Reduced requirement of mitogen-activated protein kinase (MAPK) activity for entry into the S phase of the cell cycle in Swiss 3T3 fibroblasts stimulated by bombesin and insulin.

Bombesin induced a marked and persistent activation of the mitogen-activated protein kinase kinase-1 (MEK-1), p42(mapk) and p90(rsk) in Swiss 3T3 cells by a pathway that was independent of p74(raf-1) but dependent on the activity of protein kinase C. Pretreatment of the cells with a specific inhibitor of MEK-1, PD 098059, markedly reduced the early and abolished the sustained phase of bombesin-induced p42(mapk) activation. In addition, PD 098059 prevented bombesin-induced DNA synthesis and progression of the cells through the cell cycle, indicating that the mitogenic effect of bombesin is dependent on the activation of p42(mapk). However, in the presence of insulin, which neither stimulated p42(mapk) activation nor DNA synthesis on its own in Swiss 3T3 cells, bombesin potently stimulated DNA synthesis even at concentrations of PD 098059 (15 microM) that completely abolished the mitogenic effect of bombesin alone. Furthermore, Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a marked decrease in the mitogenic effect of bombesin. In contrast, the combination of bombesin and insulin strongly stimulated DNA synthesis in these cells to levels comparable with that obtained in the wild type cells. Thus, our data demonstrate that insulin dramatically reduced the requirement for the mitogen-activated protein kinase pathway for reinitiation of DNA synthesis in bombesin-treated Swiss 3T3 cells and consequently indicate that the contribution of the mitogen-activated protein kinase cascade to mitogenesis depends on the combination of extracellular signals that are used to stimulate these cells.

Bombesin induced a marked and persistent activation of the mitogen-activated protein kinase kinase-1 (MEK-1), p42 mapk and p90 rsk in Swiss 3T3 cells by a pathway that was independent of p74 raf-1 but dependent on the activity of protein kinase C. Pretreatment of the cells with a specific inhibitor of MEK-1, PD 098059, markedly reduced the early and abolished the sustained phase of bombesin-induced p42 mapk activation. In addition, PD 098059 prevented bombesin-induced DNA synthesis and progression of the cells through the cell cycle, indicating that the mitogenic effect of bombesin is dependent on the activation of p42 mapk . However, in the presence of insulin, which neither stimulated p42 mapk activation nor DNA synthesis on its own in Swiss 3T3 cells, bombesin potently stimulated DNA synthesis even at concentrations of PD 098059 (15 M) that completely abolished the mitogenic effect of bombesin alone. Furthermore, Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a marked decrease in the mitogenic effect of bombesin. In contrast, the combination of bombesin and insulin strongly stimulated DNA synthesis in these cells to levels comparable with that obtained in the wild type cells. Thus, our data demonstrate that insulin dramatically reduced the requirement for the mitogen-activated protein kinase pathway for reinitiation of DNA synthesis in bombesin-treated Swiss 3T3 cells and consequently indicate that the contribution of the mitogen-activated protein kinase cascade to mitogenesis depends on the combination of extracellular signals that are used to stimulate these cells.
Neuropeptides stimulate DNA synthesis and proliferation in cultured cells and are implicated as growth factors in embryogenesis, tissue regeneration, and tumorigenesis (1,2). In particular, bombesin is a potent mitogen for quiescent Swiss 3T3 cells (3), a useful model to elucidate signal transduction pathways leading to cell proliferation (4). Bombesin binds to a seven-transmembrane receptor (5-7) and induces rapid polyphosphoinositide hydrolysis, Ca 2ϩ mobilization, PKC 1 ac-tivation and tyrosine phosphorylation of focal adhesion-associated proteins, including p125 FAK (8 -10). Further downstream, bombesin induces the expression of immediate early genes and subsequently stimulates DNA synthesis via PKC-dependent and -independent pathways (11).
The mitogen-activated protein (MAP) kinases are a family of highly conserved serine/threonine kinases that are activated by a range of extracellular signals (12,13). The two best characterized isoforms p42 mapk and p44 mapk are directly activated by phosphorylation on specific tyrosine and threonine residues by the dual specificity MAPK kinase (or MEK) of which at least two isoforms have been identified in mammalian cells (14 -16). Several pathways leading to MEK activation have been described. Tyrosine kinase receptors induce MAPK activation via SOS-mediated accumulation of p21 ras -GTP, which then activates a kinase cascade comprising p74 raf-1 , MEK, and MAPK (12,13). In contrast, the mechanisms by which G proteincoupled receptors induce MAPK activation remain less well defined though p21 ras -and PKC-dependent pathways have been implicated (17)(18)(19)(20)(21). MAPK has various substrates, including transcription factors (22) and other protein kinases such as p90 rsk (23). Recent reports indicate that sustained activation of p42/p44 mapk is both necessary and sufficient to induce proliferation or differentiation of various cell lines (24 -27). However, the contribution of the activation of the MAPK cascade to bombesin-stimulated DNA synthesis has as yet not been defined.
Here we report that bombesin stimulates MEK-1, p42 mapk , and p90 rsk activity in Swiss 3T3 cells through a PKC-dependent pathway. Using the selective inhibitor of MEK-1 activation, PD 098059, and Swiss 3T3 cells stably transfected with interfering mutants of MEK-1, we show that MAPK activation is essential for bombesin-stimulated DNA synthesis. However, using both experimental approaches we also show that insulin strikingly reduces the requirement of MAPK activity for reinitiation of DNA synthesis in bombesin-stimulated cells. Thus, our results demonstrate, for the first time, that the level of MAPK activity required for the transition of quiescent cells to S phase of the cell cycle depends on the combination of growth factors used to stimulate the cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 fibroblasts were maintained in DMEM supplemented with 10% FBS in a humidified atmosphere containing 10% CO 2 and 90% air at 37°C. For experimental purposes, cells were plated either in 35-mm Nunc Petri dishes at 10 5 cells/dish or in 100-mm dishes at 6 ϫ 10 5 cells/dish in DMEM containing 10% FBS and used after 6 -8 days when the cells were confluent and quiescent.
p42 mapk Mobility Shift Assays-Activation of p42 mapk can be determined by the appearance of slower migrating forms in SDS-PAGE gels (28). Quiescent cultures of Swiss 3T3 cells were treated with factors as indicated, and the cells were lysed in 2 ϫ SDS-PAGE sample buffer (200 mM Tris-HCl, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glyc-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. erol, pH 6.8) and analyzed by SDS-PAGE. After SDS-PAGE, proteins were transferred to Immobilon membranes. Membranes were blocked in PBS containing 3% non-fat milk and incubated for 1 h at 22°C with a polyclonal p42 mapk antiserum (1:1000) in PBS containing 3% non-fat dried milk. Immunoreactive bands were visualized using 125 I-labeled protein A followed by autoradiography.
Immune Complex Assay for p42 mapk and p90 rsk Activation-Quiescent and confluent Swiss 3T3 cells were treated with factors as described in the figure legends and lysed at 4°C in 1 ml of a solution containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 M Na 3 VO 4 , 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride (lysis buffer). Lysates were clarified by centrifugation at 15,000 ϫ g for 10 min at 4°C. Immunoprecipitation was performed using a polyclonal anti-p42 mapk antibody or a polyclonal anti-p90 rsk antibody, incubating the samples on a rotating wheel for 2 h. Protein A-agarose beads (40 l, 1:1 slurry) were added for the second hour. Immune complexes were collected by centrifugation and washed twice in lysis buffer and twice in kinase buffer (15 mM Tris-HCl, pH 7.4, 15 mM MgCl 2 ). The kinase reaction was performed by resuspending the pellet in 25 l of kinase assay mixture containing kinase buffer, 100 M ATP, 100 Ci/ml [␥-32 P]ATP, 100 nM microcystin LR, and either 1 mg/ml MBP-peptide (APRTPGGRR) or S6 peptide (RRRLSSLRA) for the assays of p42 mapk and p90 rsk , respectively. Incubations were performed for 10 min (linear assay conditions) at 30°C and terminated by spotting 20 l of the supernatant onto P81 chromatography paper (Whatman). Filters were washed four times for 5 min in 0.5% orthophosphoric acid, immersed in acetone, and dried before Cerenkov counting. The average radioactivity of two blank samples containing no immune complex was subtracted from the result of each sample. The specific activity of [␥-32 P]ATP used was 900-1200 cpm/pmol. p74 raf-1 Kinase and MEK Kinase Assays-Quiescent cells were treated as indicated and lysed in lysis buffer as above with the addition of 100 nM microcystin LR, 10 g/ml aprotinin, and 10 g/ml leupeptin. Immunoprecipitations were performed incubating the lysates with a polyclonal anti-p74 raf-1 antibody or a 1:1 mixture of monoclonal anti-MEK-1 and anti-MEK-2 antibody or either monoclonal anti-MEK-1 or anti-MEK-2 antibody for 2 h with 40 l of protein A-agarose (1:1 slurry) added for the second hour. Immune complexes were collected by centrifugation and washed three times in lysis buffer without phenylmethylsulfonyl fluoride and once with buffer A (50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.5 mM Na 3 VO 4 and 0.1% 2-mercaptoethanol). Pellets were then resuspended in 30 l of MEK/MAPK buffer (30 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1% 2-mercaptoethanol, 6.5 g/ml GST-MEK, 100 g/ml GST-MAPK, 0.03% Brij-35, 10 mM Mg-ATP and 20 mM N-octyl-␤-D-glucopyranoside) and incubated at 30°C for 30 min. The MEK kinase buffer did not contain GST-MEK. The reaction was then terminated by diluting the supernatant in 40 l of buffer A containing 1 mg/ml of bovine serum albumin and, after mixing, 10 l of the supernatant was removed to a fresh tube. MAPK activation was then measured using the MBP-peptide phosphorylation assay, essentially as described above. The specific activity of [␥-32 P]ATP used was 900-1200 cpm/pmol. twice with PBS and incubated in 5% trichloroacetic acid at 4°C for 30 min to remove acid-soluble radioactivity, washed with methanol, and solubilized in 1 ml of 2% Na 2 CO 3 , 0.1 M NaOH, 1% SDS. The acid-insoluble radioactivity was determined by scintillation counting in 6 ml of Ultima Gold (Packard).
For detection of BrdUrd incorporated into cellular DNA, confluent and quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated with DMEM/Waymouth's medium (1:1 (v/v)) containing 10 M BrdUrd and various additions as described in the figure legends. After 40 h of incubation at 37°C, cultures were washed twice with PBS, fixed in 70% ethanol for 20 min, and incubated with anti-BrdUrd monoclonal antibody followed by labeling the monoclonal antibody with anti-mouse Ig-fluorescein isothiocyanate. Cells were examined using a Zeiss Axiophot immunofluorescence microscope, and data are expressed as the percentage of BrdUrd-labeled nuclei.
FACS Analysis-The number of cells in G 0 /G 1 , S, G 2 , or M phase was determined by FACS analysis. After washing three times with PBS containing 4 mM EDTA, cells were detached by treatment with trypsin (0.025%), suspended in DMEM containing 10% FBS, centrifuged at FIG. 1. Effect of bombesin on p42 mapk , MEK-1, p74 raf-1 , and p90 rsk . A, quiescent Swiss 3T3 cells were treated with various concentrations of bombesin for 5 min (left) or with 10 nM bombesin for various times (right) as indicated. Cells were lysed and mobility shift assays (upper panels) or p42 mapk immune complex kinase assays (lower panels) were performed as described under "Experimental Procedures." The results shown in each case are representative of three independent experiments. The positions of nonphosphorylated p42 mapk and the slower migrating phosphorylated form of p42 mapk in the mobility shift assay are indicated. Results of the kinase assays are the means of duplicates and are expressed as percentage of the maximum bombesinstimulated p42 mapk activation (5000 -6000 cpm/1.5 ϫ 10 6 cells at 5 min); left panel, inset: Swiss 3T3 cells were treated with either 10 nM bombesin (B) or 10 ng/ml PDGF (P) for 5 min, lysed, and p42 mapk immune complex kinase assays were performed as described under "Experimental Procedures." Data shown are representative of three independent experiments each performed in duplicate and are expressed as counts/min ϫ 10 3 /1.5 ϫ 10 6 cells. B, quiescent Swiss 3T3 cells were stimulated with 10 nM bombesin (Bom) or 10 ng/ml PDGF for 3 min. Cells were lysed, and immune complex kinase assays were performed as described under "Experimental Procedures" using anti-MEK-1/MEK-2 monoclonal antibodies. Results are expressed as percentage of the maximum PDGF-stimulated activation of MEK-1/MEK-2 (19,000 -25,000 cpm/1.5 ϫ 10 6 cells at 3 min), and the data shown are the mean Ϯ S.E. of three independent experiments each performed in duplicate. C, quiescent Swiss 3T3 cells were treated with 10 nM bombesin (Bom) or 10 ng/ml PDGF for 3 min, cells were lysed, and two-step immune complex kinase assays were performed using an anti-p74 raf-1 polyclonal antibody as described under "Experimental Procedures." Results are expressed as percentage of the maximum PDGF-stimulated activation of p74 raf-1 (5000 -6000 cpm/1.5 ϫ 10 6 cells at 3 min) and are the mean Ϯ S.E. of five independent experiments each performed in duplicate. D, quiescent Swiss 3T3 cells were treated with 10 nM bombesin for various times (left) or with various concentrations of bombesin for 5 min (right) and p90 rsk immune complex kinase assays were performed as described under "Experimental Procedures." Results are the means of duplicates, are expressed as percentage of the maximum bombesin-stimulated activation (10,000 -15,000 cpm/1.5 ϫ 10 6 cells at 5 min), and are representative of three independent experiments. The specific activity of [␥-32 P]ATP used in all experiments was 900-1200 cpm/pmol. 1000 ϫ g for 5 min, and resuspended in PBS. Cells (10 6 ) in a volume of 200 l were stained by adding 800 l of staining solution containing propidium iodide (50 g/ml), sodium citrate (1 mg/ml), and Triton X-100 (0.1%). The stained chromosomal DNA was kept on ice for 15 min and analyzed on a FACStar 4 (Becton Dickinson).
Materials-The PKC inhibitor GF 109203X and microcystin LR were obtained from Calbiochem-Novabiochem Ltd., Nottingham, UK. PDGF (BB homodimer), 125 I-labeled protein A (15 mCi/mg), and [␥-32 P]ATP (370 MBq/ml) were from Amersham Corp., Amersham, UK. The MEK-1 inhibitor PD 098059 was the generous gift of Alan R. Saltiel, Department of Signal Transduction, Parke Davis Research Division, Ann Arbor, MI. The polyclonal anti-p42 mapk antibody raised against a COOH-terminal peptide (EETARFQPGYRS) and the polyclonal antibody against p90 rsk raised against a COOH-terminal peptide (IESSI-LAQRRVRKLPSTTL) were the generous gift from Dr. J. Van Lint, Katholieke Universiteit Leuven, Belgium. The polyclonal anti-p74 raf-1 antibody was obtained from Santa Cruz Biotechnology Ltd., Santa Cruz, CA. The monoclonal anti-MEK-1 and anti-MEK-2 antibodies were obtained from Affiniti Research Products Ltd., Nottingham, UK. GST-MEK and GST-MAPK fusion proteins were the generous gift from Professor C. Marshall, Institute of Cancer Research, London, UK. All other reagents were of the purest grade available.

RESULTS
Bombesin Induces Activation of MAPK, MEK-1, and p90 rsk -To examine the effects of bombesin on p42 mapk activity, lysates of Swiss 3T3 cells treated with various concentrations of bombesin for 5 min were analyzed by Western blotting using a specific polyclonal antibody against p42 mapk . Activation of p42 mapk was determined by the appearance of slower migrating forms, which results from the phosphorylation of specific threonine and tyrosine residues within subdomain VIII (28). Bombesin induced p42 mapk activation in a concentration-dependent manner as judged by the mobility shift assay (Fig. 1A, top left  panel). Half-maximum and maximum effects in immune complex kinase assays were achieved at 0.15 and 1 nM bombesin (Fig. 1A, lower left panel). The maximum effect of bombesin on p42 mapk activation was comparable with that induced by 10 ng/ml PDGF in Swiss 3T3 cells (Fig. 1A, lower left panel, inset). Activation of p42 mapk peaked after 5 min of bombesin stimulation and remained elevated above base-line levels after 3 h of stimulation (Fig. 1A, right panel). Similar results were obtained in mobility shift assays using a specific polyclonal antibody against p44 mapk (data not shown).
Bombesin also markedly stimulated the activity of p90 rsk , a major downstream target of MAPK (23,29). As shown in Fig.  1D (left panel), p90 rsk activation in response to bombesin was rapid reaching its maximum after 5 min of incubation and remained elevated to 30% of its maximum value after 1 h of stimulation. Bombesin-induced p90 rsk activity in a concentration-dependent manner; maximum stimulation in immune complex kinase assays was achieved at 1-3 nM bombesin (Fig.  1D, right panel).
Role of PKC and MEK-1 in Bombesin-induced p42 mapk Activation-Bombesin potently simulates PKC activation and Ca 2ϩ mobilization from intracellular stores (6,8), but does not promote p21 ras activation (30,31) in Swiss 3T3 cells. PKC stimulation has been identified as a major pathway leading to MAPK activation (32)(33)(34)(35), but the role of this pathway in bombesinmediated MAPK activation remains unclear (31). Here we show that pretreatment of Swiss 3T3 cells with the selective PKC inhibitor GF 109203X (9,36,37) prevented both the mobility shift of p42 mapk (Fig. 2A, upper left panel) and the increase in p42 mapk activity in immune complex kinase assays in response to bombesin. The effect of GF 109203X was concentration-dependent; a complete inhibition of bombesin-induced p42 mapk activation was observed at 5 M GF 109203X ( Fig. 2A,  left). Similar results were obtained when PKC was down-regulated by chronic treatment with PDB (results not shown), in agreement with previous data (38).
Pretreatment of Swiss 3T3 cells with GF 109203X or downregulation of PKC by prolonged pretreatment with PDB also prevented MEK-1 and p90 rsk activation in response to bombesin ( Fig. 2A and results not shown). In contrast, inhibition of Ca 2ϩ influx or mobilization from intracellular stores did not affect bombesin-mediated activation of p42 mapk or p90 rsk (results not shown). Thus, PKC activation is a major signaling pathway leading to MAPK activation in bombesin-treated cells, possibly through a MEK-1-dependent pathway.
To further examine whether MEK-1 is the main upstream regulator of bombesin-induced MAPK activity, quiescent Swiss 3T3 cells were treated with PD 098059, a recently identified compound that selectively inhibits MEK-1 activation (39,40). PD 098059 prevented the mobility shift of p42 mapk in response to bombesin (Fig. 2B, upper panel) and markedly decreased the early (5 min) and the late phase (60 min) of bombesin-stimulated p42 mapk activity in immune complex kinase assays in a concentration-dependent manner (Fig. 2B, lower panel, inset). A maximum effect was achieved at 15 M PD 098059; at this concentration PD 098059 reduced the early phase of bombesininduced p42 mapk activation by 60% and caused an almost complete inhibition of the sustained activation of p42 mapk (Fig. 2B,  lower panel and inset). These findings indicate that PD 098059 is a potent inhibitor particularly of the sustained phase of bombesin-mediated p42 mapk activation in Swiss 3T3 cells.
Role of Activation of p42 mapk in Bombesin-stimulated DNA Synthesis-It has been reported that the sustained phase of p42 mapk activation is necessary for the induction of DNA synthesis by growth factors in fibroblasts (24,27). Therefore, we examined the contribution of the MAPK pathway to bombesinstimulated reinitiation of DNA synthesis in Swiss 3T3 cells using PD 098059. Quiescent cultures of these cells were stimulated with 10 nM bombesin in the presence of increasing concentrations of PD 098059. Cumulative [ 3 H]thymidine incorporation was measured after 40 h of incubation. As shown in Fig. 3A (closed circles), PD 098059 dramatically decreased bombesin-induced DNA synthesis in a concentration-dependent manner. Half-maximum inhibition was achieved at 1 M PD 098059, and treatment of the cells with 15 M PD 098059 completely blocked DNA synthesis induced by bombesin (Fig.  3A, closed circles). We verified that [ 3 H]thymidine incorporation in response to bombesin was virtually abolished by 15 M PD 098059 at all times examined up to 48 h of incubation (results not shown). Thus, the activity of MEK-1-dependent MAPKs is essential for bombesin-induced mitogenesis.
We also examined the effect of PD 098059 on [ 3 H]thymidine incorporation in Swiss 3T3 cells stimulated by bombesin in the presence of insulin, which is not a sole mitogen for these cells, but potentiates the mitogenic activity of bombesin (3). A salient feature shown in Fig. 3A (open circles)  shown). Insulin also partially reversed the inhibition of DNA synthesis by 15 M PD 098059 in Swiss 3T3 cells stimulated with bombesin at 1 nM instead of 10 nM (results not shown).
Recently, Alessi et al. (40) demonstrated that the inhibitory effect of PD 098059 on the activation of p42 mapk depends on the strength of activation of p74 raf-1 and MEK by growth factors. We therefore examined whether insulin could synergize with bombesin in MAPK activation, thereby reversing the inhibition of p42 mapk activity and DNA synthesis by PD 098059. As shown in Fig. 3B, insulin neither induced p42 mapk activation in Swiss 3T3 cells nor potentiated p42 mapk activation induced by 10 nM bombesin. Insulin slightly reversed the inhibitory effect of PD 098059 on the early phase of p42 mapk activation induced by bombesin. However, PD 098059 inhibited the late phase of bombesin-stimulated p42 mapk activation virtually to the same degree in the absence or presence of insulin (Fig. 3B).
The results presented in Fig. 3, A and B, suggest that insulin markedly reduced the requirement of a sustained increase of MAPK activity for bombesin-stimulated DNA synthesis. To test this possibility further, parallel cultures of quiescent Swiss 3T3 cells were preincubated with various concentrations of PD 098059 and subsequently stimulated with either bombesin or bombesin and insulin for 5 or 60 min to determine p42 mapk activity and for 40 h to assess [ 3 H]thymidine incorporation. Fig.  3C shows [ 3 H]thymidine incorporation of cells stimulated with bombesin (closed circles) or bombesin and insulin (open circles) as a function of p42 mapk activity measured after 5 min (Fig. 3C,  left panel) and 60 min (Fig. 3C, right panel) of stimulation with bombesin or bombesin and insulin at each concentration of PD 098059. It can be seen that a reduction of as little as 30% of the late phase of p42 mapk activation severely impaired DNA synthesis induced by bombesin. In striking contrast, a decrease of 80% of the late phase of p42 mapk activation only slightly reduced the stimulation of [ 3 H]thymidine incorporation induced by the combination of bombesin and insulin. These results suggest that insulin induced a striking shift in the dependence of DNA synthesis on MAPK activity in bombesin-stimulated cells.
The conclusions drawn from Fig. 3, using [ 3 H]thymidine incorporation, were further substantiated by experiments in which DNA synthesis in Swiss 3T3 cells was determined using either an immunofluoresence assay to detect BrdUrd incorporated into cellular nuclei or FACS analysis. As shown in Fig.  4A, treatment with 15 M PD 098059 markedly reduced the proportion of BrdUrd-labeled nuclei in response to bombesin (from 64 to 15%). In contrast, DNA synthesis induced by bombesin and insulin (98% stained nuclei) was virtually undiminished by treatment with 15 M PD 098059 (Fig. 4, A and B).
The FACS analyses presented in Fig. 4C demonstrate that addition of 10 nM bombesin increased the proportion of the cell population moving from G 0 /G 1 to S ϩ G 2 ϩ M, an effect inhib- Interfering Mutants of MEK-1 Is Blocked in Response to Bombesin, but Not in Response to Bombesin and Insulin-Expression of interfering MEK-1 mutants with alanine substitutions at serine 217 or serine 221 have been shown to block MAPK activation in vivo (27). If, as indicated by the preceding results, bombesin induces DNA synthesis through a MEK-1-dependent MAPK pathway in Swiss 3T3 cells, expression of interfering mutants of MEK-1 should prevent bombesin-induced [ 3 H]thymidine incorporation. To test this prediction, we employed Swiss 3T3 cells overexpressing wild type MEK-1 and Ala 217 and Ala 221 mutants to comparable levels (41). To verify that the level of expression of these mutants interfered with p42 mapk activation in Swiss 3T3 cells, we performed mobility shift assays using lysates of these three cell subtypes treated with bombesin or solvent. As shown in Fig. 5, p42 mapk activation in cells overexpressing wild type MEK-1 was similar to that achieved with bombesin in untransfected cells. However, both the Ala 217 and Ala 221 mutants partially inhibited bombesininduced p42 mapk activation in the mobility shift assays.
As shown in Fig. 5 (upper panels), the levels of [ 3 H]thymidine incorporation induced by bombesin at either 2 or 4 nM in cells overexpressing the wild-type MEK-1 were comparable with those in untransfected cells and further enhanced by the presence of insulin. In contrast, the ability of bombesin to stimulate DNA synthesis was completely abolished in cells overexpressing the interfering mutants (Fig. 5, lower panels). The striking finding, however, was that in the presence of insulin, which did not induce significant [ 3 H]thymidine incorporation on its own, bombesin-stimulated [ 3 H]thymidine incorporation in these cells was comparable with that in untransfected cells or those overexpressing wild-type MEK-1 (Fig. 5, lower panels). DISCUSSION While a large number of studies have been dedicated to the dissection of the upstream pathways leading to MAPK activation by a wide variety of extracellular stimuli, the precise role of this kinase cascade in the transition of quiescent cells to the S phase of the cell cycle induced by defined mitogens added singly or in combination has been much less explored.
In the present study we examined the contribution of the MAPK pathway to the stimulation of DNA synthesis induced by bombesin in Swiss 3T3 cells using two different experimental approaches. First, we found that the specific MEK-1 inhibitor, PD 098059, dramatically inhibited bombesin-stimulated DNA synthesis and progression through the cell cycle as judged by three different assays, including FACS analysis. In fact, a modest inhibition of p42 mapk activation severely reduced bombesin-mediated DNA synthesis. Second, interfering mutants of MEK-1 stably transfected into Swiss 3T3 cells also provided convincing evidence that MEK-1 activation is essential for DNA synthesis induced by bombesin. Given that bombesin predominantly stimulates MEK-1 activity in Swiss 3T3 cells (Fig. 1) and p42 mapk and p44 mapk are the only known substrates for MEK-1 (27), the inhibitory effects on cellular DNA synthesis described above occur most likely at the level of activation of MAPK. Thus, our results show that a high level of p42 mapk activity is crucial for bombesin-stimulated mitogenesis.
Phorbol ester-sensitive PKC isoforms play a critical role in bombesin-mediated DNA synthesis (3). Since activation of MEK-1, p42 mapk , and p90 rsk is downstream of PKC (Fig. 2) and MEK-1 activation is essential for DNA synthesis (Figs. [3][4][5], it is conceivable that a major function of PKC in bombesin-stimulated mitogenesis is to activate the MAPK cascade. Interestingly, bombesin, in the presence of insulin, is known to stimulate DNA synthesis through a PKC-independent pathway (11), although insulin neither induces p42 mapk activation nor potentiates the stimulation of this pathway by bombesin (Fig. 3). These considerations raise important questions regarding the requirement of p42 mapk activity for the stimulation of DNA synthesis induced by the combination of bombesin and insulin.
Our results demonstrate that DNA synthesis induced by bombesin and insulin was only slightly inhibited by PD 098059 added at concentrations that completely abolished DNA synthesis in response to bombesin alone. Furthermore, bombesin failed to induce DNA synthesis in Swiss 3T3 cells stably transfected with interfering mutants of MEK-1, whereas addition of insulin together with bombesin produced a marked stimulation of DNA synthesis in these cells. Thus, using two independent experimental approaches, our results indicate that the requirement of the MAPK cascade for DNA synthesis is strikingly reduced in Swiss 3T3 cells stimulated with bombesin and insulin. The results presented here have several important implications. Recently, Cowley et al. (27) emphasized the importance of the kinetics of MAPK activation and the cellular context in defining the role of the MAPK pathway in the production of biological responses. Our results provide a novel insight into the role of the MAPK cascade in cellular mitogenesis, demonstrating, for the first time, that the level of MAPK activity required for the transition of quiescent cells to the S phase of the cell cycle depends on the combination of growth factors used to stimulate the cells. Since each cell in a multicellular organism is exposed physiologically to multiple growth regulatory and differentiation signals, we suggest that the level of MAPK activity required for cellular mitogenesis is likely to depend on the repertoire of extracellular signals that interact with the cell at any given time. This concept is relevant for the development of antiproliferative drugs directed against the MAPK pathway. It has been proposed that MEK-1 could be a useful target to select drugs or other agents capable of inhibiting cell proliferation (27). In view of the results presented in this study, we predict that the potency of MEK-1 inhibitors as blockers of cell proliferation will depend dramatically on the combination of mitogenic signals that interact with the target cell.