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Eur Respir J 2002; 20:353-358
Copyright ©ERS Journals Ltd 2002

ß-adrenergic receptor-mediated growth of human airway epithelial cell lines

K. Nishimura, J. Tamaoki, K. Isono, K. Aoshiba and A. Nagai

First Dept of Medicine, Tokyo Women's Medical University School of Medicine, Tokyo, Japan

CORRESPONDENCE: A. Nagai, First Dept of Medicine, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-Cho, Shinjuku, Tokyo, 162-8666, Japan. Fax: 81 353795457. E-mail: anagai@chi.twmu.ac.jp

Keywords: airway epithelium, cyclic adenosine monophosphate, deoxyribonucleic acid synthesis, mitogen-activated protein kinase, remodelling

Received: August 17, 2001
Accepted February 1, 2002

This study was supported in part by Grant No. 06670243 from the Ministry of Education, Science and Culture, Japan.


   Abstract
TOP
 Abstract
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Abnormal growth of airway epithelium and the resultant thickening of airway walls may produce narrowing of airway calibre, thereby contributing to deterioration of bronchoconstriction in chronic obstructive pulmonary disease (COPD). ß2-adrenergic agonists have been widely used for the treatment of COPD, but their effects on the growth of airway epithelial cells is unknown.

Growth of three human airway epithelial cell lines was studied in vitro. Exposure to salbutamol in serum-free medium increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide reduction and intracellular deoxyribonucleic acid (DNA) contents in 16-human bronchial epithelium (16-HBE) cells and NCI-H292 cells, but not in A549 cells. The growth-promoting effect of salbutamol in 16-HBE cells was equipotent to 10% foetal bovine serum and was inhibited by propranolol and a cyclic adenosine monophosphate (cAMP) antagonist, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). Likewise, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) caused cell growth and DNA synthesis. Western blot analysis showed that salbutamol, forskolin, and 8-Br-cAMP each induced expression of the phosphorylated form of mitogen-activated protein (MAP) kinase, and that the salbutamol-induced phosphorylation was inhibited by propranolol, Rp-cAMPS, and the MAP kinase-kinase inhibitor PD98059.

These results suggest that in certain airway epithelial cell lines stimulation of ß2-adrenergic receptors and the consequent production of cyclic adenosine monophosphate may upregulate cell growth, probably through activation of the mitogen-activated protein kinase cascade.

Mitogen-activated protein (MAP) is a family of 42 and 44 kDa cytosolic serine-threonine kinases 1, 2, and has been identified as an important regulatory component in the growth and proliferative responses of many cell types 3. There is increasing evidence that MAP kinase can be activated by a variety of signals, including growth factors 4, phorbol esters 5 and Ca2+ 6. In addition, stimulation of cell surface receptors, linked to Gi and Gq proteins, has been shown to potently activate MAP kinase 7, 8, but the effect of Gs protein-coupled receptors on the enzyme activity is poorly understood.

ß-adrenergic receptors are Gs protein-coupled receptors and stimulate the synthesis of intracellular cyclic adenosine monophosphate (cAMP). Inhaled ß2-adrenoceptor agonists have been widely used in the treatment of chronic obstructive pulmonary disease and asthma, and it is likely that airway epithelial cells are frequently exposed to these drugs in high concentrations. It has recently been demonstrated that cAMP stimulates proliferation of auditory receptor epithelial cells 9. However, the role of the cAMP-dependent signalling pathway in the proliferative responses of airway epithelial cells is unknown. Therefore, the current authors studied three human airway epithelial cell lines in vitro, in order to determine whether ß2-adrenergic agonists affect the growth of airway epithelial cells and to assess a possible involvement of cAMP-mediated stimulation of the MAP kinase cascade.


   Materials and methods
TOP
 Abstract
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Cell growth
A human bronchial epithelium (HBE) cell line, 16-HBE, was obtained from the Cardiovascular Research Institute (University of California, San Francisco, CA, USA). A human airway mucoepidermoid carcinoma cell line, NCI-H292, and an epithelial cell line derived from lung carcinoma, A549, were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were incubated in Ham's F12 medium, supplemented with 10% foetal bovine serum (FBS), 100 U·mL–1 penicillin, 100 µg·mL–1 streptomycin and 100 µg·mL–1 amphotericin B in a carbon dioxide (CO2) incubator (5% CO2:95% air) at 37°C. After growth to confluence, the cells were detached with a 0.25% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA) solution for passage. In the preliminary experiment, the cells prepared in this manner maintained the same morphology for ≥15 passages.

The cells were plated in 100 µL of Ham's F12 medium containing 10% FBS at a density of 1x104 cells per flat-bottomed well in 96-well microtitre plates, and grown to subconfluence. After washing the cells three times with phosphate-buffered saline (PBS), the cell growth was arrested by incubation in Ham's F12 medium without FBS for 72 h. The medium was then replaced with serum-free Ham's F12 medium to which salbutamol (1x10–7 M) (Sigma Chemical Co., St Louis, MO, USA) was added, and the cells were further cultured. In the control experiment, cell culture was performed in the serum-free medium alone. Growth of epithelial cells was assessed at indicated times after exposure to salbutamol, using the MTT dye technique, which relies on the specific metabolic reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) (Sigma Chemical Co.) by living cells 10. MTT (100 µL of 10 µg·mL–1 in PBS) was added to each well, and the cells were incubated for 4 h at 37°C. The medium and MTT were then removed from the wells, and 25 µL of Sorenson's glycine buffer (0.1 M glycine and 0.1 M NaCl equilibrated to pH of 10.5 with 0.1 N NaOH) and 200 µL dimethylsulfoxide were consecutively added. The optical density was determined using an immunosorbent assay plate reader (Model 450; Bio-Rad Laboratories Ltd, Watford, UK) against a reagent blank (i.e. no cells) at a test wavelength of 570 nm and a reference of 630 nm.

To confirm that the effect of salbutamol on cell proliferation is mediated by ß-adrenergic receptors and the consequent synthesis of cAMP, 16-HBE cells were cultured in serum-free medium containing both salbutamol (1x10–7 M) and the ß-adrenergic receptor antagonist propranolol (1x10–5 M) (Sigma Chemical Co.) or the cAMP antagonist Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS) (1x10–4 M), and the MTT assay was performed (BIOLOG Life Science Institute, Bremen, Germany). To assess a contribution of the cAMP-dependent signal transduction pathway, the effects on cell growth of forskolin (1x10–7 M) (Sigma Chemical Co.), a stimulator of adenylyl cyclase and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) (Sigma Chemical Co.) (1x10–7 M), a stable analogue of cAMP, were studied in a similar manner. In evaluating the potency of these drugs, the response was compared to that induced by 10% FBS at 48 h after initiation of culture.

Deoxyribonucleic acid synthesis
The deoxyribonucleic acid (DNA) contents in cultured epithelial cells were assayed by the method of Rago et al. 11. Briefly, the cells were cultured in serum-free Ham's F12 medium containing salbutamol (1x10–7 M). Then, 100 µL distilled water was added to each well, and the cells were incubated for 1 h. To destruct cell membranes, the cells were frozen at –80°C and thawed at room temperature. A stock solution of Hoechst 33258 (200 µg·mL–1 in distilled water) (Sigma Chemical Co.) was diluted 10-fold before use with TNE buffer (10 mM Tris, 2 M NaCl, pH 7.4, 1 mM EDTA), and 100 µL of diluted Hoechst 33258 was added to each well. Fluorescence was measured by a spectrofluorimeter (Ultrospec Plus; Phamacia LKB Biochrom Ltd, Cambridge, UK) at excitation and emission lengths of 350 nm and 460 nm, respectively. In addition, the DNA contents were likewise determined after exposure to forskolin (1x10–7 M), 8-Br-cAMP (1x10–7 M) and salbutamol (1x10–7 M) plus propranolol (1x10–5 M).

To determine the role of MAP kinase cascade and cAMP in the salbutamol-induced DNA synthesis, 16-HBE cells were cultured in serum-free medium containing salbutamol (1x10–7 M) alone or in combination with various concentrations of PD98059 (1, 30, and 100 µM) (New England Biolabs, Inc., Beverly, MA, USA), a specific inhibitor of MAP kinase-kinase 12, or with Rp-cAMPS (1x10–4 M), and the DNA contents were measured 48 h later. In this experiment, to block MAP kinase cascade, a MAP kinase-kinase inhibitor was used because a specific MAP kinase inhibitor was not available.

Western blotting
Cell lysates were solubilised in radioimmunoprecipitation (RIPA) buffer (0.15 M NaCl, 50 mM Tris-Cl pH 7.4, 0.5% Nonidet P40 (NP40), and 0.1% sodium dodecylsulphate (SDS)) containing 10 µg·mL–1 leupeptin, 1 mM phenylmethylsulfonylfluoride, 10 µg·mL–1 aprotinin, and 1 mM sodium vanadate, and then fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and probed with antibodies (1:1,000) against MAP kinase and phosphorylated MAP kinase (New England Biolabs, Inc.). Primary antibody (1:2,500) was visualised using enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL, USA).

Statistical analysis
All values are expressed as mean±sem. One-way analysis of variance and Newman Keuls multiple comparison test were used to determine statistically significant differences between groups, and a p<0.05 was considered significant.


   Results
TOP
 Abstract
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
Incubation with salbutamol (1x10–7 M) for 48 h in serum-free medium caused increases in both MTT reduction and intracellular DNA content in 16-HBE cells and NCI-H292 cells, but not in A549 cells (fig. 1Go). Therefore, the growth-promoting effect of salbutamol is different among epithelial cell lines, and the subsequent experiments were conducted using 16-HBE cells.



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  		Fig. 1.— a) Effect of salbutamol on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) reduction and b) intracellular deoxyribonucleic acid (DNA) contents in 16-human bronchial epithelium (16-HBE) cells, NCI-H292 cells and A549 cells. Data are expressed as optical density determined by MTT assay and fluor units using Hoechst 33258, respectively. After a 72-h incubation in serum-free medium to arrest cell growth, salbutamol (1x10–7 M, ) was added and the cells were cultured for 48 h. In the control experiment, no drug was added ({square}). Data are shown as mean±sem; n=5 for 16-HBE cells and n=6 for others. **: p<0.01, significantly different from control values.

 
As demonstrated in figure 2Go, the 16-HBE cells that had been incubated for 72 h in Ham's F12 medium, without FBS, did not grow during the next 48 h in the serum-free medium. However, addition of salbutamol (1x10–7 M) to the serum-free medium stimulated the growth of epithelial cells, the values of MTT reduction compared with controls (no salbutamol) being 197±20% (p<0.01, n=10) at 24 h and 233±27% (p<0.01, n=10) at 48 h after initiation of the culture. The potency of the effect of salbutamol on cell growth was similar to that of 10% FBS. When the cells were cultured in the serum-free medium containing salbutamol (1x10–7 M) plus propranolol (1x10–5 M), the response of growth was completely abolished. To determine the role of cAMP in epithelial cell proliferation, the effects of cAMP-generating agents were determined.



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  		Fig. 2.— Time course of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) reduction of 16-human bronchial epithelium (16-HBE) cells in response to salbutamol (1x10–7 M, •), salbutamol (1x10–7 M) plus propranolol (1x10–5 M, {blacksquare}), and 10% foetal bovine serum ({blacktriangleup}). After a 72-h incubation in serum-free medium to arrest cell growth, each drug was added and the cells were cultured for up to 48 h. In the control experiment, no drug was added ({circ}). Data are shown as mean±sem; n=10 for each point. **: p<0.01, significantly different from control values; #: p<0.01, significantly different from corresponding values for salbutamol alone.

 
As shown in figure 3Go, incubation for 48 h with forskolin (1x10–7 M) or 8-Br-cAMP (1x10–7 M) in the serum-free medium increased MTT reduction to the same degree as with salbutamol (1x10–7 M). The salbutamol-induced increase in MTT reduction was reduced by 86±10% (p<0.01, n=8) by co-incubation with Rp-cAMPS (1x10–4 M).



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  		Fig. 3.— Effects of salbutamol (SAL), forskolin (FSK), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) reduction of 16-human bronchial epithelium (16-HBE) cells, and the effect of Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS) (1x10–4 M) on salbutamol-induced MTT reduction. The response was assessed 48 h after incubation in serum-free medium alone (control) or containing each agonist at 1x10–7 M. Data are shown as mean±sem; n=8 for each column. **: p<0.01, significantly different from control values; #: p<0.01, significantly different from values for salbutamol alone.

 
The DNA content of the 16-HBE cells was not changed in the serum-free medium during the 48-h culture period, but increased in a time-dependent manner after the addition of salbutamol (1x10–7 M) to the medium (fig. 4Go). As with the finding in the MTT assay, the effect of salbutamol on DNA synthesis was as potent as forskolin, 8-Br-cAMP and 10% FBS, and was abolished in the presence of propranolol.



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  		Fig. 4.— Time course of deoxyribonucleic acid (DNA) content in 16-human bronchial epithelium (16-HBE) cells in response to salbutamol (1x10–7 M, •), salbutamol (1x10–7 M) plus propranolol (1x10–5 M, {blacksquare}), forskolin (1x10–7 M, {square}), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) (1x10–7 M, {triangleup}), and 10% foetal bovine serum ({blacktriangleup}). After a 72-h incubation in serum-free medium to arrest cell growth, each drug was added and the cells were cultured for up to 48 h. In the control experiment, no drug was added ({circ}). Data are shown as mean±sem; n=10 for each point. *: p<0.05, **: p<0.01, significantly different from control values; #: p<0.01, significantly different from corresponding values for salbutamol alone.

 
To assess the involvement of MAP kinase cascade and cAMP in salbutamol-induced DNA synthesis, the effects of the MAP kinase-kinase inhibitor PD98059 and Rp-cAMPS were examined. As shown in figure 5Go, the increase in DNA content induced by salbutamol was inhibited by PD98059 in a concentration-dependent manner, and by Rp-cAMPS.



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  		Fig. 5.— Effects of PD98059 and Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS) on salbutamol-induced deoxyribonucleic acid (DNA) synthesis in 16-human bronchial epithelium (16-HBE) cells. DNA content was determined 48 h after incubation in serum-free medium alone (control) or that containing salbutamol (SAL, 1x10–7 M) in the absence and presence of various concentrations of PD98059 or 1x10–4 M Rp-cAMPS. Data are shown as mean±sem; n=9 for each point. *: p<0.05, #: p<0.01, significantly different from values for salbutamol alone; **: p<0.01, significantly different from control values.

 
Western blot analysis showed that incubation with salbutamol (1x10–7 M) caused phosphorylation of MAP kinase in 16-HBE cells and NCI-H292 cells, but it had little effect in A549 cells (fig. 6Go).



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  		Fig. 6.— Western blotting of phosphorylated mitogen-activated protein (MAP) kinase (p-MAPK) in 16-human bronchial epithelium (16-HBE) cells, NCI-H292 cells, and A549 cells after a 5-min exposure to serum-free medium alone (C) or that containing salbutamol (SAL, 1x10–7 M).

 
In 16-HBE cells, MAP kinase activation was induced by salbutamol, forskolin, and 8-Br-cAMP, the salbutamol-induced expression of phosphorylated MAP kinase was greatly inhibited by propranolol (fig. 7Go). Moreover, the phosphorylation of MAP kinase produced by salbutamol was inhibited by PD98059 and Rp-cAMPS.



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  		Fig. 7.— Western blotting of mitogen-activated protein (MAP) kinase (MAPK) and phosphorylated MAP kinase (p-MAPK) in 16-human bronchial epithelium (16-HBE) cells after a 5-min exposure to serum-free medium alone (control) or containing various drugs. a) Effects of cyclic adenosine monophosphate (cAMP)-generating agents and the effect of ß-adrenoceptor antagonist on salbutamol-induced MAPK phosphorylation. Lane 1: control; lane 2: salbutamol plus propranolol; lane 3: salbutamol; lane 4: forskolin; lane 5: 10% foetal bovine serum (FBS). b) Effects of MAP kinase-kinase inhibitor and cAMP antagonist on salbutamol-induced MAPK phosphorylation. Lane 1: control; lane 2: salbutamol; lane 3: 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP); lane 4: salbutamol plus PD98059; lane 5: salbutamol plus Rp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS); lane 6: 10% FBS.

 

   Discussion
TOP
 Abstract
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
The present study shows that salbutamol, a ß2-adrenergic receptor agonist, stimulates the proliferation of human airway epithelial cell lines. The current authors found that, in 16-HBE and NCI-H292 cells (but not A549 cells), exposure to salbutamol caused remarkable increases in MTT reduction and intracellular DNA content in a time-dependent manner, even in the culture medium from which FBS was substituted in order to arrest cell growth. This effect in 16-HBE cells is mediated by stimulation of ß2-adrenergic receptors, because co-incubation with propranolol abolished the salbutamol-induced cell growth and DNA synthesis. Furthermore, cAMP-generating agents, including the adenylyl cyclase stimulator, forskolin, and the stable cAMP analogue, 8-Br-cAMP, also showed a growth-promoting effect. In this experiment, time course of DNA synthesis was different between salbutamol and other drugs; the response to salbutamol reached a plateau during the first 24 h, whereas forskolin and 8-Br-cAMP seemed to induce a continued increase in DNA contents over the full 48 h. The reason for this difference is unknown, but could be due to desensitisation of ß2-adrenergic receptors by salbutamol. In addition, the present authors found that the salbutamol-induced MTT reduction and DNA synthesis were inhibited by the cAMP antagonist, Rp-cAMPS. Taken together, cAMP formation may represent a mitogenic signal in certain airway epithelial cell lines.

The role of cAMP in the regulation of cell growth is controversial, probably because of the differences in cell types and experimental conditions. Previous studies have shown that the elevation of intracellular cAMP content may link to the inhibition of growth of human and guinea pig airway smooth muscle cells 13, 14. Conversely, in rat parotid cells 15, dog thyroid cells 16, and rat cardiac myocytes 17, the increases in intracellular content of cAMP have been shown to stimulate cell growth. In addition, according to the recent study on cochlear sensory epithelium, forskolin and 8-Br-cAMP stimulate cell growth, an effect that can be blocked by the inhibitors of protein kinase A 9. These findings are in accordance with the present results and suggest a stimulatory role for the cAMP-dependent signalling pathway in the proliferation of epithelial cells. However, this experiment was conducted using only airway epithelial cell lines, and the effect of salbutamol was different between the cell lines. Thus, it remains to be determined whether such a signalling pathway is also present in primary culture of human airway epithelium.

The MAP kinases actively participate in converting extracellular stimuli detected by cell surface receptors to intracellular signals, leading to the expression of genes necessary for cell growth and differentiation 18. For example, there is ample evidence that stimulation of Gi and Gq protein-coupled receptors potently activates MAP kinase 7, 8. Regarding the effect of Gs protein-coupled receptor stimulation, isoproterenol has been shown to activate and inhibit the enzymatic activity in cardiac myocytes 17 and COS-7 cells 19, respectively. In the present study, the induction of phosphorylated MAP kinase was observed in 16-HBE and NCI-H292 cells, but not A549 cells after incubation with salbutamol. In 16-HBE cells, other cAMP-generating agents also caused activation of MAP kinase, and the salbutamol-induced MAP kinase phosphorylation was abolished by Rp-cAMPS. These results indicate that there may be a Gs-induced stimulatory signal mediated by cAMP for MAP kinase activity in certain human airway epithelial cells.

The major pathway involved in mitogen-activated protein kinase activation generally requires the sequential activation of Ras, Raf-1, and mitogen-activated protein kinase-kinase 20, 21. The present authors found that a specific inhibitor of mitogen-activated protein kinase-kinase, PD98059 12, inhibited the mitogen-activated protein kinase phosphorylation induced by salbutamol, and the salbutamol-induced deoxyribonucleic acid synthesis in a concentration-dependent fashion. Thus, activation of mitogen-activated protein kinase-kinase is likely to be involved in the observed growth of 16-human bronchial epithelium cells. However, the link between the cyclic adenosine monophosphate-dependent pathway and the initially activated molecule that localises proximal to mitogen-activated protein kinase-kinase in the mitogen-activated protein kinase cascade is still unknown and warrants further investigation.


   Acknowledgements
TOP
 Abstract
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors would like to thank M. Shino and Y. Sugimura for their technical assistance.


   References
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 Abstract
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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