β-Catenin plays a dual role in cellular signaling by stabilizing cadherin-mediated cell-cell contact and by regulating gene transcription associated with cell cycle progression. Nonetheless, its presence and function in airway smooth muscle have not been determined. We hypothesized a central role for β-catenin in mitogenic signaling in airway smooth muscle in response to growth factor stimulation. Immunocytochemical and biochemical analysis revealed that human airway smooth muscle cells indeed express abundant β-catenin, which was localized primarily to the plasma membrane in quiescent cells. Treatment of airway smooth muscle cells with PDGF or FBS induced sustained phosphorylation of glycogen synthase kinase-3 (GSK-3), a negative regulator in its unphosphorylated form that promotes β-catenin degradation. GSK-3 phosphorylation was also increased in airway smooth muscle cells with a proliferative phenotype compared with quiescent airway smooth muscle cells with a mature phenotype. Parallel with the increase in GSK-3 phosphorylation, growth factor treatment induced an increased expression and nuclear presence of β-catenin and activated promitogenic signaling in airway smooth muscle, including the phosphorylation of retinoblastoma protein, DNA synthesis ([3H]thymidine incorporation), and cell proliferation. Importantly, small interfering RNA knockdown of β-catenin strongly reduced retinoblastoma protein phosphorylation, [3H]thymidine incorporation, and cell proliferation induced by PDGF and FBS. Collectively, these data reveal the existence of a GSK-3/β-catenin signaling axis in airway smooth muscle that is regulated by growth factors and of central importance to mitogenic signaling.
- airway remodeling
- growth factor
airway smooth muscle thickening is a pathological feature that likely contributes significantly to airflow limitation and airway hyperresponsiveness in asthma and chronic obstructive pulmonary disease (COPD; Ref. 1). Ongoing research efforts aimed at identifying the mechanisms responsible for the increased airway smooth muscle mass have indicated that hyperplasia of airway smooth muscle, due in part to airway myocyte proliferation, is likely an important contributing factor, although the molecular and cellular mechanisms that regulate this response are still largely unknown (19, 20, 31).
Recently, we identified a key repressive role for glycogen synthase kinase-3 (GSK-3) in airway smooth muscle cell proliferation (12). Thus, pharmacological inhibition of GSK-3 increased cyclin D1 abundance in airway smooth muscle cells and, although not sufficient for concomitant induction of cell proliferation by itself, augmented the proliferative response to PDGF (12). Moreover, we demonstrated that GSK-3 is regulated by G protein-coupled receptors and receptor tyrosine kinases that mediate PKC and Akt-dependent GSK-3 inhibition (phosphorylation of Ser 9 on GSK-3β and Ser 21 on GSK-3α), permitting the induction of retinoblastoma protein (Rb) phosphorylation and cell proliferation (12). Inhibition of GSK-3 could therefore represent a novel intracellular mechanism that regulates airway smooth muscle thickening.
GSK-3 associates with axin, adenomatous polyposis coli, and casein kinase 1 to form a cytosolic multiprotein complex that regulates the intracellular stability of β-catenin (2). β-Catenin is a membrane-associated protein localized to adherens junctions where it is associated with and stabilizes cadherin-mediated cell-cell contact (9). GSK-3 phosphorylates β-catenin when released into the cytosol and targets it for intracellular breakdown (2, 30). As such, GSK-3 likely plays a crucial role in cellular homeostasis: when targeted to the nucleus, β-catenin promotes transcription of T-cell factor (TCF)/lymphoid enhancer factor-dependent genes, which include a number of genes that are involved in cell proliferation, including several growth factors (e.g., VEGF) and cyclin D1 (26, 32). These data collectively suggest that β-catenin is an important transducer of promitogenic signaling regulated by growth factors that can promote GSK-3 inhibition through its phosphorylation. A role for β-catenin in airway smooth muscle and fibroblasts in lung health and disease and to what extent it may modulate their proliferation in response to physiological mediators have, however, not been reported.
In the present study, we hypothesized the existence of a GSK-3/β-catenin signaling axis in airway smooth muscle cells that is regulated by growth factors and mediates cell proliferation. To this aim, we determined the effects of PDGF and FBS on GSK-3 phosphorylation, β-catenin expression, and β-catenin localization in immortalized human airway smooth muscle cell lines. Moreover, we used pharmacological inhibition of GSK-3 and small interfering RNA (siRNA) knockdown of β-catenin to investigate their functional role in mitogenic signaling.
MATERIALS AND METHODS
Antibodies and reagents.
Mouse anti-β-actin antibody, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody, and HRP-conjugated goat anti-rabbit antibody were purchased from Sigma (St. Louis, MO). Rabbit anti-GSK-3 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cy3 and FITC conjugated secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA). Mouse anti-β-catenin antibody was from BD Biosciences (San Jose, CA). Rabbit anti-phospho-Ser9/21-GSK-3 antibody and rabbit anti-phospho-Ser807/811 polyclonal antibody were from Cell Signaling Technology (Beverly, MA). An anti- β-catenin (CTNNB1) siRNA (SI02662478) was obtained from Qiagen (Missisauga, Canada), and siRNA transfection reagent (Genesilencer) was from Genlantis (San Diego, CA). [methyl-3H]thymidine (specific activity of 25 Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). PDGF-AB was purchased from Bachem (Weil am Rhein, Germany), and SB216763 was from Tocris Cookson (Bristol, UK). All other chemicals were of analytical grade.
Four human bronchial smooth muscle cell lines, immortalized by stable expression of human telomerase reverse transcriptase (hTERT), were used for all experiments. The primary cultured human bronchial smooth muscle cells used to generate each cell line were prepared as we have described previously (11, 12, 14) from macroscopically healthy segments of second to fourth generation main bronchus obtained after lung resection surgery from patients with a diagnosis of adenocarcinoma (H. Unruh, Section of Thoracic Surgery, University of Manitoba, Canada). All procedures were approved by the Human Research Ethics Board of the University of Manitoba.
As we have detailed previously (11, 14, 27, 28), each cell line was thoroughly characterized to passage 10 and higher and was shown to express a number of smooth muscle contractile phenotype marker proteins (e.g., smooth muscle-myosin heavy chain, smooth muscle-α-actin, and desmin). For all experiments, passages 10–25 myocytes grown on uncoated plastic dishes in DMEM supplemented with 50 U/ml streptomycin, 50 μg/ml penicillin, and 10% vol/vol FBS were used.
Preparation of cell lysates.
Unless otherwise specified, cells were grown on sixwell cluster plates to confluence and serum starved for 3 days in DMEM supplemented with antibiotics (50 U/ml streptomycin, 50 μg/ml penicillin, and 1.5 μg/ml amphotericin B) and ITS (5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium) before each experiment. Cells were then subjected to different treatments in DMEM supplemented with antibiotics. To obtain total cell lysates, cells were washed once with ice-cold PBS then lysed in ice-cold RIPA buffer (composition: 40 mM Tris, 150 mM NaCl, 1% vol/vol Igepal CA-630, 1% wt/vol deoxycholic acid, 1 mM NaF, 1 mM Na3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 7 μg/ml pepstatin A, pH 8.0). Lysates were then sonicated, and protein concentration was determined according to Bradford. Lysates were stored at −20°C.
Cells were seeded onto precleaned, uncoated glass coverslips in sixwell cluster plates. Cells were grown to subconfluence and then maintained in DMEM supplemented with antibiotics and ITS for 3 days. Cells were then fixed, permeabilized, and immunolabeled using anti-β-catenin, phosho-GSK-3, or total GSK-3. FITC and Cy3-conjugated secondary antibodies were used to detect primary antibody bound to labeled cells. Nuclei were labeled with Hoechst 33342 (10 μg/ml). Coverslips were mounted using anti-fade medium and digitally imaged using an epifluorescence microscope as described previously (17).
Western blot analysis.
Equal amounts of protein (10–20 μg/lane) were subjected to electrophoresis on 8% (β-catenin and phospho-Rb) or 10% (GSK-3, phospho-GSK-3, and β-actin) polyacrylamide gels, transferred to nitrocellulose membranes, and analyzed for the proteins of interest using specific primary and HRP-conjugated secondary antibodies. Bands were subsequently visualized on film using enhanced chemiluminescence reagents. Band intensities were quantified by densitometry using Totallab software (Nonlinear Dynamics, Newcastle, UK).
Isolation of membrane and nuclei enriched fractions.
Cells were grown to confluence on uncoated 100-mm dishes and then maintained in DMEM supplemented with antibiotics and ITS for 3 days. Cells were then 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, and 7 μg/ml pepstatin A. After 20 strokes in a Potter homogenizer, the homogenate was centrifuged for 5 min at 500 g. The resulting nuclei-enriched pellet was resuspended in 200 μl RIPA buffer and solubilized by sonification. The nuclear fraction was then further purified by treatment with 1% vol/vol Triton X-100 to solubilize the nuclei and remove the insoluble cytoskeletal fraction by centrifugation (5 min at 16,100 g). The supernatant obtained in the first centrifugation step was transferred to a new tube and centrifuged for 30 min at 16,100 g. The membrane pellet was resuspended in 200 μl RIPA buffer, sonicated, and stored at −20 °C.
Cells were grown to ∼70% confluence in 6- or 24-well cluster plates and transiently transfected with a 21-bp, double-stranded siRNA targeted against the β-catenin transcript. Cells were transfected in serum-free DMEM supplemented with antibiotics and ITS using 2.5 μg/ml of siRNA in combination with Genesilencer siRNA transfection reagent. In all studies, control transfections were performed using equal amounts of transfection reagent (vehicle control). After 24 h of transfection, cells were washed with fresh DMEM supplemented with antibiotics and ITS to reduce toxicity effects of the transfection reagent.
[3H]thymidine incorporation assay.
Cells were grown to ∼70% confluence in 24-well cluster plates and then maintained in DMEM supplemented with antibiotics and ITS for 3 days. Cells were then subjected to different treatments in fresh DMEM supplemented with antibiotics for 28 h, the last 24 h of which in the presence of [3H]thymidine (0.25 μCi/ml). Cells were then washed twice with PBS at room temperature and once with ice-cold 5% trichloroacetic acid. Cells were treated with this trichloroacetic acid solution on ice for 30 min; subsequently, the acid-insoluble fraction was dissolved in 1 ml NaOH (1 M). Incorporated [3H]thymidine was quantified by liquid-scintillation counting.
Alamar blue proliferation assay.
Cells were grown to ∼70% confluence in 24-well cluster plates and then maintained in DMEM supplemented with antibiotics and ITS for 3 days. Cells were then incubated with mitogens for 4 days in DMEM supplemented with antibiotics. Thereafter, 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 quantified by fluorimetric analysis, as indicated by the manufacturer.
Values reported for all data are means ± SE. The statistical significance of differences between means was determined by an unpaired two-tailed Student's t-test or one-way ANOVA, where appropriate. Differences were considered to be statistically significant when P < 0.05.
β-Catenin expression in airway smooth muscle.
As a role for β-catenin in airway smooth muscle has not yet been reported, we first confirmed its expression in airway smooth muscle. Immunocytochemistry of subconfluent airway smooth muscle cell cultures revealed a ubiquitous expression pattern; cells negative for β-catenin immunoreactivity were not observed, and its expression level among cells appeared similar (Fig. 1A). The β-catenin staining showed a distribution pattern within the cell reminiscent of a membrane-associated protein. This was confirmed in biochemical studies that showed enrichment of β-catenin in the membrane fraction compared with its expression in the cytosol (Fig. 1B). This distribution was similar among all the cell lines used for this experiment.
Effects of growth factor treatment on GSK-3 phosphorylation in airway smooth muscle.
β-Catenin expression and localization are tightly regulated by a cytosolic multiprotein complex containing GSK-3, which in its unphosphorylated form targets free cytosolic β-catenin for intracellular breakdown (2). To investigate if the GSK-3/β-catenin signaling axis is modulated by growth factor treatment, we first investigated the effects of PDGF (30 ng/ml) on GSK-3 phosphorylation, a signaling event that uncouples β-catenin from its degradation cycle. Immunocytochemical analysis indicated that GSK-3 phosphorylation was induced in most cells and that its localization was primarily cytosolic (Fig. 2A). Furthermore, Western analysis of cells treated with PDGF (30 ng/ml) and FBS (10% vol/vol) indicated that both growth-promoting stimuli induced profound phosphorylation of GSK-3, particularly of 47 kDa GSK-3β isoform (Fig. 2, B and C). This phosphorylation was sustained for both stimuli and amounted to 220 ± 20% (P = 0.026) and 405 ± 37% (P < 0.001) of basal for PDGF and FBS, respectively (n = 3).
GSK-3 phosphorylation and concomitant β-catenin expression are induced in proliferating myocytes.
We next determined GSK-3 phosphorylation and consequent β-catenin expression in different phenotypes of airway smooth muscle cells. To investigate the changes in GSK-3/β-catenin signaling during myocyte proliferation and maturation, cells were grown to 50% confluence in serum-rich media (proliferating phenotype) and compared with confluent cultures that were serum deprived for 7 days in insulin-supplemented media (mature phenotype; Refs. 16, 18). In this experiment, we observed a single band of β-catenin in the proliferating cells (Fig. 3, A and B). In contrast, in 7-day serum-deprived cells, multiple bands of lower molecular mass appeared, suggestive of degradation (Fig. 3, A and B). In addition, full-length β-catenin expression and GSK-3 phosphorylation were decreased significantly in quiescent, mature mycoytes (Fig. 3, C and D). Collectively, these results suggest that during airway smooth muscle cell proliferation elevated GSK-3 phosphorylation blocks β-catenin degradation, resulting in enhanced β-catenin abundance.
To test this hypothesis further, we treated airway smooth muscle cell cultures with FBS for varying time points and evaluated its effects on β-catenin protein expression and subcellular localization. Cultures treated with 10% vol/vol FBS showed a gradual increase in β-catenin expression from 16 h of treatment onwards that reached statistical significance at 24 h (Fig. 4A). Fractionation of cells into an insoluble membrane fraction and an insoluble fraction containing nuclei indicated that the increase in β-catenin expression that we observed at 24 h of FBS treatment localized primarily to the nuclear fraction, whereas β-catenin content in the plasma membrane remained unchanged, irrespective of the time point studied (Fig. 4B).
Functional role of β-catenin in mitogenic signaling.
We next determined the functional role of β-catenin in growth factor-induced Rb phosphorylation, DNA synthesis ([3H]thymidine incorporation), and cell proliferation. The functional role of β-catenin was determined using β-catenin specific siRNA to silence the expression of the protein. Initial investigations indicated that after transfection of the cells with β-catenin siRNA, optimal reduction in the expression of β-catenin was achieved at 3 and 4 days of treatment (Fig. 5A). Therefore, in subsequent studies, cells were transfected with β-catenin siRNA concomitant with serum deprivation and treated with growth-promoting stimuli 3 days thereafter to ensure optimal β-catenin knockdown during the window of growth factor treatment. After siRNA treatment, β-catenin expression was reduced to 53 ± 3% (P < 0.001) in control cultures that were maintained in serum-free media (Fig. 5, B and C). This reduction was comparable with that observed in cultures that were simultaneously treated with PDGF or FBS for the last 24 h (Fig. 5, B and C). Although the induction of β-catenin expression by PDGF was not statistically significant, 24 h of FBS treatment increased the expression of β-catenin in control cultures not exposed to β-catenin siRNA, which is in agreement with our earlier findings (compare Figs. 4A and 5C).
We then tested the functional effects of β-catenin siRNA treatment on growth factor-induced mitogenic signaling in airway smooth muscle. Importantly, silencing of β-catenin expression markedly reduced Rb phosphorylation by PDGF and FBS (Fig. 6A). This strong reduction suggests that β-catenin plays an important functional role in these cells. In agreement with this contention, pharmacological inhibition of GSK-3 using SB 216763 (10 μM) induced the opposite effects and strongly augmented PDGF- and FBS-induced Rb phosphorylation (Fig. 6B). This is in agreement with our previously published data that showed SB 216763 augmented growth factor-induced Rb phosphorylation and DNA synthesis in airway smooth muscle (12). Furthermore, the induction of DNA synthesis and cell proliferation by PDGF and FBS was dependent on β-catenin, as siRNA knockdown of the protein reduced PDGF-induced responses to baseline and partially reduced the responses to FBS (Fig. 7). Transfection of the cells with a nonsilencing siRNA control did not reduce basal or FBS-induced mitogenic responses, indicating that the effects of the β-catenin siRNA were specific (FBS-induced cell proliferation amounted 453 ± 27 and 455 ± 44% of baseline for vehicle transfection and nonsilencing siRNA control transfection, respectively; n = 3). Collectively, these data indicate the existence of a GSK-3/β-catenin signaling axis in airway smooth muscle that is regulated by PDGF and FBS treatment and necessary for growth factor-induced mitogenic signaling.
The results of this study identify the GSK-3/β-catenin signaling axis as a central signal transduction pathway in airway smooth muscle cells that can be stimulated by growth factors and that promotes cell proliferation. We demonstrate that growth-promoting stimuli induce GSK-3 phosphorylation, which is paralleled by enhanced expression and nuclear localization of β-catenin. Also, β-catenin expression and GSK-3 phosphorylation were highest in myocytes with a proliferative phenotype. The temporal profile of β-catenin induction by FBS parallels the progression of airway smooth muscle cells into the S phase of the cell cycle (12). Thus, previous studies from our laboratory indicate that 24 h of FBS and PDGF treatment induces DNA synthesis (assessed by flow cytometry and [3H]thymidine incorporation) and Rb phosphorylation, a signaling event necessary for G1 to S phase traversal (12–14; see also Figs. 6 and 7 of the present study). Indeed, silencing of cellular β-catenin reveals that β-catenin is required for mitogen-induced Rb phosphorylation, DNA synthesis, and cell proliferation. Collectively, our studies indicate the existence of a GSK-3/β-catenin signaling axis in airway smooth muscle that is necessary for mitogenic signaling. These observations are important as they suggest that GSK-3/β-catenin signaling is involved in airway smooth muscle thickening, a pathological feature observed in obstructive airways diseases, including asthma and COPD (20).
A central functional role for the GSK-3/β-catenin signaling axis in airway smooth muscle is indicated by our current and previous experiments that indicate that GSK-3 phosphorylation is induced by a diversity of agonists known to act on distinct receptor classes. For example, FBS, PDGF, and agonists for G protein-coupled muscarinic receptors all induce GSK-3 phosphorylation and the combination of muscarinic receptor stimulation with PDGF receptor stimulation is synergistic (12). In fact, since GSK-3 phosphorylation is regulated by several kinases, including PKC isoenzymes, Akt, and integrin-linked kinase (4, 5, 10), and since several agonists acting on G protein-coupled receptors, receptor tyrosine kinases, integrins, and cytokine receptors all induce GSK-3 phosphorylation in cell types other than airway smooth muscle (6, 8), GSK-3 should be considered a central point of convergence for multiple signaling molecules. Indeed, unpublished observations from our group indicate that GSK-3 phosphorylation is also regulated by transforming growth factor-β and IL-1β in airway smooth muscle. Whether β-catenin-dependent gene transcription is central to all of these stimuli and receptor classes mentioned as well is at present not yet clear, and future studies are clearly needed in this area.
Our studies indicate a clear relationship between GSK-3 phosphorylation and β-catenin expression in myocytes with a proliferating phenotype. In addition, FBS treatment induced concomitant GSK-3 phosphorylation and β-catenin expression and was more effective in each of these responses than PDGF, which correlates with the strong functional effects of FBS on Rb phosphorylation, DNA synthesis, and cell proliferation. These findings are in agreement with a substantial amount of evidence that indicates that GSK-3 is responsible for phosphorylation and downregulation of β-catenin (6, 8, 15), which suggests that phosphorylation of GSK-3, leading to its inactivation, induces cell proliferation as a direct result of enhanced β-catenin expression. Nonetheless, the results of the present study and our previous study (12) indicate that the relationship between GSK-3 phosphorylation, β-catenin signaling, and cell proliferation is likely more complex. We demonstrate that pharmacological GSK-3 inhibition using SB 216763 is not sufficient to induce Rb phosphorylation or DNA synthesis (12); only after combined treatment with PDGF or FBS is the inhibitory role for GSK-3 in these promitogenic signaling processes revealed. Also, PDGF had no significant effects on β-catenin expression even though PDGF clearly increased GSK-3 phosphorylation and even though β-catenin was functionally required for PDGF-induced Rb phosphorylation, DNA synthesis, and cell proliferation induced by PDGF. These findings are supported by recent studies (22, 29) in vascular smooth muscle that indicate that PDGF treatment induces TCF-dependent gene transcription without significantly affecting total cellular β-catenin content. Clearly, the regulation of β-catenin expression and function in smooth muscle is complex, and future studies are needed to clarify this process in more detail.
Our siRNA studies indicate a functional role for β-catenin in airway smooth muscle cell proliferation and suggest that this pathway is regulated by a variety of promitogenic stimuli. These findings are supported by a recent study (22) in vascular smooth muscle cells that showed PDGF- and FBS-induced bromodeoxyuridine (BrdU) incorporation was suppressed by overexpression of a dominant negative TCF-4 construct. TCF-4 mediates β-catenin dependent gene transcription and regulates vascular smooth muscle cell proliferation by inducing the cell cycle regulatory protein cyclin D1 and by reducing the cell cycle inhibitor p21 (22). A reduction in mitogen-induced BrdU incorporation in these cells was also achieved by overexpressing the inhibitor of β-catenin and T cell factor (ICAT), a protein that interferes with the interaction of β-catenin with TCF, which substantiates the role for β-catenin signaling further (22). Likewise, a role for β-catenin was recently implicated in IL-5-mediated eosinophil survival. Thus IL-5 treatment induced GSK-3 phosphorylation and promoted the nuclear localization of β-catenin in human eosinophils (24). Collectively, our studies and these recent studies strongly suggest a broad physiological role for GSK-3/β-catenin signaling in regulating cell survival and proliferation in a variety of cells that are present in the lungs.
The functional role of the GSK-3/β-catenin signaling axis in airway smooth muscle thickening in asthma and COPD is presently unknown. Our studies provide novel insights in this area, as they reveal that the β-catenin/GSK-3 signaling axis is operative in airway smooth muscle and regulates mitogenic signaling in response to growth factors. Furthermore, recent studies suggest that β-catenin integrates signals associated with tissue damage and repair during remodeling. β-Catenin is bound to cadherins, which are sensitive to the presence of extracellular proteases, including a disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (9, 21, 23). Thus, disruption of cadherin function by matrix metalloproteinase-dependent cleavage induces β-catenin nuclear translocation and cyclin D1 accumulation (25, 29). The presence of extracellular proteases and growth factors (e.g., during inflammation) can thus activate genes associated with cell proliferation. Indeed, enhanced expression and nuclear localization of β-catenin have been demonstrated in idiophatic pulmonary fibrosis (3) and in a murine model of acute lung injury (7). Therefore, β-catenin could play a role in tissue remodeling in the lung, suggesting that targeting β-catenin-dependent gene transcription holds promise as a therapeutic intervention.
In conclusion, the results of the present study indicate that airway smooth muscle cells express β-catenin, which is primarily localized at the plasma membrane in quiescent cells. Growth factors regulate GSK-3 phosphorylation and activate β-catenin signaling, which is required for Rb phosphorylation and DNA synthesis. These data suggest that the GSK-3/β-catenin signaling axis may play a key role in airway smooth muscle thickening, a common pathological feature in asthma and COPD. Future studies are needed to investigate the role of β-catenin signaling in airway remodeling in more detail.
This work was supported by a grant from the Netherlands Asthma Foundation (3.2.07.023). R. Gosens is the recipient of a Veni Grant (916.86.036) from the Netherlands Organisation for Scientific Research. M. Schmidt is the recipient of a Rosalind Franklin Fellowship from the University of Groningen. The immunocytochemistry studies were supported by an operating grant to A. J. Halayko from the Canadian Institutes of Health Research and in part by the Canada Foundation for Innovation and the Canada Research Chairs Program.
A. J. Halayko holds a Canada Research Chair in Airway Cell and Molecular Biology.
We thank W. T. Gerthoffer (University of Nevada-Reno) for preparation of the hTERT cell lines used in the study.
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.
- Copyright © 2008 the American Physiological Society