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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 61-68, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4715

Increased Expression of p21waf Cyclin-Dependent Kinase Inhibitor in Asthmatic Bronchial Epithelium

Sarah M. Puddicombe, Carlos Torres-Lozano, Audrey Richter, Fabio Bucchieri, James L. Lordan, Peter H. Howarth, Bart Vrugt, Rene Albers, Ratko Djukanovic, Stephen T. Holgate, Susan J. Wilson and Donna E. Davies

The Brooke Laboratory, Division of Infection, Inflammation and Repair, School of Medicine, Southampton General Hospital, Southampton, United Kingdom

Address correspondence to: Dr. S. M. Puddicombe, RCMB Division, Mailpoint 810, Southampton General Hospital, Southampton SO16 6YD, UK. E-mail: smp2{at}soton.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because the asthmatic bronchial epithelium is characterized by widespread damage, we postulated that this is associated with expression of cell cycle inhibitors that control proliferation. Using bronchial biopsies, the epithelium was the major site of expression of the cyclin-dependent kinase inhibitor, p21waf. Immunostaining usually occurred in the cytoplasm of columnar cells; however, in severe asthma, nuclear staining was also evident in the proliferative, basal cell compartment. p21waf expression was significantly higher in asthmatic versus nonasthmatic epithelium and was unaffected by corticosteroid treatment; proliferating cell nuclear antigen was not significantly different in any group. p21waf, but not p27kip1, mRNA and protein were induced by treatment of bronchial epithelial cells in vitro with transforming growth factor (TGF)-ß or H2O2, but not by dexamethasone, which induced p57kip2. TGF-ß and dexamethasone inhibited epidermal growth factor (EGF)-induced DNA synthesis, whereas low concentrations of H2O2 synergized with EGF; at higher doses, growth inhibition and induction of apoptosis occurred. TGF-ß caused p21waf to become nuclear, suggesting interaction with the replicative machinery; however, in oxidant-stressed cells, p21waf was predominantly cytoplasmic, where it has been linked to cell survival. We conclude that p21waf overexpression in asthma influences cell proliferation and survival. This may cause abnormal repair responses that contribute to airway inflammation and remodeling.

Abbreviations: bronchial epithelial basal medium, BEBM • bronchial epithelial growth medium, BEGM • bronchial hyperresponsiveness, BHR • cyclin-dependent kinase, CDK • cystic fibrosis, CF • chronic obstructive pulmonary disease, COPD • epidermal growth factor, EGF • fetal bovine serum, FBS • glyceraldehyde phosphate dehydrogenase, GAPDH • glycol methacrylate, GMA • poly ADP ribose polymerase, PARP • proliferating cell nuclear antigen, PCNA • serum-free medium, SFM • transforming growth factor, TGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Asthma is a chronic inflammatory disorder, characterized by exacerbations and remissions and an underlying bronchial hyperresponsiveness (BHR) of the airways to a wide variety of environmental factors. In established disease, a major part of the BHR can be separated from the inflammatory response and most likely represents structural changes in the airways. These include extensive epithelial damage, deposition of extracellular matrix proteins throughout the airways, and other features of remodeling such as epithelial goblet cell metaplasia, smooth muscle hypertrophy, and increases in nerves and blood vessels (1). Whether these features occur as a consequence of, or in parallel with, the inflammatory response is not known, but increasingly it is being appreciated that many of the airway's structural elements are themselves altered to produce cytokines, growth factors, and mediators that may contribute to sustaining the inflammatory response (2).

The bronchial epithelium acts as the physical barrier to separate the external environment and the internal milieu of the lung. Therefore, maintaining its integrity is an important component of airways defense. Impairment of this protective function in asthma was originally suggested by the extent of epithelial damage that was evident in asthmatic airways at autopsy (3). That epithelial shedding is an important feature of asthma is supported by the presence of increased numbers of epithelial cell clumps (Creola Bodies) in sputum (4), and by the disruption of the bronchial epithelium that is frequently observed in mucosal biopsies obtained from asthmatic airways by either rigid (5) or fiberoptic (6) bronchoscopy. The type of epithelial disruption seen in asthma is not observed in other inflammatory diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), even though these conditions are closely associated with microbial infections and high concentrations of irritant tobacco smoke, respectively.

We have previously reported a disease-related increase in expression of the epidermal growth factor (EGF) receptor (EGFR) in asthmatic bronchial epithelium (7), suggesting induction of a normal reparative response in response to injury. However, this does not appear to be associated with increased proliferation, as expression of proliferating cell nuclear antigen (PCNA) has not been found to be increased in asthmatic bronchial epithelium (8), even though {bsim} 60–70% of the specimens examined showed epithelial shedding. This finding markedly contrasted with that observed in chronic bronchitis, where increased proliferation was a consistent finding (8). Thus, the nature of the response of the asthmatic bronchial epithelium to injury appears to differ markedly from that resulting from chronic exposure to cigarette smoke.

Cell division is, by necessity, a highly regulated process with checkpoints to ensure that DNA is faithfully copied and that identical chromosomal copies are distributed equally to each daughter cell (9). These checkpoints also respond to damage, enabling the cell cycle to be arrested to provide time for transcription and activation of genes that facilitate repair. G1 progression differs from transit through S, G2, and M phases because it normally depends on stimulation by mitogens that stimulate synthesis of the D-type cyclins and their assembly with cyclin-dependent kinase (CDK)4 or CDK6 (10, 11). Passage through the restriction point and entry into S phase is controlled by CDKs that are sequentially regulated by the cyclins D, E, and A. CDK activity requires cyclin binding, can be modulated by phosphorylation, and can be inhibited by CDK inhibitory proteins (12). One such CDK inhibitor is p21waf (p21CIP1), which interacts with cyclins D and E to block the G1 to S phase transition by inhibiting phosphorylation of retinoblastoma protein (13). P21waf also interacts with PCNA to prevent its activation of DNA polymerase {delta} (13).

In addition to the tumor suppressor gene, p53, p21waf can be induced by antiproliferative cytokines such as transforming growth factor (TGF)-ß and interferon-{gamma}, by corticosteroids and by stress and injury (14). In asthma, we have reported elevated levels of TGF-ß in bronchoalveolar lavage fluid (BALF) of subjects with asthma (15), whereas STAT-1, a transcription factor activated by interferon-{gamma}, has also been reported to be active in asthmatic bronchial epithelium (16). The predominance of factors that negatively regulate the cell cycle, together with lack of any change in PCNA expression in asthma (8), led us to postulate that there was a block in the G1 phase of the cell cycle and that this is caused by expression of p21waf. To test our hypothesis, we compared the expression of p21waf in bronchial biopsies from normal subjects and from subjects with asthma and undertook in vitro analyses to compare the effects of TGF-ß, oxidant stress, and corticosteroids on p21waf expression and the functional consequences of its expression on cell proliferation and survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Archival glycol methcrylate (GMA)-embedded bronchial biopsies collected from 11 control subjects without asthma (6 male; age [median (range)] = 24.5 [20–46]), 14 subjects with mild asthma (12 male; age = 26 [19–54]), and 10 subjects with severe asthma (1 male; age = 28.6 [13–53]) were used for the immunohistochemical study. The asthma groups were defined according to the GINA guidelines (17); the severe group had a mean FEV1 of 75 (range 53–95)% predicted, which was significantly less than that of the mildly asthmatic group (FEV1 = 94% [range 82–107]). The subjects with mild asthma were receiving Ventolin only, whereas those with severe asthma were on a mean inhaled steroid dose of 3,060 µg/d (range 2,000–4,000) and an oral steroid dose of 37.5 mg/d (range 2.5–100). The study was approved by the Southampton Joint University and Hospitals Ethics Committee, and all subjects gave their written consent after being fully informed about the nature and purpose of the study.

Sample Processing and Immunohistochemistry
Processing of tissue into GMA and the immunohistochemical method have been described previously (7, 18). Primary mouse monoclonal antibodies were used for detection of p21waf (SantaCruz, 10 µg/ml; Autogen Bioclear, Calne, UK) and PCNA (1 µg/ml; Sigma Immunochemicals, Poole, UK); isotype matched and no primary antibody controls were routinely included in every staining run. Sections were counterstained with Mayer's haematoxylin and mounted in p-xylene-bis-pyridium bromide. Airway epithelial expression of immunoreactive p21waf and PCNA was quantified by computer-assisted image analysis (Colorvision 1.7.6; Improvision, Coventry, UK). For each biopsy specimen, the entire intact epithelium, excluding the brush border, was systematically assessed in two nonserial sections based on red, blue, green (RGB) color balance. At the beginning of each session, the image analysis system was standardized as previously described (7) using the same section of bronchial mucosa stained for each antigen, to ensure reproducibility of analysis. Measurements were performed by an observer who was unaware of the clinical group from which the biopsy specimen was derived. Comparisons between clinical groups for immunostaining of p21waf and PCNA were made using the Mann-Whitney U test .

Epithelial Cultures
Primary bronchial epithelial cells were established from epithelial brushings and grown in bronchial epithelial growth medium (BEGM) (Biowhittaker UK Ltd, Wokingham, UK), as previously described (19). For immunocytochemical staining, cells were rendered quiescent by culture for 24 h in bronchial epithelial basal medium (BEBM) (Biowhittaker UK Ltd) containing 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 µg/ml sodium selenite, and 1% bovine serum albumin. Exposures were performed in fresh BEBM in the absence or presence of H2O2 (200 µM), TGF-ß2 (200 pM) or dexamethasone (1 µM) for 18 h. NCI-H292 bronchial epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown to 90% confluence in RPMI containing 10% fetal bovine serum (FBS), 50 IU/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine, and serum depleted by growth in Ultraculture (Biowhittaker UK Ltd) for 24 h. The medium was replaced with fresh Ultraculture in the absence or presence of H2O2 (50 or 200 µM), TGF-ß2 (200 pM) or dexamethasone (1 µM) for 8 h. For analysis of target mRNA expression, the RNA was extracted using TRIzol reagent (Life Technologies, Paisley, Scotland, UK) in accordance with manufacturer's instructions. Total RNA was quantitated using GeneQuant spectrophotometer (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK).

RNase Protection Assays
The hCC-2 multiprobe template set (Becton Dickinson UK Ltd, Cowley, Oxford, UK) containing DNA probes to the cell cycle regulatory proteins: p130, Rb, p107, p53, p57, p27, p21, p19, p18, p16, p14/15, L32, and glyceraldehydes phosphate dehydrogenase (GAPDH) were used as templates for in vitro transcription using 32P-dUTP (Amersham Pharmacia Biotech) and RiboQuant In vitro Transcription Kit (Becton Dickinson UK Ltd). High specific activity hCC-2 riboprobes were hydridized to 10 µg total RNA and then RNase-treated to leave "protected" double-stranded probes using the RiboQuant RPA kit (Becton Dickinson UK Ltd). Control human RNA (2 µg) and total yeast RNA (2 µg) were included as positive and negative controls, respectively. The probes and "protected" target RNA were separated by denaturing PAGE (5% acrylamide/bis-acrylamide [19:1] gel containing 8 M urea/0.5x Tris borate EDTA at 50 W for 2 h). The gel was dried and exposed to a Phosphor screen (Molecular Dynamics Ltd, Chesham, Bucks, UK) which was scanned on a Storm 840 Phosphorimager (Molecular Dynamics Ltd). Images were analyzed using the FragmeNT Analysis package (Molecular Dynamics Ltd). The pixel density of the bands corresponding to the expected sizes for protected probes were normalized with respect to expression of the house keeping genes, L32 and GAPDH, in the sample. In each analysis, data were normalized with each housekeeping gene separately and then the mean of both housekeeping genes. As the data were closely matched in each analysis, those presented are derived from analysis using the mean of two the housekeeping gene signals. Results were analyzed using the Students t test for paired data.

Mitogenesis Assay
The effects of TGF-ß, dexamethasone, and H2O2 on EGF-induced DNA synthesis were evaluated using confluent and quiescent H292 bronchial epithelial cells in a modification of a standard mitogenesis assay (7). Cells were grown to confluence in 96-well opaque cell culture trays in RPMI/10% FBS and rendered quiescent by serum reduction. Growth factors, H2O2, dexamethasone, or tyrphostin AG1478 (the latter two dissolved in DMSO) were added to the cells in mitogenesis assay buffer and DNA synthesis was determined 18 h later by incorporation of [125I]UdR over a 2-h pulse period. The cells were fixed and washed with 5% trichloroacetic acid followed by methanol. After drying, acid-insoluble material was dissolved in 40 µl/well of 0.2 M NaOH and radioactivity determined on a Topcount Scintillation counter (Canberra Packard, Pangbourne, Berks, UK) after addition of 150 µl of Microscint-40 (Canberra Packard) to each well.

Western Blotting
H292 bronchial epithelial cells were seeded into 24-well dishes in RPMI 1640/10% FCS and grown to 70–80% confluence. After serum starvation for 24 h, the cells were treated in serum-free medium (SFM) without or with 10 ng/ml TGF-ß, or in the presence H2O2 (50 or 200 µM) for 0, 2, 4, 8, or 24 h. As a positive control, cells were treated with 200 µM H2O2 in the presence of 10 ng/ml TNF-{alpha}, which is known to induce apoptosis in H292 cells (19). At each time point, cells were lysed into SDS sample buffer and analyzed by SDS-PAGE and Western blotting with p21waf, p27kip1 (SantaCruz; Autogen Bioclear), or p85 poly ADP ribose polymerase (PARP) (Promega, Southampton, UK) antibodies, using previously described protocols (7).

Analysis of Apoptosis by Flow Cytometry
Apoptosis was measured by externalization as previously described (19). Briefly, H292 bronchial epithelial cells were seeded into 24-well plates at a density of 5 x 104/well and allowed to grow to 80% confluence. The cells were rendered quiescent for 24 h before exposure to pro-apoptotic stimuli for up to 24 h, as detailed in RESULTS. Adherent cells were harvested with trypsin in Ca2+ and Mg2+-free Hanks' balanced salts solution and combined with nonadherent cells for analysis. After washing twice in cold PBS, the cells were resuspended at a density of 1 x 105 cells/100 µl of binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; Becton and Dickinson UK Ltd) in 5 ml propylene FACS tubes. Annexin-V fluorescein isothiocyanate conjugate (AxV-FITC) (1 µg/ml) and 7-aminoactinomycin D (7-AAD) (2.5 µg/ml) (Becton and Dickinson UK Ltd) were added to the tubes, which were then incubated in the dark. After 15 min, 400 µl of cold binding buffer was added to the tubes and cells analyzed using a FACScan flow cytometer (Becton and Dickinson UK Ltd). In each case, control tubes lacking AxV-FITC, 7-AAD or both were included for the acquisition. Analysis of dot plots of FL1 (Annexin V-FITC) versus FL2 (7-AAD) was performed using WinMDI 2.8.

Immunocytochemistry
Primary bronchial epithelial cultures were grown in 8-well chamber slides and exposed for 18 h to TGF-ß or H2O2, as described in EPITHELIAL CULTURES. p21waf was detected by immunoperoxidase staining with mouse anti p21waf as above using diaminobenzidine (DAB) in TrisHCl buffer containing 0.01% (vol/vol) H2O2. Slides were counterstained with Mayer's haematoxylin and mounted in p-xylene-bis-pyridium bromide.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of p21WAF and PCNA Expression in Bronchial Biopsies from Normal Subjects and from Subjects with Asthma
Immunohistochemical analysis of bronchial biopsies showed that columnar epithelial cells were the major sites of expression of p21waf. However, in subjects with severe asthma, staining was also evident in the basal cells, which are considered to be the proliferative compartment of the epithelium. On closer inspection, the distribution of p21waf within each cell was complex. In normal subjects, much of the staining in the differentiated columnar cells was around the nuclear region (Figure 1A), whereas in the severe asthma group, there was a more diffuse cytoplasmic distribution (Figure 1C). However, nuclear staining was also clearly detectable, with dense staining of nucleolar regions (Figure 1C), even in damaged areas of epithelium (Figure 1D), as indicated by the arrows. In 60–70% of biopsies (including some from normal subjects), immunostaining of the brush border for p21waf was also evident. Although we have occasionally observed some nonspecific staining of the brush border with other antibodies (18), this did not appear to be the case for p21waf, as staining with the isotype control antibody was minimal (data not shown). However, in view of our previous findings and the variable nature of the brush border staining, this region of the epithelium was excluded during the image analysis and does not contribute to the quantitative data described below.



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  		Figure 1. Immunohistochemical analysis of p21waf expression in bronchial biopsies from normal subjects and from subjects with asthma. Tissue sections were stained for the presence of p21waf using a streptavidin–biotin peroxidase detection system as described in MATERIALS AND METHODS. The plate shows sections from a normal subject (A), a subject with mild asthma (B), and a subject with severe asthma (C and D). D shows region of damaged bronchial epithelium from a subject with asthma, demonstrating the cytoplasmic and nuclear distribution of p21waf (nuclear and nucleolar staining indicated by small arrows).

 
Quantitation of staining using computerized image analysis showed that there was a significant increase in p21waf expression (% epithelial staining median [range]) in mild (7.3 [1.0–19.5]; n = 14, P = 0.008) and severe (13.0 [0.8–21.9]; n = 10, P = 0.002) asthma when compared with the nonasthmatic control group (2.2 [0.0–7.7]; n = 11) (Figure 2). There was also a tendency for p21waf expression to be higher in severe asthma when compared with mild asthma; however, this failed to reach statistical significance (P = 0.08). Although corticosteroids have been reported to induce p21waf expression (20), there was no obvious relationship between the dose of corticosteroids used by the subjects with severe asthma and the amount of p21waf expressed within the bronchial epithelium (Figure 2). This was further assessed in a group of seven subjects who were studied before and after treatment with 400 µg of inhaled budesonide twice daily for 8 wk. However, no significant difference in epithelial p21waf expression was found before (% epithelial staining median [range] = 4.9 [2.5–9.3]) or after (5.7 [2.7–13.9]; P = 0.67) treatment.



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  		Figure 2. Quantitative analysis of p21waf expression in bronchial epithelium. Immunohistochemical staining was quantified by computerized image analysis as described in MATERIALS AND METHODS. The figure shows % epithelial staining for each group, the line represents the median value, and error bars denote the 5% and 95% percentile range. Data were analyzed using Mann-Whitney U test. The figures on the right of the plot refer to the dose of oral (mg/d) and inhaled (µg/d) corticosteroid taken by the corresponding subject with severe asthma.

 
To determine the relationship between p21waf expression and epithelial proliferation, sections from the same subjects were stained for expression of the proliferation marker, PCNA. No difference in staining was found in any of the groups even in those subjects treated with corticosteroids. The % epithelial staining (median [range]) was found to be normal: 0.4 (0–1.4) (n = 11) versus mild asthma: 0.8 (0–2.1) (n = 14) versus severe asthma: 0.7 (0–2.0) (n = 10).

Induction of p21WAF Expression by TGF-ß, H2O2, and Dexamethasone
To gain some insight into the regulation of p21waf expression in bronchial epithelial cells, we used RNase protection assays to compare the effects of TGF-ß, H2O2, and dexamethasone on induction of expression of a range of CDK inhibitors, including p21waf. As shown in Figure 3A, H2O2 and TGF-ß both induced p21waf expression; densitometry revealed baseline expression of 0.086 (0.050–0.090) (median [range]), which increased to 0.133 (0.043–0.290) after treatment with 200 µM H2O2 (P = 0.05) and to 0.105 (0.090–0.170) with 200 pM TGF-ß (P = 0.003), respectively (Figure 3B). In contrast, dexamethasone failed to affect p21waf expression, but induced an increase in expression of p57kip2, a related CDK inhibitor from a baseline of 0.003–0.126 with 1 µM dexamethasone (P = 0.03) (Figure 3B). Expression of p27kip1, a related cyclin-dependent kinase, was unaffected by treatment with TGF-ß, H2O2, and dexamethasone. Western blot analysis confirmed that p21waf protein was markedly elevated in the presence of H2O2, with a much smaller change being evident in the presence of TGF-ß (Figure 3C, left panel); there was minimal effect on p27kip1 protein (Figure 3C, middle panel), even though it has been reported that it is regulated at the level of protein stability, rather than at the level of transcription (21). In these same experiments, there was no evidence of caspase activation, as detected using an antibody to p85 (Figure 3C, right panel), the caspase cleavage product of PARP.



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  		Figure 3. Regulation of p21waf expression in vitro by TGF-ß, H2O2, and dexamethasone. H292 bronchial epithelial cells were exposed to TGF-ß, H2O2, or dexamethasone as described in MATERIALS AND METHODS. RNA was extracted using TRIzol and analyzed for expression of CDK inhibitors using an RNase protection assay. A shows a representative phosphorimage for unprotected probe (lane 1), control yeast RNA (lane 2), untreated (lane 3), 200 µM H2O2 (lane 4), 200 pM TGF-ß (lane 5), and 1 µM dexamethasone (lane 6) treated cells. The expected positions of "protected" probes for each of the cell cycle regulators are indicated on the left. The graph in B shows quantitation of the data for expression of p21waf (white bars), p27kip (light gray bars), and p57kip2 (dark gray bars) obtained using FragmeNT Image Analysis software and normalizing using both GAPDH and L32 as housekeeping genes. Data are representative of four experiments, which were analyzed using the Student's t test for paired data *P <= 0.05, **P < 0.005. The Western blots in C show p21waf (left panel), p27kip1 (middle panel), and p85 PARP (right panel) protein levels in control cells (lane 1), or cells treated with 200 pM TGF-ß (lane 2), 50 µM H2O2 (lane 3), or 200 µM H2O2 (lane 4) for 0, 2, 4, 8, and 24 h. Lane C shows cells treated in the presence of 200 µM H2O2 plus 10 ng/ml TNF-{alpha} for 0, 4, and 24 h, which was used as a positive control to confirm the ability of the p85 PARP antibody to detect induction of apoptosis. Data are representative of three individual experiments.

 
Effects of TGF-ß, H2O2, and Dexamethasone on EGF-Induced DNA Synthesis
To relate p21waf expression to function, H292 bronchial epithelial cells were tested in a standard mitogenesis assay, which measures the ability of growth factors such as EGF to cause quiescent cells to re-enter the cell cycle and progress into S-phase to undergo DNA replication. In this system, EGF caused a dose-dependent increase in DNA synthesis that was maximal between 0.5 and 1.0 nM EGF, with an 8-fold increase in thymidine incorporation (Figure 4A, inset). Simultaneous exposure of the cells to TGF-ß or dexamethasone caused a dose-dependent decrease in DNA synthesis (Figures 4A and 4B), consistent with the induction of p21waf and p57kip2 expression, respectively. The responses of the cells to H2O2 were more complex. At doses below 200 µM, H2O2 stimulated DNA synthesis and was additive with EGF (Figures 5A and 5B). This effect appeared to be mediated by the EGFR, because it became phosphorylated upon treatment with H2O2 (Figure 5C) and the mitogenic response was blocked by the EGFR selective inhibitor tyrphostin, AG1478 (Figure 5B) as reported in other systems (22). Above 200 µM, H2O2 inhibited DNA synthesis (Figure 5A) and caused a gradual induction of apoptosis as measured by externalization of phosphatidylserine to the outer leaflet of the plasma membrane (Figure 5D).



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  		Figure 4. The effects of TGF-ß, H2O2, and dexamethasone on epithelial proliferation. Confluent and quiescent H292 bronchial epithelial cells were tested in a mitogenesis assay by monitoring incorporation of the thymidine analog 5'-iodo deoxyuridine into acid insoluble material. The inset in A shows a typical dose–response curve obtained with EGF, whereas A and B show the growth-inhibitory effect of dexamethasone and TGF-ß, respectively.

 


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  		Figure 5. The effects of H2O2 on epithelial proliferation and survival. H292 bronchial epithelial cells tested in a mitogenesis assay. The plot in A shows the biphasic mitogenic response to H2O2 where doses of H2O2 up to 200 µM stimulated DNA synthesis, and the plot in B shows that the effects were additive with EGF and blocked by the EGFR-selective tyrosine kinase inhibitor, AG1478. C shows phosphorylation of the EGF receptor (170 kD) in response to SFM (Con), 0.5nM EGF (E), 100 µM or 200 µM H2O2 observed in cell lysate after SDS-PAGE and Western blotting with anti-phosphotyrosine antibody (PY-20). D shows induction of apoptosis caused by increasing doses of H2O2.

 
Cellular Distribution of p21WAF
To determine whether primary bronchial epithelial cells responded similarly to TGF-ß, H2O2, and dexamethasone, immunocytochemical staining for p21waf expression was performed. In untreated cells, p21waf immunoreactivity was weak and cytoplasmic (Figure 6A); however, upon treatment with TGF-ß, it was localized in the nuclei and was particularly concentrated in nucleolar regions (Figure 6B), as was also observed in the tissue sections. In the case of H2O2, the staining intensity also increased; however, it remained diffuse and cytoplasmic (Figure 6C), resembling the staining observed in the columnar cells of the bronchial epithelium from subjects with severe asthma. In contrast, the staining observed for dexamethasone was weak and cytoplasmic (Figure 6D).



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  		Figure 6. Immunocytochemical distribution of p21waf. Primary bronchial epithelial cells were untreated (A) or treated with TGF-ß (B), H2O2 (C), or dexamethasone (D) for 18 h and then stained for the presence of p21waf by immunocytochemistry using DAB as chromogen. Note the brown nuclear stain in B and the cytoplasmic stain in C. Magnification: x253.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent attention has focused on the central role played by the bronchial epithelium as a key regulator of inflammation and remodeling in asthma (1). This altered phenotype has been attributed to increased stress (i.e., subcytotoxic damage) and physical injury, which characteristically involves widespread activation of transcription factors such as NF-{kappa}B (23) and STAT1 (16), and loss of columnar cells (6), respectively. The type of epithelial disruption seen in asthma is not observed in other inflammatory diseases such as CF and COPD. Although these differences may reflect the quality of inflammation, this cannot be the sole explanation, as airway eosinophilia can be observed in the absence of asthma or BHR (24) and, like COPD and CF, neutrophils may dominate inflammation in severe asthma (25). This raises the possibility that the extent of epithelial damage in asthma is due to increased susceptibility to a damaging agent and/or an inadequate repair response.

Although a considerable amount of attention has been paid to the proinflammatory nature of the bronchial epithelium in asthma, little is known the factors that regulate its maintenance and repair. The EGF signaling pathway appears to play a role in the response to injury; however, as previously reported (8) and confirmed in the present study, it does not appear to be functionally linked to a proliferative response. Because restoration of the damaged epithelium is essential for maintenance of its barrier function, it is not surprising to find that the extent of epithelial damage correlates with epithelial permeability (26) and BHR (27).

Cellular homeostasis is regulated by a balance between proliferation, growth arrest, and apoptosis. p21waf is known to play a vital role in these processes, and its protein levels increase in response to a variety of growth-inhibitory signals, such as DNA damage, nutrient deprivation, growth inhibitors, and cell differentiation (28). Recent work has demonstrated that p21waf has different functional roles depending on its intracellular distribution and on the stage of differentiation. The cell cycle inhibitory activity of p21waf is intimately associated with its nuclear localization, as observed in our studies upon TGF-ß treatment. Nuclear localization is also associated with induction of differentiation (29), explaining its expression in the nuclear region of columnar epithelial cells. Expression of p21waf is also particularly important following tissue injury, where it is critically involved in processes that control cell survival or commitment to apoptosis, depending on the extent of cell damage. Using a model of monocyte differentiation, p21waf has recently been shown to protect against cytotoxic stimuli by a mechanism that involves its cytoplasmic localization (29). This enables binding to and inhibition of the stress-activated kinase, ASK-1. This anti-apoptotic function of p21waf appears compatible with the prominent cytoplasmic localization of p21waf in oxidant-treated bronchial epithelial cells. Because the increase in p21waf observed in the columnar cells in severe asthmatic epithelium follows a more cytoplasmic distribution, this suggests that the p21waf is engaged in an anti-apoptotic function. This proposal is consistent with the increased expression of hsp27 (30) and bcl-2 (31) in asthmatic bronchial epithelium. However, in spite of increased expression of these markers of survival, our recent finding that asthmatic bronchial epithelium contains significantly more of the p85 peptide generated upon cleavage of PARP by caspase 3 (19), suggests that some asthmatic epithelial cells are at the limit of survival and are commencing the effector stage of apoptosis.

Environmental and endogenous oxidant and antioxidant status have been associated with asthma induction and exacerbation (32). In the present study, we found that H2O2 was a potent inducer of p21waf expression, which, as already discussed, may have an anti-apoptotic role in the columnar cells, which form the barrier to the external environment. In our recent studies, we have found that primary cultures of asthmatic bronchial epithelial cells are more susceptible to oxidant-induced apoptosis than those from normal subjects (19). In being preserved through several generations in vitro, this increased oxidant sensitivity is unlikely to be a consequence of inflammation. Therefore, we have postulated that this intrinsic sensitivity to oxidants may be a triggering mechanism that translates changes in the environment into excessive epithelial injury, establishing the appropriate microenvironment for persistent airways inflammation, chronic damage, and tissue remodeling (19). The high level of p21waf expressed in asthmatic biopsies seen under ambient conditions may be a reflection of an abnormal sensitivity to oxidants leading to induction of survival mechanisms.

In addition to diffuse cytoplasmic staining for p21waf in severe asthma, we also noted strong immunostaining for p21waf just below the brush border. This pattern of staining was observed in all severe asthmatic biopsies, but was also evident in some biopsies from normal subjects and from subjects with mild asthma, although staining was usually less pronounced. Although not included in our quantitative analysis, this staining appeared to be specific, as sections stained with an isotype control antibody were negative. One possible reason for accumulation of p21waf at the brush border is in relation to mucociliary clearance. Because providing the motive power for ciliary beating makes a substantial demand on the mitochondria for provision of ATP, this may create a significant local oxidative burden, as evidenced by the accumulation of the antioxidant, zinc, in the mitochondria-rich cytoplasm below the cilia (33). Localization of p21waf to this oxidant-rich environment is consistent with its anti-apoptotic function and may be particularly important in severe asthma, where the mucus is very tenacious and difficult to clear.

In marked contrast with the basal cells in normal epithelium, those in severe asthmatic biopsies frequently exhibited p21waf immunoreactivity. In these cells, the pattern of staining was both perinuclear and occasionally nucleolar, suggesting that p21waf may be affecting proliferation in the basal cell compartment. This suggestion is consistent with our failure to observe any evidence of increased proliferation in asthmatic biopsies. One possible candidate for this growth-inhibitory activity is TGF-ß, whose levels we have reported to be elevated in bronchial alveolar lavage fluid from subjects with asthma (15). As illustrated in Figure 5, TGF-ß is antiproliferative, probably acting via the SMAD proteins to induce expression of cell cycle inhibitors including p21waf. Although the level of p27kip1 protein was minimally affected by oxidant damage, a small change in the amount of this protein was evident in the presence of TGF-ß (Figure 3C). As p27kip1 also regulates cell cycle progression, further investigation of its expression in asthmatic biopsies may be warranted.

In addition to induction by TGF-ß, other mechanisms may contribute to p21waf expression in the basal cell compartment. For example, in ductal carcinoma in situ of the breast, the tumor is surrounded by a layer of myoepithelial cells, which exert a tumor-suppressive effect. This occurs through production of soluble mediators from the myoepithelial cells that cause an increase in p21waf transcription leading to a G2/M block and a 3-fold increase in apoptosis in the breast carcinoma cells (34). The antiproliferative effects mediated by myoepithelial cells do not have an autocrine component and are not mediated by TGF-ß1 or p53-dependent pathways. Because we have postulated that bronchial epithelial cells and the underlying myofibroblast sheath act as a trophic unit in asthma (1), the relationship between the mesenchymal-derived growth factors and epithelial survival merits further investigation.

Although corticosteroids have also been reported to induce cell cycle arrest in endothelial cells, we were unable to find any effect of budesonide on p21waf expression in subjects before and after an 8-week trial on corticosteroids, and we were also unable to find any change in p21waf expression in bronchial epithelial cells upon treatment with dexamethasone. However, because dexamethasone induced expression of p57kip2, expression of this CDK inhibitor may explain the lack of induction of PCNA in the patients with severe asthma and effect of dexamethasone in the in vitro mitogenesis assays. These observations, together with those demonstrating that corticosteroids cause apoptosis of bronchial epithelial cells in vitro (35), suggest that although corticosteroids may have a beneficial effect with respect to reducing inflammation, they may suppress epithelial repair, leading to a prolonged period during which tissue remodeling may occur.

In summary, we have shown that there is abnormally high expression of the CDK inhibitor, p21waf, in asthmatic bronchial epithelium. This may reflect the abnormally stressed phenotype of the asthmatic epithelium and may affect the ability of the cells to proliferate to replace cells that have been shed into the airways lining fluid. This data, together with our observation of increased apoptosis in asthmatic bronchial epithelium (19), suggest that there is an imbalance in the inter-relationship between survival, proliferation, and apoptosis, leading to abnormal regulation of epithelial homeostasis. This epithelial abnormality may contribute to persistent inflammation and airways remodeling in asthma.


   Acknowledgments
 
This work was supported by Programme Grant number G8604034 from the Medical Research Council (UK) and by the Sir Jules Thorn Charitable Trust.

Received in original form September 4, 2001

Received in final form July 2, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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