Address correspondence and reprint requests to Marina Guizzetti, Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE #100 Seattle, WA 98105, USA. E-mail: firstname.lastname@example.org
The signal transduction pathways that mediate the mitogenic response of muscarinic acetylcholine receptors in astroglial cells have not been fully elucidated. In this study we investigated the activation of p70S6 kinase (p70S6K) by carbachol in 1321 N1 astroctyoma cells. Carbachol induced a dose- and time-dependent activation of p70S6K, as evidenced by increased phosphorylation at Thr-389, Thr-421 and Ser-424, by increased p70S6K activity, and by a shift in its molecular weight. Activation of p70S6K was mediated by M3 muscarinic acetylcholine receptors (mAChRs) and was inhibited by two phosphatidylinositol-3-kinase (PI3-K) inhibitors, by a pseudosubstrate to protein kinase C (PKC) ζ, and by the p70S6K inhibitor rapamycin. Carbachol-induced DNA synthesis was strongly inhibited by rapamycin, suggesting that p70S6K activation plays an important role in carbachol-induced cell proliferation. Ethanol (25–100 mm) has been shown to inhibit carbachol-induced proliferation of astroglial cells. In the same range of concentrations, ethanol also inhibits carbachol-induced activation of PKCζ and of p70S6K. On the other hand, inhibition of PI3-kinase was only observed at higher ethanol concentrations. These results indicate that activation of the PKCζ→ p70S6K pathway by M3 mAChRs may play a role in the increased DNA synthesis and may represent a target for ethanol-induced inhibition of astroglial cell proliferation.
A variety of cells expressing muscarinic acetylcholine receptors (mAChRs) proliferate in response to stimulation by cholinergic agonists (reviewed in Costa et al. 2001). The mAChR family consists of five genetically defined subtypes which preferentially couple to the GTP-binding (G) proteins Gαq/Gα11 (M1, M3, and M5 mAChRs) and to Gαi/Gαo (M2 and M4 mAChRs; Bonner 1992; Wess 1993). Studies carried out in cells transfected with different subtypes of mAChRs indicated that only M1, M3, and M5 mAChRs induce cell proliferation, whereas the M2 and M4 receptors do not (Gutkind et al. 1991).
The 70-kDa ribosomal S6 kinase (p70S6K) is an ubiquitously expressed serine/threonine protein kinase that phosphorylates the 40S ribosomal protein S6 in response to mitogen stimulation (Dufner and Thomas 1999). S6 phosphorylation up-regulates translation of mRNAs with 5′-oligopyrimidine tracts (Jefferies et al. 1997). The regulation of p70S6K is complex and requires phosphorylation at multiple sites for full activation of the kinase. Recent studies have shown that various signaling pathways influence p70S6K. One pathway involves PI3-K, its downstream effector phosphoinositide-dependent protein kinase-1 (PDK-1) and, perhaps, Akt (Alessi et al. 1998; Pullen et al. 1998). An additional pathway essential for p70S6K activation involves the mammalian target of rapamycin (mTOR; Burnett et al. 1998). Other phosphorylation sites involved in the activation of p70S6K are located in its C-terminal domain and are followed by proline residues, suggesting they may be targets for proline-directed kinases such as MAPK. However, though p70S6K can be phosphorylated in vitro by MAPK (Mukhopadhyay et al. 1992), p70S6K appears to reside on a signaling pathway distinct form the Ras/MAPK pathway (Ballou et al. 1991). More recently, the atypical PKC isoforms ζ and λ, which can be regulated by PI3-K and PDK-1, have also been implicated in p70S6K regulation, although it is not yet known whether they directly phosphorylate p70S6K (Akimoto et al. 1998; Romanelli et al. 1999).
In the present study we investigated whether stimulation of mAChRs in astrocytoma cells could lead to activation of p70S6K and whether its activation plays a role in astroglial cell proliferation.
Ethanol has been found to be a potent inhibitor of muscarinic receptor-stimulated proliferation of astroglial cells, with an IC50 of 10–25 mm (Guizzetti and Costa 1996). As astrocyte proliferation is a major event occurring during the brain growth spurt, a period that is exquisitely sensitive to the developmental neurotoxic effect of ethanol, such inhibition of muscarinic receptor-induced astrocyte proliferation has been suggested to be relevant in the causation of the microencephaly present in the fetal alcohol syndrome (Guizzetti et al. 1997; Costa and Guizzetti 1999). Thus, the second aim of this study was to determine whether activation of p70S6K by mAChRs may represent a target for ethanol, responsible for its inhibition of astroglial cell proliferation.
Materials and methods
Dulbecco's modified Eagle medium (DMEM), fetal bovine serum and trypsin were purchased from Gibco (Grand Island, NY, USA). [Methyl-3H]thymidine (6.7 Ci/nmol) was from New England Nuclear (Boston, MA, USA). The phospho(Thr-389) p70S6K, the phospho(Thr-421/Ser-424) p70S6K, the phospho(Ser-473) Akt, the Akt, the p70S6K, and the phospho(Thr-410) PKCζ polyclonal antibodies, and the 10× lysis buffer were from Cell Signaling (Beverly, MA, USA); the PKCζ polyclonal antibody, the protein A agarose, and the protease inhibitor cocktail was from Roche (Indianapolis, IN, USA). The p70S6K activity kit was from Upstate Biotechnology (Lake Placid, NY, USA). The enhanced chemiluminescence (ECL) detection kit was from Amersham Corp (Arlington, IL, USA). Polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, MA, USA). 4-Diphenylacetoxy-N-methylpiperidine (4-DAMP), gallamine, and pertussis toxin (PTX) were from RBI (Matick, MA, USA). Myristoylated peptides corresponding to the pseudosubstrate regions of PKCζ (peptide Z: positions 116–124; sequence: myrRRGARRWRK) and PKCα (peptide A: positions 22–30; sequence: myrRKGALRQKN) were custom-synthesized from United Biochemical Research, Inc. (Seattle, WA, USA). LY294002, PD98058 and GF109203X were from Calbiochem (La Jolla, CA, USA), while UO126 was from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA).
The human astrocytoma cell line 1321 N1 was maintained in DMEM, low glucose, supplemented with 5% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin in 75-cm2 flasks under a humidified atmosphere of 5% CO2/95% air at 37°C. For the proliferation experiments, cells were seeded in 24-well plates; for western blot experiments cells were seeded in 100-mm dishes. Forty-eight hours before each experiment cells were shifted to the same medium without serum, supplemented with 0.1% bovine serum albumin.
Pertussis toxin (100 ng/mL) was added to cells 20 h before treatment with mitogens. 12-O-tetradecanoyl phorbol-13-acetate (PMA; 300 nm) was added to cells 24 h before agonist treatment. All other inhibitors were added 15 min prior to agonists. In order to reduce ethanol evaporation during short-term exposure to ethanol (1 h or less), Parafilm was put around two-thirds of the perimeter of the Petri dishes, closing part of the space between the lid and the dish without totally stopping the air exchange. We found that in these conditions the ethanol loss does not exceed 10%, cells do not suffer from lack of oxygen, and no pH changes occur. Ethanol levels were determined randomly in the culture medium before and after the incubation, using a kit (Sigma) for the spectrophotometric detection of the reduction of NAD to NADH in presence of ethanol and alcohol dehydrogenase.
After agonist treatment, cells were scraped in buffer containing Tris–HCl 20 mm (pH 7.4), EGTA 2 mm, EDTA 10 mm, β-mercaptoethanol 0.1%, and a protease inhibitor cocktail. Cells were sonicated in five 10-s bursts using the W-220 cell disruptor (Heat Systems Ultrasonics, Inc., Farmingdale, NY, USA) at power setting 3. After 5 min of centrifugation at 1000 g, the supernatant was centrifuged at 100 000 g for 20 min. The supernatant was collected as cytosol. The membrane pellet was resuspended in the sonication buffer containing 1% sodium dodecyl sulfate (SDS) and shaken at 4°C for 30 min. Proteins were quantified by the Bradford's method, and a 5× sample buffer was added; 50 µg proteins were loaded on a 8% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gel.
Immunoprecipitation and measurement of p70S6K activity
At the end of the treatment with agonists and/or inhibitors, cells were washed with ice-cold PBS and dissolved in cell lysis buffer (20 mm Tris pH 7.5, 150 mm NaCl, 1.0 mm EDTA, 1.0 mm EGTA, 1.0% Triton X-100, 50 mm sodium fluoride, 10 mm sodium pyrophosphate, 10 mm sodium β-glycerophosphate, 1.5 mm sodium orthovanadate, and a protease inhibitor cocktail). After sonication of the samples four times for 5 s each, the cell lysates were centrifuged at 14 000 g for 15 min at 4°C and the supernatant was collected. Proteins were quantified by the Bradford's method, the cell lysate (1.0–1.5 mg proteins) was incubated with 4 µg polyclonal antibody against p70S6K and 25 µL protein A agarose, and incubated over night at 4°C under mixing by inversion. The immune complexes were pelleted at 4°C by centrifugation at 14 000 g for 10 min. The beads were washed twice with 1 mL cell lysis buffer and twice with 200 µL of kinase reaction buffer (20 mm MOPS, pH 7.2, 25 mmβ-glycerol phosphate, 5 mm EGTA, 1 mm sodium orthovanadate, and 1 mm dithiothreitol). The pellet was resuspended in 100 µL of kinase reaction buffer (about 10 µg protein/µL). p70S6K activity was assessed by the S6 kinase assay kit (Upstate Biotechnology) following the instructions of the manufacturer. The kit is based on the phosphorylation of a specific substrate (AKRRRLSSLRA) modeled after the major phosphorylation site in S6 kinase in the presence of [γ-32P]ATP, a PKC inhibitor peptide, a PKA inhibitor peptide, and phosphatase inhibitors. The samples were incubated for 10 min at 30°C, 25 µL of the mixture was spotted in numbered paper squares, the reaction was stopped by washing the paper squares with 0.75% phosphoric acid followed by a wash in acetone. Samples were and counted in a Beckmann LS 5000 CE scintillation counter.
Cell extracts were prepared as described in the previous section and 50 µg proteins were loaded on a 7.5% SDS–PAGE gel. After separation, proteins were transferred to PVDF membranes, which were incubated in the presence of polyclonal antibodies to phospho(Thr-389) p70S6K, phospho(Thr-421/Ser-424) p70S6K, phospho(Ser-473) Akt, or phospho(Thr-410) PKCζ (dilution 1 : 1000) polyclonal antibodies. p70S6K exhibits a retarded electrophoretic mobility characteristic of the phosphorylated form of this enzyme. The electrophoretic mobility of p70S6K was assessed by Western blot analysis using a polyclonal antibody against p70S6K; Akt and PKCζ levels were assessed by western blot analysis using a polyclonal antibody against Akt or PKCζ. Protein molecular weight markers were run with each gel.
Incorporation of [methyl-3H]-thymidine into cell DNA was measured as described previously (Guizzetti et al. 1996). Briefly, cells were treated with agonists and/or inhibitors for 24 h. One µCi/well [methyl-3H]-thymidine was included for the last 6 h of incubation. The monolayer was fixed in methanol and the DNA precipitated with 10% trichloroacetic acid, dissolved in 500 µL of 1 N NaOH and counted in a Beckmann LS 5000 CE scintillation counter.
One way analysis of variance (mnova), followed by the Fisher's least significance difference test was used to determine statistically significant differences from controls.
The cholinergic agonist carbachol (1 mmnova) induced phosphorylation of Thr-389 on p70S6K, a key phosphorylation site for the activation of this enzyme (Pullen and Thomas 1997; Weng et al. 1998), in 1321 N1 astrocytoma cells, with a maximal effect between 15 min and 1 h (Fig. 1a). Carbachol, in the same time frame, also induced a shift in the p70S6K molecular weight, indicating that other phosphorylation sites are also phosphorylated (Fig. 1b). Concentration–response experiments indicated that phosphorylation of Thr-389 on p70S6K and the shift in molecular weight shift occurred at carbachol concentrations of 100 nmnova to 1 mmnova (Figs 1c and d). Furthermore, phosphorylation of Thr-421 and Ser-424, which lie within a Ser-Pro rich region located within the pseudosubstrate region (Pullen and Thomas 1997), was also observed upon carbachol stimulation (Fig. 1e); phosphorylation at these sites is thought to contribute to p70S6K activation via relief of pseudosubstrate suppression (Pullen and Thomas 1997; Weng et al. 1998). Carbachol also induced an increase in the in vitro phosphorylation of a synthetic substrate in samples immunoprecipitated with p70S6K polyclonal antibody (Fig. 1f).
These results clearly indicate that carbachol activates p70S6K in 1321 N1 astrocytoma cells. In subsequent experiments we chose to measure the phosphorylation of Thr389 as an index of p70S6K activation, as this phosphorylation site is one of the last to be phosphorylated during the modular activation of p70S6K (Pullen and Thomas 1997), and is the one which most closely correlates with p70S6K activation in vivo (Weng et al. 1998).
We previously observed that carbachol-induced proliferation of 1321 N1 astroctyoma cells is primarily mediated by activation of the M3 subtype of mAChRs (Guizzetti et al. 1996). Figure 2 shows the effects of the non-selective muscarinic antagonist atropine, the M3-selective antagonist 4-DAMP, the M2-selective antagonist gallamine, and the Gi protein inhibitor pertussis toxin (PTX) on carbachol-induced p70S6K phosphorylation. Atropine and 4-DAMP strongly inhibited carbachol-induced p70S6K phosphorylation, while gallamine and PTX had no effect, suggesting the involvement of the Gq-coupled M3 muscarinic receptor in p70S6K activation by carbachol.
Carbachol-mediated proliferation of 1321 N1 astrocytoma cells is only partially dependent on the activation of conventional and novel PKCs (Guizzetti et al. 1998), while it is largely dependent on the activation of atypical PKCζ (Guizzetti and Costa 2000b), which has been proposed to bind and contribute to p70S6K activation (Romanelli et al. 1999). We thus investigated the role of the three PKC isozymes expressed in this cell line (α, ε, and ζ; Post et al. 1996) in p70S6K activation. Down-regulation of PMA-sensitive PKCs by prolonged treatment with PMA did not affect the activation of p70S6K by carbachol, nor did the PKC inhibitor GF109203X (2 µmnova; Fig. 3a). On the other hand, a pseudosubstrate to PKCζ (peptide Z), but not peptide A, a pseudosubstrate to PKCα, inhibited p70S6K activation by carbachol (Fig. 3b).
The activation of p70S6K is a complex event; there are at least seven different phosphorylation sites which require to be phosphorylated in order to obtain a fully activated enzyme and therefore several kinases are involved in its activation. The PI3-kinase inhibitors LY294002 (2 µmnova) and wortmannin (100 nmnova), as well as rapamycin (1 nmnova), the most potent inhibitor of p70S6K described, blocked the activation of p70S6K by carbachol (Fig. 3c). Some phosphorylation sites involved in the activation of p70S6K lie in its C-terminal domain and are followed by proline residues, suggesting that they may be targets for proline-directed kinases. However, though p70S6K can be phosphorylated in vitro by MAPK (Mukhopadhyay et al. 1992), other studies indicate that p70S6K resides on a signaling pathway distinct from the Ras/MAP kinase pathway (Ballou et al. 1991). We found that Thr389 phosphorylation is not sensitive to the MEK inhibitor PD98058 (up to 60 µmnova), while it is inhibited by another MEK inhibitor, UO126 (5 µmnova) (Fig. 3d).
To determine whether p70S6K activation is involved in carbachol-mediated proliferation, we investigated the effect of the p70S6K inhibitor rapamycin on carbachol-induced [3H]-thymidine proliferation. Figure 4 shows that rapamycin inhibited carbachol-induced proliferation, with a 50% inhibition observed at the concentration of 0.5 nmnova. On the other hand, 5 nmnova rapamycin alone did not affect either basal [3H]-thymidine incorporation or cell morphology after 24 h incubation (not shown), suggesting that the effect of rapamycin on carbachol-induced astrocytoma cell proliferation was not due to cytotoxicity. Altogether, these results indicate that in 1321 N1 astrocytoma cells stimulation of M3 mAChR leads to activation of p70S6K through a pathway that involves PKCζ and PI3-K, and that p70S6K plays a role in carbachol-induced DNA synthesis.
We had previously observed that ethanol strongly inhibits carbachol-induced 1321 N1 astrocytoma cell proliferation (Guizzetti and Costa 1996). Ethanol also caused a strong, concentration-dependent (20–200 mmnova) inhibition of carbachol-induced p70S6K (Fig. 5a), which was not due to protein degradation (Fig. 5b).
As p70S6K activation is mediated by PKCζ and by PI3-kinase (Fig. 3), we investigated the effect of ethanol on these two enzymes. Both kinases are activated by carbachol in 1321 N1 astrocytoma cells, and their activation is important for carbachol-induced astrocytoma cell proliferation (Guizzetti and Costa 2000b, 2001). Ethanol potently inhibited carbachol-induced translocation of PKCζ from the cytosol to membranes in astrocytoma cells (Fig. 6a), as well as carbachol-induced phosphorylation of Thr410, an important site of phosphorylation in PKCζ (Chou et al. 1998) (Fig. 6b). On the other hand, ethanol could inhibit phosphorylation of Akt, a substrate of PI3-K, only at higher concentrations (≥ 100 mmnova; Fig. 6c).
p70S6K is the physiological kinase phosphorylating the S6 protein of 40S ribosomal subunits. Phosphorylation of the S6 protein is a highly conserved response of animal cells to treatment with growth factors, steroid hormones, phorol esters, and oncogenes (Ferrari and Thomas 1994). S6 phosphorylation correlates with a selective up-regulation in expression of a family of mRNAs containing polypyrimidine tracts at their 5′ untranslated regions. Most of these mRNAs encode ribosomal proteins and elongation factors (such as eEF1a and eEF2), whose production is required for efficient transit through the G1 phase of the cell cycle (Grammer et al. 1996).
Carbachol-induced p70S6K activation in 1321 N1 astrocytoma cells was demonstrated by assessing the carbachol-induced phosphorylation of specific sites (Thr389 and Thr421/Ser424), the molecular weight shift of the kinase, and the ability of immunoprecipitated p70S6K to phosphorylate a synthetic substrate (Fig. 1). Carbachol-induced p70S6K activation was due to stimulation of M3 mAChR, and was mediated by PI3-kinase and PKCζ, but not by PKCε or α. The role played by MAPK in carbachol-induced p70S6K activation is unclear, as the two MEK inhibitors used in this study gave opposite results: UO126 potently inhibited p70S6K activation while PD98058 was ineffective even at high concentrations (60 µmnova; Fig. 3d); on the other hand, both these compounds (1 µmnova and 10 µmnova, respectively) were effective in inhibiting MEK-mediated ERK1/2 activation (Yagle et al. 2001). Whether p70S6K is directly phosphorylated by MAPK is still controversial: some phosphorylation sites involved in the activation of p70S6K lie in its C-terminal domain and are followed by proline residues, suggesting that they may be targets for proline-directed kinases. However, though p70S6K can be phosphorylated in vitro by MAPK (a proline-directed enzyme; Mukhopadhyay et al. 1992), another study indicated that p70S6K resides on a signaling pathway distinct from the Ras/MAP kinase pathway (Ballou et al. 1991). It was recently shown that a newly described member of the MAPK family, ERK5, is inhibited by the MEK inhibitor UO126, but not by PD98058 (Dong et al. 2001). M1 muscarinic receptors have been shown to activate ERK5 (Fukuhara et al. 2000), and PKCζ can activate ERK5 (Diaz-Meco and Moscat 2001). Thus, a possible explanation of our findings is that ERK5 may be involved in the p70S6K activation induced by carbachol, perhaps downstream to PKCζ, though this hypothesis needs to be verified by further experiments.
Rapamycin, an immunosuppressant which inhibits p70S6K activation (Burnett et al. 1998), was a potent inhibitor of carbachol-induced DNA synthesis, suggesting that activation of p70S6K may play a role in the mitogenic response associated with stimulation of mAChRs. However, at the concentration of 5 nmnova, which completely inhibited p70S6K activation, only 65% of DNA synthesis was inhibited, suggesting that other pathway(s) may be involved in carbachol-induced cell proliferation. Indeed, we previously described that PKCε (Guizzetti et al. 1998) and MAPK (Yagle et al. 2001) were activated by carbachol and played a role in carbachol-induced cell proliferation independently from the PI3-K PKCζ p70S6K pathway. Activation of p70S6K by mAChRs had been previously reported in pancreatic acini (Bragado et al. 1997) and in human airway smooth muscle cells (Krymskaya et al. 2000); however, this effect was not further characterized. A lack of activation of p70S6K by M1 mAChRs transfected into CCL39 cells has also been reported (Kahan et al. 1992); however, in these cells, carbachol failed to induce cell cycle progression (Seuwen et al. 1990), thus substantiating, albeit indirectly, the hypothesis of a central role for p70S6K in muscarinic receptor-induced cell proliferation.
We had previously found that ethanol (25–100 mmnova) strongly inhibits carbachol-induced proliferation (Guizzetti and Costa 1996). Carbachol-induced calcium mobilization and activation of PKCε were inhibited by ethanol only at high concentrations (> 100 mmnova; Catlin et al. 2000a; Guizzetti and Costa 2000a), while activation of MAPK (ERK1/2) was not affected by ethanol (Yagle et al. 2001). In the present study we investigated whether ethanol would inhibit carbachol-induced activation of p70S6K and the two possible upstream activators of this kinase, PKCζ and PI3-K. We found that ethanol caused a strong inhibition of p70S6K in the same range of concentrations (25–100 mmnova) that inhibit carbachol-induced DNA synthesis. At these concentrations, ethanol also caused a robust inhibition of PKCζ translocation and phosphorylation (Fig. 6a,b). On the other hand, Akt phosphorylation, measured as an index of PI3-K activation, was minimally affected by ethanol at 100 mmnova (Fig. 6c,d). Thus, PI3-K, which through PDK-1 can phosphorylate p70S6K, does not appear to be a relevant target for ethanol. Furthermore, though PDK-1 can phosphorylate PKCζ, this must be preceded by PKCζ translocation to the membrane (Chou et al. 1998). Inhibition of PKCζ phosphorylation by ethanol thus appears to occur as a result of inhibition of PKCζ translocation, rather than inhibition of PI3-K. Such inhibition of PKCζ translocation by ethanol may in turn be due to inhibition of phospholipase mnova-produced phosphatidic acid, which has been shown to activate PKCζ (Limatola et al. 1994; Guizzetti and Costa 2000b).
In summary, our results show that, in human astrocytoma cells, stimulation of M3 mAChRs by carbachol causes activation of p70S6K, which is primarily mediated by PKCζ and PI3-K, and that such activation is involved in the mitogenic effect of muscarinic agonists in these cells. Inhibition of carbachol-induced DNA synthesis by ethanol appears to be associated with inhibition of the sequential activation of PKCζ and p70S6K, rather then PI3-K.
This work was supported in part by Grants AA-08154 and ES-07033 from the National Institute of Health. We thank Ms. Khoi Dao for her assistance in some of the experiments.