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Am J Physiol Lung Cell Mol Physiol 292: L1163-L1172, 2007. First published January 19, 2007; doi:10.1152/ajplung.00471.2006
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p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Reinoud Gosens,1,2,3 Gordon Dueck,1,2 William T. Gerthoffer,4 Helmut Unruh,5 Johan Zaagsma,3 Herman Meurs,3 and Andrew J. Halayko1,2

1Departments of Physiology and Internal Medicine, University of Manitoba, and 2Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; 3Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands; 4Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; and 5Section of Thoracic Surgery, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 7 December 2006 ; accepted in final form 19 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M2 and M3 receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.

epidermal growth factor receptor; muscarinic receptor; airway smooth muscle; p42/p44 mitogen-activated protein kinase

CAVEOLAE ARE FLASK-LIKE SPHINGOLIPID- and cholesterol-enriched invaginations of the plasma membrane that are abundant in many cell types including smooth muscle cells (4). In smooth muscle, caveolae are enriched in caveolin-1 and caveolin-2 proteins; caveolin-1 possess a unique 21-amino acid caveolin-scaffolding domain (CSD) that binds to, and may regulate, a number of receptors and signaling proteins that are known to control smooth muscle contraction, proliferation, and migration (1, 21, 43). Consequently, it has been suggested that caveolae may function as "signalosomes," playing a central role in transmembrane processing of signals by orchestrating the subcellular organization of receptors and signal effectors (18, 21, 36, 40, 42).

Signaling proteins and receptors involved in cell proliferation show a distinct enrichment in caveolae (10, 29); in addition, several reports demonstrate that growth factor-induced activation of p42/p44 MAP kinase, a central signaling effector for receptor-mediated myocyte proliferation, is localized in and facilitated by caveolae (3, 9, 29, 30). Our (18) previous studies using quiescent human airway smooth muscle cells demonstrate that as much as ~95% of platelet-derived growth factor (PDGF) receptor-beta (PDGFRbeta) is sequestered in caveolae. Paradoxically, although these findings support an important role for caveolae in organizing and facilitating signaling events associated with mitogen-induced smooth muscle cell proliferation, disruption of caveolae using either methyl-beta-cyclodextrin or small interfering RNA (siRNA) knockdown of caveolin-1 significantly activates p42/p44 MAP kinase and promotes airway smooth muscle cell proliferation in both the presence and absence of exogenous mitogen (18). Furthermore, our studies also showed p42/p44 MAP kinase is absent from caveolae in quiescent airway smooth muscle cells. These results confirm that the spatial association of receptor tyrosine kinases (RTKs) with p42/p44 MAP kinase activation is highly regulated, but at present, the precise role of caveolae and caveolin-1 in RTK-induced activation of p42/p44 MAP kinase is unclear.

In the current study, we investigated changes that occur in the distribution of RTKs and the p42/p44 MAP kinase-signaling cascade in caveolae after airway smooth muscle cells were exposed to mitogens. We used immortalized human airway smooth muscle cell lines in conjunction with sucrose density gradient centrifugation to fractionate caveolae before and after exposure to EGF or methacholine, agonists that are known to induce p42/p44 MAP kinase activation in human airway myocytes via RTK and G protein-coupled receptors, respectively. We demonstrate that the p42/p44 MAP kinase-signaling cascade is localized to noncaveolae plasma membrane fractions, and that this distribution is unchanged by activation after mitogen exposure. Notably, in a temporal manner that paralleled p42/p44 MAP kinase activation, EGFR exhibited rapid and dramatic relocalization from caveolae to p42/p44 MAP kinase-enriched noncaveolae membrane fractions after EGF exposure. Thus our studies demonstrate that although caveolae appear to be sites for RTK sequestration in quiescent airway myocytes, translocation of receptors from caveolae occurs upon growth factor receptor activation, a process that facilitates the activation of p42/p44 MAP kinase in caveolae-free membrane fractions.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and reagents. HRP-conjugated goat anti-mouse antibody and HRP-conjugated goat anti-rabbit antibody were purchased from Sigma (St. Louis, MO). Mouse anti-caveolin-1 monoclonal antibody and mouse anti-tyrosine-14 phosphorylated caveolin-1 monoclonal antibody were obtained from BD Biosciences (San Jose, CA). FITC- and Cy3-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Rabbit anti-caveolin-1 polyclonal antibody, anti-phosphotyrosine conjugated agarose beads (clone PY20), rabbit anti-EGFR polyclonal antibody, rabbit anti-PDGFRbeta polyclonal antibody, goat anti-muscarinic M2 receptor polyclonal antibody, goat anti-muscarinic M3 receptor polyclonal antibody, mouse anti-Raf-1 monoclonal antibody, and mouse anti-Grb2 monoclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-phospho-Thr202/Tyr204-p42/p44 MAP kinase monoclonal antibody, rabbit anti-p42/p44 MAP kinase polyclonal antibody, and rabbit anti-Src antibody were all from Cell Signaling Technology (Beverly, MA). All other chemicals were of analytical grade.

Cell culture. As we have previously described, human bronchial smooth muscle cell lines were immortalized by stable expression of human telomerase reverse transcriptase (hTERT), which has been shown to extend the lifespan of endothelial cells, fibroblasts, and smooth muscle cells (2, 5, 15, 18, 32, 49). The primary cultured human bronchial smooth muscle cells used to generate each cell line were prepared as we have previously described (35, 39) from macroscopically healthy segments of second to fourth 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 we (18) have detailed previously, each cell line was thoroughly characterized to passage 10 and higher and was shown to express a number of smooth muscle (sm) mature phenotype marker proteins (e.g., sm-myosin heavy chain, sm-{alpha}-actin, and desmin; Ref. 45). In addition, these cell lines were characterized for their proliferative responses to PDGF-BB, sensitivity to which was comparable to primary human airway smooth muscle cells (18). For all experiments, passages 10-16 myocytes 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) were used. Unless otherwise specified, cells were grown to subconfluence (~70%) 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.

Isolation of caveolae-enriched and caveolae-deficient membrane fractions. Plasma membrane fractions were isolated as we (18) have previously described. Briefly, cells grown on uncoated 150-mm dishes were washed with ice-cold phosphate-buffered saline (PBS) and lysed in 500 mM sodium carbonate (pH 11.0) supplemented with 1 mM Na3VO4, 1 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, and 1 mM PMSF. After homogenization by sonification, 2 ml of homogenate was mixed with an equal volume of a solution containing 150 mM NaCl, 25 mM MES, and 80% (wt/vol) sucrose (pH 6.5) and placed in the bottom of a centrifuge tube. A stepwise sucrose density gradient (30, 20, and 5%) was then carefully layered on top of the homogenate. Thereafter, the samples were centrifuged at 210,000 g for 18 h at 4°C, and 1-ml fractions were collected from the top of the gradient. Samples were stored at –80°C until further use.

Isolation of total membrane-enriched fractions. Cells grown on uncoated 150-mm dishes were washed with ice-cold PBS and lysed for 10 min on ice in 50 mM Tris (pH 7.4), supplemented with 1 mM Na3VO4, 1 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, and 1 mM PMSF. After 20 strokes in a Dounce homogenizer, the homogenate was centrifuged for 5 min at 1,000 g. The supernatant was transferred to a new tube and centrifuged for 60 min at 210,000 g. The pellet-containing membrane was resuspended and stored at –80°C until further use.

Preparation of whole cell lysates. As we (18) have previously described, after cultured cells were subjected to different treatments, cells were washed once with ice-cold PBS and then lysed by scraping in ice-cold lysis buffer (composition: 40 mM Tris, 150 mM NaCl, 1% Igepal, 1% deoxycholic acid, 1 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, 1 mM PMSF, pH 8.0). Lysates were stored at –80°C until further use.

Western blot analysis. Equal amounts of protein were subjected to electrophoresis, transferred to nitrocellulose membranes, and analyzed for the proteins of interest using specific primary and horseradish peroxidase (HRP)-conjugated secondary antibodies as we (18–20) have previously described. Protein bands were subsequently visualized on film using enhanced chemiluminescence reagents and quantified by scanning densitometry using TotalLab software (Nonlinear Dynamics, Newcastle, United Kingdom)

Immunocytochemistry. Cells were seeded onto precleaned, uncoated glass coverslips in culture dishes and subsequently grown to confluence and then maintained in serum-free medium for 3 days. Cells were then fixed with 3% paraformaldehyde, permeabilized using 0.3% Triton X-100, and immunolabeled as we have previously described (20). For the present study, primary antibodies were used to assess cellular distribution of caveolin-1 and phospho-p42/p44 MAP kinase. FITC-or Cy3-conjugated secondary antibodies were used to detect primary antibody bound to labeled cells. Coverslips were mounted using anti-fade medium and digitally imaged as described previously (20).

Immunoprecipitation. Polyclonal rabbit anti-caveolin-1 IgG, directed against the NH2-terminal sequence of caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA), was conjugated to protein A sepharose beads immediately before use by incubating for 1 h (4°C) in 30 µl of a 50% sepharose slurry. Beads were then washed 3 times with lysis buffer, blocked with 10% BSA, and transferred to 500 µg of cell lysate. For immunoprecipitation of phosphotyrosine (pY), anti-pY-conjugated agarose beads (Santa Cruz Biotechnology) were transferred directly to 500 µg of cell lysate. After 1-h incubation at 4°C, beads were washed 4 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.

Data analysis. Values reported for all data represent means ± SE. For all studies, replicate data from three different cell lines were obtained. The statistical significance of differences between means was determined by an unpaired two-tailed Student's t-test. Differences were considered to be statistically significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Receptor localization in caveolae is not required for p42/p44 MAP kinase induction. To best assess the role of caveolae in receptor-mediated signaling, we first used sucrose density gradient centrifugation, a technique widely employed to isolate caveolae from caveolae-free cell membranes (41), to profile the distribution of a panel of receptors and proximal signaling proteins previously shown to mediate activation of p42/p44 MAP kinase in airway smooth muscle cells (17, 26). In our protocols, we lysed cells using detergent-free carbonate buffer, which facilitated subsequent enrichment of caveolae membrane fractions; these appeared as an opaque band at the 5-to-20% sucrose density interface after centrifugation. Immunoblot analyses of fractions collected from this region (generally fractions 3–5) were highly enriched in caveolin-1 (Fig. 1). As we (18) have previously shown, caveolae-enriched membranes lacked clathrin heavy chain, which instead appears in fractions with caveolae-free membranes that isolate in higher density sucrose regions (Fig. 1). We found that the RTKs PDGFRbeta and EGFR were highly enriched in caveolae, where ~95% of the total receptor population was sequestered. In contrast, G protein-coupled muscarinic M2 and M3 receptors were mainly retrieved in caveolae-deficient membrane fractions that were enriched in clathrin heavy chain. Gi and Gq proteins, which are the G protein {alpha}-subunits associated with M2 and M3 receptors, respectively, fractionated in approximately equal abundance to both caveolae-enriched and caveolae-free fractions.


Figure 1
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  		Fig. 1. Differential localization of G protein-coupled muscarinic receptors and receptor tyrosine kinases in caveolae microdomains. Cells were grown to subconfluence and serum-deprived 3 days before homogenization in carbonate buffer for caveolae isolation using sucrose-density gradient centrifugation. After centrifugation, 1-ml fractions were collected from the top (i.e., buoyant density increases with increasing fraction number). Equal volumes from each fraction were subjected to Western blot analysis for the indicated proteins. M3R, muscarinic M3 receptor; M2R, muscarinic M2 receptor; Gq{alpha}, {alpha}-subunit of trimeric Gq proteins that couple to M3R; Gi{alpha}, {alpha}-subunit of trimeric Gi proteins that couple to M2R; EGFR, epidermal growth factor receptor; PDGFRbeta, platelet-derived growth factor receptor-beta; clathrin HC, clathrin heavy chain. Results shown are representative for all cell lines used in this study.

 
We next tested whether the differential localization of EGFR and muscarinic receptors in caveolae and noncaveolae membranes of human airway smooth muscle cell lines correlated with any difference in capacity or kinetics of p42/p44 MAP kinase induction. As expected, EGF induced rapid and profound p42/p44 MAP kinase phosphorylation that decayed gradually but remained ~20% higher than baseline even after 2 h (Fig. 2A). Interestingly, the muscarinic receptor agonist methacholine also rapidly induced phospho-p42/p44 MAP kinase, and this was temporally similar to the response elicited by EGF (Fig. 2B). Methacholine-induced phospho-p42/p44 MAP kinase, however, decayed more rapidly than we observed after EGF exposure. Notably, methacholine did not induce EGFR or PDGFRbeta phosphorylation, confirming that p42/p44 MAP kinase activation by this agonist did not occur as the result of RTK transactivation (Fig. 2C). Collectively, these observations indicate that in quiescent airway smooth muscle cells before agonist exposure, the differential distribution of receptor subtypes in caveolae-containing and caveolae-free plasma membrane domains is not a critical determinant of coupling to signaling cascades that result in p42/p44 MAP kinase activation.


Figure 2
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  		Fig. 2. Localization of receptors in caveolae is not required for p42/p44 MAP kinase induction. Time-dependent phospho-T202/Y204-p42/p44 MAP kinase accumulation in human airway myocytes treated with EGF (A; 10 ng/ml) or methacholine (MCh; B; 10 µM). Cell lysates were analyzed by Western blotting for phospho-T202/Y204-p42/p44 MAP kinase and total p42/p44 MAP kinase. Blots shown are representative for all human airway smooth muscle cell lines used in this study. The graphs shown below each set of blots depict densitometric data normalized to maximal agonist-induced p42/p44 MAP kinase-phosphorylation. Baseline p42/p44 MAP kinase phosphorylation levels were generally below detection limits in control cultures. Data are expressed as means ± SE obtained from 3 to 4 experiments. C: muscarinic receptor stimulation activates p42/p44 MAP kinase independent of EGFR or PDGFRbeta transactivation. Cell cultures were treated with PDGF-BB (30 ng/ml), EGF (10 ng/ml), or methacholine (10 µM) for 15 min. Whole cell lysates were subjected to immunoprecipitation (IP) for phosphotyrosine (pY); then, precipitates were analyzed by Western blotting for EGFR, PDGFRbeta, and caveolin-1. Results shown are representative for all cell lines used in this study. C, control.

 
Caveolin-1 phosphorylation is not required for p42/p44 MAP kinase induction. We next investigated whether the differential plasma membrane distribution of EGFR and G protein-coupled muscarinic receptors was associated with differences in modulation of caveolin-1 in the events leading to p42/p44 MAP kinase activation. Through its CSD, caveolin-1 can bind and may inactivate growth factor receptors, such as EGFR and PDGFRbeta, and {alpha}-subunits of G proteins (6, 7, 34, 48). Upon growth factor exposure, phospho-Y14-caveolin-1 is induced, and we (18) have shown that this correlates temporally with the disassembly of caveolin-1-PDGFRbeta complexes. Phospho-Y14-caveolin-1 is also implicated to have direct signaling functions, including the recruitment of adaptor proteins such as Grb7; binding of Grb7 to phospho-Y14-caveolin-1 stimulates cell growth and migration (28). We therefore measured the temporal pattern of caveolin-1 phosphorylation in cultures treated with EGF or methacholine. Despite our previous observation that both EGF and methacholine induced rapid, profound p42/p44 MAP kinase phosphorylation (Fig. 2), only exposure with EGF resulted in significant phosphorylation of caveolin-1 at tyrosine-14 (Fig. 3, A-C). Notably, phospho-Y14-caveolin-1 remained localized to caveolae-rich membrane fractions in EGF-treated cultures (Fig. 3D). Collectively, our observations strongly suggest that phosphorylation of caveolin-1 at tyrosine-14 is not a fundamental step in the activation of p42/p44 MAP kinase by all receptor types.


Figure 3
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  		Fig. 3. Caveolin-1 phosphorylation is not required for p42/p44 MAP kinase induction. Time-dependent caveolin-1 phosphorylation in response to EGF (A; 10 ng/ml) or methacholine (B; 10 µM) was measured. Cell cultures were treated with agonists and then subjected to Western blot analysis for caveolin-1 and phospho-Y14-caveolin-1. Blots shown are representative for all cell lines used in this study. C: densitometric data for comparing the maximal level of caveolin-1 phosphorylation induced by EGF (10 ng/ml) or methacholine (10 µM). Results shown are normalized to basal caveolin-1-Y14-phosphorylation levels present in cultures before ligand exposure. Data are expressed as means ± SE obtained from 3 to 5 experiments. *P < 0.05 compared with control (Student's t-test for unpaired observations). D: subcellular localization of phospho-Y14-caveolin-1 in EGF (10 ng/ml, 15 min)-treated cell cultures. Caveolae microdomains were isolated using sucrose density gradient centrifugation; equal volumes of each fraction were analyzed by Western blotting for caveolin-1 and phospho-Y14-caveolin-1. Blots shown are representative of all cell lines used in this study.

 
p42/p44 MAP kinase activation is not localized to caveolae. Our (18) previous studies demonstrated that p42/p44 MAP kinase is not sequestered to plasma membrane caveolae in quiescent human airway smooth muscle cells. In the present study, we extended these observations by assessing whether receptor-mediated activation of p42/p44 MAP kinase altered the distribution of the p42/p44 MAP kinase-signaling cascade in cell membrane subfractions. As expected, in quiescent cultures before growth factor exposure, p42/p44 MAP kinase was retrieved chiefly in caveolae-free fractions; the adaptor protein Grb2 and the MEK kinase, Raf-1, also primarily localized to caveolae-free fractions of the gradient (Fig. 4A). At time points that corresponded to the onset of (2 min) and maximal (15 min) p42/p44 MAP kinase activation after EGF exposure (Fig. 4B), p42/p44 MAP kinase, Grb2 and Raf-1 were all retrieved almost exclusively in caveolae-deficient fractions, a pattern that was indistinct from myocytes before EGF exposure (Fig. 4, A-C). Thus exposure of the cells to EGF did not induce any translocation of the p42/p44 MAP kinase-signaling cascade to caveolae membrane domains. We further characterized the cellular distribution of phosphorylated p42/p44 MAP kinase in human airway myocytes using immunocytochemistry by comparing its localization with that of caveolin-1 (Fig. 5). In cells that were treated with EGF (10 ng/ml, 15 min), phosphorylated p42/p44 MAP kinase appeared in a diffuse, cytoplasmic pattern with the strongest signal observed closest to nuclei. In contrast, caveolin-1 staining was punctate and clearly marked the edges of each cell. Colabeling for phospho-p42/p44 MAP kinase and caveolin-1 indicated distinct differences in staining patterns for phospho-p42/p44 MAP kinase and caveolin-1 (Fig. 5), which is highly consistent with data we obtained using biochemical fractionation that shows activated p42/p44 MAP kinase is not concentrated in caveolae-enriched membrane microdomains.


Figure 4
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  		Fig. 4. p42/p44 MAP kinase activation is not localized to caveolae in human airway smooth muscle. A: subcellular localization of Grb2, Raf-1, p42/p44 MAP kinase, and phospho-T202/Y204-p42/p44 MAP kinase was assessed in membrane microdomains isolated using sucrose density gradient centrifugation. Equal volumes of each fraction were analyzed by Western blotting for the indicated proteins. Top: distribution of the p42/p44 MAP kinase-signaling cascade in cultures before exposure to EGF (10 ng/ml, 15 min). A blot for basal phosphorylated p42/p44 MAP kinase is not shown for control cells, as levels were generally below detection limits. Bottom: distribution of the p42/p44 MAP kinase-signaling cascade after EGF exposure (10 ng/ml, 15 min) in human airway smooth muscle cell cultures. Blots shown are representative for all cell lines used. B: time-dependent phospho-T202/Y204-p42/p44 MAP kinase accumulation in human airway myocytes treated with EGF (10 ng/ml). Whole cell lysates were analyzed by Western blotting for phospho-T202/Y204-p42/p44 MAP kinase and total p42/p44 MAP kinase. C: subcellular distribution of Grb2, Raf-1, and p42/p44 MAP kinase was assessed after EGF exposure at time points corresponding to the activation phase of p42/p44 MAP kinase, as assessed in B. Panels show distribution of Grb2, Raf-1, caveolin-1, and p42/p44 MAP kinase in caveolae (black bars) and caveolae-free fractions (white bars). No translocation of the p42/p44 MAP kinase-signaling to caveolae was observed during the p42/p44 MAP kinase activation.

 

Figure 5
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  		Fig. 5. Immunocytochemistry showing staining for caveolin-1 (green) and phospho-T202/Y204-p42/p44 MAP kinase (red) after EGF exposure (10 ng/ml, 15 min). Areas marked by arrows and the composite image indicate clearly the distinct subcellular localization of caveolin-1 and phospho-p42/p44 MAP kinase in human airway smooth muscle cells.

 
EGFR-mediated p42/p44 MAP kinase activation is localized to caveolae-free cell membranes. Despite observing that EGF dramatically induced p42/p44 MAP kinase activation, our data clearly demonstrate that EGFR and the p42/p44 MAP kinase-signaling cascade are differentially distributed in membrane microdomains of quiescent cells. Furthermore, we (18) have previously shown that PDGFRbeta binding to caveolin-1 is rapidly reduced after PDGF exposure. Thus we next tested whether disassociation of EGFR from caveolin-1 and trafficking from caveolae domains occurred during EGF-induced activation of p42/p44 MAP kinase in caveolae-free cell fractions.

We first used immunoprecipitation to monitor the kinetics of EGFR phosphorylation in response to EGF exposure and observed rapid receptor activation that was temporally similar to EGF-induced p42/p44 MAP kinase activation (Figs. 6A and 2A). We next assessed the cell membrane distribution of EGFR in myocytes during maximum receptor activation (10 ng EGF, 15 min). EGF exposure induced a marked depletion of EGFR from caveolae microdomains and a concomitant increase of EGFR in caveolae-free fractions where the p42/p44 MAP kinase-signaling cascade is enriched (Fig. 6B). Although there was some variability in the magnitude of EGFR trafficking that we observed after EGF exposure, ~70% of the receptor that was associated with caveolae in quiescent myocytes relocalized to caveolae-free membrane fractions (Fig. 6C). As a control, we measured caveolin-1 distribution and found that is was unchanged by EGF exposure, with most of the protein detected in fractions 3–5 of the sucrose gradient, corresponding to the location of the visible opaque band characteristic of caveolae-rich membrane fractions. As these observations suggest that activated EGFR dissociates from caveolin-1 and caveolae, we next used immunoprecipitation to compare the relative abundance of phosphorylated EGFR in caveolae-rich and caveolae-free membranes in myocytes after EGF exposure. Indeed, tyrosine-phosphorylated EGFR was primarily retrieved from caveolae-free fractions that corresponded with those where p42/p44 MAP kinase was localized (Fig. 6D).


Figure 6
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  		Fig. 6. EGFR activates p42/p44 MAP kinase in noncaveolae membrane. A: time-dependent EGFR phosphorylation induced by EGF (10 ng/ml). Human airway smooth muscle cell lysates were immunoprecipitated for phosphotyrosine, and then precipitates were subjected to Western blotting (IB) for EGFR. The blot shown is representative of all cell lines used in this study. The graph shown below the blot represents densitometric data, normalized to maximal EGF-induced EGFR phosphorylation. Baseline EGFR phosphorylation levels were generally below detection limits in control cultures. Data are expressed as means ± SE obtained from 5 experiments. B: subcellular distribution of EGFR. Caveolae microdomains were isolated using sucrose density gradient centrifugation; equal volumes of each fraction were analyzed for EGFR by Western blotting. Top: EGFR distribution in control cultures before growth factor exposure. Bottom: EGFR distribution in EGF-treated (10 ng/ml, 15 min) cell cultures. C: EGFR physically dissociates from caveolae microdomains after EGF exposure. Caveolae microdomains were isolated from cultures before growth factor exposure (Control, white bars) and after EGF treatment (10 ng/ml, 15 min; black bars); equal volumes of fractions obtained after sucrose density gradient centrifugation were analyzed for caveolin-1 and EGFR by Western blotting. Graphs shown depict densitometric data from cell fractions enriched with caveolae (fractions 3–5) of distribution of EGFR and caveolin-1. Data are expressed as means ± SE obtained from 4 to 5 experiments. *P < 0.05 compared with control (Student's t-test for unpaired observations). D: subcellular distribution of phospho-EGFR. Caveolae microdomains were isolated using sucrose density gradient centrifugation; the pooled caveolae fractions (fractions 3–5) and the pooled fractions toward which EGFR trafficking was primarily directed (fractions 8–10) were immunoprecipitated for phosphotyrosine; precipitates were then analyzed for EGFR by Western blotting. Results were obtained using EGF-treated (10 ng/ml, 15 min) cell cultures. Baseline EGFR phosphorylation was generally below detection limits before EGF exposure. The blot shown is representative of all cell lines used in this study. E: EGF-induced p42/p44 MAP kinase activation is localized to the cell membrane. Total membrane preparations were prepared from control (C) cultures before and after EGF exposure (10 ng/ml, 15 min) and were then subjected to Western blotting for EGFR and phosphorylated and total p42/p44 MAP kinase, as indicated. The blot shown is representative of those obtained for all cell lines used.

 
As caveolae have been implicated in the process of receptor internalization, to determine whether the dramatic decrease we observed in caveolae-associated EGFR might be due to a general loss of caveolae from the cell membrane in response to agonist, we compared EGFR abundance in total cell membrane fractions prepared from myocytes before and after EGF exposure. Importantly, we clearly observed that plasma membrane EGFR abundance was unaffected by EGF exposure (Fig. 6E). Moreover, as caveolin-1 content in low buoyant density fractions was also unchanged after EGF exposure, growth factor treatment did not alter caveolae abundance over the time course of our experiment (Fig. 6C). Notably, in our total membrane preparations, we were able to detect p42/p44 MAP kinase, indicating that even though we observed little or none of the enzyme in caveolae-enriched membrane fractions, at least a proportion of it must be associated with caveolae-free membranes. Furthermore, EGF treatment did not change the content of p42/p44 MAP kinase in total membrane fractions, but we did observe a marked increase in the abundance of activated p42/p44 MAP kinase in total membrane fractions collected after EGF exposure (Fig. 6E). Collectively, these data indicate that upon growth factor activation, EGFR dissociates from caveolae and subsequently initiates p42/p44 MAP kinase activation in caveolae-free membrane compartments of airway smooth muscle cells.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of the present study demonstrate that in human airway smooth muscle cells, the primary site of p42/p44 MAP kinase activation occurs in cell compartments that are caveolae-free. Moreover, this occurs despite clear evidence that both EGFR and PDGFRbeta (18) are tightly sequestered to caveolae in myocytes before mitogen exposure. Our studies further reveal that p42/p44 MAP kinase is virtually absent from caveolae in both quiescent cells and myocytes treated with growth factor. Furthermore, p42/p44 MAP kinase activation by EGF occurs concomitantly with phosphorylation of caveolin-1 and is associated with a rapid and dramatic relocation of EGFR from caveolae to caveolae-free cell compartments. This is consistent with our (18) previous observations that caveolin-1 is required for suppression of autonomous p42/p44 MAP kinase activity in quiescent human airway myocytes and that PDGFRbeta rapidly dissociates from caveolin-1 after ligand exposure. Collectively, our studies reveal the existence of a dynamic association of RTKs with caveolae whereby receptor trafficking between membrane microdomains is a component of transduction events that lead to activation of intracellular signaling that is critical to promote myocyte proliferation.

Our findings that p42/p44 MAP kinase, Grb2, and Raf-1 are not associated with caveolae membranes and do not migrate to caveolae upon agonist exposure is consistent with observations from other studies using HUVEC cells, in which p42/p44 MAP kinase, MEK, and Raf-1 are primarily localized to noncaveolae compartments (3). Our results from cytochemistry experiments and studies using crude membrane fractionation further suggest that p42/p44 MAP kinase is likely distributed both in the cytoplasm and to some extent in noncaveolae membranes. Notably, we show evidence for a significant increase in the association of phosphorylated p42/p44 MAP kinase with total membrane fractions after EGF exposure. This confirms that although caveolae microdomains are likely not involved, dynamic association of p42/p44 MAP kinase with the cell membrane can occur as the result of growth factor stimulation, and this could be a critical step leading to p42/p44 MAP kinase activation.

Our muscarinic receptor studies provide further evidence that direct activation of p42/p44 MAP kinase is not localized to caveolae in human airway smooth muscle cells. Muscarinic M2 and M3 receptors are the only muscarinic receptor subtypes expressed by airway smooth muscle (31). Notably, the immortalized cell lines used for the studies retain expression of muscarinic M3 receptor expression and function, as assessed by the induction of calcium mobilization in response to methacholine (data not shown). This is a significant advantage over primary airway smooth muscle cells, where muscarinic M3 receptor expression in culture is either diminished or lost (16, 46). These G protein-coupled receptors were primarily localized in caveolae-free membranes, although their corresponding G protein {alpha}-subunits were distributed in both caveolae-rich and caveolae-free cell fractions. We noted that methacholine, a selective muscarinic receptor agonist, did not induce caveolin-1 phosphorylation, yet still induced p42/p44 MAP kinase phosphorylation. Moreover, this proceeded completely independent of RTK transactivation. We (8, 17, 23) and others previously demonstrated that p42/p44 MAP kinase activation by muscarinic receptor agonists in airway smooth muscle is mediated via PKC and Ras, suggesting the combined involvement of muscarinic M2 and M3 receptor subtypes. Collectively, these results confirm that receptor systems that are not directly associated with caveolae or caveolin-1 can potently induce p42/p44 MAP kinase activation and, as such, support the existence of p42/p44 MAP kinase signal transduction in caveolae-free cell compartments.

There is some variability in reports concerning the localization and activation of the p42/p44 MAP kinase cascade with caveolae. On one hand, most components required for growth factor-induced p42/p44 MAP kinase signaling, including RTKs, non-RTKs, adaptor proteins, and guanine nucleotide exchange factors, Ras, Raf-1, MEK, and p42/p44 MAP kinase, have been reported to be enriched in caveolae of fibroblasts (29, 30). In fibroblasts, PDGF exposure has been associated with p42/p44 MAP kinase activation in caveolae (29, 30), and EGF can induce the recruitment of activated Raf-1 to caveolae (33). In contrast, we (18) and other investigators (12) have shown that disruption of caveolae in airway smooth muscle cells and fibroblasts potentiates RTK-induced activation, facilitating cell proliferation despite the nearly complete relocation of several key signaling components of EGF-induced p42/p44 MAP kinase activation to noncaveolae membrane. This is entirely consistent with multiple studies showing that caveolin proteins are themselves potent inhibitors of RTK activity and components of the p42/p44 MAP kinase cascade (7, 9, 11, 13, 48). These results demonstrate that RTK-signaling machinery is hyperactivated when disengaged from caveolae, supporting the idea that caveolae inhibit but do not directly facilitate the molecular interactions that drive functional p42/p44 MAP kinase activation. Thus it appears that the primary role of caveolae in p42/p44 MAP kinase activation and cell proliferation of human airway smooth muscle is, in fact, to sustain mitogenic quiescence.

The differentiation state of cells may also be a determinant of the role of caveolae in MAP kinase signaling. In freshly isolated bovine parathyroid cells, p42/p44 MAP kinase is primarily localized and activated in caveolae (27). In these cells, nuclear translocation of p42/p44 MAP kinase is low, as is cell proliferation. Conversely, when the same cells are cultured, colocalization of p42/p44 MAP kinase with caveolin-1 is greatly reduced and cell proliferation is enhanced (27). This is consistent with observations by us (18) and others (38, 44) showing caveolin-1 expression and caveolae number are highest in smooth muscle cells of a more contractile phenotype, which exhibit minimal proliferative activity. This suggests that differences in cell phenotype could, in part, contribute to the diversity in published reports of the role of caveolae in mitogen-induced signaling. In the current study, myocytes were subjected to 3-day serum deprivation before growth factor exposure; we (18) have shown that this is sufficient to significantly induce caveolin-1 expression in these cells.

Several chronic airways diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis, are characterized by exaggerated airway smooth muscle growth (22, 25, 47). Since the expression of growth factors and their receptors is increased in these diseases (24), insights in the basic mechanisms that drive growth factor-induced airway smooth muscle proliferation are important. It is established that p42/p44 MAP kinase plays a central role in growth factor-induced airway smooth muscle proliferation (26, 37) and has additional roles in the regulation of airway smooth muscle cell migration, contraction, and synthetic function (14). From the present study and our previous work (18), it appears that caveolins and caveolae play an important role in maintaining airway smooth muscle cells in a quiescent mitogenic state. It is yet to be established whether caveolin expression and caveolae number are altered in these diseases.

In conclusion, the results from this study demonstrate that p42/p44 MAP kinase activation is localized to noncaveolae compartments of the cell membrane in airway smooth muscle. Although caveolae contain growth factor receptors in quiescent cells, relocation of these receptors to caveolae-free compartments occurs during p42/p44 MAP kinase activation and appears to be a key step leading to mitogenic activation of myocytes. Moreover, caveolae and caveolins appear to function primarily to sustain mitogenic quiescence, perhaps, in the case of RTKs, by preventing their association with downstream signaling effectors of p42/p44 MAP kinase signal transduction.


   GRANTS
TOP
 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. 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-2005-008823). This research was undertaken, in part, thanks to funding from the Canada Research Chairs Program. A. J. Halayko holds a Canada Research Chair in Airway Cell and Molecular Biology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Gosens, Dept. of Physiology, Univ. of Manitoba, 715 McDermot Ave., Rm. 547, John Buhler Research Centre, Winnipeg, Manitoba, Canada, R3E 3P4 (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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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