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Am J Physiol Lung Cell Mol Physiol 290: L485-L491, 2006. First published October 14, 2005; doi:10.1152/ajplung.00233.2005
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The beta-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells

Bradley J. Schnackenberg,1,3 Stacie M. Jones,1,2,3 Crystal Pate,1 Brian Shank,1 Laura Sessions,1 Laura M. Pittman,1 Lawrence E. Cornett,2 and Richard C. Kurten2,3

Departments of 1Pediatrics and 2Physiology and Biophysics, University of Arkansas for Medical Sciences, and 3Arkansas Children's Hospital Research Institute, Little Rock, Arkansas

Submitted 31 May 2005 ; accepted in final form 7 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although beta-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that beta-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the beta-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing beta2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.

beta2-adrenergic receptor; epidermal growth factor receptor; asthma; bronchospasm

UNDER NORMAL CONDITIONS, the airway epithelium functions as a protective barrier and serves as a first line of defense against the external environment. However, injury of the airway epithelium has been described in a number of pulmonary diseases. In asthma, epithelial denudation and chronic airway inflammation lead to airway remodeling and thickening of the airway wall. The major medications used to treat the bronchospasm associated with asthma are the beta2-adrenergic agonists that bind beta2-adrenergic receptors (beta2-AR) localized throughout the airway (18). The beta2-AR is a G protein-coupled receptor (GPCR) and is the predominant AR in the lung (19, 23). Activation of beta2-ARs induces association of Gs with adenylyl cyclase resulting in cAMP production (23). Although beta2-AR activation mediates a variety of cellular functions in the lung, which includes airway smooth muscle relaxation, increased mucociliary action, and ion transport, the effects of beta-adrenergic agonists on wound healing are unknown.

Immediately after wounding, the epithelial barrier function is reestablished by cells at the wound edge spreading and migrating into the denuded area (34, 35). Peptide growth factors, such as epidermal growth factor (EGF), have been shown to affect cell motility and enhance wound-healing responses. EGF binds EGF receptor (EGFR), a single-pass transmembrane domain receptor in the family of receptor tyrosine kinases, and enhances wound healing in a variety of tissues including airway (9, 16, 24). Elevated EGFR expression appears related to epithelial repair because EGF has been shown to accelerate the repair of scrape-wounded 16HBE 14o- bronchiolar epithelial cell monolayers (24). Interestingly, in asthmatic bronchial epithelium, the repair process is abnormal, and EGFR expression is elevated compared with normal epithelium (24).

Upon binding of EGF, the EGFR becomes activated by autophosphorylation of tyrosine residues on the cytoplasmic domain of the EGFR (30). This initiates activation of the ERK/MAPK pathways via interactions with mediator proteins such as Grb2, Ras, and Raf (20, 26) and ultimately leads to the activation of nuclear transcription factors to initiate a functional response, such as wound closure.

A number of studies have shown that tyrosine kinase receptors, such as the EGFR, can be transactivated by GPCRs. For example, the beta2-AR has been shown to transactivate the EGFR upon isoproterenol (ISO) stimulation in cardiac fibroblasts (13) and COS-7 cells (21), resulting in the activation of ERK2. These reports illustrate that beta-agonists, which are used therapeutically for bronchorelaxation in asthma, can modulate EGFR signaling (12, 13, 21). Therefore, we hypothesized that ISO would influence wound healing in asthmatic airways by modulating EGFR signaling pathways. The purpose of this study was to determine the effect of ISO on EGF-stimulated closure of scrape wounds using 16HBE epithelial cells as an in vitro model for airway epithelium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. Human recombinant EGF, ISO, and forskolin (FOR) were purchased from Sigma (St. Louis, MO). MEK inhibitor (U0126) was obtained from Promega (Madison, WI). Antibodies to phospho-tyrosine (PY20), EGFR (12020), and ERK2 were purchased from BD Transduction Laboratories (San Diego, CA). Antibodies to phospho-p44/42 MAPK (Thr202/Tyr204) were purchased from Cell Signaling Technology (Beverly, MA). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cell culture. Human bronchial epithelial cells (16HBE 14o-) were generously provided by Dr. D. C. Gruenert (Univ. of Vermont, Burlington, VT). 16HBE cells were cultured in MEM with Earle's salt supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO2 incubator.

Scrape wound assay. 16HBE cells were cultured in six-well plates (Corning, Corning, NY). Confluent 16HBE cultures were serum starved for 36–48 h before being wounded to minimize the effect of growth factors in serum. With the use of a 200-µl yellow pipette tip, cultures were wounded with three parallel scrapes that extended the full diameter of the well. Debris were removed from the culture by washing with HBS (20 mM HEPES, 120 mM NaCl, 1 mM glucose, 6 mM Na2HPO4·7 H2O, 1.25 x 10–4% phenol red, pH 7.5), and the cultures were then incubated in MEM plus 1% FBS supplemented with or without 10 nM EGF and 10 µM ISO for 6 h. In experiments using U0126 or FOR, the cultures were pretreated for 60 min before being wounded.

Images were collected using a COHU charge-coupled device camera fitted to a Zeiss Axiovert 135 microscope equipped with a Plan-NEOFLUAR x2.5/0.075 numerical aperture objective lens. Twelve images were collected immediately after wounding (original wound) and at the end of 6 h (final wound). The areas of the original and final wounds were measured manually using Scion Image (Scion, Frederick, MD), and the percent wound closure was calculated. To control for inconsistencies in wound sizes, only experimental sets in which the original wound areas varied by ≤10% were used.

Cell extracts, SDS-PAGE, and Western blots. Whole cell extracts were prepared from 16HBE cultures treated with 10 nM EGF or 10 µM ISO over a 24-h time course using an SDS-lysis buffer as previously described (10). The protein concentrations of the extracts were determined using the BCA Protein Assay (Pierce, Rockford, IL), and the extracts were loaded onto 12% SDS-polyacrylamide gels on the basis of equal protein. Proteins were separated by SDS-PAGE (17) and transferred to nitrocellulose (28). Blots were blocked with 5% milk in TBST (150 mM NaCl, 10 mM Tris, pH 7.5, 0.05% Tween 20) for 1 h followed by incubation with primary antibodies for 1 h. The blots were washed in TBST, incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, washed, and developed. Detection was performed using the Super Signal West Pico chemiluminescence kit (Pierce) followed by exposure to Hyperfilm ECL (Amersham Biosciences, Piscataway, NJ).

For densitometry analysis, films were scanned using a flatbed scanner, and images were analyzed using Scion Image (Scion). Intensities were determined for all bands and for the corresponding local background. Final intensities were obtained by background subtraction.

Clones. The EGFR-green fluorescent protein (GFP) expression plasmid (2) was generously provided by Dr. A. Sorlein (Univ. of Colorado Health Science Center, Boulder, CO). To create a human beta2-AR gene with no stop codon, human genomic DNA (Clontech, Palo Alto, CA) was PCR amplified with the oligonucleotides 5'-CCGGAATTCCAGTGCGCTTACCTGCCAGA-3' and 5'-CAGCAGTGAGTCATTTGTACTACAATTC-3'. The PCR product was digested with EcoRI, and the 1274-bp fragment was purified from an agarose gel slice using the gel extraction kit from Qiagen (Valencia, CA). The EcoRI-digested PCR product was ligated into the multiple cloning site of pEYFP-N1 (Clontech). For the ligation of this PCR product, the pEYFP-N1 was digested with BamHI, treated with Klenow enzyme, and then digested with EcoRI. The insert was sequenced and found to have the R16G polymorphism. A 0.24-kb fragment that encodes the R16G mutation was removed with HindIII and BstEII and replaced with a 0.24-kb HindIII-BstEII fragment excised from an adeno-associated virus-cloned beta2-AR gene known to have an arginine at position 16 (11).

Transfections. 16HBE cells were transfected with EGFR-GFP or beta2-AR-yellow fluorescent protein (YFP) using the calcium phosphate method (3). Briefly, 15 µg of plasmid were mixed with calcium phosphate and HEPES-buffered saline (pH 7.05) and incubated for 60 min. Transfection mix (400 µl) was then added to 16HBE cells grown in 6-cm dishes containing 3 ml of MEM plus 10% FBS and incubated overnight at 37°C. The media were then aspirated, and the cultures were washed with HBS and incubated in MEM plus 10% FBS.

To create 16HBE cell lines that stably express beta2-AR-YFP, transfected cells were selected by growth in MEM plus 10% FBS containing 400 µg/ml G418 and cloned by limiting dilution. Stable cell lines were maintained in MEM plus 10% FBS containing 200 µg/ml G418.

Fluorescence time-lapse microscopy. 16HBE cells were grown in {Delta}T glass-bottom culture dishes (Bioptechs, Butler, PA) and maintained at 37°C using a Bioptechs {Delta}TC4 controller. Media were equilibrated with 5% CO2-95% air and warmed to 37°C before use. 16HBE cultures transiently transfected with EGFR-GFP were overlaid with mineral oil to prevent evaporation and were then treated with 10 nM EGF or 10 µM ISO. Images of cells expressing EGFR-GFP were collected every 2 min for 3 h.

Statistical analysis. SigmaStat for Windows (version 3.0; SPSS, Rochester, MN) was used to analyze the means from replicate experiments. One-way analysis of variance and post hoc testing of all pairwise comparisons using the Holm-Sidak method were used with P < 0.05 as the criterion for significance. Power calculations were also performed as part of the statistical analysis. Experiments were conducted in triplicate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ISO attenuates EGF-induced scrape wound closure in 16HBE cells. To determine the effect of ISO on wound healing, we used an in vitro scrape wound assay with 16HBE human airway epithelial cells. Serum-starved confluent cultures were scrape wounded, and images were collected of the original wound and after 6 h of culture (Fig. 1A) to determine percent wound closure (Fig. 1B).


Figure 1
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  		Fig. 1. Isoproterenol (ISO) attenuates wound healing in 16HBE cells. A: confluent serum-starved 16HBE cells were scrape wounded and cultured for 6 h in FBS, FBS+ISO, FBS+EGF, or FBS+EGF+ISO. Twelve images were collected immediately after wounding (original wound) and again after 6 h (A). B: total wound areas were measured using Scion Image, and the % wound closure was calculated and graphed. Means ± SD from 4 independent experiments were plotted. *P = 0.014, **P = 0.041.

 
Consistent with previous reports (16, 24), the addition of EGF significantly (P = 0.014) stimulated wound healing over FBS alone. Wounds treated with FBS alone closed 67% (SD 9), whereas wounds treated with FBS + EGF closed 90% (SD 8). ISO had an inhibitory effect on wound healing, as wounds treated with FBS + ISO only closed 56% (SD 13). Although these differences were not statistically significant (P = 0.184), it should be noted that in each individual experiment, the wounds treated with FBS + ISO did not close as much as those treated with FBS alone. However, ISO did significantly (P = 0.041) attenuate the enhancement of closure observed using EGF: wounds treated with FBS + EGF + ISO closed 72% (SD 15) compared with FBS + EGF, which closed 90% (SD 8). These findings demonstrate that ISO attenuates EGF-stimulated wound healing in 16HBE cells.

ISO does not transactivate EGFRs. After activation, the EGFR is internalized by endocytosis and downregulated by degradation. To determine whether ISO is capable of transactivating the EGFR and initiating its internalization and downregulation, we used a combination of time-lapse microscopy and Western blot analysis.

Upon EGF binding, EGFRs undergo autophosphorylation. To determine whether EGFRs are autophosphorylated in response to ISO, we treated 16HBE cells with EGF or ISO for 6 h and made extracts at various time points throughout the treatment period. The extracts were then analyzed by Western blot using antibodies to phospho-tyrosine (Fig. 2A). In EGF-treated cultures, EGFR phospho-tyrosine reactivity increased between 0 and 30 min of treatment and declined thereafter. By contrast, the ISO-treated cultures showed no such increase in EGFR phospho-tyrosine reactivity.


Figure 2
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  		Fig. 2. ISO does not transactivate the EGF receptor (EGFR). A: cells were treated with EGF or ISO, and extracts were prepared at 0 min, 5 min, 30 min, 2 h, and 6 h. Extracts were analyzed by Western blot using antibodies to phospho-tyrosine to measure phosphorylated EGFR (pEGFR). Because of the intense phospho-tyrosine signal, the blot shown for the EGF-treated extracts was from a shorter exposure than that for the ISO-treated extracts. B–E: 16HBE cells were transiently transfected with EGFR-GFP and treated with EGF (B and C) or ISO (D and E). Expressing cells were followed by time-lapse microscopy from time 0 (B and D) through 60 min (C and E) to monitor EGFR internalization. F: the same extracts in A were analyzed by Western blot using antibodies to EGFR to assay for receptor downregulation.

 
To test for internalization of the EGFR, 16HBE cells were transiently transfected with an EGFR-GFP expression plasmid (2). The cultures were treated with EGF (Fig. 2, B and C) or ISO (Fig. 2, D and E), and the green fluorescence in EGFR-GFP-expressing cells was followed using time-lapse microscopy. Before treatment, EGFR-GFP signal was diffusely distributed throughout the periphery of the cell (plasma membrane), with some signal in the interior (Fig. 2, B and D). EGF treatment initiated the internalization of EGFR-GFP as evidenced by the formation of bright punctuate spots throughout the cell within 30–60 min of treatment (Fig. 2C). By contrast, the EGFR-GFP signal remained diffuse, and no internalization could be detected after ISO treatment (Fig. 2E).

After internalization, the EGFR is downregulated by lysosomal degradation. To determine whether ISO initiated the downregulation of the EGFR, we analyzed the same extracts by Western blots using an antibody to the EGFR (Fig. 2F). If the receptor was downregulated, one would expect to see a reduction in the EGFR signal over time. As expected, EGF treatment initiated EGFR downregulation as the signal was significantly reduced by 2 h posttreatment. By contrast, EGFR levels remained constant throughout the entire 6-h treatment with ISO.

This set of experiments shows that ISO does not initiate the autophosphorylation, internalization, or downregulation of the EGFR. Therefore, ISO does not directly affect the activation of the EGFR and must exert its effects to attenuate EGF-stimulated wound healing downstream of, or in parallel to, EGFR signaling.

A MEK inhibitor blocks EGF-induced scrape wound closure. In part, the EGFR exerts its control through the MAPK signaling pathways. To determine the role of the MAPK pathway in wound healing in 16HBE cells, we utilized the MEK inhibitor U0126 in the in vitro scrape wound assay. Confluent cultures were scrape wounded and treated with FBS, FBS + U0126 (10 µM), FBS + EGF, FBS + EGF + U0126, FBS + EGF + ISO, or FBS + EGF + ISO + U0126 for 6 h. The wounds were then measured and analyzed for percent wound closure (Fig. 3).


Figure 3
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  		Fig. 3. The MEK inhibitor U0126 attenuates wound healing in 16HBE cells. Confluent cultures were scrape wounded and treated with FBS, FBS+U0126 (10 µM), FBS+EGF, FBS+EGF+U0126, FBS+EGF+ISO, or FBS+EGF+ISO+U0126 for 6 h. Cultures treated with U0126 were pretreated for 60 min before wounding. The total wound areas were measured, and percent wound closure was calculated. Means ± SD from 3 independent experiments were plotted. *P ≤ 0.001, **P = 0.003.

 
When added to FBS alone, U0126 had no significant effect on wound closure (P = 0.530), as wounds treated with FBS closed 53% (SD 4) and wounds treated with FBS + U0126 closed 50% (SD 9). However, addition of U0126 to media supplemented with EGF significantly reduced wound healing (P < 0.001), as wounds treated with FBS + EGF closed 77% (SD 5) and wounds treated with FBS + EGF + U0126 closed 48% (SD 1). Consistent with previous experiments, ISO also significantly (P = 0.001) attenuated EGF-enhanced wound healing, as wounds treated with FBS + EGF + ISO closed 57% (SD 9). Additionally, treatment of wounds with FBS + EGF + ISO + U0126 further inhibited wound healing (P = 0.003) compared with the results obtained with FBS + EGF + ISO and reduced wound closure to 40% (SD 3). Therefore, MEK activity is important for EGF-stimulated wound healing, but the additive effect of ISO and U0126 suggests that ISO attenuates wound healing through at least one other pathway that is not the ERK pathway.

EGF and ISO activate ERK in 16HBE cells. To define the potential effect of ISO on the MAPK signaling pathway, we analyzed the EGF- and ISO-treated extracts for MAPK activation using a phospho-p44/42 MAP kinase (ERK1/2) antibody to measure the phosphorylation of ERK (the substrate for MEK) in Western blot assays. The Western blot signals for phospho-ERK1/2 (Fig. 4A) were analyzed by densitometry and normalized to the signal intensity for ERK2 (Fig. 4A). The resulting intensities were plotted vs. time (Fig. 4B). EGF treatment initiated a rise in MAPK phosphorylation that peaked at 30 min posttreatment and then gradually decreased. ISO treatment also resulted in MAPK phosphorylation, but it peaked at 5 min posttreatment with an intensity two to three times less than that observed with EGF treatment and decreased rapidly thereafter.


Figure 4
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  		Fig. 4. ISO initiates a rapid, low-level activation of MAPK. A: extracts were prepared from cultures treated with ISO (top 2 panels) or EGF (bottom 2 panels) for 0 min, 5 min, 30 min, 2 h, and 6 h. Extracts were analyzed by Western blot using antibodies to phospho-p44/42 MAPK (pERK1/2) and ERK2. B: Western blots were scanned, and band intensities were measured using Scion Image. Normalized intensities (pERK/ERK) were plotted against time in minutes.

 
ISO does not alter MAPK activation by EGF. Having shown that MAPK activity is involved in timely wound healing and that the time course of MAPK activation by ISO differs from the activation by EGF, we sought to determine how ISO would affect the activation of EGFR and MAPK in the presence of EGF. Therefore, cultures were treated with FBS, FBS + EGF, FBS + ISO, or FBS + EGF + ISO, and extracts were prepared 5 and 30 min after treatment. Controls were cultured in serum-free medium. The extracts were analyzed by Western blot using antibodies to phospho-tyrosine (PY20) to judge EGFR phosphorylation (Fig. 5A) and phospho-p44/42 MAP kinase to judge ERK phosphorylation (Fig. 5B). The blots were scanned, and band intensities were measured using image analysis.


Figure 5
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  		Fig. 5. ISO does not alter the EGF-stimulated activation of EGFR or MAPK. Serum-starved cultures were treated with serum-free media (SF), FBS, FBS+EGF, FBS+ISO, or FBS+EGF+ISO, and extracts were prepared at 5 and 30 min. Extracts were analyzed by Western blot using antibodies to phospho-tyrosine (phospho-EGFR; A) and phospho-p44/42 MAPK (phospho-MAPK; B).

 
As seen in Fig. 5A, FBS addition initiates an approximate sevenfold increase in band intensity for phospho-EGFR at 5 min that falls to four times the serum-free level by 30 min. However, when EGF was added, a 215-fold increase in EGFR phosphorylation was measured at 5 min, and this had fallen to 150-fold by 30 min. Conversely, the intensities of phospho-EGFR after 5 and 30 min of ISO treatment were ~20 and 4 times the levels for serum-free treatment, respectively, which is similar to the effects of FBS alone. Additionally, treatment with FBS + EGF + ISO resulted in intensities that were ~180 and 130 times the serum-free levels at 5 and 30 min, respectively.

As observed in Fig. 5B, treatment of cells with FBS resulted in a 3-fold increase in MAPK phosphorylation by 30 min, whereas treatment with FBS + EGF induced a 10-fold increase in the level of phosphorylation by 30 min. However, treatment with FBS + ISO resulted in phosphorylation of MAPK that peaked at 5 min and was only five times the level of the serum-free control. Interestingly, when cells were treated with EGF and ISO in combination (FBS+EGF+ISO), the phosphorylation of MAPK closely resembled that of the FBS plus EGF treatment with a ~7-fold increase at 5 min and a maximal phosphorylation that was ~10-fold over the serum-free control occurring at 30 min. Since the time course of MAPK activation in the presence of FBS + EGF + ISO essentially mirrors the response observed with FBS + EGF alone, this indicates that ISO does not have an appreciable effect on the kinetics or intensity of EGF-stimulated activation of the ERK/MAPK pathway. Therefore, alteration of ERK/MAPK signaling is likely not the mechanism through which ISO attenuates wound healing.

Effects of FOR on EGF-induced scrape wound closure. cAMP is an important downstream messenger for beta2-ARs. To determine whether elevated cAMP levels were involved in the attenuation of wound healing, we treated wounded cultures with FOR, an activator of adenylyl cyclase. Confluent cultures were scrape wounded and treated with FBS, FBS + FOR (10 µM), FBS + EGF, FBS + EGF + FOR, FBS + EGF + ISO, or FBS + EGF + ISO + FOR for 6 h and were analyzed for wound healing as before (Fig. 6). Again, ISO significantly (P = 0.008) attenuated the EGF-stimulated wound healing response. Wounds treated with FBS + EGF closed 80% (SD 5), whereas wounds treated with FBS + EGF + ISO closed 63% (SD 11). Treatment with FOR significantly (P = 0.005) reduced wound closure as wounds closed 36% (SD 5) compared with the 55% (SD 6) closure in FBS alone. Moreover, FOR also inhibited EGF-stimulated wound closure (P ≤ 0.001), and this inhibition was greater than observed with ISO, as wounds treated with FBS + EGF + FOR closed 52% (SD 6) compared with the 80% (SD 5) in FBS + EGF. Finally, wounds treated with FBS + EGF + ISO + FOR only closed 24% (SD 7), and this was significantly less (P ≤ 0.001) than with either FBS + EGF + ISO or FBS + EGF + FOR. These results indicate that elevated cAMP levels have a negative impact on wound healing and could be involved in the mechanism by which ISO attenuates wound healing in 16HBE cells. However, the cumulative inhibitory effect of ISO and FOR suggests the involvement of at least one other pathway.


Figure 6
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  		Fig. 6. Forskolin (FOR) attenuates wound healing in 16HBE cells. Confluent serum-starved cells were scrape wounded and cultured for 6 h in FBS, FBS+FOR (10 µM), FBS+EGF, FBS+EGF+FOR, FBS+EGF+ISO, or FBS+EGF+ISO+FOR. Cultures treated with FOR were pretreated for 60 min before wounding. Images were collected and analyzed as before. Means ± SD from 3 independent experiments were plotted. *P ≤ 0.001, **P = 0.005, ***P = 0.008.

 
Effects of ISO in HBE cell lines stably expressing beta2-AR-YFP. It has been shown that overexpression of beta2-AR can increase the production of cAMP upon addition of agonist (8, 11). Therefore, based on the results with FOR, we hypothesized that overexpression of beta2-AR in 16HBE cells would enhance the inhibitory effects of ISO on EGF-stimulated wound healing. To test this hypothesis, confluent cultures of 16HBE cells stably expressing beta2-AR-YFP were prepared and serum starved for 36–48 h and scrape wounded as previously described (Fig. 7). beta2-AR-YFP-expressing 16HBE cells treated with FBS closed 68% (SD 4) as opposed to 42% (SD 10) when treated with FBS + ISO. By contrast to the results in untransfected cells where the attenuation of serum-induced wound closure was not statistically significant, in beta2-AR-YFP-expressing 16HBE cells, ISO did significantly attenuate serum-induced wound closure (P < 0.001). A significant difference (P = 0.013) was also observed when comparing cultures treated with FBS + EGF and FBS + EGF + ISO with wound closures of 85% (SD 5) and 58% (SD 10), respectively. Therefore, overexpression of beta2-AR attenuates wound healing to an even greater extent than is observed in cells expressing only endogenous beta2-AR. This further confirms that the attenuation of EGF-stimulated wound closure by ISO is a consequence of beta2-AR activation and involves the accumulation of cAMP.


Figure 7
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  		Fig. 7. Overexpression of beta2-adrenergic receptor (AR) accentuates the effect of ISO. Confluent serum-starved cells stably expressing beta2-AR-yellow fluorescent protein were scrape wounded and cultured for 6 h in FBS, FBS+ISO, FBS+EGF, or FBS+EGF+ISO. Images were collected and analyzed as previously described. Means ± SD from 3 independent experiments were plotted. *P < 0.001, **P = 0.007, ***P = 0.013.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Asthma is an inflammatory disease that presents clinically as a reversible airway obstruction. Airway epithelial injury is expected to facilitate the initiation of the smooth muscle contractile responses underlying these obstructions due to enhanced exposure of the underlying tissue to environmental irritants. Altered wound healing and the airway remodeling that occurs with chronic inflammation is another important aspect of asthma that can contribute to less reversible airway obstruction with aging. A significant class of medications used for the treatment of asthma is the beta-adrenergic agonist. These are used as "quick relievers" to bring about the relaxation of airway smooth muscle, as well as "long-term controllers" for chronic treatment, typically in combination with steroids. Although beta-agonists are well documented bronchodilators, their effects on wound healing are not defined.

We chose to use human bronchial epithelial cells (16HBE) in this study because they maintain many characteristics of airway epithelium. Under the appropriate conditions, 16HBE cells in culture demonstrate tight junctions and cilia, the monolayers generate transepithelial resistance and polarize, and the cells retain beta2-adrenergic stimulation of cAMP-dependent chloride ion transport (5, 14, 15, 31, 33). Therefore, 16HBE cells provide a valuable in vitro model for determining the effects of beta-agonist on airway epithelial cell wound healing.

We have shown for the first time that a beta-agonist, ISO, is capable of attenuating the EGF-stimulated wound healing in 16HBE cell cultures. ISO has previously been reported to reduce migration and wound healing in primary human keratinocytes (4, 25). However, this effect was shown to be independent of cAMP (4) and to occur through the serine-threonine protein phosphatase 2A inhibition of ERK (25).

We demonstrate that either treatment of cells with FOR or overexpression of beta2-AR results in an even greater attenuation of EGF-stimulated wound healing than observed in ISO-treated cells. Both of these results indicate the involvement of adenylyl cyclase activity and elevated cAMP levels in the mechanism for this attenuation. Conflicting results have been published on the effects of cAMP-dependent protein kinase (PKA) activity on cell migration. PKA has been implicated in inhibition of cell migration in NIH/3T3 cells, and this was shown to be due to inhibition of a factor other than ERK (6). Conversely, PKA was shown to have an activating effect on migration in primary bovine bronchial cells (27). Additionally, our data show an additive effect on the attenuation of wound closure when ISO and FOR are combined. This indicates the involvement of one or more pathways in addition to an elevation of intracellular cAMP that are activated by ISO.

Our data show a clear involvement of ERK activity in EGF-stimulated wound closure since the MEK inhibitor (U0126) significantly inhibited wound closure in the presence of EGF. Although inhibition of ERK activity attenuates wound healing, it does not appear to be the mechanism by which ISO exerts its effect. We show that the time course of ERK1/2 phosphorylation upon ISO stimulation differs from that of EGF stimulation. However, when cells are treated with ISO and EGF in combination, the time course of ERK1/2 phosphorylation is similar to that of EGF treatment alone. Therefore, it is unlikely that ISO attenuates wound healing through alteration of ERK signaling. Alternatively, ISO could influence components of parallel signaling pathways, such as the JNK and/or the p38 pathways. This is supported by our results, which show a significant attenuation of wound closure in cells treated with FBS + EGF + ISO + U0126 in addition to that observed with FBS + EGF + ISO treatment.

It has recently been shown that wounding activates the MAPK pathways ERK, JNK, and p38 in primary human keratinocytes (29) and in 16HBE monolayers (32). Inhibition of JNK and p38 kinases in 16HBE cells results in attenuated cell migration and wound healing (32). Evidence for JNK and p38 involvement comes from other sources as well. JNK activation was shown to contribute to cell migration in a gene expression-independent manner in MDCK cells (1), and p38 has been shown to be activated by Src kinase in response to EGF leading to cell migration in young adult mouse colon cells (7). Interestingly, elevating the intracellular levels of cAMP by FOR treatment has been shown to inhibit the EGF-stimulated activation of both p38 and JNK, but not ERK, in human keratinocytes (22).

Based on the present results, we propose that ISO attenuates wound healing by altering the activity of multiple signaling pathways. An initial event in this attenuation is the activation of adenylyl cyclase and an increase in intracellular levels of cAMP. These elevated cAMP levels could then interfere with one or both of the remaining MAPK pathways (JNK and p38), thus attenuating wound healing. Which of these pathways are inhibited and the molecular interactions that occur remain to be discerned. Our studies also define a problem of potential therapeutic relevance whereby medications (beta-agonists) that are frequently used to relieve acute bronchospasm may attenuate the wound closure response, further contributing to airway remodeling in the long term. Future studies to determine the effects of adjunctive therapies, such as inhaled corticosteroids, on this beta-agonist-induced attenuation of wound healing are also necessary.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Allergy and Infectious Diseases Grant K23-AI-01818 (S. M. Jones), the Arkansas Biosciences Institute (S. M. Jones/R. C. Kurten), the University of Arkansas for Medical Sciences College of Medicine Dean's Research Development Fund (B. J. Schnackenberg/S. M. Jones), and the Marion B. Lyon New Scientist Development Award from the Arkansas Children's Hospital Research Institute (B. J. Schnackenberg).


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. J. Schnackenberg, Dept. of Pediatrics, Univ. of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, 1120 Marshall St., Slot 512-13, Little Rock, AR 72202 (e-mail: SchnackenbergBradley{at}uams.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
 REFERENCES
 

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