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Am J Physiol Lung Cell Mol Physiol 293: L1348-L1358, 2007. First published September 14, 2007; doi:10.1152/ajplung.00346.2007
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Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation

Reinoud Gosens,1,2,3 Gordon Dueck,1,2 Edward Rector,2,4 Raquel O. Nunes,3,5 William T. Gerthoffer,6 Helmut Unruh,7 Johan Zaagsma,3 Herman Meurs,3 and Andrew J. Halayko1,2

1Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg; 2Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg; 3Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands; 4Flow Cytometry Laboratory, University of Manitoba, Winnipeg; 5Escola Superior de Tecnologia da Saúde de Lisbon, Lisbon, Portugal; 6Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; 7Section of Thoracic Surgery, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 23 August 2007 ; accepted in final form 8 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Muscarinic receptors and platelet-derived growth factor (PDGF) receptors synergistically induce proliferation of airway smooth muscle (ASM), but the pathways that regulate these effects are not yet completely identified. We hypothesized that glycogen synthase kinase-3 (GSK-3), a kinase that represses several promitogenic signaling pathways in its unphosphorylated form, is cooperatively inhibited by PDGF and muscarinic receptors in immortalized human ASM cell lines. PDGF or methacholine alone induced rapid GSK-3 phosphorylation. This phosphorylation was sustained only for PDGF; however, methacholine potentiated PDGF-induced sustained GSK-3 phosphorylation. Synergistic effects of methacholine also were observed on PDGF-induced retinoblastoma protein (Rb) phosphorylation and cell proliferation. Suppression of GSK-3 inhibitory function using SB 216763 also augmented PDGF-induced Rb phosphorylation and cell cycle progression; this synergy was similar in magnitude to that seen for methacholine with PDGF. GSK-3 phosphorylation induced by methacholine required PKC, since it was abolished by GF 109203X and Gö 6976; however, inhibition of PKC had no effect on cell responses to PDGF. PKC inhibition also specifically abolished the synergistic effect of methacholine on PDGF-induced GSK-3 phosphorylation and cell proliferation. Collectively, these results show that GSK-3 plays a key repressive role in ASM cell proliferation. Moreover, muscarinic receptors mediate PKC-dependent GSK-3 inhibition, and this appears to be a primary mechanism underpinning augmentation of PDGF-induced cell growth.

asthma; airway hyperresponsiveness; cell cycle; G protein-coupled receptor; airway remodeling

ALTHOUGH AIRWAY SMOOTH MUSCLE THICKENING, due in part to airway myocyte hyperplasia and hypertrophy, contributes to the pathophysiology of chronic airways diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, the mechanisms that underpin this pathological response are not clear (19, 23, 36). In vitro studies indicate that airway smooth muscle cell proliferation is induced by several agonists for receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) (22); moreover, synergistic interactions between GPCR and RTK agonists resulting in enhanced airway myocyte proliferation have been reported and are known to occur even for GPCR agonists that alone are not promitogenic (5). For example, although muscarinic and CysLT1 receptor stimulation are insufficient to induce airway smooth muscle proliferation in mitogen-deficient conditions, they do have positive synergistic effects on cell proliferation and cyclin D1 accumulation in combination with peptide growth factors such as EGF or PDGF (14, 26, 30). Cooperative regulation of airway smooth muscle proliferation by GPCR agonists and growth factors likely contributes to allergen-induced airway smooth muscle remodeling, since airway myocyte hyperplasia in situ can be effectively inhibited by muscarinic (12) or CysLT1 receptor antagonists (20, 21).

Recent studies by Billington et al. (2) indicate that a pathway involving phosphatidylinositol 3-kinase (PI3K), Akt, and p70 S6 kinase is synergistically activated by GPCR agonists and EGF in airway smooth muscle. Activation of this pathway by GPCR agonists is primarily dependent on Gq/11-derived beta{gamma}-subunits (2, 25), which supports observations from several other groups that show synergistic regulation of growth factor-induced airway myocyte proliferation by a number of Gq/11-coupled receptors (13, 14, 25, 26, 30). Although these studies have significantly extended our understanding of functional GPCR-RTK interactions, there also is evidence that it is unlikely proliferation synergy is regulated exclusively by p70 S6 kinase-dependent pathways. For instance, although synergistic regulation of p70 S6 kinase by thrombin and EGF is not dependent on PKC (2), PKC-dependent pathways are crucial for synergy of bradykinin and EGF (13) and of endothelin-1 with PDGF in airway smooth muscle proliferation (37). These discrepancies suggest that multiple parallel intracellular signaling pathways, which to date have not been fully elucidated, regulate the functional responses of myocytes induced by synergistic GPCR-RTK interaction.

Glycogen synthase kinase-3 (GSK-3) is a central signaling intermediate in multiple pathways that control a broad spectrum of cell responses including proliferation (7). GSK-3 is constitutively active in its unphosphorylated form in mitotically quiescent cells (7). The role GSK-3 plays in airway smooth muscle proliferation has not yet been described; however, unphosphorylated/active GSK-3 represses cell proliferation of nonmuscle cells by negatively regulating transcription factors and cell cycle regulatory proteins, including c-Myc, cyclin D1, and c-Jun (7). In these cells, mitogen stimulation reverses the repressive role of GSK-3 by inducing PKC- and Akt-dependent phosphorylation of the kinase at Ser9 (GSK-3beta) and Ser21 (GSK-3{alpha}), which blocks the binding pocket and active site of the enzyme (4, 9, 11). Since both PKC and Akt are implicated in proliferation synergy of GPCR agonists with RTKs in airway smooth muscle, we hypothesized the existence of a signaling pathway involving GSK-3, acting in parallel with p70 S6 kinase, which is cooperatively regulated by GPCR agonists and RTKs. To test this hypothesis in human airway myocytes, we investigated signaling, cross talk, and proliferative responses induced by combinations of agonists for muscarinic and PDGF receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and reagents. Mouse anti-beta-actin antibody, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody, and HRP-conjugated goat anti-rabbit antibody were purchased from Sigma (St. Louis, MO). Anti-phosphotyrosine-conjugated agarose beads (clone PY20), rabbit anti-GSK-3 polyclonal antibody, rabbit anti-p70S6K polyclonal antibody, and rabbit anti-PDGFRbeta polyclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-phospho-Thr202/Tyr204-p42/p44 MAPK monoclonal antibody, rabbit anti-p42/p44 MAPK polyclonal antibody, rabbit anti-phospho-Ser9/21-GSK-3 antibody, rabbit anti-phospho-Thr389-p70S6K antibody, mouse anti-cyclin D1 monoclonal antibody, and rabbit anti-phospho-Ser807/811 polyclonal antibody all were obtained from Cell Signaling Technology (Beverly, MA). GF 109203X, Gö 6976, and LY-294,002 were obtained from Calbiochem (Darmstadt, Germany). SB 216763 was acquired from Tocris Cookson (Bristol, UK). All other chemicals were of analytical grade.

Cell culture. As previously described by our group (15), human bronchial smooth muscle cell lines were immortalized by stable expression of human telomerase reverse transcriptase (hTERT). The primary cultured human bronchial smooth muscle cells used to generate each cell line were prepared from macroscopically healthy segments of 2nd- to 4th-generation main bronchus obtained after lung resection surgery from patients with a diagnosis of adenocarcinoma. All procedures were approved by the Human Research Ethics Board (University of Manitoba). As detailed previously by our group (15), each cell line was thoroughly characterized to passage 10 and higher; hTERT cell lines retain responsiveness to mitogens and capacity to express protein markers of the contractile phenotype, including smooth muscle myosin heavy chain (MHC), smooth muscle {alpha}-actin, and desmin, in a manner that mimics primary human bronchial smooth muscle cultures (15, 33, 34). For all experiments, passage 10–19 myocytes were used. Cell lines were grown on uncoated plastic dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 50 U/ml streptomycin, 50 µg/ml penicillin, and 10% fetal bovine serum (FBS). Unless otherwise specified, cells were grown to ~80% confluence and then serum-starved for 3 days in DMEM supplemented with 50 U/ml streptomycin, 50 µg/ml penicillin, 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium before the start of each experiment.

Cell proliferation assay. Cells were grown to subconfluence in 24-well cluster plates and then serum-starved for 3 days in DMEM supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium. Cells were then incubated with mitogens for 4 days in DMEM supplemented with 50 U/ml streptomycin and 50 µg/ml penicillin. Thereafter, cells were either trypsinized and counted manually using a hemocytometer or assayed using an Alamar blue proliferation assay. Briefly, cells were washed two times with HBSS and incubated with HBSS containing 10% (vol/vol) Alamar blue solution (Biosource, Camarillo, CA). Conversion of Alamar blue into its reduced form by mitochondrial cytochromes was then assayed by dual-wavelength spectrophotometry at wavelengths of 562 and 630 nm. As indicated by the manufacturer, the degree of Alamar blue conversion is proportional to cell number; this was confirmed by our previous experiments using our human airway myocyte cultures (15).

Preparation of cell lysates. To obtain total cell lysates for assessing protein abundance and phosphorylation, cells were washed once with ice-cold PBS then lysed in ice-cold RIPA buffer (composition: 40 mM Tris, 150 mM NaCl, 1% Igepal CA-630, 1% deoxycholic acid, 1 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, and 1 mM PMSF, pH 8.0). Lysates were stored at –80°C until further use.

Western blot analysis. Equal amounts of total protein lysates were subjected to electrophoresis, transferred to nitrocellulose membranes, and analyzed by immunoblotting using specific primary and HRP-conjugated secondary antibodies. Bands were subsequently visualized on film using enhanced chemiluminescence reagents and quantified by scanning densitometry using Totallab software (Nonlinear Dynamics, Newcastle, UK).

Immunoprecipitation. For immunoprecipitation of phosphotyrosine (pY), anti-pY-conjugated agarose beads (SantaCruz Biotechnology) were transferred directly to 500 µg of cell lysate. After 1 h of incubation at 4°C, beads were washed four times with Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and once with PBS. Beads with immunoprecipitated proteins were then stored at –80°C until used for Western blot analysis.

Flow cytometric cell cycle analysis. Cells were grown to subconfluence in six-well cluster plates and then serum-starved for 3 days in DMEM supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium. Cells were then incubated with mitogens for up to 36 h in DMEM supplemented only with streptomycin (50 U/ml) and penicillin (50 µg/ml). For control conditions, cells were maintained in medium lacking mitogens. At preselected time points after mitogen stimulation, cells were harvested for flow analyses first by being washed twice with warmed PBS, and then cells were lifted by trypsinization and diluted in DMEM/10% FBS. Cells were washed three times thereafter by centrifugation (800 g, 5 min) and subsequent resuspension in ice-cold PBS. After the final wash, cells were fixed by transfer to ice-cold 70% ethanol. Cell samples were stored at –20°C before cell cycle analysis by flow cytometry using protocols our group (18) described previously. Briefly, cells were then washed in PBS and then resuspended in a solution containing 5 µg/ml propidium iodide (PI), 0.1% Triton X-100, and 50 µg/ml RNase. Cell cycle analysis was assessed based on PI staining in cells using a Beckman Coulter EPICS ALTRA flow cytometer (Beckman Coulter Canada, Mississauga, Canada) with a 488-nm line from an argon laser and a 610-nm long-pass emission filter. Fluorescence histograms of 1,024-channel resolution were collected for at least 10,000 cells, satisfying light scatter and doublet discrimination criteria, using the EXPO32 MultiCOMP acquisition software (V1.2B; Beckman Coulter Canada) provided with the instrument. The cell cycle distributions were subsequently analyzed by employing ModFit LT flow cytometry modeling software (ModFit LT, version 3.1) obtained from Verity Software House (Topsham, ME).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Muscarinic receptor stimulation augments the proliferative response to PDGF. We first characterized proliferation synergy caused by coincident exposure of myocytes to methacholine and PDGF-BB, ligands for muscarinic receptors and PDGF receptors in the human airway smooth muscle cells. Of note, these cell lines retain the functional expression of muscarinic M3 receptors, as assessed by Western blot analysis and Ca2+ mobilization assays (data not shown). As our group (14) has observed previously for bovine tracheal smooth muscle cells, methacholine alone (10 µM) did not induce cell proliferation in mitogen-deficient conditions but did augment cell proliferation of airway smooth muscle cells in the presence of PDGF (Fig. 1) (P < 0.001, 2-way ANOVA). This effect was most profound at the highest concentration of PDGF we tested (30 ng/ml). Concomitant with its effects on myocyte proliferation, methacholine also synergistically augmented PDGF-induced phosphorylation of retinoblastoma (Rb) protein, a signaling event that is necessary for G1-to-S phase traversal and cell proliferation (Table 1).


Figure 1
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  		Fig. 1. Muscarinic receptor stimulation augments PDGF-induced airway smooth muscle cell proliferation. A: subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with methacholine (MCh; 10 µM) and increasing concentrations of PDGF-BB (0, 3, 10, or 30 ng/ml). After 4 days of treatment, cell number was quantified using an Alamar blue assay (15). Data are means ± SE of 9–12 experiments. B: subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM) and PDGF-BB (30 ng/ml). After 4 days of treatment, cells were counted manually. Data are means ± SE of 3 experiments. Differences between data were analyzed using 2-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; ***P < 0.001.

 

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  		Table 1. Phosphorylation of receptors and signaling proteins in response to muscarinic and PDGF receptor stimulation in airway smooth muscle

 
Muscarinic receptor stimulation augments GSK-3 phosphorylation in response to PDGF. We next profiled the activation of known intracellular signaling pathways associated with myocyte proliferation to assess their involvement in methacholine-PDGF synergy. To test our main hypothesis that GSK-3 may be involved in the synergistic response, we used lysates from cells treated with methacholine and PDGF, either alone or in combination, to measure inactive (phospho-Ser9/21) and total GSK-3 content both acutely (15 min) and 4 h after cell stimulation (Table 1). For all treatments, phosphorylation of the 47-kDa GSK-3beta species was consistently higher than phosphorylation of the 51-kDa GSK-3{alpha} isoform. Since GSK-3{alpha} phosphorylation was undetectable in some blots, we only included GSK-3beta phosphorylation in the quantitative densitometry we performed. Both methacholine and PDGF induced significant GSK-3beta phosphorylation, but with different kinetics, since only the PDGF response was sustained after 4 h of treatment (Fig. 2). Notably, although methacholine alone did not induce sustained phosphorylation of GSK-3beta, in combination with PDGF it did augment the late-phase GSK-3beta phosphorylation (P = 0.01, 2-way ANOVA). Synergy was not observed acutely, however, since 15 min after dual agonist treatment, phospho-GSK-3beta was not different from that induced by PDGF alone (Fig. 2). Collectively, these results indicate that cross talk between GPCR and RTK signaling converges at GSK-3beta to produce enhanced sustained phosphorylation.


Figure 2
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  		Fig. 2. Muscarinic receptor stimulation augments PDGF-induced glycogen synthase kinase-3beta (GSK-3beta) phosphorylation. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM) and increasing concentrations of PDGF-BB (0, 3, or 30 ng/ml). After 15 min (A) or 4 h (B), cell lysates were prepared and Western blot analysis performed for phospho-(Ser9/21)-GSK-3 and total GSK-3 to correct for differences in protein loading. Phospho-GSK-3beta (pGSK3beta) was quantified using densitometry and normalized to the response induced by PDGF-BB (30 ng/ml; 15 min), which was set at 100%. Data are means ± SE of 4–5 experiments. Differences between data were analyzed using 2-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.

 
We next investigated whether targeted phosphorylation of GSK-3beta is a unique point of convergence for signaling cross talk induced by methacholine and PDGF. Initial studies were directed at the effect of muscarinic receptor stimulation on PDGF-induced PDGF receptor beta (PDGFRbeta) phosphorylation, since in some cell types, RTK transactivation is central to mitogenic signaling of GPCR agonists (38). After cells were treated with methacholine and PDGF alone or in combination, we used whole cell lysates for immunoprecipitation of total phosphotyrosine; immunoprecipitates were then analyzed for PDGFRbeta content. As expected, PDGF-BB induced a robust increase in PDGFRbeta phosphorylation within 15 min (Table 1); this response returned to baseline after 4 h of treatment (data not shown). Methacholine treatment did not change baseline PDGFRbeta phosphorylation and did not augment PDGFRbeta activation 15 min or 4 h after PDGF exposure. Collectively, these data indicate that muscarinic receptors do not mediate PDGF receptor transactivation (Table 1).

We next studied the phosphorylation status of p70 S6 kinase and p42/p44 MAP kinase, since both of these intracellular signaling targets have previously been associated with signaling and cross talk of GPCR and RTK agonists in airway smooth muscle (2, 10, 29). For p70 S6 kinase, the Thr389 phosphorylation site was selected for analysis on the basis of observations that cooperative p70 S6 kinase phosphorylation by GPCR and RTK agonists in airway smooth muscle is site specific (2). Phosphorylation of both p42/p44 MAP kinase and p70 S6 kinase in response to methacholine or PDGF alone showed a temporal profile similar to that measured for GSK-3beta, with both agonists eliciting a robust response acutely (15 min), whereas only PDGF induced sustained phosphorylation (4 h) (Table 1). Moreover, only at the latter time point did methacholine significantly augment PDGF-induced phosphorylation of both kinases (Table 1). Collectively, these results indicate that muscarinic receptor stimulation augments intracellular signaling induced by PDGFR at several sites, including, but not limited to, GSK-3beta.

GSK-3 inhibition augments the proliferative response to PDGF. Since a functional role for GSK-3 has not yet been described in airway smooth muscle, we next designed experiments to confirm the repressive role of unphosphorylated (active) GSK-3 in cell proliferation. GSK-3 represses cell proliferation by negatively regulating transcription factors and cell cycle regulatory proteins, including c-Myc, cyclin D1, and c-Jun (7). To this purpose, we investigated the effects of the GSK-3 inhibitor SB 216763 (10 µM) on promitogenic signaling and cell cycle progression induced by PDGF. SB 216763 alone was sufficient to induce cyclin D1 expression, but it did not change baseline or PDGF-induced phosphorylation of p42/p44 MAP kinase or p70 S6 kinase (Thr389) and did not affect baseline phospho-Rb (Fig. 3). Conversely, in combination with PDGF stimulation of myocytes, GSK-3 inhibition did significantly augment phospho-Rb. Of note, the synergistic induction of phospho-Rb induced by SB 216763 in combination with PDGF was of a magnitude similar to that observed in earlier experiments for methacholine with PDGF (Fig. 3D and Table 1).


Figure 3
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  		Fig. 3. Effects of GSK-3 inhibition on PDGF-induced promitogenic signaling. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with 10 µM SB 216763 (SB) and PDGF-BB (30 ng/ml) for 15 min. Cell lysates were analyzed by immunoblotting for phospho-(Thr202/Tyr204)-p42/p44 MAP kinase and total p42/p44 MAP kinase (A), phospho-(Thr389)-p70 S6 kinase (p70S6K) and total p70S6K (B), cyclin D1 (C), and phospho-(Ser807/811)-retinoblastoma protein (Rb) (D). beta-Actin expression was also determined to correct for differences in protein loading. Data are means ± SE of 4 experiments. Differences between data were analyzed using 1-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.

 
On the basis of these observations, we next tested whether SB 216763 augments PDGF-induced airway smooth muscle cell proliferation by assessing cell cycle progression using flow cytometry in combination with PI staining of DNA (Fig. 4). Treatment with SB 216763 alone was not sufficient to induce cell cycle progression; however, in cultures treated with PDGF and GSK-3 inhibitor in combination, a significantly greater number of myocytes were induced to proliferate, as indicated by the increased fraction of cells (52%) that moved from G0/G1 into S and G2/M phase at 24 h compared with that seen with PDGF alone (37%). In similar experiments using methacholine in combination with PDGF, we observed virtually identical results, in which 50% of myocytes were induced to traverse the cell cycle. Thus the synergy we observed between methacholine and PDGF was the same as that measured with GSK-3 inhibition during PDGF exposure.


Figure 4
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  		Fig. 4. Effects of GSK-3 inhibition on PDGF-induced cell cycle progression. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with either 30 ng/ml PDGF-BB, PDGF-BB + 10 µM SB, or PDGF plus 10 µM MCh. For each experiment, fluorescence histograms were collected for at least 10,000 cells, satisfying light scatter and doublet discrimination criteria (top). Untreated cells from the start of the experiment were used as controls (t = 0). At t = 16 (bottom left), 24 (bottom center), and 30 h (bottom right), cells were harvested, stained with propidium iodide (PI), and analyzed for DNA content by flow cytometry. Histograms represent cell cycle analyses of 10,000 cells and show typical distribution of cells in G0/G1 (1st peak in histogram), G2/M (2nd peak in histogram), and S phase (intermediate distribution). Experiments were completed in triplicate. Histograms from a single, typical experiment are shown.

 
Interestingly, although the fraction of cells induced to proliferate was increased by addition of methacholine or SB 216763, our data suggest this difference was not likely associated with a concomitant change in the kinetics of cell cycle transit time of activated cells. First, little evidence for S-phase entry was evident after 16 h of stimulation with PDGF alone, PDGF/methacholine, or PDGF/SB 216763, since ~78% of cells remained in G0/G1; this was unchanged from the cell cycle distribution measured before mitogen addition (time 0: 70%). Second, despite the fact that there were differences between treatments in the number of cells that entered S phase by 24 h, for all conditions virtually all responding myocytes had reached G2/M phase 30 h after stimulation; thus there were no differences in cell cycle distribution at that time point (Fig. 4). Last, for all treatment groups, cell cycle transit was completed in parallel, 36 h after stimulation, as indicated by a return to the cell cycle distribution we measured before mitogen/inhibitor exposure (data not shown for 36-h time point). Collectively, the comparable synergistic effects on PDGF-induced myocyte cell cycle entry, resulting from either methacholine exposure or selective pharmacological inhibition of GSK-3, implicates a role for GSK-3 in muscarinic receptor-PDGF receptor cross talk leading to myocyte proliferation.

Cell proliferation and GSK-3 phosphorylation induced by muscarinic receptor stimulation are PKC dependent. Our group (13) previously reported a role for PKC in proliferation synergy induced by bradykinin and EGF in airway smooth muscle; therefore, we next determined whether cell proliferation and GSK-3beta phosphorylation induced by PDGF and methacholine are PKC dependent. For this purpose, we used GF 109203X, an inhibitor of conventional and novel PKC isoenzymes. Additional studies employed the PI3K inhibitor LY-294,002 to evaluate the involvement of the PI3K/Akt signaling axis, which has been previously implicated in GPCR-RTK synergy (2). Interestingly, PKC inhibition completely abrogated GSK-3beta phosphorylation induced by methacholine but was without effect on PDGF-induced targeting of GSK-3beta (Fig. 5). In contrast, although PI3K inhibition did quench GSK-3beta phosphorylation induced by both agonists, this effect was far less than that observed for PKC inhibition (Fig. 5). To further confirm the role of PKC in methacholine-stimulated GSK-3beta phosphorylation, we also measured the effects of the conventional PKC subclass inhibitor Gö 6976. In agreement with the results obtained using GF 109203X, Gö 6976 also inhibited methacholine-induced GSK-3beta phosphorylation but had no effect on PDGF-stimulated GSK-3beta inhibition (Fig. 6). Moreover, the PKC activator PMA (0.1 µM) induced a 2.0 ± 0.3-fold increase in GSK-3beta phosphorylation in these cells (data not shown). These data confirm a primary role for PKC, likely of the conventional isoforms subclass, in GSK-3beta phosphorylation induced by muscarinic receptors.


Figure 5
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  		Fig. 5. Effects of PKC and phosphatidylinositol 3-kinase (PI3K) inhibition on GSK-3beta phosphorylation by methacholine and PDGF. Subconfluent 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM; A) or PDGF-BB (30 ng/ml; B) for 15 min in the presence or absence of either the pan-PKC inhibitor GF 109203X (GF; 3 µM) or the PI3K inhibitor LY-294,002 (LY; 10 µM). Inhibitors were administered to the cells 30 min before the addition of methacholine or PDGF-BB. Cell lysates were obtained and analyzed by immunoblotting for phospho-(Ser9/21)-GSK-3 and total GSK-3 to correct for differences in protein loading. Phospho-GSK-3beta was quantified using densitometry and normalized to the response induced by MCh (10 µM; 15 min) or PDGF-BB alone (30 ng/ml; 15 min). Data are means ± SE of 4 experiments. Differences between data were analyzed using 1-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. ***P < 0.001. C, control.

 

Figure 6
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  		Fig. 6. Effects of the conventional PKC isoform inhibitor Gö 6976 on GSK-3beta phosphorylation by MCh and PDGF. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM; A) or PDGF-BB (30 ng/ml; B) for 15 min in the presence or absence of the PKC inhibitor Gö 6976 (Go; 300 nM). Cell lysates were obtained and analyzed by immunoblotting for phospho-(Ser9/21)-GSK-3 and total GSK-3 to correct for differences in protein loading. Phospho-GSK-3beta was quantified using densitometry and normalized to the response induced by MCh (10 µM, 15 min) or PDGF-BB (30 ng/ml; 15 min). Data are means ± SE of 6 experiments. Differences between data were analyzed using 1-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001.

 
The main hypothesis of the current study was that synergistic effects of methacholine and PDGF are mediated, in part, by parallel inactivation of GSK-3 through phosphorylation at Ser9/21. Since our earlier experiments showed that methacholine or PDGF alone could induce GSK-3beta phosphorylation, we next completed experiments to determine whether increased GSK-3beta phosphorylation induced by methacholine and PDGF in combination occurred via muscarinic receptor-mediated activation of PKC. To address this issue, we measured the effects of PKC inhibition using GF 109203X on GSK-3beta phosphorylation induced by methacholine (10 µM) in combination with increasing concentrations of PDGF-BB (0–30 ng/ml). All measurements were made 4 h after myocyte stimulation, because our earlier data (Fig. 2) showed synergy was greatest at that time point. Indeed, GF 109203X selectively abrogated the synergistic augmentation of GSK-3beta phosphorylation induced by methacholine with PDGF but was without effect on the response to PDGF alone (Fig. 7). Collectively, these results reveal that GSK-3beta signaling and cross talk induced by muscarinic receptor stimulation is predominantly mediated by PKC.


Figure 7
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  		Fig. 7. Effects of PKC inhibition on the cooperative regulation of GSK-3beta by MCh and PDGF. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM) and increasing concentrations of PDGF-BB (0, 3, and 30 ng/ml) for 4 h in the presence and absence of the PKC inhibitor GF (3 µM). Cell lysates were obtained and analyzed by immunoblotting for phospho-(Ser9/21)-GSK-3 and total GSK-3 to correct for differences in protein loading. Phospho-GSK-3beta was quantified using densitometry and normalized to the response induced by PDGF-BB (30 ng/ml), which was set at 100%. Data are means ± SE of 4 experiments. Differences between data were analyzed using 2-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; ***P < 0.001.

 
Since these results implicate a crucial role for PKC in the proliferation synergy induced by muscarinic and PDGF receptors, we next determined the effect of GF 109203X on cell proliferation induced by PDGF and methacholine (Fig. 8). GF 109203X itself induced a modest reduction in cell number compared with the controls that were treated with serum-free medium alone (Fig. 8). In the presence of GF 109203X, PDGF-BB-induced cell proliferation was maintained, since the relative increase in cell number induced by PDGF was not different between cells treated with and without the PKC inhibitor. This shows that PKC is not a primary mediator of PDGF-induced proliferation. Nonetheless, the synergistic effect induced by combined treatment with methacholine and PDGF was almost completely abrogated by GF 109203X, indicating that proliferation synergy of muscarinic and PDGF receptors is PKC dependent (Fig. 8).


Figure 8
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  		Fig. 8. Effects of PKC inhibition on the cooperative regulation of cell proliferation by MCh and PDGF. Subconfluent, 3-day serum-deprived airway smooth muscle cells were treated with MCh (10 µM) and PDGF-BB (30 ng/ml) for 4 days in the presence and absence of the PKC inhibitor GF (3 µM). Cell number was quantified using an Alamar blue assay. Data are means ± SE of 3 experiments each performed in triplicate. Differences between data were analyzed using 2-way ANOVA, with post hoc Bonferroni's t-test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. CTR, control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study indicate that synergistic regulation of airway smooth muscle cell proliferation by muscarinic and PDGF receptors is, in large part, mediated by cooperative phosphorylation of GSK-3 via PKC and PI3K pathways, respectively. These studies are the first to describe a functional role for GSK-3 in airway smooth muscle cell proliferation. Moreover, our studies also are the first to show that the GSK-3 pathway can be regulated synergistically in response to GPCR and RTK agonists. Our studies indicate that PKC, downstream from muscarinic receptors, targets GSK-3 and thereby mediates positive synergistic effects on airway smooth muscle growth. This mechanism appears to operate in parallel with previously identified cross talk mechanisms in which GPCR beta{gamma}-subunits increase activation of p70 S6 kinase, downstream from PI3K (2, 25). Furthermore, the involvement of PKC in synergistic pro-proliferative signaling axes is highly consistent with published results by our group (13) and others (37) showing that pharmacological inhibitors of PKC abrogate GPCR-potentiated proliferation but are without effect on RTK-mediated myocyte growth. These observations are important, because they provide some important insights into the mechanisms that likely underpin muscarinic receptor-mediated accumulation of airway smooth muscle in airway remodeling associated with allergic airways diseases (16, 17).

Cooperative GSK-3 phosphorylation by PDGF and methacholine, as demonstrated in our studies, provides a plausible mechanism for the synergistic effects of other RTK and GPCR agonists on cell proliferation; nonetheless, this hypothesis clearly needs to be confirmed in separate studies. Active (unphosphorylated) GSK-3 inhibits cell proliferation by negatively regulating several promitogenic signaling effectors, including cyclin D1, which is targeted for proteolysis and nuclear export by GSK-3 in NIH-3T3 fibroblasts (6). Our studies are consistent with a constitutively repressive role of GSK-3 in airway smooth muscle cells, because they demonstrate that cyclin D1 accumulates approximately twofold in cells treated with a selective GSK-3 inhibitor alone. Interestingly, our results indicated that despite effects on cyclin D1 abundance, suppression of GSK-3 activity alone was not sufficient to induce cell cycle entry. However, pharmacological GSK-3 inhibition concomitantly potentiated both PDGF-induced Rb protein phosphorylation and cell cycle entry of airway smooth muscle. Phosphorylation of Ser9/21 is well established to reduce GSK-3 activity by blocking access to the active site of the enzyme (4, 7), which results in abrogation of its repressive effects on cell proliferation. Previous work in several cell types indicates that Ser9/21 are targeted by several protein kinases, including PKC, Akt, and integrin-linked kinase (ILK), in response to individual RTK or GPCR agonists (1, 4, 9). Our experiments greatly extend this understanding and reveal for the first time cooperative regulation of Ser9/21 phosphorylation sites on GSK-3 by muscarinic and PDGF receptors. This paradigm clearly has significant potential in dissecting cross talk between other receptor systems that induce growth responses including mitosis, cell hypertrophy, and apoptosis in different cell types.

As previously stated, GSK-3 inhibition is unlikely to be the only mechanism responsible for cross talk between muscarinic and PDGF receptors. First, we found that in addition to GSK-3, parallel increases in PDGF-induced phosphorylation of p42/p44 MAP kinase and p70 S6 kinase were induced by methacholine. The synergistic effects observed on p70 S6 kinase phosphorylation were expected in view of the wide body of evidence that PI3K signaling, which is upstream of p70S6K phosphorylation, is critical to GPCR-RTK cross talk leading to airway smooth muscle growth (2, 25, 26). The effects on p42/p44 MAP kinase phosphorylation were not expected based on the current understanding of upstream regulatory pathways (2, 8, 10, 13, 32); nonetheless, a previous study (26) already has suggested that additive/synergistic regulation of p42/p44 MAP kinase phosphorylation by GPCR and RTK agonists can occur, although this was not required for the cooperative effects. Second, we found that pharmacological inhibition of GSK-3 was not sufficient to induce Rb phosphorylation and cell cycle entry in mitogen-deficient cell culture conditions. GSK-3 inhibition also failed to increase baseline p42/p44 MAP kinase phosphorylation, despite recent findings in HT29 cells that GSK-3 inhibition may activate p42/p44 MAP kinase via a pathway involving PKC{delta} (35). This indicates that in addition to GSK-3, parallel pathways exist, such as interaction with cell membrane caveolin-1 (15), to repress spontaneous p42/p44 MAP kinase activation and cell cycle progression in airway smooth muscle. Thus GSK-3 inhibition alone is not sufficient to overcome these mechanisms and induce cell proliferation. Collectively, our experiments strongly indicate that inhibition of GSK-3 by targeted phosphorylation of Ser9/21 contributes to, but is not solely responsible for, proliferation synergy of methacholine with PDGF.

Our observations using the conventional and novel PKC subclass inhibitor GF 109203X indicate that methacholine-induced GSK-3 phosphorylation, both alone and in combination with PDGF, is primarily mediated by PKC, whereas PDGF-induced GSK-3 phosphorylation occurs entirely independently of PKC. The additional PKC input appears to be of less consequence at earlier time points but becomes critical at later time points. This is consistent with previous work showing that nonmitogenic factors such as methacholine alone induce early-phase phosphorylation of p42/p44 MAP kinase and p70 S6 kinase but that mitogenic activity of biomolecules relies on sustained effects on promitogenic signaling effectors (24). Our studies using the conventional PKC subfamily-specific inhibitor Gö 6976 (27) further suggest that Ca2+-dependent PKC isoenzymes are primarily involved in the methacholine response. Clearly, future experiments using selective small RNA interference (sRNAi) technology are necessary to fully elucidate the principal PKC isoforms targeting methacholine-induced GSK-3beta phosphorylation. The effects we observed with Gö 6976 are nonetheless in agreement with in vitro studies showing that the Ca2+-dependent PKC isoenzymes are particularly effective in inducing serine phosphorylation of GSK-3 (9, 11). Furthermore, the involvement of PKC in GPCR-induced but not RTK-induced GSK-3 phosphorylation, as demonstrated in the present study and reported by others (9), is in full agreement with our studies showing that GF 109203X selectively inhibits proliferation synergy induced by methacholine and PDGF without reducing the response to PDGF alone. Our previous study also showed that the PKC inhibitor Gö 6976 effectively inhibits proliferation synergy of bradykinin with EGF but has no effect on the mitogenic response to EGF alone (13). This indicates that GSK-3 acts as a point of convergence for multiple signaling cascades, including PKC and Akt1, which is a downstream effector of PI3K (9). Moreover, it appears that targeting of GSK-3 by multiple signaling axes significantly potentiates its inactivation and is manifest, in the case of airway myocyte proliferation, in a synergistic fashion. In combination with previous reports (2, 13), our present studies strongly suggest a mechanism in which GqPCR regulation of PKC and PI3K combines with RTK induction of PI3K to synergistically regulate several key downstream effectors in airway myocyte proliferation, including GSK-3 and PI3K, whereas RTK regulation of PKC is not key to the synergistic response (Fig. 9). The crucial role for Gq is further supported by previous studies by our group (14) showing that muscarinic M3 receptors mediate the synergistic effect despite the functional presence of muscarinic M2 receptors in airway smooth muscle. Moreover, multiple studies have shown that GPCR agonists that activate both Gi and Gq, such as thrombin and lysophosphatidic acid, rely primarily on Gq and less on Gi for their synergistic interaction with RTK ligands (2, 3, 25).


Figure 9
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  		Fig. 9. Proposed signaling mechanism involving GSK-3 in Gq protein-coupled receptor (GqPCR)-receptor tyrosine kinase (RTK) proliferation synergy in airway smooth muscle cells. In combination with previous reports (2, 3, 13, 14, 25), our present studies strongly suggest a mechanism in which GqPCR regulation of PKC and PI3K/Akt1 combines with RTK induction of PI3K to synergistically regulate several key downstream effectors including p70 S6 kinase and GSK-3 in airway myocyte proliferation. PLCbeta1, phospholipase Cbeta1; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; cPKC, conventional PKC isoforms.

 
Our studies provide some novel insights into the mechanisms that may regulate the effects of muscarinic receptor stimulation on airway remodeling. Our group (12) recently demonstrated beneficial effects of the long-acting anticholinergic agent tiotropium bromide on allergen-induced airway smooth muscle accumulation, subserved by myocyte hyperplasia and increased smooth muscle-specific protein expression, in guinea pigs. These beneficial effects suggest the involvement of endogenous acetylcholine, acting on muscarinic receptors in the pathophysiology of chronic allergic airways disease (17, 31). Moreover, in vitro studies indicate that muscarinic receptor stimulation induces lung fibroblast proliferation (28) and enhances airway myocyte proliferation in combination with peptide growth factors, as demonstrated in the current and previous studies (14, 26). Although the involvement of p70 S6 kinase and muscarinic M3 receptors in these effects of muscarinic receptor stimulation already has been established (26), our current studies suggest important additional roles for PKC and GSK-3 in this pathology. It is unclear at this point whether similar mechanisms underpin the effects of muscarinic receptor stimulation on other pathophysiological events in the airways (17, 31), and future studies are clearly warranted in this area.

In conclusion, we describe a mechanism for muscarinic receptors to augment airway myocyte proliferation by enhancing PDGF-induced serine phosphorylation of GSK-3. This mechanism may contribute to recently described effects of endogenous acetylcholine on airway myocyte hyperplasia in animal models of allergic asthma and provides important insights into the mechanisms that regulate and repress airway smooth muscle proliferation.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by grants from the Sick Kids Foundation/Institute of Human Development, Child and Youth Health (XG05-011), Canadian Institutes of Health Research, and the Manitoba Institute of Child Health, Inc. R. Gosens received support from the National Training Program in Allergy and Asthma and is currently the recipient of a Marie Curie Outgoing International Fellowship from the European Community (MOIF-CT-2005-008823).

This work was undertaken, in part, thanks to funding from the Canada Research Chairs Program and Canada Foundation for Innovation. A. J. Halayko holds a Canada Research Chair in Airway Cell and Molecular Biology. The research was conducted using facilities of the Manitoba Institute of Child Health, Inc., in the John Buhler Research Center.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Gosens, Dept. of Molecular Pharmacology, Univ. of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands (e-mail: r.gosens{at}rug.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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