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Am J Physiol Lung Cell Mol Physiol 294: L131-L138, 2008. First published November 9, 2007; doi:10.1152/ajplung.00381.2007
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Inhibition of human airway smooth muscle cell proliferation by β2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Karen M. Kassel,1 Todd A. Wyatt,2 Reynold A. Panettieri, Jr.,3 and Myron L. Toews1

1Department of Pharmacology and Experimental Neuroscience and 2Pulmonary, Critical Care, Sleep, and Allergy Section, University of Nebraska Medical Center, Omaha, Nebraska; and 3Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania

Submitted 12 September 2007 ; accepted in final form 3 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Mechanisms by which β-adrenergic receptor (βAR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that βAR agonists would inhibit mitogenesis in HASM cells via the β2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by ~10-fold. The nonselective βAR agonist isoproterenol and the β2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A β2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a β1AR-selective antagonist, confirming β2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. β2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10–100 µM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 µM). These data show that β2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.

exchange protein directly activated by cAMP; lysophosphatidic acid; epidermal growth factor; asthma

ASTHMA IS A DISEASE CHARACTERIZED by hypercontractility of the airway smooth muscle cells and by the largely irreversible structural remodeling of the airway. This remodeling is thought to result from improper repair of airway epithelial cell damage, increased collagen and fibronectin deposition in the airways causing a thickening of the basement membrane, mucus gland hyperplasia resulting in increased mucus synthesis and secretion, and increased airway smooth muscle cell mass (12, 16). Multiple studies have shown that there is an increase in the thickness of the smooth muscle layer in the lungs of patients with asthma, with both hyperplasia and hypertrophy contributing (1, 6, 7). This increased thickness of airway smooth muscle and the airway wall has also been shown to contribute to the hypercontractility of the airways to physical and chemical stimuli that is the hallmark of asthma (21, 26, 35). To date, no effective therapies are available for preventing and/or reversing the thickening of the airway wall. A better understanding of the mechanisms for both the stimulation and inhibition of airway smooth muscle proliferation may reveal new therapeutic targets to control hyperplasia of the airway smooth muscle layer.

EGF plays an important role in many normal physiological processes within the lung (25), but it has also been implicated in the pathological airway remodeling seen in asthmatic airways (1, 25). Among the best characterized effects of EGF are those on lung epithelial cells, including epithelial repair (25), mucus synthesis and secretion (31), and its role in lung cancer epithelial cell metastasis (4, 27). However, EGF also contributes to the hyperplasia and hypertrophy of the airway smooth muscle layer during airway remodeling in asthma (12, 16). EGF has been shown to stimulate the proliferation of isolated human airway smooth muscle (HASM) cells on its own; in addition, it synergizes with several G protein-coupled receptor (GPCR) agonists, including lysophosphatidic acid (LPA), leukotriene D4, carbachol, endothelin-1, sphingosine-1-phosphate, and thrombin (3, 8, 24). This synergistic response to multiple mediators present in asthmatic airways likely contributes to the inappropriate proliferation seen in asthma, leading to both remodeling and hyperreactivity of the airways.

Both EGF and LPA have been shown to be elevated in asthmatic airways (1, 10). In addition to its ability to synergize with EGF to stimulate proliferation of HASM cells, the lipid mediator LPA also enhances contractility, stimulates proliferation of lung fibroblasts, and stimulates fibronectin secretion and filopodia extension in airway epithelial cells, suggesting potential pathological roles for LPA in multiple aspects of airway remodeling in asthma and other airway diseases (32). The presence of elevated levels of LPA and EGF in the lung, along with their abilities to enhance asthma-related responses and perhaps contribute to the progression of the disease, underscores the importance of studying potential mechanisms whereby the effects of EGF and LPA on HASM proliferation could be prevented or reversed.

β2-adrenergic receptor (β2AR) agonists are widely used to treat the airway smooth muscle-mediated bronchoconstriction associated with asthma. β2ARs are GPCRs that activate the heterotrimeric G protein Gs and the effector enzyme adenylyl cyclase to stimulate the production of the second messenger cAMP. Several previous studies have shown that these and other cAMP-elevating agents, including PGE2, can inhibit airway smooth muscle cell proliferation stimulated by various agonists (9, 20, 33); however, downstream signaling pathways for this cAMP effect have not yet been established.

PKA and EPAC (exchange protein directly activated by cAMP) are the two best established downstream effectors of cAMP action. PKA has been the classic cAMP effector, with EPAC having been identified only in 1998 (5). Thus, most studies to date have somewhat "assumed" the role of PKA in cAMP-mediated effects; in particular, there appear to be no studies exploring the potential role(s) of EPAC in the regulation of airway smooth muscle. The purpose of this study was to more definitively establish the mechanisms for βAR agonist-mediated inhibition of EGF-stimulated airway smooth muscle cell proliferation, with a focus on the involvement of cAMP and on the roles of PKA and/or EPAC as downstream mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Reagents. Reagents were purchased from the following sources: EGF from Biosource International (Camarillo, CA); forskolin and PGE2 from Calbiochem (San Diego, CA); DMEM, penicillin/streptomycin, and FBS from Invitrogen (Carlsbad, CA); phosphocellulose P-81 paper from Whatman (Clifton, NJ); heptapeptide substrate for PKA (LRRASLG) from Peninsula Laboratories (San Carlos, CA); [3H]thymidine and [3H]adenine from PerkinElmer Life Sciences (Boston, MA); [{gamma}-32P]ATP from National Diagnostics (Atlanta, GA); Bradford protein assay dye reagent from Bio-Rad Laboratories (Hercules, CA); Econosafe scintillation cocktail from Research Products International (Mt. Prospect, IL); and nonaqueous scintillation cocktail from MP Biomedicals (Solon, OH). Other reagents were gifts: salmeterol xinafoate from GlaxoSmithKline (Middlesex, UK) and ICI 118551 and ICI 89406 from ICI Pharmaceuticals (London, England). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Fairlawn, NJ).

Cell culture. HASM cells were isolated from human trachea by enzymatic dissociation as described previously, with Institutional Review Board approval (23). Cells were grown in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified 5% CO2 incubator at 37°C. Cells were passaged weekly and typically used through passage 12, which is common for these cells (3, 22, 28). The experiments presented here were performed using a single isolation of cells for consistency; however, key experiments were performed in cells from a different donor to confirm the results.

[3H]thymidine incorporation assay. HASM cells were plated at 20,000 cells/well in 12-well plates. On day 6 after plating, confluent cells were starved overnight in serum-free DMEM before being stimulated with 60 ng/ml EGF, 10 µM LPA, or 60 ng/ml EGF plus 10 µM LPA in the absence or presence of cAMP-elevating agents for an additional 24 h at 37°C. All experiments included control wells that were treated with appropriate vehicle solutions. After 22 h of treatment, [3H]thymidine (2 µCi/well) was added for the last 2 h of stimulation, to assess DNA synthesis. Cells were then washed once with 1 ml of PBS and twice with 1 ml of 10% TCA. The TCA precipitates were dissolved in 0.5 ml of 0.2 N NaOH, and [3H]thymidine incorporation was analyzed by liquid scintillation spectroscopy. Previous studies have documented that data from a 2-h [3H]thymidine pulse nicely parallels the data for number of cells in S-phase as determined by flow cytometry (8).

Cell counting. HASM cells were plated at 20,000 cells/well in 12-well plates. On day 3 after plating, nonconfluent cells were treated in serum-free medium with 60 ng/ml EGF in the absence or presence of cAMP-elevating agents for 3 days at 37°C. All experiments included control wells that were treated with appropriate vehicle solutions. Cells were then washed with PBS, trypsinized, and resuspended in DMEM containing 10% FBS. Cell number was determined using a Coulter counter (Coulter Electronics, Hialeah, FL).

cAMP assay. cAMP formation was measured using a modification of the Shimizu method (29) as previously described (19). HASM cells were plated at 40,000 cells/well in six-well plates. Confluent cells were washed with 1 ml of HEPES-buffered DMEM and labeled with 5 µCi/ml [3H]adenine for 1 h at 37°C. Cells were then washed with 1 ml of HEPES-buffered DMEM and stimulated with the indicated concentrations of βAR agonists or Fsk analogs in the presence of 1 µM IBMX for 5 min. Stimulation medium was aspirated, and 1 ml of 5% TCA containing 1 mM non-radioactive cAMP was added. Samples were passed over 50WX8-400 Dowex columns; the eluate was collected as the ATP fraction, and [3H]ATP in this fraction was quantified by liquid scintillation spectroscopy. The Dowex columns were placed over type WN-3 neutral alumina columns and washed with 3 ml of H2O to elute cAMP. The alumina columns were washed with 3 ml of 50 mM Tris, pH 8.0, and the eluate was collected as the cAMP fraction. Absorbance of the eluate at 259 nm was determined to quantify cAMP recovery for each column. Scintillation fluid was then added to the eluate to quantify [3H]cAMP formation by liquid scintillation spectroscopy. Data are expressed as the percentage of [3H]ATP converted to [3H]cAMP during the assay.

PKA activation assay. HASM cells were plated at 80,000 cells/dish in 60-mm dishes. Confluent cells were starved overnight in serum-free DMEM before stimulation for 15 min with cAMP-elevating agents. Lysis buffer containing 35 mM Tris·HCl, 0.4 mM EGTA, 10 mM MgCl2, 0.1% protease inhibitor cocktail, and 0.1% Triton X-100 was added to the cells, which were then flash frozen. PKA activity was determined using a modification of procedures previously described (13). Briefly, thawed cells were scraped, sonicated, and centrifuged for 30 min at 10,000 g at 4°C. Samples (20 µl) were added to 50 µl of reaction mix [130 µM PKA substrate heptapeptide (LRRASLG), 0.9 mg/ml BSA, 0.2 mM IBMX, 20 mM Mg-acetate, and 0.2 mM [{gamma}-32P]ATP in a 40 mM Tris·HCl buffer, pH 7.5] and incubated at 30°C for 10 min. Incubations were halted by spotting 60 µl of each sample onto P-81 phosphocellulose papers. Papers were then washed five times for 5 min each in 75 mM phosphoric acid, washed once in ethanol, dried, and analyzed by scintillation spectroscopy. Kinase activity is expressed as picomoles of phosphate incorporated per minute per milligram of protein. Total protein was determined by the Bradford protein assay. All samples were assayed in duplicate or triplicate in at least three separate experiments.

Data analysis. Data were analyzed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Statistical significance was determined using repeated measures ANOVA with the Bonferroni post test to compare selected pairs of data. Pairs were considered significantly different at P ≤ 0.05. All experiments were performed in triplicate, and data are presented as means ± SE from replicate experiments. The n values for each figure and value presented indicate the number of replicate experiments with cells from one donor. As indicated above, key findings were replicated with cells from at least one additional donor.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Isoproterenol inhibition of proliferation. Treatment of HASM cells for 24 h with 60 ng/ml EGF stimulated proliferation by 9.1 ± 1.2-fold (n = 22), as assessed by [3H]thymidine incorporation (Fig. 1A). The prototypical βAR agonist isoproterenol inhibited this EGF-stimulated proliferation by 39 ± 12% (Fig. 1A). Isoproterenol also consistently decreased the low level of basal proliferation, but this inhibition was not statistically significant. The effect of isoproterenol to inhibit EGF-stimulated proliferation was concentration dependent (Fig. 1B), with an EC50 of 4.8 ± 1.0 nM (n = 4). To confirm that this effect was mediated by β2ARs, the predominant βAR subtype in airway smooth muscle, HASM cells were stimulated with 60 ng/ml EGF in the presence of 100 nM isoproterenol and various concentrations of the β2AR-selective antagonist ICI 118551 or the β1AR-selective antagonist ICI 89406. ICI 118551 was 100-fold more potent than ICI 89406 for preventing the isoproterenol-mediated inhibition of proliferation (Fig. 1C), and the IC50 of 14 ± 5 nM (n = 3) for ICI 118551 is in agreement with its known potency at β2ARs. Although ICI 89406 inhibited the isoproterenol effect at higher concentrations, the IC50 of 1.4 ± 0.5 µM (n = 3) is again consistent with this antagonist's much lower potency at the β2AR and not with its potency at its preferred β1AR. Thus, inhibition of proliferation by isoproterenol is mediated via the β2AR subtype.


Figure 1
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  		Fig. 1. Isoproterenol inhibits EGF-stimulated human airway smooth muscle (HASM) cell proliferation by activating the β2-adrenergic receptor (β2AR). HASM cells were treated for 24 h with vehicle (CTL), 10 µM isoproterenol (Iso), 60 ng/ml EGF, or 10 µM Iso + 60 ng/ml EGF (A), the indicated concentrations of Iso in the presence of 60 ng/ml EGF (B), or 100 nM Iso + 60 ng/ml EGF in the presence of the indicated concentrations of the β2AR-selective antagonist ICI 118551 or the β1AR-selective antagonist ICI 89406 (C). [3H]thymidine ([3H]Thy) was added for the final 2 h of treatment to measure cell proliferation. In A, data are expressed as fold of control and are from 22 separate experiments, each in triplicate. In B and C, data are expressed as fold of EGF stimulation and are from 3 separate experiments, each in duplicate. **P < 0.01, ***P < 0.001.

 
Inhibition of proliferation by albuterol and salmeterol. To further confirm β2AR involvement, the effects of isoproterenol were compared with those of two β2AR-selective agonists that are widely used to treat asthma, the short-acting agent albuterol and the long-acting agent salmeterol. Both of these β2AR-selective agonists dose dependently decreased the EGF-stimulated increase in proliferation (Fig. 2A). Albuterol inhibited by 49 ± 11% with an EC50 of 110 ± 37 nM (n = 4), and salmeterol inhibited by 56 ± 3% with an EC50 of 6.7 ± 1.5 nM (n = 3).


Figure 2
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  		Fig. 2. β2AR-selective agonists are full agonists for inhibiting EGF-stimulated HASM cell proliferation but partial agonists for cAMP production and PKA activation. A: HASM cells were treated with the indicated concentrations of isoproterenol, albuterol (Alb), or salmeterol (Sal) in the presence of 60 ng/ml EGF for 24 h. [3H]thymidine was added for the final 2 h of treatment to measure cell proliferation. Data are expressed as fold of EGF stimulation and are from 3 separate experiments, each in duplicate. B: HASM cells were treated with 10 µM Iso, 10 µM Alb, or 10 µM Sal in 5-min assays of cAMP accumulation (left). Data are expressed as a percent of maximal conversion of ATP to cAMP and are from 5 separate experiments, each in triplicate. *P < 0.05, ***P < 0.001 compared with control values. Bars at right represent the percent inhibition of proliferation by the 10 µM concentration of each agonist from A. C: HASM cells were treated with the indicated concentrations of Iso, Alb, and Sal for 15 min and assayed for PKA activation. Data are expressed as picomoles of phosphate incorporated per minute per milligram of total protein and are from at least 3 separate experiments, each in duplicate or triplicate.

 
Comparison of inhibition of proliferation with stimulation of cAMP and PKA activation. The abilities of albuterol and salmeterol to stimulate cAMP synthesis and PKA activation were compared with those of the full agonist isoproterenol. Both albuterol and salmeterol were relatively low-efficacy partial agonists for both cAMP production and PKA activation [Fig. 2B, P < 0.05, Fig. 2C], as has been reported previously (28). Salmeterol was much more potent than either isoproterenol or albuterol for activating PKA, with an EC50 of 350 ± 130 pM for salmeterol compared with EC50 values of 290 ± 110 nM and 64 ± 25 nM for isoproterenol and albuterol, respectively. Interestingly, although albuterol and salmeterol were only partial agonists for cAMP production and PKA activation, they were nonetheless able to inhibit EGF-stimulated proliferation to a similar extent as isoproterenol (Fig. 2, A and B), indicating that significant signal amplification takes place between cAMP production and the regulation of DNA synthesis, as has been shown previously for these cells (28).

Inhibition of HASM cell proliferation by cAMP-elevating agents. Because the extent of cAMP stimulation did not correlate with the extent of the inhibition of proliferation, other cAMP-elevating agents were tested to further explore cAMP involvement. The Gs-coupled GPCR agonist PGE2 has been shown previously to inhibit serum-, thrombin-, and PDGF-stimulated HASM cell proliferation (14, 17, 33), and it prevents allergen-induced bronchoconstriction and reduces airway hyperreactivity and inflammation in bronchial asthma (34). This makes the endogenous mediator PGE2 a good candidate for comparison with the synthetic β2AR agonists used in clinical therapy. As expected, treatment of HASM cells with 10 µM PGE2 (Fig. 3) inhibited EGF-stimulated proliferation by 58 ± 5% (n = 3), similar to the results with the β2AR agonists.


Figure 3
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  		Fig. 3. PGE2 and forskolin (Fsk) inhibit EGF-stimulated HASM cell proliferation. HASM cells were treated for 24 h with vehicle (CTL), 10 µM PGE2, or 33 µM Fsk in the absence and presence of 60 ng/ml EGF. [3H]thymidine was added for the final 2 h of treatment to measure cell proliferation. Data are expressed as fold of control and are from at least 3 separate experiments, each in triplicate. **P < 0.01, ***P < 0.001.

 
To establish whether cAMP elevation alone was sufficient to decrease proliferation, the direct adenylyl cyclase activator forskolin was tested. Forskolin (33 µM) inhibited HASM cell proliferation to a greater extent than any of the GPCR agonists, with a 75 ± 3% (n = 4) inhibition of EGF-stimulated HASM cell proliferation (Fig. 3). Forskolin has been shown to exhibit effects on other targets in addition to adenylyl cyclase (15), perhaps accounting for its greater inhibition. Alternatively, rapid desensitization of the β2AR after agonist treatment during the 24-h assays could perhaps decrease their effects. Regardless, the ability of multiple Gs-coupled agonists and forskolin to inhibit EGF-stimulated proliferation strongly suggests that this inhibition is mediated by an increase in cAMP.

Inhibition of proliferation by cAMP analogs. To directly establish the role of cAMP, cell-permeable and nonhydrolyzable cAMP analogs were tested for their ability to inhibit HASM cell proliferation. The two main effectors for cAMP are PKA and EPAC; accordingly, agents selective for PKA and/or EPAC were included in these studies. The PKA-selective cAMP analogs dibutyryl-cAMP (db-cAMP), 8-bromo-cAMP (8Br-cAMP), and N6-benzoyl-cAMP (6Bnz-cAMP) inhibited EGF-stimulated proliferation only at 1 mM, the highest concentration achievable (Fig. 4A); their steep dose-response curves suggest the possibility that their effects may be nonspecific rather than true PKA-mediated responses. In contrast, the EPAC-selective cAMP analog 8-chlorophenylthio-2'-O-methyl-cAMP (8CPT-2Me-cAMP) caused a clearly concentration-dependent inhibition of EGF-stimulated proliferation at much lower concentrations (Fig. 4A), with an EC50 of 1.6 ± 0.1 µM (n = 5). The cAMP analog 8-chlorophenylthio-cAMP (8CPT-cAMP), which activates both PKA and EPAC, inhibited EGF-stimulated proliferation with an EC50 of 16 ± 1 µM, a much lower concentration than for the PKA-selective cAMP analogs, although not as potent as the EPAC-selective cAMP analog (Fig. 4A). These studies suggest that cAMP does in fact inhibit proliferation of these cells. More importantly, these data strongly suggest EPAC rather than PKA as the relevant effector of cAMP-mediated inhibition of proliferation of HASM cells.


Figure 4
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  		Fig. 4. EPAC-activating cAMP analogs inhibit EGF-stimulated HASM cell proliferation. A: HASM cells were treated for 24 h with the indicated concentrations of dibutyryl-cAMP (db-cAMP), 8-bromo-cAMP (8Br-cAMP), N6-benzoyl-cAMP (6Bnz-cAMP), 8-chlorophenylthio-cAMP (8CPT-cAMP), or 8-chlorophenylthio-2'-O-methyl-cAMP (8CPT-2Me-cAMP) in the presence of 60 ng/ml EGF. [3H]thymidine was added for the final 2 h of treatment to measure cell proliferation. Data are expressed as fold of EGF stimulation and are from at least 3 separate experiments, each in duplicate or triplicate. B: HASM cells were treated with the indicated concentrations of db-cAMP, 8Br-cAMP, 6Bnz-cAMP, 8CPT-cAMP, or 8CPT-2Me-cAMP for 15 min and assayed for PKA activation. Data are expressed as picomoles of phosphate incorporated per minute per milligram of total protein and are from at least 3 separate experiments, each in duplicate or triplicate.

 
Stimulation of PKA by cAMP analogs. Because PKA-selective cAMP analogs did not inhibit proliferation except at very high concentrations, we performed PKA activation assays to confirm that these analogs did stimulate PKA activation at the expected lower concentrations. The PKA-selective cAMP analogs db-cAMP, 8Br-cAMP, and 6Bnz-cAMP were all able to activate PKA, with maximal activity of ~2,000 pmol·min–1·mg–1 (Fig. 4B). In addition, the nonselective cAMP analog 8CPT-cAMP was also able to maximally activate PKA. 8Br-cAMP and 6Bnz-cAMP were the most potent analogs, with EC50 values of 290 ± 60 nM and 520 ± 110 nM, respectively. 8CPT-cAMP was slightly less potent, with an EC50 of 11 ± 2 µM, followed by db-cAMP with an EC50 of 18 ± 1 µM. The EPAC-selective cAMP analog 8CPT-2Me-cAMP had a very low potency for activating PKA and showed essentially no PKA activation until concentrations of 100 µM. Most importantly, PKA-selective cAMP analogs maximally activated PKA at concentrations that had no effect on proliferation (10–100 µM), indicating that PKA activation cannot explain the inhibition of proliferation. In contrast, the EPAC-selective cAMP analog maximally inhibited proliferation at a concentration that had no effect on PKA activation (10 µM), again indicating a PKA-independent mechanism for inhibition of proliferation and pointing to EPAC as the more likely mediator.

Inhibition of EGF-stimulated increase in cell number by cAMP-elevating agents. The [3H]thymidine assay used in our routine assays measures DNA synthesis and has been found to be an appropriate marker for cell proliferation in previous studies (3). To confirm that the [3H]thymidine data accurately reflect true cell proliferation, cell proliferation was measured more directly using a Coulter counter to quantify cell number. Previous studies from our laboratory have shown that EGF significantly increases the cell number after 3 days of treatment (3), although this increase in cell number is much smaller than the fold increase in [3H]thymidine incorporation. To determine whether cAMP-elevating agents could inhibit this increase in cell number, HASM cells were treated with 60 ng/ml EGF in the absence and presence of various cAMP-elevating agents. As in previous studies, EGF significantly increased cell number, by 25 ± 9% (P < 0.05). In contrast, EGF was not able to significantly increase cell number in the presence of 10 µM isoproterenol (–7 ± 7%), 10 µM albuterol (9 ± 8%), or 33 µM forskolin (6 ± 2%). In addition, EGF did not increase cell number in the presence of 10 µM 8CPT-2Me-cAMP (–5 ± 19%) but did increase cell number in the presence of 100 µM db-cAMP (17 ± 10%), which maximally activates PKA.

Inhibition of LPA- and LPA+EGF-stimulated proliferation by cAMP-elevating agents. We have shown previously that LPA not only stimulates proliferation on its own but also enhances EGF stimulation to synergistically increase proliferation (3, 8). This synergism may play a role in the hyperproliferation of the smooth muscle layer and contribute to the remodeling of the airway (32). Cells were treated with 10 µM LPA alone, 60 ng/ml EGF alone, or 10 µM LPA plus 60 ng/ml EGF, to determine if β2AR agonists and other cAMP-elevating agents could inhibit LPA-stimulated proliferation and the synergistic stimulation by LPA+EGF. Isoproterenol, albuterol, and salmeterol were able to inhibit LPA-stimulated proliferation to a similar extent as their inhibition of EGF-stimulated proliferation (Fig. 5, A and B). Although all agonists were also able to inhibit LPA+EGF-stimulated proliferation, salmeterol inhibited this synergistic proliferation to a much greater extent than either isoproterenol or albuterol. Forskolin and 8CPT-2Me-cAMP also inhibited LPA- and LPA+EGF-stimulated proliferation to an even greater extent than the β2AR agonists (Figs. 5C and 6A). The effect of 8CPT-2Me-cAMP was concentration dependent (Fig. 5D), with an EC50 of 2–4 µM. The PKA-selective cAMP analog dibutyryl-cAMP was much less potent for inhibiting LPA- and LPA+EGF-stimulated proliferation (Fig. 5D), similar to its low potency for the inhibition of EGF stimulation.


Figure 5
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  		Fig. 5. cAMP-elevating agents inhibit lysophosphatidic acid (LPA)- and LPA+EGF-stimulated proliferation of HASM cells. HASM cells were treated for 24 h with vehicle (CTL), 10 µM LPA, 60 ng/ml EGF, or 10 µM LPA + 60 ng/ml EGF in the absence or presence of 10 µM isoproterenol (A; Iso), 10 µM albuterol (B; Alb) or 10 µM salmeterol (B; Sal), or 10 µM 8CPT-2Me-cAMP (C). D: cells were also treated with 10 µM LPA or 10 µM LPA + 60 ng/ml EGF in the presence of the indicated concentrations of db-cAMP or 8CPT-2Me-cAMP. [3H]thymidine was added for the final 2 h of treatment to measure cell proliferation. Data are expressed as fold of CTL (A–C) or as fold of EGF stimulation (D) and are from 3 separate experiments, each in duplicate or triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.

 

Figure 6
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  		Fig. 6. The complete inhibition of LPA+EGF-stimulated proliferation by forskolin may include by both cAMP-dependent and cAMP-independent mechanisms. HASM cells were treated for 24 h with vehicle (CTL), 10 µM LPA, 60 ng/ml EGF, or 10 µM LPA + 60 ng/ml EGF in the absence or presence of 33 µM Fsk (A), 10 µM NKH477 (B), or 10 µM 1,9-dideoxy-forskolin (ddFsk) (C). [3H]thymidine was added for the final 2 h of treatment to measure cell proliferation. Data are expressed as fold of CTL and are from 3 separate experiments, each in triplicate. D: HASM cells were treated with 33 µM Fsk, 10 µM NKH477, or 10 µM ddFsk in 5-min assays of cAMP accumulation. Data are expressed as a percent of maximal conversion of ATP to cAMP and are from 3 separate experiments, each in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.

 
cAMP-dependent and cAMP-independent effects of forskolin. Forskolin completely inhibited [3H]thymidine incorporation, including the synergistic stimulation by LPA+EGF (Fig. 6A), whereas other cAMP-elevating agents had only partial effects. Because of this unexpected result, the greater effects of forskolin compared with β2AR agonists and other cAMP-elevating agents for inhibiting LPA-, EGF-, and LPA+EGF-stimulated proliferation were investigated further using forskolin analogs. The adenylyl cyclase-inactive forskolin analog 1,9-dideoxy-forskolin (ddFsk) did not inhibit proliferation by LPA or EGF alone (Fig. 6C), but the adenylyl cyclase-activating analog NKH477 was as effective as forskolin itself (Fig. 6B). However, the ability of forskolin to completely inhibit synergistic proliferation by LPA+EGF appeared to involve both cAMP-dependent and cAMP-independent effects, because NKH477 was able to completely inhibit synergism similarly to forskolin, but ddFsk was only able to partially inhibit synergism (Fig. 6, B and C). Interestingly, ddFsk did not inhibit stimulation by either LPA or EGF alone, but it did reduce their synergistic interaction. To confirm that the inhibition of synergism by ddFsk was not a result of contamination of the ddFsk with forskolin, cAMP assays were performed for the forskolin analogs. Both forskolin and NKH477 significantly increased cAMP production, whereas ddFsk did not stimulate cAMP production and in fact slightly decreased cAMP production (Fig. 6D). These results suggest that forskolin may inhibit stimulation by EGF or LPA alone by a cAMP-dependent mechanism but may also affect an adenylyl cyclase-independent mechanism uniquely involved in the synergism between LPA and EGF.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The studies presented here clearly show that β2AR agonists, including β2AR-selective agonists used clinically in airway disease, can inhibit EGF-, LPA-, and LPA+EGF-stimulated proliferation of HASM cells. Other cAMP-elevating agents, specifically PGE2 and forskolin, also inhibit proliferation, implicating cAMP in this process. The ability of direct cAMP analogs to inhibit proliferation further suggests cAMP involvement. However, the failure of PKA-selective cAMP analogs to inhibit proliferation at concentrations that maximally activate PKA indicates that PKA activation cannot be the mechanism for the inhibition by cAMP. Three widely used PKA inhibitors, KT5720, the myristoylated PKI peptide, and the cAMP antagonist R(p)-cAMP(S), all failed to inhibit PKA effectively in HASM cells (data not shown), in agreement with a previous study of these compounds in HASM cells (11). In addition, the PKA inhibitors KT5720 and H89 both greatly inhibited EGF-stimulated proliferation on their own, through mechanisms apparently unrelated to PKA inhibition (data not shown).

In contrast to PKA-selective cAMP analogs, the ability of EPAC-selective cAMP analogs to inhibit proliferation without activating PKA points to EPAC or an EPAC-like cAMP effector as the key mediator for inhibition of proliferation in these cells. This is the first study showing the potential importance of EPAC proteins in airway smooth muscle cells. Other studies with EPAC in lung cells have shown that EPAC suppresses phagocytosis, bactericidal activity, and H2O2 production in alveolar macrophages (2).

β2AR agonists are the mainstay of bronchodilator therapy for asthma, including short-acting agonists such as albuterol for acute bronchodilation (18) and long-acting agonists such as salmeterol and formoterol used together with anti-inflammatory glucocorticoids for long-term therapy (30). The widespread clinical use of these agents warrants a full understanding of the molecular basis for each of their multiple effects. Our studies implicating EPAC-related mechanisms rather than PKA in cAMP-mediated inhibition of proliferation suggest that possible roles of EPAC proteins in bronchodilation and other airway cell effects of β2AR agonists should also be investigated. If EPAC proteins mediate a wider range of β2AR agonist and cAMP effects in the airway, more extensive studies of EPAC would become critical. Alternatively, if EPAC proteins are not involved in bronchodilation, then therapies specifically targeting EPAC proteins and pathways might allow for selective modulation of proliferation without interfering with contractile effects. Details of the targets and mechanisms further downstream in the pathway(s) to inhibition of HASM cell proliferation will also be required, and these may reveal additional novel therapeutic targets for controlling inappropriate airway smooth muscle proliferation.

Increased concentrations of both EGF and LPA have been detected in airway fluids from diseased airways (1, 10), and together they exhibit a markedly synergistic stimulation of HASM cell proliferation (3, 8). Both agents may contribute to the structural remodeling seen in asthma and other airway diseases, and their synergism may be particularly important for hyperproliferation of the smooth muscle layer (32). Like EGF and LPA, several other agents are known to stimulate HASM proliferation, and many of these agents also synergize with EGF to greatly enhance proliferation and may also contribute to the remodeling of the airway during disease. All three of the β2AR agonists that were tested inhibited both EGF- and LPA-stimulated proliferation in vitro, but the short-acting agonists isoproterenol and albuterol were less effective for inhibiting the stronger synergistic stimulation of proliferation by LPA+EGF. This could explain why β2AR agonists have not been reported to inhibit smooth muscle proliferation in vivo in diseased airways, which likely are exposed to elevated levels of many proliferation-stimulating agonists acting together.

Importantly, the EPAC-selective cAMP analog is more effective for inhibiting the synergistic proliferation between LPA and EGF than short-acting albuterol and as effective as long-acting salmeterol. Furthermore, forskolin and some of its analogs completely inhibit even the strong proliferation by LPA+EGF that may be most relevant to the pathological setting in asthma. The ability of an adenylyl cyclase-inactive forskolin analog to inhibit the synergistic stimulation of proliferation by LPA+EGF but not by either agent alone points to both cAMP-dependent and cAMP-independent pathways for inhibiting proliferation. cAMP-independent effects of forskolin include inhibition of glucose transport, inhibition of ion flux through nicotinic receptors and enhancement of nicotinic receptor desensitization, and modulation of voltage-dependent K+ channels (15), although these and other possible cAMP-independent effects of forskolin have not been well studied. Together, the differential effects of these agents on synergism may help to reveal the interactive mechanisms through which synergism between mitogens occurs. These findings also raise the hope that new drugs targeted at EPAC proteins and pathways and/or selective forskolin analogs could be effective for preventing or reversing the hyperproliferation of airway smooth muscle, providing therapeutic benefit beyond that attainable with the current therapies.

In conclusion, cAMP-elevating agents, including the β2AR agonists used clinically to induce relaxation of the airways, inhibit EGF-, LPA-, and LPA+EGF-stimulated proliferation of HASM cells. This effect is mediated by cAMP, but it cannot be explained by PKA activation and appears to be mediated through EPAC or an EPAC-like effector instead. Our data are the first to implicate EPAC proteins as candidate cAMP effectors for clinically important drug effects in the lung. Further studies of the multiple and interactive signaling pathways that regulate the stimulation as well as the inhibition of HASM cell proliferation, both in vitro and then in vivo, will lead to a better understanding of the pathogenesis of asthma. More importantly, such studies are likely to reveal additional new therapeutic targets for prevention and/or reversal of the multiple components of the pathology of asthma and other proliferative airway diseases.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by American Heart Fellowship 0415367Z and a University of Nebraska Medical Center Skala Fellowship (K. M. Kassel) and by Nebraska Department of Health and Human Services Grant 2005-29 and American Heart Association Grant-In-Aid 0750108Z (to M. L. Toews). T. A. Wyatt is an American Lung Association Career Investigator.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
M. L. Toews was the recipient of a research grant from GlaxoSmithKline for an unrelated research project.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Stephen Rennard for helpful discussions and Nancy Schulte, Jane DeVasure, and Jacqueline Pavlik for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. L. Toews, 985800 Nebraska Medical Center, Omaha, NE 68198-5800 (e-mail: mtoews{at}unmc.edu)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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