bFGF (10 ng/ml) in any of the recombinant cell lines (Table 1). However, both the sst₂ and the sst₄ receptor-mediated inhibition of the bFGF-induced proliferative effect was abolished by the p38 inhibitor (Table 1). Cell numbers determined after incubation of CHOsst₃ cells in the presence of bFGF and somatostatin with PD 169316 were not significantly different from values obtained for treatment with bFGF alone (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of the p38 kinase inhibitor on the induction of the cell cycle inhibitor p21cip1, and phosphorylation of Rb. Immediately after denudation, cell monolayers were incubated with incomplete media (CON), bFGF (10 ng/ml; FGF), somatostatin (100 nM; SRIF), or somatostatin and bFGF (S+F) either in the presence or absence of PD 169316 (10 µM). Whole cell protein extracts were prepared 24 h later from human recombinant sst2 and sst4 receptors and analyzed by Western blotting using an anti-p21cip1 antibody (A) or from all three somatostatin receptor-expressing cells analyzed by phosphospecific Rb antibodies (B). Detection was also made with an anti- actin antibody to demonstrate consistency of protein loading.

Effect of Somatostatin on Tyrosine Phosphorylation of STAT3 in the Recombinant Cell Lines. Tyrosine phosphorylation of STAT3 is required for dimerization of the transcription factor and subsequent binding to DNA (Darnell et al., 1994). Western analysis of CHOsst₂, CHOsst₃, and CHOsst₄ cells incubated for 10 min after partial denudation in the presence of incomplete media or somatostatin (100 nM) showed similar levels of STAT3 protein expression (Fig. 7A). Regeneration in the presence of somatostatin resulted in a marked increase in the tyrosine phosphorylation of STAT3 compared with basal levels for CHOsst₄ cells but had no effect in CHOsst₂ or CHOsst₃ cells (Fig. 7B). Multiple products with discrete electrophoretic mobilities were detected by the anti-phosphospecific antibody, although the increased immunoreactivity observed after sst₄ receptor activation was primarily of the band with the greatest mobility.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Changes in the phosphorylation status at tyrosine-705 of STAT3 during initial events in the repopulation of CHOsst2, CHOsst3, or CHOsst4 cells. Whole cell extracts were prepared from partially denuded confluent monolayers allowed to recover for 10 min in the presence of incomplete media (CON) or somatostatin (SRIF; 100 nM). Top, Western analysis using the anti-STAT3 antibody; bottom, the immunoreactivity obtained with the antibody recognizing the phosphorylated form (P-Y705).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Considerable attention is currently being focused on the role played by MAP kinase cascades in the control of cell survival or programmed death mechanisms. Several G protein-coupled receptors, including the human somatostatin sst₁ (Florio et al., 1999) and sst₄ (Sellers, 1999) receptor types have been shown to stimulate the ERK pathway, and in this report we demonstrate that the human sst₂ and sst₃ receptors similarly activate this cascade through a pertussis toxin-sensitive mechanism. However, no correlation could be made with the activation of the ERK pathway by the somatostatin receptor types and the proliferative outcome, a finding that is in accord with the role of ERK in the signaling pathways of both mitogenic and antimitogenic agents (Cospedal et al., 1999; Wang et al., 2000). ERK phosphorylation of similar intensity and with identical kinetics was obtained after sst₂ and sst₄ receptor activation, although only the latter receptor induced a proliferative response. In contrast, the sst₂ and sst₃ receptors both lacked mitogenic activity, but showed differences in both the maxima as well as the duration of the induced ERK phosphorylation.

One aspect that is fundamental to the resultant cellular effect induced by the activation of the ERK cascade will be which transcription factors are present and whether they are restricted in localization to the nucleus. In every case examined thus far, sustained ERK activation is associated with translocation of the kinases to the nucleus (Dikic et al., 1994; Traverse et al., 1994). Transient activation, which does not induce cytoplasmic-nuclear migration, will therefore have very different consequences for gene expression compared with that of sustained ERK activity. However, because both sst₂ and sst₄ receptor types induce prolonged activation of ERK, it is unlikely that differential activation of nuclear-located transcription factors can account for their contrasting effects on cell proliferation. Stimulation of sst₄ receptors has been shown to phosphorylate the transcription factor STAT3, which is present in a latent form in the cytoplasm and becomes phosphorylated on a single tyrosine, obligatory for STAT activation (Darnell et al., 1994). Phosphorylation of STAT3 and prolonged activation of ERK have both been shown to be critical for the proliferative activity of sst₄ receptors (Sellers et al., 1999). In this study, the sst₂ receptor had no effect on the tyrosine phosphorylation status of STAT3, which may account for the lack of proliferative activity exhibited by this receptor type despite its ability to induce sustained ERK phosphorylation. In addition, the sst₃ receptor, which also has no proliferative activity, similarly failed to phosphorylate STAT3.

Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated (Widmann et al., 1999), the mechanism of activation of p38 and SAPKs is poorly understood. This is particularly so for members of the G protein-coupled receptor family, which have only recently been shown to use these alternative MAP kinase cascades for transduction purposes. Activation of p38 (Yamauchi et al., 1997) and SAPKs (Coso et al., 1996) has been demonstrated after stimulation of the G_q/G₁₁-coupled m₁ and G_i-coupled m₂ muscarinic acetylcholine receptors and a role for p38 in directly controlling cell growth has recently been demonstrated for the rat somatostatin sst_2(a) receptor splice variant (Sellers et al., 2000). In the current study we show the antiproliferative effect of both the human sst₂ and sst₄ receptors as well as the induction of the cell cycle inhibitor p21^cip1 to be dependent on p38 activation, which was blocked by pertussis toxin. ERK activation of G_i-coupled somatostatin receptors is dependent on -release (Sellers, 1999) and it may be that the p38 kinase cascade is similarly activated, consistent with the demonstration that overexpression of G can stimulate p38 activity in human embryonic kidney 293 cells (Yamauchi et al., 1997) and expression of transducin can inhibit the p38-dependent antiproliferative activity of the rat sst_2(a) receptor (Sellers et al., 2000). The pertussis toxin sensitivity of p38 phosphorylation in the current study is also in agreement with the known coupling of G_i proteins to the rat sst₂ receptor homolog (Sellers et al., 2000).

The proliferative effect of both sst₄ and bFGF receptors was unaffected after inhibition of p38 MAP kinase, suggesting that this kinase cascade is not involved in the mitogenic effects observed in this study. There are few reports demonstrating p38 activation through bFGF receptors, although it has been implicated in bFGF-mediated tube formation by endothelial cells (Tanaka et al., 1999) and in bFGF-induced interleukin-6 synthesis in osteoblasts (Kozawa et al., 1999). Here we demonstrate that bFGF, despite having no effect on p38 phosphorylation in CHO-K1 cells, can enhance the phosphorylation of p38 and its substrate ATF-2 after stimulation of either the sst₂ or the sst₄ receptor types. The abolition of the somatostatin-induced ATF-2 phosphorylation by PD 169316 confirms that p38 activity is increased after sst₂ and sst₄ receptor stimulation. In addition, the antiproliferative function of both somatostatin receptors against bFGF-induced growth was shown to be critically dependent on this kinase activity. The inability of the sst₃ receptor to phosphorylate p38 either in the presence or absence of bFGF, may account for its lack of antiproliferative activity.

As well as prolonging and amplifying the activity of p38 by the concomitant effect of somatostatin and the growth factor in CHOsst₂ and CHOsst₄ cells, an enhanced phosphorylation of ERK during the sustained phase of its activity profile was also observed. In contrast, the transient ERK activation by sst₃ receptor types was unaffected by the presence of the growth factor. Because bFGF, sst₂, and sst₄ receptors have the capacity to stimulate a prolonged activation of ERK1 and ERK2, the amplification of this signal as observed in the presence of somatostatin and the growth factor was perhaps expected. However, the mechanism by which bFGF increases the intensity of somatostatin-induced p38 phosphorylation is unclear. It is possible that bFGF in addition to activating ERK1 and ERK2 may additionally inhibit members of the dual-specificity phosphatase family, which reverse MAP kinase activities, enabling high-intensity signals to be observed for both p38 and ERK in the presence of somatostatin. Taken together, these data demonstrate that a complex interplay exists in the transduction cascades activated by distinct receptor types.

A further example of the influence of stimulating two receptor types on the net activity of a particular signaling pathway was also demonstrated in this study for the induction of the cell cycle inhibitor p21^cip1. The increased accumulation of p21^cip1 required a sustained activation of both p38 and ERK with a critical signal strength that was provided in this system by the cooperative effects of both the growth factor and sst₂ or sst₄ receptor activities. The importance of p38 activity in mediating the induction of p21^cip1 was further supported by the lack of effect on the expression of this protein by activated sst₃ receptors. The role of ATF-2 in regulating the accumulation of p21^cip1 remains to be determined, although it should be noted that in contrast to the abolition of the induced accumulation of the cell cycle inhibitor by PD 98059, ATF-2 phosphorylation was unaffected. In addition to the sustained activity of p38, ERK is also necessary for the increased accumulation of p21^cip1 by sst₂ and sst₄ receptors. Although PD 98059 blocked the induction of p21^cip1 by bFGF and somatostatin in CHOsst₂ and CHOsst₄ cells, it only partially inhibited the transient phase of ERK phosphorylation and abolished the sustained phase. This is in accord with previous reports showing that amplification of the ERK cascade is necessary for increased accumulation of this cell cycle inhibitor and growth arrest (Sewing et al., 1997) and consistent with observations from this study that the duration of ERK activation is also critical for p21^cip1 induction.

Further support for the involvement of the p38 cascade in mediating the antiproliferative effect of somatostatin was demonstrated by determining changes in the phosphorylation of the retinoblastoma tumor suppressor protein Rb. Phosphorylation of Rb by cyclin-cyclin-dependent kinase complexes prevents binding to various regulatory target proteins such as the E2F family of transcription factors and regulates cell proliferation by controlling progression through the restriction point within the G₁ phase of the cell cycle (Sherr and Roberts, 1995). The ability of bFGF to induce Rb phosphorylation is in agreement with other studies demonstrating that various mitogens that can sustain ERK1 and ERK2 activation can also inactivate Rb (Lavoie et al., 1996). Activation of sst₂ or sst₄ receptor types, however, inhibited Rb phosphorylation by the growth factor, which was blocked by the p38 kinase inhibitor. The p38 kinase cascade has also been shown to inhibit the expression of cyclin D1, required for cdk4 activity and progression through the restriction point (Lavoie et al., 1996). Therefore, in the present study, it is possible that cells are arrested in late G₁ before Rb phosphorylation by a combined effect of the reduction of cyclin D1 proteins, which could lead to a redistribution of sequestered p21^cip1 to cdk2 complexes, together with the increased total levels of p21^cip1. The expression of p21^cip1 is transcriptionally regulated by p53 and its function is critical for p53-dependent G₁ growth arrest (Kachnic et al., 1999). The p53 gene is mutated in approximately half of all human cancers (Ullrich et al., 1993) and it is possible that activation of somatostatin receptors in certain tumors may not result in the induction of this potent antiproliferative activity. This could perhaps explain the poor effects of somatostatin analogs observed in the clinical setting, to effectively treat the growth of some cancers cells (Macaulay et al., 1991). However, the loss of an antiproliferative activity in certain cancers such as colorectal and pancreatic adenocarcinomas has been show to correlate with the decreased expression of sst₂ receptor types (Benali et al., 2000).

There is an increasing number of examples in the literature where the functional outcome in response to a mitogenic agent is not only determined by the strength but also the duration of the stimulus and small differences in signal input can generate large differences in transcriptional response. Such quantitative variations can control physiological decisions and has been demonstrated in this study by the switch from a proliferative to an antiproliferative activity, as observed for the sst₄ receptor. This seems to be caused by coupling to transduction cascades that culminate in the activation of STAT3, a sustained activation of ERK1 and ERK2, and a weak activation of p38. However, with an additional signal from bFGF that amplifies and prolongs the MAP kinase cascades, induction of p21^cip1 is increased and an antiproliferative activity results, demonstrating the importance of defining the functional outcome in the context of the activity status of the cell.

We show in this study that the duration of the p38 MAP kinase cascade, as has been well documented for ERK activation (Marshall, 1995), is also critical for dictating functional responses. The contribution of several input signals, such as that from bFGF and somatostatin receptors, can generate large differences in the duration of the p38 MAP kinase activity and the subsequent regulation of transcriptional events such as ATF-2 phosphorylation and protein expression. The induction of p21^cip1, for example, requires activation of both the p38 and ERK cascades mediated by the interplay of bFGF and sst₂ or sst₄ receptors. The dependence on p38 for p21^cip1 expression also suggests that p38 activity may have a dual role in not only mediating apoptotic processes but also inhibiting cell proliferation. This is analogous to that of ERK activation, which can promote mitogenesis and provide protection against apoptosis (Berra et al., 1997).