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Published ahead of print on January 10, 2003, doi:10.1165/rcmb.2002-0074OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 12-18, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0074OC

Effects of Transforming Growth Factor-ß and Budesonide on Mitogen-Activated Protein Kinase Activation and Apoptosis in Airway Epithelial Cells

Girolamo Pelaia*, Giovanni Cuda*, Alessandro Vatrella, Donatella Fratto, Rosa D. Grembiale, Pierosandro Tagliaferri, Rosario Maselli, Francesco S. Costanzo and Serafino A. Marsico

Department of Experimental and Clinical Medicine, University "Magna Græcia" of Catanzaro, Catanzaro; and Department of Cardiothoracic and Respiratory Sciences, Second University of Naples, Naples, Italy

Address correspondence to: Dr. Girolamo Pelaia, M.D., Policlinico Universitario "Mater Domini," Via T. Campanella, 88100 Catanzaro, Italy. E-mail: pelaia{at}unicz.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cells play a central role in the inflammatory, apoptotic, and remodeling processes associated with asthma. Within this context, a key function is exerted by transforming growth factor-ß (TGF-ß), whose biological effects are mediated at least in part by mitogen-activated protein kinases (MAPKs). The aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the effects of TGF-ß (10 ng/ml) on both MAPK activation and apoptosis, in the presence or absence of a pretreatment with budesonide (10-8 M). MAPK activation was detected by Western blotting, using anti–phospho-MAPK monoclonal antibodies, which specifically recognize the phosphorylated, active forms of these enzymes. Apoptosis was assayed by caspase-3 activation and fluorescence microscopy, using annexin-V (An-V) and propidium iodide (PI) as markers of cell death. Our results show that TGF-ß induced a marked ({bsim} 9-fold) increase in p38 MAPK phosphorylation, and also dramatically enhanced cell death, which was completely prevented by specific MAPK inhibitors. Both MAPK activation and apoptosis were effectively inhibited by budesonide (BUD), thereby suggesting that the powerful antiapoptotic action of inhaled glucocorticoids may be very important for their protective role against epithelial injury, which represents a key pathogenic event in asthma.

Abbreviations: Annexin-V, An-V • budesonide, BUD • extracellular signal–regulated kinases, ERK • human bronchial epithelial cells, HBECs • c-Jun N-terminal kinases, JNK • mitogen-activated protein kinase, MAPK • propidium iodide, PI • sodium dodecyl sulfate, SDS • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the past few years, it has been well established that transforming growth factor-ß (TGF-ß) is crucially involved in airway remodeling associated with asthma, and higher levels of TGF-ß are detectable in the airways of patients with asthma in comparison with normal subjects (1, 2). Bronchial epithelial cells represent one of the most important sources of this growth factor, and it is now recognized that such cells play a pivotal role in driving and amplifying the airway inflammatory and remodeling processes which characterize asthma pathogenesis and evolution. Indeed, damage of bronchial epithelium is a hallmark of this disease, and recent evidences indicate that apoptosis represents a key pathogenic event responsible for epithelial shedding (3). Epithelium repair follows inflammatory injury, thus leading to an increased expression of repairing molecules such as epidermal growth factor and TGF-ß (4, 5).

Recent studies performed in epithelium–fibroblast co-cultures have shown that airway epithelial cells significantly affect the underlying extracellular matrix and fibroblasts, thereby providing a structural and functional framework for the so-called epithelial–mesenchimal trophic unit, which plays a central role in fetal lung development and is reactivated during the airway remodeling response (6). Within this context, TGF-ß stimulates the synthesis of extracellular matrix components such as tenascin and fibronectin, upregulates the expression of the {alpha}5-ß1 integrin (which is a fibronectin receptor), and promotes the transformation of fibroblasts into contractile myofibroblasts (4). The increased release of fibronectin from airway epithelial cells significantly contributes to fibroblast chemoattraction and growth. Moreover, TGF-ß induces the production of collagen types I and III by fibroblasts and of collagen type IV by bronchial epithelial cells, thereby leading to the subepithelial fibrosis that characterizes the airway wall thickening occurring in chronic asthma (4, 7). This profibrogenic action of TGF-ß is further enhanced by its capability of modulating extracellular matrix degradation by inhibiting the synthesis of matrix metalloproteinases, as well as by inducing the activity of the tissue inhibitors of such proteolytic enzymes (4). In addition to these stimulatory effects, TGF-ß can also inhibit the proliferation of airway epithelial cells, and negatively regulates several inflammatory cells such as T lymphocytes, dendritic cells, mast cells, and eosinophils (8).

The biological effects of TGF-ß are mediated by signal transduction pathways including both Smad proteins and mitogen-activated protein kinases (MAPKs) (9–13). The latter operate through an articulated phosphorylation cascade involving TGF-ß–activated kinase 1, a member of the MAPK kinase kinase family. TGF-ß–activated kinase 1 phosphorylates MAPK kinases 3 and 6 (MKK3 and MKK6), which in turn are responsible for phosphorylation and activation of the p38 subgroup of MAPKs (14). p38 exerts a key function in cellular stress and inflammation by phosphorylating several different substrates, mainly including transcription factors such as Chop (also known as GADD153), Max, MEF2C, and activating transcription factor-2 (12, 13). It has also been suggested that other MAPK subfamilies such as c-jun N-terminal kinase (JNK) may be activated by TGF-ß (15).

Our group has recently shown that dexamethasone is able to inhibit phosphorylation-dependent activation of p38, JNK, and extracellular signal–regulated kinases (ERK) in human pulmonary endothelial cells stimulated with either hydrogen peroxide (H2O2), or tumor necrosis factor-{alpha}, or interleukin-1ß (16). Moreover, an inhibitory effect of corticosteroids on MAPK activation has also been detected by other authors in several different cell types (17–21).

Therefore, we decided to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the cellular effects of TGF-ß and the signal transduction mechanisms activated by this growth factor. In addition, because airway epithelium is the first and one of the most important targets of inhaled corticosteroids (22), a further objective of the present study was to verify whether budesonide (BUD) may interfere, in these airway epithelial cells, with MAPK signaling pathways or with the biological effects of TGF-ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
BUD was purchased from Sigma (St. Louis, MO). Recombinant human TGF-ß1 was purchased from PeproTech (Rocky Hill, NJ). Anti–phospho-p38, anti–phospho-ERK1/2, and anti–phospho-JNK monoclonal antibodies were purchased from New England Biolabs (Beverly, MA); anti–(total)-p38, anti–(total)-ERK1/2, and anti–(total)-JNK polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti–caspase-3 polyclonal antibody was purchased from BD-Pharmingen (Palo Alto, CA). Annexin-V (An-V) and propidium iodide (PI) were from Clontech Laboratories (Basel, Switzerland). MAPK inhibitors PD98059 and SB203580 were from Calbiochem (San Diego, CA); the MAPK inhibitor SP600125 was from Tocris Cookson Inc. (Ellisville, MO).

Primary Cultures of Human Bronchial Epithelial Cells
HBEC were obtained from fresh surgical specimens taken from two patients who underwent either lobectomy or pneumonectomy for lung cancer at "V. Monaldi" University Hospital (Naples, Italy). Lung segments away from and free of the tumor were used. Bronchial mucosal biopsy samples were dissected from the underlying tissues and soaked in 0.1% protease solution (Type XIV, Streptomyces griseus; Sigma) overnight at 4°C (23, 24). The following day, samples were flushed with Eagle's minimum essential medium containing 10% fetal calf serum; the resulting suspension was filtered through a 100-µm sterile Nitex mesh to remove mucus, and centrifuged for 5 min at 1,500 x g. Bronchial epithelial cells were then harvested and cultured at 37°C, 5% CO2 in bronchial epithelial growth medium (BEGM; Clonetics, San Diego, CA) with added antibiotics (100 U/ml of penicillin and 100 µg/ml of streptomycin; Sigma) and Fungizone (1 µg/ml; GIBCO BRL, Gaithersburg, MD). For assays, cells (passage 3 or 4) were seeded into 24-well trays (1 ml BEGM/well containing 5 x 104 cells/ml) and cultured until {bsim} 80% confluent. The medium was then replaced by 1 ml/well of bronchial epithelial basal medium (BEBM; Clonetics) containing 1% of insulin, transferrin, and sodium selenite (ITS) media supplement for 24 h to render the cells quiescent. The medium was then replaced with 1 ml/well of BEBM/ITS, and cells were incubated for a further 12-h period in the absence or presence of 10-8 M BUD. Cell stimulation was performed by addition of TGF-ß (10 ng/ml) for 2 h; after 2 h, the medium was removed and the cells were processed for protein extraction and immunoblotting.

Protein Extraction and Immunoblot Analysis
For Western blotting, HBEC were grown to confluence and, following stimulation, lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1.5 mM MgCl2, 10 mM NaF, 10% glycerol, 4 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% deoxycholate, 50 mM Hepes, pH 7.4, plus PPIM, 25 mM ß-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Protein extracts were then separated on a 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes (Amersham Pharmacia, Little Chalfont, UK). Immunoblotting was performed using anti–phospho-p38, anti–phospho-ERK1/2, and anti–phospho-JNK monoclonal antibodies. After being "stripped," the membranes were reprobed with polyclonal antibodies against total (phosphorylated and unphosphorylated) p38, ERK1/2, and JNK. Antibody binding was visualized by enhanced chemiluminescence (ECL-Plus; Amersham Pharmacia), and intensities of experimental bands were analyzed by computer-assisted densitometry.

Apoptosis Assay
Detection of cell death in vitro was performed by three distinct approaches. HBEC were grown to 80% confluence and stimulated with TGF-ß for 2 h. When needed, cells were preincubated for 12 h with the MAPK inhibitors PD98059 (40 µM), SB203580 (1 µM), or SP600125 (20 µM). Six hours after removal of the stimulus (i.e., 8 h after the initial addition of TGF-ß), cell death was detected by either fluorescence microscopy or fluorescence-activated cell sorting analysis (FACSCalibur) using An-V/PI double staining. An-V is a phosphatidylserine-binding protein used to detect phosphatidylserine translocation from the inner to the outer plasma membrane leaflet, which is assumed to be an early feature of programmed cell death. PI is a marker for cell membrane permeability.

Programmed cell death was further investigated by measurement of caspase-3 activity. Briefly, cells were cultured under the same conditions. Two hours after addition of TGF-ß, cells were collected and lysed in appropriate buffer. Total cell extracts (30–50 µg) were electrophoresed onto a 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated with a polyclonal anti–caspase-3 antibody, and the presence of total and active caspase-3 was revealed by chemiluminescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of TGF-ß on MAPK Phosphorylation
Evaluation of the results obtained in three independent sets of experiments showed that exposure of HBEC for 2 h to TGF-ß (10 ng/ml) induced a marked ({bsim} 9-fold) increase, with respect to baseline levels, in the amount of phosphorylated p38 (Figure 1). TGF-ß also enhanced, although to a much lesser extent than p38, the phosphorylation pattern of JNK and ERK1/2 (Figure 1). Because the monoclonal antibodies (anti–phospho-p38, anti–phospho-ERK1/2, and anti–phospho-JNK) used in this study specifically recognize the phosphorylated, active forms of MAPKs, the remarkable increase in the phosphorylation pattern of the p38 subgroup can be considered as a reliable marker of its highly efficient activation elicited by TGF-ß.



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  		Figure 1. TGF-ß–induced phosphorylation of p38, ERK1/2, and JNK was inhibited by BUD. Left panel: incubation of primary cultures of human bronchial epithelial cells with TGF-ß (10 ng/ml, for 2 h) enhanced ERK1/2, JNK, and, especially, p38 phosphorylation, which was inhibited by a pretreatment with BUD (10-8 M, for 12 h). Both TGF-ß and BUD did not affect the total expresion of these MAPK subgroups. Phosphorylation of p38, ERK1/2, and JNK was detected by immunoblotting using anti–phospho-p38, anti–phospho-ERK1/2, and anti–phospho-JNK monoclonal antibodies, respectively. The total expression of these enzymes was detected by anti–total-p38, anti–total-ERK1/2, and anti–total-JNK polyclonal antibodies, respectively. Right panel: densitometric analysis of experimental bands referring to phosphorylated (black bars) and total (shaded bars) MAPK is shown. AU: arbitrary units. All data are expressed as mean ± SEM of three independent experiments.

 
Effects of Pretreatment with BUD on TGF-ß–Induced MAPK Phosphorylation
HBEC pretreatment for 12 h with 10-8 M BUD inhibited the effects of TGF-ß on MAPK phosphorylation, which was maintained at almost baseline levels (Figure 1). Because of the highly inducible phosphorylation profile of p38, the inhibitory effect of BUD was much more evident for this MAPK subgroup, in comparison with both JNK and ERK1/2. BUD exerted its effects uniquely on phosphorylation-dependent activation of MAPKs induced by TGF-ß, without affecting their total expression, as shown by the unchanged binding patterns of the anti-(total)MAPK polyclonal antibodies (Figure 1).

Effects of TGF-ß and BUD on HBEC Apoptosis
TGF-ß highly enhanced HBEC apoptosis with respect to control levels, as shown by the marked increase in An-V/PI staining (Figure 2) and by caspase-3 activation (Figure 3). BUD exerted a complete protection against the pro-apoptotic effect of TGF-ß (10 ng/ml) (Figure 2).



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  		Figure 2. BUD exerted a protective effect against TGF-ß–induced cell death. Left panel: light microscope morphology of primary cultures of HBEC. Right panel: An-V/PI staining, performed 6 h after TGF-ß removal (i.e., 8 h after TGF-ß addition), shows that this growth factor induced a remarkable increase in the amount of apoptotic cells, as evidenced by fluorescence microscopy. Apoptotic cells were visualized by an intense red staining (PI), surrounded by a scanty green fluorescence (An-V). BUD completely inhibited the proapoptotic effect of TGF-ß. Three independent sets of experiments were performed.

 


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  		Figure 3. Caspase-3 pathway is activated by TGF-ß in HBEC. Immunoblot obtained using a polyclonal antibody against caspase-3, in the absence or presence of TGF-ß stimulation (for 2 h). Diagram shows densitometric analysis of active caspase-3. AU: arbitrary units. All data are expressed as mean ± SEM of three independent experiments.

 
Effects of MAPK Inhibitors on TGF-ß–Induced HBEC Apoptosis
To further investigate the involvement of MAPK signaling pathways in the cell death induced by TGF-ß, before being exposed to this growth factor, HBEC were incubated for 12 h with different concentrations of specific MEK-ERK1/2 (PD98059, 40 µM), p38 (SB203580, 1 µM), and JNK (SP600125, 20 µM) inhibitors, respectively (Figure 4). Under these conditions, the apoptotic response elicited by TGF-ß was fully blocked, thus suggesting that MAPKs, and especially the p38 subfamily, play a central role in transducing the apoptotic signal. The solvent used to dissolve the MAPK inhibitors did not exert any effect on TGF-ß–induced apoptosis (data not shown).



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  		Figure 4. MAPK inhibitors prevent the pro-apoptotic effect produced by TGF-ß. FACS analysis of HBEC stained with An-V–cy3 in the presence or absence of the MAPK inhibitors PD98059 (40 µM), SB203580 (1 µM), and SP600125 (20 µM). Preincubation (for 12 h) with specific MEK/ERK1/2 (PD98059), p38 (SB203580), and JNK (SP600125) inhibitors was able to prevent the apoptosis induced by exposition to TGF-ß. Histograms represent the percentage of An-V–positive cells, determined in three independent experiments. All data are expressed as mean ± SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using primary cultures of HBEC freshly isolated from lobectomy or pneumonectomy specimens, we herein demonstrate that TGF-ß remarkably stimulated MAPK phosphorylation, which was effectively prevented by BUD. The latter drug was also able to completely inhibit the MAPK-dependent, pro-apoptotic effect exhibited by TGF-ß in these cell cultures.

TGF-ß, whose levels are increased in bronchoalveolar lavage fluid and epithelial cell culture supernatants from patients with asthma (1, 2), can inhibit activation of many cell types (e.g., T lymphocytes, eosinophils, mast cells, and dendritic cells) involved in airway inflammation, thereby contributing to suppress the asthmatic phenotype (8). On the other hand, this growth factor is a key player in the bidirectional, functional cross-talk connecting airway epithelium and subepithelial fibroblasts, thus having a prominent role in the reactivation of the epithelial–mesenchimal trophic unit occurring in asthma (25, 26). In particular, TGF-ß enhances collagen gene expression, extracellular matrix accumulation, fibroblast proliferation, and myofibroblast differentiation (25, 27).

One of the most important airway targets of TGF-ß is represented by the bronchial epithelium, where this growth factor acts via an autocrine loop thus promoting its own synthesis and release. A persistent, self-maintained expression of TGF-ß in bronchial epithelial cells of subjects with asthma may lead to a profibrogenic phenotype that characterizes airway remodeling and the resulting chronic airflow limitation. This is also suggested by the stimulatory effect of TGF-ß on the expression of fibronectin and tenascin in human bronchial epithelial cells (28). Indeed, TGF-ß1 is currently considered as a promising candidate gene for contributing to asthma severity (29).

Our results highlight a very relevant aspect of the biological activity of TGF-ß, characterized by its remarkable proapoptotic effect on HBEC, which resulted to be fully prevented by pretreatment with specific MAPK inhibitors. This finding may significantly contribute to clarify the pathogenic role of TGF-ß, because apoptosis is emerging as a key event for development of the epithelial shedding detectable in the airways of patients with asthma (3). Of course, activation of apoptotic programs may dramatically impair the repair of a damaged bronchial epithelium, thus making it more vulnerable to the deleterious effects of allergens, pollutants, and other noxious agents. We also showed that budesonide was very effective in inhibiting TGF-ß–induced, MAPK-dependent apoptosis of HBEC, thereby suggesting that such an anti-apoptotic action may be mainly responsible for the protective role of inhaled corticosteroids against airway epithelial injury. Interestingly, this effect was observed at a concentration of budesonide (10-8 M) which probably approximates drug levels included within the range achieved in the airways after administration of therapeutic doses by inhalation (30). However, conflicting results have been reported about the effects of glucocorticoids on HBEC apoptosis. In this regard, our findings are quite consistent with those published a few years ago by Wen and coworkers, who described a protective effect of dexamethasone against apoptosis of lung epithelial cells induced by interferon-{gamma} (31). Conversely, Dorscheid and colleagues have more recently observed in airway epithelial cells a proapoptotic action of dexamethasone, beclomethasone, budesonide, and triamcinolone, thus surprisingly deducing that these drugs may have a deleterious role in airway remodeling and epithelial damage characterizing chronic asthma (32); such assertions are, however, in sharp contrast with the widely recognized beneficial effects of corticosteroids in asthma treatment, which largely depend on their positive action on bronchial epithelium (22).

Our study provides valuable information about the signaling pathways operating downstream of TGF-ß receptor stimulation in HBEC. Furthermore, we evidenced that budesonide exerted a powerful inhibitory effect on MAPK activation triggered by TGF-ß. In particular, the negative interference with phosphorylation of the proinflammatory p38 MAPK may represent a key feature of the molecular mechanisms underlying the pharmacologic action of inhaled glucocorticoids, thus allowing them to target a crucial step in the complex TGF-ß signaling cascade. Indeed, a corticosteroid-dependent functional inhibition of p38 has been recently demonstrated by us and others in various cell types stimulated by different agents (16, 21). This negative effect is rather specific, in that other steroid hormones such as testosterone do not seem to be able to affect MAPK phosphorylation (16, 33).

For all these reasons, we think that our present findings may contribute to explain the mechanisms underlying the results obtained a few years ago by Olivieri and colleagues, who showed that a 6-wk treatment with inhaled fluticasone was able to partly reverse airway remodeling, as evidenced by the significant decrease in basement membrane thickness (34). Such observations were also consistent with those of Trigg and coworkers, who had previously found that a longer, 4-mo treatment with inhaled beclomethasone can reduce type III collagen deposition under bronchial epithelium (35). However, despite a treatment with high doses of corticosteroids, some patients with chronic severe asthma may experience both a persistence of symptoms and a progressive decline in pulmonary function (36). This suggests that glucocorticoids may not be adequate to completely block the biochemical events induced by activation of the epithelial–mesenchimal trophic unit and leading to airway remodeling. With regard to TGF-ß signaling in HBEC, it is therefore possible that corticosteroids, though being very effective in inhibiting p38 phosphorylation, are not able to thoroughly suppress the TGF-ß–induced transcriptional program, characterized by an increased expression of several genes including those encoding fibronectin, tenascin, collagen, adhesion molecules, and TGF-ß itself. On the other hand, the intracellular signal transduction mechanisms triggered by TGF-ß are not only dependent on MAPK activation, but also involve other pathways, mainly coordinated by Smad proteins, which act as effectors of the transcriptional response elicited by TGF-ß and related factors (9, 10).

In bronchial epithelial cells, Smads and MAPKs interact effectively, thus originating a very dynamic cross-talk at crucial regulatory points within intracellular signal transduction, where different pathways converge to coordinate and integrate the cellular responses to TGF-ß and to other very important growth factors such as epidermal growth factor. In addition to MAPKs, which have been identified by us and others as suitable molecular targets for pharmacologic modulation by corticosteroids (16–21), the latter may also interfere with Smad activity. In this regard, it has been shown that glucocorticoid receptors are able to interact with Smad-3, a TGF-ß–activated DNA-binding protein, thus inactivating its transcriptional function (37).

Taken together, these considerations point out that the biology of HBEC in both normal subjects and in subjects with asthma still needs to be further investigated. Indeed, it is now well known that the bronchial epithelium is not merely a physical barrier interposed between the respiratory system and the external environment, but is rather a very active structure which participates in lung development and airway branching, as well as in inflammatory processes and repair responses to tissue injury (25). In asthma, the bronchial epithelium is oriented toward a proinflammatory phenotype, characterized by an increased production of autacoids, cytokines, chemokines, adhesion molecules, and growth factors. Such a phenotype may be activated by several different stimuli including allergens, pollutants, viruses, bacteria, reactive oxygen species, and mechanical stress (38–42). It is likely that airway epithelial cells of individuals with asthma, because of a genetic predisposition, are intrinsically hypersensitive to environmental factors, thus being biased to activate a transcriptional program which significantly contributes to drive, propagate, and amplify the inflammatory and remodeling responses.

Therefore, the bronchial epithelium represents a main focus of the current efforts aimed at gaining a better knowledge of asthma pathogenesis, as well as of the molecular targets of antiasthma therapies. Within this context, we think that our findings concerning the proapoptotic effect of TGF-ß and the antiapoptotic action of BUD detected in primary HBEC cultures are very interesting. In particular, it is intriguing that MAPK activation and inhibition elicited by TGF-ß and BUD, respectively, were closely paralleled by their opposite effects on HBEC apoptosis. These observations assume a relevant importance in view of the recently emerging key role of HBEC apoptosis in the pathogenesis of asthma (3). On the other hand, an involvement of the p38 signaling pathway in TGF-ß–dependent programmed cell death has been documented in various cell types such as macrophages, B lymphocytes, and hepatocytes (43–45). Therefore, the existence of a tight link between MAPK phosphorylation and HBEC apoptosis may reasonably explain our results, thus contributing to highlight some crucial molecular events underlying both the development of asthma and its therapeutic control by inhaled corticosteroids.

In conclusion, the present study shows that within the various MAPK subfamilies expressed by primary cultures of HBEC, TGF-ß preferentially activates the p38 pathway, which is notably involved in the synthesis of proinflammatory cytokines and chemokines, as well as in the induction of apoptosis. Furthermore, both phosphorylation-dependent MAPK activation and HBEC apoptosis induced by TGF-ß were, in the final results, powerfully inhibited by BUD. We think that these findings may contribute to better understand the intracellular events leading to the biological effects of TGF-ß, as well as the molecular mechanisms responsible for the antiasthma action of inhaled corticosteroids. In fact, because of its strategic anatomical position and biological role, the bronchial epithelium is a prominent target of antiinflammatory drugs administered through the inhalational route. Finally, we hope that such studies may help to unveil new therapeutic strategies directed to modulation of MAPK signaling cascades. Among the different MAPK subgroups, p38 is currently considered as the most suitable target for pharmacologic intervention aimed at counteracting airway inflammation (46–48). With regard to the operative role of the epithelial–mesenchimal trophic unit in asthma pathophysiology, in particular, the future research efforts should be focused on reverting and/or preventing the detrimental clinical and functional consequences of airway remodeling and HBEC apoptosis.


   Acknowledgments
 
The authors gratefully thank Drs. Nunzia Montuori and Pia Ragno (Center for Experimental Endocrinology and Oncology, National Research Council, Naples, Italy) for preparing the primary cultures of human bronchial epithelial cells.


   Footnotes
 
* These authors contributed equally to the work presented in this article. Back

Received in original form May 22, 2002

Received in final form December 24, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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