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The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst2 receptor isoforms, which couple to identical Gα subunit pools (Gαi3 > Gαi2 >> Gα0), were both inhibited following βγ sequestration. The sst2(a) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70rsk induced by sst2(b) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst2(a) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21cip1 was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst2(a) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst2(a) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst2 receptor isoforms may determine the selection of appropriate βγ-pairings necessary for interaction with distinct kinase cascades.
Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that play important roles as mediators of cellular responses to a variety of stimuli such as growth factors, cytokines, hormones, and environmental stresses (18, 23). MAP kinases in mammalian cells have been classified into at least four subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases/c-Jun NH2-terminal kinase (SAPKs/JNK), p38 kinases, and BMK1/ERK5 (51). ERK is activated by many growth factors and cytokines and is implicated in cell growth as well as differentiation (32). Various stressors such as chemical agents and UV irradiation, tumor necrosis factor, interleukin-1, CD40 ligand, and Fas/CD95 ligand stimulate the activities of SAPKs and p38 (10, 24) which appear to play a decisive role in the control of cell death. Thus, the SAPK pathway is critical during ceramide-induced (49) and stress-induced (56) apoptosis as well as in the Daxx-mediated Fas cascade (55), whereas transfection of a constitutively active mutant of MKK3/6, the physiological activator of p38, is sufficient to induce apoptosis in PC-12 cells (53). In contrast, overexpression of ERK in NIH 3T3 cells impairs a large part of the UV-induced apoptotic response and the inhibition of ERK below a basal threshold level triggers apoptosis (2), suggesting that besides its well-established role in cell cycle progression, ERK controls survival. BMK1 is a redox-regulated kinase and phosphorylates the transcription factor MEF2C, although its physiological role has remained unclear (23).
Recently, considerable attention has also been focused on the role of phosphatidylinositol 3-kinase (PI 3-K) in protecting against apoptosis and promoting cell proliferation (21). Studies indicate that insulin supports the survival of primary cerebellar neurons by activation of the serine/threonine protein kinase Akt (also known as PKB-α) (4, 14). Akt is a widely expressed kinase that is activated by a PI 3-K-dependent mechanism (5), and it has been shown to phosphorylate and inactivate the Bcl-XL/Bcl-2-associated death promoter (11) as well as caspases (48). Another downstream component of the PI 3-K pathway required for G1 cell cycle progression is p70 S6 kinase (p70rsk) (6, 37). This kinase phosphorylates the 40S subunit of ribosomal protein S6 and is involved in the translational control of 5′-oligopyrimidine tract mRNAs. Thus, in addition to the two regulatory pathways mediated by SAPKs and p38 which culminate in apoptotic processes, the ERK and PI 3-K cascades can evoke the induction of cell survival and proliferative events.
In the present study, we have examined the ability of two G-protein-coupled receptors to differentially activate these kinase pathways in order to explain the opposing effects of the receptors on cell proliferation. The somatostatin sst2(a) and sst2(b) receptor splice variants differ only in length and composition of their intracellular COOH termini and inhibit adenylate cyclase activity with similar potency when recombinantly expressed in Chinese hamster ovary (CHO-K1) cells (38). However, initial findings revealed that only the sst2(b) receptor mediated an increase in cell number while having no effect on the proliferation induced by basic fibroblast growth factor (bFGF), which was potently inhibited following activation of sst2(a) receptors.
Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated (51), 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 utilize these alternative MAP kinase cascades for transduction purposes. Activation of p38 (54) and JNK (7) has been demonstrated following stimulation of the Gq/G11-coupled m1 and Gi-coupled m2 muscarinic acetylcholine receptors, and the integration of signals transduced by both these MAP kinase family members appears to be necessary for the m1 muscarinic receptor to activate the c-jun promoter (31). Although the expression of the c-jun proto-oncogene is rapidly induced in response to numerous mitogens and the resulting functional activity of c-Jun proteins appears to be critical for cell proliferation, a role for SAPKs or p38 in controlling cell growth through G-protein-coupled receptors has yet to be demonstrated. Part of this study was to determine if the sst2(a) and sst2(b) receptor isoforms can differentially regulate these alternative MAP kinase cascades and thus subsequently modulate transcription factor activation, cell proliferation, and the expression of the cell cycle inhibitor p21cip1, which has been suggested to play a pivotal role (15) in mediating the well-established antiproliferative effect of somatostatin (3, 35). Changes in proliferative responses with the activation of a particular kinase cascade including that of PI 3-K were also correlated for bFGF. In addition, the time course of the kinase activity was determined. There is much evidence to suggest that the duration of ERK activity is critical for determining the proliferative outcome (32), and in every case examined thus far, only sustained ERK activation induces cytoplasmic-nuclear migration (12, 46). Prolonged stimulation of ERK will therefore have very different consequences for gene expression than will transient activation. Part of this study was thus designed to determine if the duration of the other MAP kinase cascades is similarly important for controlling proliferative events. Our data suggest that prolonged p38 MAP kinase activity plays an essential role in mediating the induction of p21cip1 and the concomitant antiproliferative function of the sst2(a) receptor isoform whereas proliferation induced by the sst2(b) receptor is dependent on Akt and a sustained ERK activity.
The cDNA encoding the rat sst2(a) or sst2(b) receptors was subcloned into the mammalian expression vector pAlphaCA12 harboring a neomycin resistance gene as a selection marker, and stable cell lines expressing the recombinant receptors were prepared as described previously (38). Receptor expression was assessed by binding of 125I-Tyr11-somatostatin. The estimated Bmax values for the two clonal lines were similar, at 2.2 ± 0.6 and 1.9 ± 0.4 pmol/mg of membrane protein for CHOsst2(a) and CHOsst2(b) cells, respectively (n = 3 for both data sets). Recombinant cells were cultured in Dulbecco's modified Eagle's medium-Ham's F12 medium (1:1) containing 10% (vol/vol) fetal calf serum, 0.5 mg of G418 sulfate per ml, and 1 mM Glutamax I. To assess the effect of various treatments on cell number, the clonal lines were grown to confluence in complete medium on Thermanox coverslips. Multiple denuded areas (400 μm wide) were produced by a method described previously (40). The Perspex comb was designed so that 50% of the confluent monolayer was removed by the partial denudation process. Repopulation of the denuded areas was investigated by placing the coverslip into a fresh well containing drug or vehicle in medium without serum. Cells were harvested following incubation for 24 h by washing the coverslip in phosphate-buffered saline and adding 0.05% (wt/vol) trypsin–0.02% (wt/vol) EDTA solution for 2 to 5 min, and the single-cell suspension was counted using a Coulter Counter model Z1. Results are expressed as the mean cell number (± standard error of the mean) harvested from a single coverslip (n = 3, three replicates per test group). Statistical analysis was carried out by Student's t test.
To analyze changes in the phosphorylation status of the MAP kinase family members, ATF-2, p70rsk, and Akt at various stages during the repopulation processes following partial denudation, whole-cell protein extract was combined from four coverslips for each treatment group. Termination of the phosphorylation events following the appropriate investigative period was achieved by washing the clonal CHO-K1 cell monolayers in ice-cold phosphate-buffered saline before applying sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer as previously described (40). Total-cell protein for each of the extracts was measured by the microBCA (Pierce) method, and equivalent amounts of protein were electrophoretically resolved on 10% polyacrylamide gels. Following electrophoretic transfer onto nitrocellulose (pore size, 0.22 μm) using a semidry blotter, the membrane was washed briefly in Tris-buffered saline (TBS) and saturated overnight in TBS supplemented with 0.1% (vol/vol) Tween 20 and 5% (wt/vol) dried milk. For detection of the phosphorylated forms of the kinases, the nitrocellulose membrane was incubated with a 1:1,000 dilution of the antiphosphospecific antibodies (New England Biolabs, Inc.). Antibodies recognizing the kinases independent of their phosphorylation state (New England Biolabs, Inc.) were also used at a 1:1,000 dilution, except for those specific to ERK1 and ERK2 (Santa Cruz Biotechnology, Inc.), which were used at a 1:2,000 dilution (1:1 mix of ERK1 and ERK2). Primary incubations were carried out for 1 h at 22°C in TBS containing 0.1% (vol/vol) Tween 20 (TBST), and the membranes were washed five times for 10 min each in TBST. They were then incubated for 1 h at 22°C with a 1:3,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody in TBST containing 5% (wt/vol) dried milk. Excess antibody was removed by washing as above, and immunocomplexes were visualized using enhanced chemiluminescence detection as specified by the manufacturer (Amersham Life Science). The Western blots shown are representative of three independent experiments, and each panel is taken from a single immunoblot.
Whole-cell protein extracts were prepared 24 h following partial denudation and analyzed by Western blotting using an anti-p21cip1 antibody (Upstate Biotechnology, Inc.) following separation on 15% polyacrylamide gels. The cDNA containing sequences corresponding to amino acids 1 to 11 of avian c-Src at the 5′ end and a Myc-His tag at the 3′ end of the mouse Akt1 open reading frame was inserted into the Klenow-blunted NheI and PmeI sites of pUSEamp (Upstate Biotechnology, Inc.). The eukaryotic expression vector pCDNA3 and that incorporating transducin cDNA were kind gifts of Alan Wise, Receptor Systems, GlaxoWellcome Medicines Research Centre, Stevenage, United Kingdom. Transfections were performed with 2 μg of DNA following complex formation with LipofectAMINE reagent as specified by the manufacturer (Life Technologies). The DNA-containing medium was removed following incubation for 3 h at 37°C, and the cells were incubated for an additional 24 h in complete medium before being transferred onto Thermanox coverslips. Gene expression using immunoblot analysis as described above was determined immediately prior to partial denudation, approximately 48 h posttransfection, using a primary-antibody concentration of 1:1,000. An appropriate antibody for monitoring expression levels of transducin was purchased from NEN Life Science Products. Expression of the myristylated, constitutively active Akt1 was determined using anti-c-Myc Tag antibody.
To determine if the contrasting effects on cell growth could be a consequence of the somatostatin receptor isoforms coupling to different G proteins, immunoprecipitation experiments were performed using Gα subunit-selective antibodies following somatostatin-induced labeling with [35S]GTPγS. For experiments involving immunoprecipitation of Gαs, Gα13, and Gαq/11, cell monolayers were pretreated with Bordetella pertussis toxin (100 ng/ml) for 18 h. Membrane fractions from CHOsst2(a) and CHOsst2(b) cells were prepared by Dounce homogenization in ice-cold lysis buffer (50 mM Tris HCl, 5 mM MgCl2, 10 μg of leupeptin per ml, 1 μg of soybean trypsin inhibitor per ml, 100 μg of saponin per ml, 0.2 mg of bacitracin per ml, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride [pH 7.5]). Membrane protein was adjusted to a concentration of 75 μg/50 μl of assay buffer (50 mM NaCl, 10 mM MgCl2, 10 mM HEPES [pH 7.4]) and incubated for 2 min at 30°C with [35S]GTPγS (2 nM) following a preincubation (2 min at 30°C) with somatostatin (300 nM). The reactions were terminated by the addition of 500 μl of ice-cold assay buffer and subsequent centrifugation at 18,000 × g for 5 min at 4°C. Membrane pellets were vortexed in solubilization buffer (100 mM Tris HCl, 200 mM NaCl, 1 mM EDTA, 1.25% [vol/vol] Nonidet P-40, 0.2% [wt/vol] SDS [pH 7.4]) and precleared for 1 h at 4°C with 20 μl of rabbit serum (1:100 final dilution) and then added as a 20% (vol/vol) protein G-bead suspension in solubilization buffer (without SDS and supplemented with 2% [wt/vol] bovine serum albumin and 0.1% [wt/vol] NaN3). The beads were pelleted, and 100 μl of the supernatant was added to tubes containing protein G suspension (40 μl) and a 1:200 final dilution of the Gα protein antibody, supplied by NEN Life Science Products (polyclonal EC/2 cross-reacting with Gαi3 and Gα0 and polyclonal AS/7 cross-reacting with Gαi1 and Gαi2) or Santa Cruz Biotechnology (for Gαs [K-20], Gα0 [K-20], Gα13 [A-20], and Gαq/11 [C-19]). Samples were gently agitated for 2 to 3 h at 4°C. Washed immunocomplexes were suspended in scintillant and counted. The results are expressed (arithmetic mean ± standard error of the mean) as the percent stimulation over the basal level (n = 3). Statistical analysis was by Student's t test. [35S]GTPγS (specific activity, 1,000 to 1,100 μCi/mmol) and GammaBind G Sepharose beads were from Amersham.
We assessed the ability of various treatments to modulate the proliferative outcome of both recombinant sst2(a) [CHOsst2(a)] and sst2(b) [CHOsst2(b)] receptor-expressing lines by determining the repopulation of denuded areas in confluent monolayers by directly counting viable cells. Parallel denuded areas were created by dragging a Perspex comb across cell monolayers grown on coverslips (40). Application of somatostatin (100 nM) immediately following denudation, in the absence of other exogenously added mitogenic factors, had no significant effect on the number of viable CHOsst2(a) cells counted 24 h later compared to the basal value (Fig. (Fig.1A).1A). In contrast, somatostatin (100 nM) caused a significant increase in CHOsst2(b) cell numbers (Fig. (Fig.1B),1B), which was comparable to that induced by bFGF (Fig. (Fig.1B),1B), using a concentration (10 ng/ml) which produced 80% of its maximal response. The specific inhibitors of MEK1 and p38 kinase, PD 98059 (20 μM) and PD 169316 (10 μM), respectively, had no significant effect on basal proliferation of CHOsst2(a) cells in either the presence or absence of somatostatin (Fig. (Fig.1A).1A). However, the increase in proliferation elicited by somatostatin in CHOsst2(b) cells was abolished by PD 98059, while PD 169316 was without effect (Fig. (Fig.1B).1B). The PI 3-K inhibitor, LY 294002 (100 μM), also blocked the sst2(b) receptor-mediated proliferative response (Fig. (Fig.1B)1B) while having no significant effect on basal proliferation in either the sst2(a) or sst2(b) receptor-expressing lines (Fig. (Fig.1A1A and B).
To correlate the effects observed on CHOsst2(a) and CHOsst2(b) cell proliferation with the activation of a particular kinase cascade, we analyzed whole-cell protein extract by Western blotting using antibodies specific for the phosphorylated and hence active forms of MAP kinases and Akt. A time course of the immunoreactivity detected with the antiphosphospecific antibodies over the initial 4 h of basal repopulation and that in the presence of somatostatin (100 nM) is shown in Fig. Fig.1.1. During this period and irrespective of drug treatment, there was no detectable change in the expression of the kinases examined and the immunoreactivity obtained using phosphorylation state-independent pan antibodies to ERK1 and ERK2 was provided for both recombinant lines (Fig. (Fig.1C1C and D). However, electrophoretic mobility shifts for both ERK1 and ERK2 could be observed in the treatment groups where a marked change in the phosphorylation status of these proteins had occurred. Similar observations were apparent using antibodies to p38, SAPKs, and Akt in both cell lines.
Before and immediately following partial denudation, phosphorylated forms of ERK1, ERK2, p38, and Akt were undetectable in either recombinant line (Fig. (Fig.1C1C and D). Under basal repopulation conditions, a small and transient increase in the phosphorylation of ERK1 and ERK2 was observed for both CHOsst2(a) (Fig. (Fig.1C)1C) and CHOsst2(b) (Fig. (Fig.1D)1D) cells, falling to undetectable levels by 60 min postdenudation. However, no immunoreactivity was detected in either cell line using antibodies to the phosphorylated forms of p38 or Akt, at any time point investigated during the initial basal repopulation processes (Fig. (Fig.1C1C and D). This suggests that the partial denudation process, possibly through disruption of zonular adheren sites, can selectively trigger some signaling pathways which may contribute to basal repopulation. However, the apparent ineffectiveness of the MEK1 inhibitor on this process also suggests that multiple, parallel pathways are involved in basal repopulation and that a blockade of any individual cascade can be effectively circumvented.
Application of somatostatin (100 nM) to either CHOsst2(a) or CHOsst2(b) cells immediately after partial denudation evoked a marked increase in the phosphorylation of ERK1 and ERK2 with a maximal response at 10 min, and although this level subsequently declined, that observed at 4 h postdenudation was increased over basal (Fig. (Fig.1C1C and D). At no time point during the initial 4 h of repopulation processes could phosphorylated Akt be detected in CHOsst2(a) cells treated with somatostatin (Fig. (Fig.1C).1C). In CHOsst2(b) cells, however, somatostatin induced a transient phosphorylation of Akt, which peaked 20 min following partial denudation and had declined to basal levels by approximately 2 h (Fig. (Fig.1D),1D), suggesting that the onset of this pathway is slower than that for ERK activation. Phosphorylation of p38 by somatostatin was apparent in both recombinant lines, but temporal differences were observed between phosphorylation mediated by the different receptor types. Activation of sst2(a) receptors induced a persistent phosphorylation of p38 (Fig. (Fig.1C),1C), whereas activation of sst2(b) receptors caused a transient phosphorylation that had declined to undetectable levels by 30 min postdenudation (Fig. (Fig.11D).
At no time point during the initial 4 h of repopulation processes could phosphorylated forms of the SAPKs be detected in either cell line treated under basal conditions or with somatostatin (100 nM) (determination following incubation for 10 min is shown in Fig. Fig.2).2). Western analysis, however, revealed CHO-K1 cells to express both the p54 and p46 forms of the SAPKs (Fig. (Fig.2A).2A). Phosphorylation of these proteins was detected following application of UTP (100 nM) for 10 min immediately after partial denudation (Fig. (Fig.2B),2B), although there was no detectable activation of Akt by UTP at this time point, in contrast to that mediated by sst2(b) receptors, and the UTP-induced phosphorylation of p38 was not as marked as that evoked by sst2(a) receptors (Fig. (Fig.2B).2B). It is possible that UTP is acting through endogenous P2Y2 receptors that exhibit a widespread distribution.
In CHOsst2(a) cells, the proliferative effect induced by bFGF (10 ng/ml) was abolished on coapplication with somatostatin (100 nM) to values not significantly different from basal (Fig. (Fig.3A)3A) whereas activated sst2(b) receptors were without effect (data not shown). The increase in cell number induced by bFGF was partially inhibited by PD 98059 or LY 294002 and unaffected by PD 169316 (Fig. (Fig.3A).3A). The sst2(a) receptor-mediated antiproliferative effect of somatostatin, however, was blocked by the p38 inhibitor (Fig. (Fig.3A).3A).
In the presence of bFGF (10 ng/ml), both the CHOsst2(a) and CHOsst2(b) cell lines showed a marked increase in the phosphorylation status of ERK1 and ERK2 (Fig. (Fig.3B3B and C). However, in contrast to the activation induced by somatostatin treatment, the time profile of ERK activation by the growth factor appeared biphasic; a transient increase occurred between 10 and 20 min with a second peak following between 4 and 7 h postdenudation (Fig. (Fig.3D).3D). Concomitant application of somatostatin and bFGF to partially denuded CHOsst2(a) (Fig. (Fig.3B)3B) or CHOsst2(b) (Fig. (Fig.3C)3C) cells induced a strong phosphorylation of ERK1 and ERK2 with similar kinetics to that obtained in the presence of somatostatin alone. Application of bFGF to repopulating cell monolayers failed to induce an increase in the phosphorylation of either p38 or Akt at any time point investigated (Fig. (Fig.3B3B and C). The induced phosphorylation of Akt by somatostatin in CHOsst2(b) cells was unaffected by the presence of bFGF in terms of both the duration of the detected immunoreactivity (Fig. (Fig.3C)3C) and its intensity (Fig. (Fig.4).4). Application of somatostatin and bFGF to CHOsst2(a) cells could not induce the phosphorylation of Akt (Fig. (Fig.3B),3B), although the phosphorylation of p38 by somatostatin alone appeared to be elevated above basal levels for longer in the presence of the growth factor (Fig. (Fig.3B).3B).
This latter observation was confirmed by analyzing samples from the various treatment groups on the same immunoblot (Fig. (Fig.4).4). The detection with phosphorylation state-independent antibodies to Akt, ERK1, ERK2, and p38 shows that the expression of these kinases was unaffected by the various treatments and unchanged over the time course analyzed (Fig. (Fig.4).4). The intensity of the immunoreactivity detected with the anti-phospho-p38 antibody was greater at all time points examined during the initial 4 h of repopulation processes in the presence of somatostatin with bFGF than for CHOsst2(a) cells treated with somatostatin alone (Fig. (Fig.4A).4A). This enhancement of the phosphorylation of p38 by the addition of bFGF to somatostatin-treated samples was also evident for CHOsst2(b) cells (Fig. (Fig.4B).4B). However, in marked contrast to the prolonged activation by sst2(a) receptors, the phosphorylation of p38 induced by somatostatin in CHOsst2(b) cells, irrespective of the presence of bFGF, was transient (Fig. (Fig.4B).4B). Figure Figure44 also demonstrates that for both cell lines, the level of phosphorylated ERK1 and ERK2 induced by somatostatin in the presence of bFGF was greater than that for either drug alone at all time points examined.
To correlate the p38 dependency of the sst2(a) receptor-mediated antiproliferative effect with the observed time-related immunoreactivity changes induced by somatostatin in CHOsst2(a) and CHOsst2(b) cells, we examined the effect of PD 169316 on the phosphorylation status of activating transcription factor 2 (ATF-2), a known substrate for p38 kinase. Activation of this transcription factor requires dual phosphorylation at threonine 69 and threonine 71, enabling subsequent binding to both AP-1 and CRE DNA response elements (16). Although both SAPK and p38 MAP kinases phosphorylate ATF-2 at these sites, we can rule out any contribution from SAPKs in this study, since no observable change in the phosphorylation status of SAPKs could be detected under basal conditions or following somatostatin and bFGF treatments at any time point investigated throughout the initial 4 h of repopulation processes for either cell line (Fig. (Fig.22).
The time points investigated were chosen to represent both the transient (10 min) and sustained (120 min) phases of the kinase activity profiles. The p38 inhibitor had no effect on the phosphorylation of ERK1 or ERK2 (Fig. (Fig.5A)5A) or of p38 (data not shown) induced by somatostatin (100 nM) in CHOsst2(a) cells in either the presence or absence of bFGF (10 ng/ml), 10 min following partial denudation. In contrast, ERK phosphorylation induced by somatostatin at 120 min was enhanced by the application of the p38 inhibitor, suggesting that cross talk between the p38 and ERK cascades exists (Fig. (Fig.5A).5A). ATF-2 phosphorylation could not be detected under basal conditions or in the presence of bFGF at either time point examined (Fig. (Fig.5A).5A). However, ATF-2 phosphorylation was evoked by somatostatin during the sustained phase of p38 activation and was abolished by PD 169316 (Fig. (Fig.5A).5A). The phospho-ATF-2 immunoreactivity obtained with somatostatin was also amplified by the presence of bFGF (Fig. (Fig.5A),5A), consistent with the enhanced phosphorylation of p38 in the presence of the growth factor. In contrast, ATF-2 phosphorylation could not be detected at either time point by somatostatin treatment in CHOsst2(b) cells (data not shown) and would be in accord with the sst2(b) receptor exhibiting only transient activation of p38.
There is accumulating evidence (41, 52) suggesting that high-intensity Ras stimulation in a number of cell types can evoke prolonged ERK activation leading to the induction of the cell cycle inhibitor p21cip1. Data provided in this study would also suggest that a strong and sustained activation of ERK1 and ERK2, as observed in both CHOsst2(a) and CHOsst2(b) cells, cannot alone be responsible for the induction of such an effective antiproliferative activity. In addition, ERK phosphorylation was shown to be amplified in the presence of bFGF following activation of either receptor isoform (Fig. (Fig.4),4), and an intense ERK activity in CHOsst2(a) cells resulted following inhibition of p38 (Fig. (Fig.5A),5A), despite the ability of this agent to abolish the antiproliferative effect of somatostatin. We thus investigated the involvement of p38 in the induction of p21cip1. In CHOsst2(a) cells, p21cip1 protein expression was elevated over basal levels following treatment for 24 h with somatostatin (100 nM) in the presence of bFGF (10 ng/ml) (Fig. (Fig.5B),5B), whereas in CHOsst2(b) cells no change was observed in the immunoreactivity detected with the p21cip1 antibody in the equivalent treatment group (Fig. (Fig.5B).5B). The increased expression of p21cip1 was reduced in CHOsst2(a) cells by the MEK1 inhibitor (Fig. (Fig.5B).5B). However, the p38 inhibitor not only abolished p21cip1 protein expression induced by somatostatin with bFGF but also reduced basal levels (Fig. (Fig.5B).5B). There was no effect on p21cip1 protein expression levels in CHOsst2(a) or CHOsst2(b) cell lines by the application of somatostatin or bFGF alone in either the presence or absence of PD 98059 or PD 169316 (Fig. (Fig.55B).
The most obvious differential transduction event between activated sst2(a) and sst2(b) receptors, which might explain their distinct proliferative functions in the absence of exogenously added mitogenic agents, is the phosphorylation of Akt, which, together with a concomitant ERK activity, may promote cell cycle progression in sst2(b) receptor-expressing cells. The somatostatin (100 nM)-induced transient phosphorylation of both Akt and p70rsk was markedly attenuated by the presence of the PI 3-K inhibitor and unaffected by PD 98059 or PD 169316 (Fig. (Fig.6A).6A). However, the PI 3-K inhibitor had no effect on the transient or sustained phases of ERK phosphorylation mediated by sst2(b) receptors (Fig. (Fig.6A).6A). In contrast, PD 98059 abolished the sustained ERK phosphorylation and partially inhibited the transient phase (Fig. (Fig.6A).6A). The sst2(a) receptor had no effect on the phosphorylation of p70rsk (Fig. (Fig.5A),5A), in keeping with its lack of effect on Akt.
To demonstrate the importance of Akt as opposed to other substrates of PI 3-K in mediating the proliferative function of the sst2(b) receptor type, we transfected CHOsst2(a) cells with a constitutively active mutant of Akt. There was no significant difference between basal cell counts determined 24 h following partial denudation of confluent CHOsst2(a) cells transfected with either pUSEamp or pUSEamp containing cDNA for tagged Akt1 with c-src-derived residues required for myristylation (Fig. (Fig.6B).6B). Application of somatostatin (100 nM) had no significant effect compared to the basal level on the repopulation of mock-transfected cells or those expressing active Akt (Fig. (Fig.6B).6B). However, somatostatin in the presence of the p38 inhibitor PD 169316 increased cell counts compared to those obtained with either drug alone for CHOsst2(a) cells expressing active Akt but not for mock-transfected cells (Fig. (Fig.6B).6B). PD 169316 on its own was without effect (Fig. (Fig.66B).
To explain the differential abilities of the somatostatin receptor isoforms to stimulate the PI 3-K and p38 pathways, we examined whether the sst2(a) or sst2(b) receptors exhibited preferential coupling to distinct Gα protein pools. This was attempted using an immunoprecipitation strategy following somatostatin-stimulated labeling of the coupled subunits with [35S]GTPγS. In the presence of 1 μM GDP, somatostatin (300 nM) increased total [35S]GTPγS (0.2 nM) binding to CHOsst2(a) membranes by 644% ± 24% over basal (pEC50 [the negative logarithm of EC50], 8.9 ± 0.1) and increased binding to CHOsst2(b) membranes by 501% ± 17% (pEC50, 8.7 ± 0.1) (n = 5 for both data sets). Optimal agonist-stimulated [35S]GTPγS binding following immunoprecipitation with Gαi1/2, Gαi3, or Gα0 antibodies could be resolved in the presence of 100 μM GDP. Binding and immunoprecipitation with antibodies specific for Gαs, Gαq/11, or Gα13 was performed in the presence of 1 μM GDP using membranes prepared from pertussis toxin-treated cells (100 ng/ml for 18 h).
There was no significant difference in the level of somatostatin (300 nM)-induced labeling between G-protein α subunits immunoprecipitated from either CHOsst2(a) and CHOsst2(b) cells, and the activated receptor isoforms showed the same preference of coupling, Gαi3 > Gαi2 >> Gα0 (Fig. (Fig.7A).7A). It should be noted that CHO-K1 cells do not express Gαi1. In addition, both the antiproliferative and proliferative effects of the sst2(a) and sst2(b) receptor types, respectively (24 h following application of 100 nM somatostatin to partially denuded monolayers), were abolished by pertussis toxin pretreatment (100 ng/ml, 18 h), whereas the proliferative activity induced by bFGF (10 ng/ml) in either cell line was unaffected (Fig. (Fig.7B).7B). Phosphorylation of ERK1, ERK2, p38, and Akt by somatostatin (100 nM for 10 min) at the appropriate receptor type was also blocked in cells pretreated with pertussis toxin, whereas the level of ERK1 and ERK2 phosphorylation mediated by bFGF was unaffected (Fig. (Fig.7C).7C).
The proliferative effect of the sst2(b) receptor and the antiproliferative effect of the sst2(a) splice variant were both inhibited by overexpression of transducin (Fig. (Fig.8A),8A), whereas the proliferative activity induced by bFGF (10 ng/ml) in either cell line was unaffected. Akt-induced phosphorylation in CHOsst2(b) cells and the somatostatin-stimulated (100 nM) phosphorylation of ERK in both recombinant lines was diminished following transducin overexpression (Fig. (Fig.8B).8B). The induced phosphorylation of p38 mediated by sst2(a) receptors was also reduced by the expression of the βγ sequestrant (Fig. (Fig.8B).8B). However, phosphorylation of ERK1 and ERK2 induced by both bFGF (10 ng/ml) or UPT (100 nM) 10 min following application to partially denuded monolayers was unaffected by transducin overexpression (Fig. (Fig.8C).8C).
Upon ligand stimulation, G-protein-coupled receptors transduce their effects through both the GTP-bound Gα and the dissociated Gβγ component of the heterotrimeric G protein, directly regulating downstream effectors (33) including adenylate cyclases, phospholipase C isoforms, ion channels, PI 3-K (42), and Tec family tyrosine kinases (25). Several G-protein-coupled receptors, including the somatostatin sst1 (15) and sst4 (39) receptor types, stimulate the ERK pathway through a variety of G-protein subunits (17). For m1 muscarinic acetylcholine and α1-adrenergic receptors, the activation of ERK is mediated by Gαq/11. In contrast, Gi-coupled m2 muscarinic acetylcholine, α2-adrenergic and somatostatin sst4 receptors, and the Gs-coupled β-adrenergic receptor all induce ERK activation through Gβγ. In this report, we demonstrate that both sst2 splice variants similarly stimulate ERK through βγ release from a pertussis toxin-sensitive G protein.
Many studies suggest that a signal transduction pathway from Gβγ to ERK starts at the direct activation of PI 3-Kγ (26), which increases the activities of Src family tyrosine kinases (13, 50), in turn leading to tyrosine phosphorylation of Shc (28). Subsequent recruitment of the Grb2-Sos complex to plasma membranes promotes the exchange of GDP with GTP on Ras and activates a sequential kinase cascade that includes Raf, MAP kinase kinase (MEK), and ERK. Data from this study suggest that the sst2(a) and sst2(b) isoforms have differential abilities to activate PI 3-K, in that Akt and p70rsk phosphorylation was observed following sst2(b) but not sst2(a) receptor stimulation. However, it should be noted that bFGF also failed to phosphorylate Akt, although its proliferative function was partially dependent on a PI 3-K activity. This suggests that the PI 3-K required for bFGF-induced proliferation, in contrast to that for sst2(b) receptors, is not able to stimulate the phosphoinositide-dependent kinase present in CHO-K1 cells and required for Akt activation (1). The growth factor-stimulated PI 3-Kα and the G-protein-coupled receptor-activated PI 3-Kγ forms (42) both have protein kinase activity in addition to their lipid kinase function, and it is possible that distinct signals may be generated through differential activation of their intrinsic kinase domains.
The observed blockade of ERK and PI 3-K by the respective inhibitors in CHOsst2(b) cells is consistent with the dependency of the proliferative function mediated by this receptor type on both these effector activities. However, the lack of effect of LY 294002 on the somatostatin-induced ERK phosphorylation and the ineffectiveness of PD 98059 on Akt and p70rsk activation suggest that these kinase cascades activated by the sst2(b) receptor are parallel but distinct. This is in contrast to the cross talk that has been demonstrated between the ERK and PI 3-K pathways for other G-protein-coupled receptors (39, 43). Although, ERK and PI 3-K activities are critical for somatostatin to induce a proliferative function in CHOsst2(b) cells, it appears that a cooperative effect from both cascades is required since the abolition of either prevents an increase in cell number. The partial dependency on both PI 3-K and ERK activities for the growth factor-induced proliferative response, in contrast to that mediated by sst2(b) receptors, is consistent with the ability of the bFGF receptor to recruit a multitude of secondary effectors and initiate a number of distinct yet parallel signaling pathways with noncooperative functional responses.
The proliferative effect of both the sst2(b) and bFGF receptors was unaffected following inhibition of p38 MAP kinase. In addition, the sst2(b) receptor induced only a transient activation of p38 and the growth factor receptor had no effect on the activity status of either the SAPKs or p38. There are very few reports demonstrating an activation of p38 through bFGF receptors. Its activation has been implicated in bFGF-mediated tube formation by endothelial cells (44) and in bFGF-induced interleukin-6 synthesis in osteoblasts (22) but not in the mechanism controlling neurite outgrowth (36). A transient activation of p38 by bFGF has been shown to occur in PC12 cells, whereas a stronger and more sustained activation has been observed in fibroblasts (30). Here we show that the sst2(a) receptor can induce a marked and sustained phosphorylation of p38, and its antiproliferative function against bFGF-induced growth was critically dependent on this kinase activity. Both the antiproliferative effect and the induced p38 phosphorylation were mediated through Gβγ release, consistent with the demonstration that Gβγ can stimulate p38 activity in HEK293 cells (54) and JNK activity in COS-7 cells (7, 27). The inability of sst2(a) receptors to mediate a proliferative effect in the presence of the p38 inhibitor despite the induced high-intensity ERK stimulation suggests that Akt activation is essential for somatostatin-induced proliferation. This was supported by the demonstration that transient expression of constitutively active Akt in sst2(a) receptor-expressing cells enabled a proliferative function to be detected in response to somatostatin, providing that the p38 cascade was blocked.
An interesting observation from this study was the enhanced phosphorylation of both ERK and p38 MAP kinase by the concomitant effect of somatostatin and the growth factor. Since bFGF and sst2 receptors have the capacity to stimulate ERK1 and ERK2, the amplification of this signal as observed in the presence of both ligands was perhaps expected. However, the mechanism by which bFGF increases the intensity of somatostatin-induced p38 phosphorylation is unclear. It is possible that bFGF may 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. However, despite the amplification of somatostatin-induced p38 by bFGF in CHOsst2(b) cells, the time profile of its activity status remained transient (>30 min), in marked contrast to the sustained p38 activity induced by sst2(a) receptors (<4 h). An enhancement of the prolonged phosphorylation of ERK induced by somatostatin through the sst2(a) receptor was also observed by inhibiting p38, suggesting that cross talk between the p38 and ERK cascades exists. Taken together, these data demonstrate that a complex interplay exists not only between the transduction cascades activated by a single receptor type but also between those activated by distinct receptors types.
Further examples of the influence of stimulating two receptor types on the net activity of a particular signaling pathway were also demonstrated in this study for the induction of the cell cycle inhibitor p21cip1 and the level of activation of the transcription factor ATF-2. The increased expression of p21cip1 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(a) receptor activities. The importance of a sustained p38 activity in mediating the induction of p21cip1 was further supported by the lack of effect on the expression of this protein by activated sst2(b) receptors in the presence of bFGF. This transduction network combination evoked only transient activation of p38, although a sustained ERK activity was observed, and the inclusion of the PI 3-K inhibitor to prevent any involvement by this kinase also failed to induce p21cip1 (data not shown). This is the first report of p38 MAP kinase being involved in the induction of this cell cycle inhibitor, and it is possible that the antiproliferative function of sst2(a) receptors is mediated through this pathway. However, in addition to the sustained activity of p38, ERK is necessary for the increased expression of p21cip1.
Although this study highlights a correlation between the induction of p21cip1 and the activity status of ATF-2, we have not demonstrated a direct involvement of this transcription factor in the regulation of the cell cycle inhibitor protein. However, it was apparent, as shown for the induction of p21cip1, that a prolonged activation of p38 was also required to phosphorylate ATF-2, since this transcription factor was not stimulated by either sst2(b) or bFGF receptors. An increase in the activity of ATF-2 was observed only during the sustained phase of p38 phosphorylation and was abolished on application of the p38 inhibitor. The activity of ATF-2 was also amplified by the presence of bFGF, consistent with the increased stimulation of p38 by the combined effects of the sst2(a) and growth factor receptors. These data suggest that varying the duration of the p38 stimulus can induce differential transcription factor activation. Phosphorylation of ATF-2 and inhibition of the growth factor-induced proliferative response by somatostatin are both critically dependent on p38 activity, suggesting that the prolonged p38 activity mediated by sst2(a) receptors and not sst2(b) receptors can account for their differential antiproliferative effects.
The contrasting growth responses evoked by the sst2 splice variants can be correlated with their abilities to differentially activate the p38 or Akt pathways, possibly as a consequence of coupling to distinct G-protein pools. The effects on cell growth and the induced changes in the phosphorylation status of ERK1, ERK2, p38, and Akt by the respective somatostatin receptor types were all abolished following pertussis toxin pretreatment, suggesting that the receptor isoforms coupled to Gi proteins. The somatostatin-activated receptor isoforms also exhibited the same preference of coupling to Giα3 over Giα2 subunits, with no significant coupling to Gαs, Gαq/11 or Gα13. It thus seems unlikely that different α subunit coupling can account for the diversity of transductional and functional responses exhibited by these receptor types. However, since all the distinct effects mediated by the splice variants were antagonized by overexpression of transducin, it remains possible that coupling to Gαi3 with different βγ partners may allow the receptor types to selectively activate transduction pathways as well as those that are common to both receptors such as adenylate cyclase inhibition.
Recent reports have suggested a role for chronic ERK activation in mediating the exit from the cell cycle and cellular differentiation (8, 45), whereas in other cell types it is associated with proliferation (9). Such observations indicate the importance of a sustained or transient activation of this particular transduction pathway, as well as cell phenotype, in determining the functional outcome (32). We show in this study that the duration of the p38 MAP kinase cascade, in addition to that of ERK activation, is also critical for dictating functional responses. The p38 and ERK cascades exhibit negative cross talk that may have significant consequences for regulating cellular processes, and the contribution of other input signals, such as that from bFGF receptors, can generate large differences in transcriptional events and subsequent protein expression (Fig. (Fig.9).9). The induction of p21cip1, for example, requires a critical signal strength from the p38 and ERK cascades mediated by the interplay of bFGF and sst2(a) receptor activation, although it has been shown that when Rho is active, induction of p21cip1 by Ras is suppressed (34). The dependency on p38 for p21cip1 expression also suggests that p38 activity may play a dual role not only in mediating apoptotic processes but also as an inhibitor of cell proliferation. This is analogous to that of ERK activation, which can promote mitogenesis as well as providing protection against apoptosis (2). The expression of p21cip1 is transcriptionally regulated by p53 and its function is critical for p53-dependent G1 growth arrest (19). The p53 gene is mutated in approximately half of all human cancers (47), and it is possible that activation of sst2(a) receptors in certain tumors may not result in the induction of this potent antiproliferative activity. This could perhaps explain the poor effects of somatostatin analogues in treating the growth of some cancer cells in the clinical setting (29).
The switch from an antiproliferative to a proliferative activity, as observed for the sst2(b) receptor, appears to be the consequence of poor coupling to the p38 cascade and the selective activation of PI 3-K (Fig. (Fig.9).9). Since the difference between the sst2 receptor isoforms is restricted to their COOH termini, it would imply that this region determines the selection of the appropriate βγ pairings necessary for interaction with the distinct kinase cascades; importantly, these results also demonstrate that even more marked functional outcomes can be derived from the small differences in receptor isoforms than has hitherto been shown (20).
We express our gratitude to John Scott (Vollum Institute) and Peter Parker (ICRF) for helpful comments.