1 Division of Pediatric Pulmonary Diseases, Duke University Medical Center, Durham, North Carolina 27710; and 2 Children's Research Institute, Washington, District of Columbia 20010
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ABSTRACT |
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Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.
mucin; messenger ribonucleic acid; protease; airway epithelium
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INTRODUCTION |
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MUCUS HYPERSECRETION is a major pathological feature of several inflammatory airway diseases including cystic fibrosis (CF), chronic bronchitis, and asthma. The excessive mucus in the airways overwhelms the normal mucociliary clearance mechanisms, leading to obstruction and impaired pulmonary function. In addition, in CF, mucus obstruction of airways is associated with recurrent airway inflammation and infection, resulting in pulmonary fibrosis and respiratory failure. In these diseases, the pathological findings of hypertrophy and hyperplasia of mucous cells, mucus obstruction of airways, and neutrophil-predominant inflammation (18, 40, 47) suggest that mucus hypersecretion may be associated with neutrophilic inflammation.
Neutrophils are present in high concentrations in airway surface fluid (ASF) in infants with CF (23) and persist in patients with varying severity of disease (10, 25). During acute exacerbations, asthmatic patients have a high percentage of neutrophils in ASF (12, 18). In addition, exposure to several air pollutants including ozone and fine particulates (16) and cigarette smoke (39) results in increased neutrophil levels in the airway.
Neutrophils release several mediators during inflammation, and one, neutrophil elastase (NE; EC 3.4.21.37), is a serine protease that impairs mucociliary clearance by several mechanisms. NE injures cilia and decreases ciliary function (3), stimulates mucin secretion (19, 24, 31), induces secretory cell hyperplasia and hypertrophy (13, 15), and increases mucin production (14, 15). The molecular mechanism(s) by which NE stimulates mucin production is unknown. We hypothesize that as part of this mechanism, NE upregulates expression of mucin genes, leading to increased production of mucin glycoproteins.
Mucin glycoproteins, the major macromolecular constituents of mucus, impart viscoelastic qualities to mucus. They are large, heavily O-glycosylated molecules and have been difficult to characterize biochemically. By molecular technology, several mucin genes have been identified and are expressed as mRNA in the respiratory tract. Of the mucin genes expressed in respiratory epithelium, MUC5AC appears to be one of the major respiratory mucins (reviewed in Ref. 35). MUC5AC is expressed at greater levels than MUC1 or MUC2 in nasal cells (46), nasal polyp tissue (45), nasal turbinates (7), and primary bronchial epithelium (6). In addition, MUC5AC glycoprotein was recently shown to be a major component of respiratory secretions from a subject with bronchial asthma (32) and normal subjects (42). Therefore, our studies have focused on regulation of MUC5AC gene expression.
To examine the effect of NE on MUC5AC gene regulation, two different models of airway epithelia were used to induce mucociliary differentiation: A549, a lung adenocarcinoma cell line, which has been used extensively as a model of respiratory epithelium and expresses both MUC5AC mRNA and glycoprotein (9), and normal human bronchial epithelial (NHBE) cells grown in air-liquid interface culture (2, 21). In this report, we demonstrate that NE upregulated MUC5AC gene expression in both culture systems, thus providing a link between chronic neutrophilic inflammation and increased mucin production in airway diseases.
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METHODS |
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Reagents. A549 cells were obtained
from the American Type Culture Collection (Manassas, VA). Ham's F-12K
medium, DMEM, fetal bovine serum, penicillin, streptomycin, and
glutamine were from Biofluids (Rockville, MD). NHBE cells, bronchial
epithelial basic medium, and SingleQuot supplements were from Clonetics
(San Diego, CA). Rat tail collagen type I was purchased from
Collaborative Biochemical (Bedford, MA). Transwell filters were
purchased from Corning Costar (Cambridge, MA). Epidermal growth factor
and bovine serum albumin were from Intergen (Purchase, NY). Retinoic
acid, N-methoxysuccinyl-Ala-Ala-Pro-Val
chloromethyl ketone (AAPV-CMK), bovine pancreatic trypsin, papain,
actinomycin D, and ribonucleotides were from Sigma (St. Louis, MO). NE
(875 U/mg of protein) and methoxysuccinyl-Ala-Ala-Pro-Val
p-nitroanilide were from Elastin Products (Owensville, MO).
1-Antitrypsin was from
Calbiochem (San Diego, CA). Collagenase was from Worthington
Biochemical (Freehold, NJ). Nylon filter (Nytran Plus) was from
Schleicher & Schuell (Keene, NH). The pBluescript II SK(
) was
purchased from Stratagene (La Jolla, CA). X-OMAT AR film was purchased
from Kodak (Rochester, NY). The camera used for densitometry was from Fotodyne (Hartland, WI).
[
-32P]UTP and
[
-32P]dCTP were
from Amersham (Arlington Heights, IL). RNasin was obtained from Promega
(Madison, WI). RNAzol B was from Cinna/Biotex Laboratories
(Friendswood, TX). Biospin columns were from Bio-Rad (Hercules,
CA). RNase A, RNase T1, and proteinase K were from Boehringer Mannheim
(Indianapolis, IN). Milk blocking agent was from Kirkegaard & Perry
Laboratories (Gaithersburg, MD). Horseradish peroxidase-conjugated goat
anti-rabbit IgG and bicinchoninic acid protein assay were from Pierce
(Rockford, IL).
Cell culture. A549, a lung carcinoma
cell line, was cultured in Ham's F-12K medium supplemented with 10%
fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml),
and glutamine (2 mM). Cells were grown at 37°C in a humidified 5%
CO2 atmosphere. NHBE cells were
seeded on rat tail collagen type I-coated microporous membranes
(Transwell filters) in a serum-free 1:1 mixture of bronchial epithelial
cell basic medium and DMEM with SingleQuot supplements, bovine
pituitary extract (13 mg/ml), epidermal growth factor (0.5 ng/ml),
bovine serum albumin (1.5 µg/ml), and all
trans-retinoic acid (5 × 108 M) in place of
SingleQuot retinoic acid. When cells were 65% confluent, culture
conditions were changed to an air-liquid interface (2, 21). Medium was
removed from the apical surface, and medium in the basolateral chamber
was changed daily for 7 days. NHBE cells were then used for experiments.
Cell stimulation. All studies were
carried out when A549 cells were 90-95% confluent. Cells were
changed to serum-free medium. Cells were exposed to NE at doses and
times specified in figure legends. A549 cells were treated for 24 h
with elastase in the presence and absence of elastase inhibitors
1-antitrypsin or AAPV-CMK.
1-Antitrypsin or AAPV-CMK was
incubated for 15 min at room temperature with NE and then diluted
1,000-fold in medium for final concentrations of 0.65 U/ml of NE
(equivalent to 25 nM), 125 nM
1-antitrypsin, and 1 µM
AAPV-CMK. In addition to the use of elastase inhibitors, NE was boiled
for 15 min and added to the cell culture medium for 24 h (final
concentration 25 nM). All inhibitors were tested by a
spectrophotometric assay with the use of the NE-specific substrate
methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide according to the
manufacturer's instructions to ensure that elastase activity was
ablated. Control conditions included resting A549 cells or cells
treated with 50 µM sodium acetate (pH 5)-100 µM sodium chloride (NE
buffer) or elastase inhibitors alone. A549 cells were also treated with
0.65-65 U/ml of bovine pancreatic trypsin, 0.65-65 U/ml of
collagenase, and 0.65-6.5 of U/ml papain for 24 h. Cell counts
were determined for adherent and nonadherent cells, and viability was
assessed by trypan blue dye exclusion. Control conditions included
resting cells or cells treated with NE buffer or buffers for other
proteases: 0.1 µM HCl (bovine pancreatic trypsin) or 50 µM sodium
acetate, pH 4.5 (papain).
RNA isolation and Northern analysis.
RNA was isolated from cell cultures as previously described (45) with
the guanidinium thiocyanate-cesium chloride method. Total RNA (10 µg)
was separated by 1.2% agarose-formaldehyde gel electrophoresis and
transferred by capillary blot to a nylon filter (Nytran Plus) in 1 M
ammonium acetate. After ultraviolet cross-linking, the filters were
hybridized at 62°C as previously described with
32P-labeled probes (specific activity > 108
counts · min1 · µg
1)
for MUC5AC,
-actin, or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously
described (45). Filters were washed twice with 250 ml of 2×
saline-sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at
room temperature for 30 min and then with 250 ml of 0.1× SSC and
0.1% SDS at 62°C for 15 min. Filters were exposed for
autoradiography at
80°C for 4 h (
-actin or GAPDH) or 24 h
(MUC5AC). Band density on
autoradiographs was determined by digitalization with the Foto/Eclipse
camera and quantitation with National Institutes of Health Image software.
Nuclear runoff assay. Nuclei were
isolated from 1.2 × 107 A549
cells at rest or stimulated with 25 nM NE for 2, 4, or 24 h. The nuclei
were incubated with 2.7 mM ATP, 1.0 mM CTP, 1.0 mM GTP (all from
Sigma), 330 µCi of
[-32P]UTP, and 0.2 U of RNase inhibitor in 20 mM HEPES buffer (pH 7.6)-90 mM KCl-5 mM
MgCl2 at 37°C for 30 min (8).
RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform
method (RNAzol B). 32P-labeled
nascent RNA was purified by Bio-Gel P-30 column chromatography (Biospin
columns) and hybridized at 52°C for 36 h to DNA targets (10 µg)
immobilized on Nytran filter by ultraviolet cross-linking. DNA targets
included plasmids containing MUC5AC
cDNA, human
-actin cDNA, and, as a negative control, the plasmid
pBluescript II SK(
). Filters were washed three times
in 2× SSC for 15 min and then exposed to RNase A (5 µg/ml) and
RNase T1 (5 U/ml) in 2× SSC and 10 mM
Tris · HCl, pH 7.3, at 37°C for 30 min, followed
by treatment with 50 µg/ml of proteinase K in 2× SSC, 10 mM
Tris · HCl, and 0.5% SDS at 37°C for 45 min.
Filters were exposed for autoradiography at
80°C for 3 days.
RNA stability assay. A549 cells were resting or stimulated with NE (50 nM, 16 h), and then transcription was stopped by treatment with actinomycin D (5 µg/ml) for 4, 8, and 24 h (33). Total cellular RNA was extracted, and MUC5AC and GAPDH mRNA levels were evaluated by slot blot analysis and quantitated as described in RNA isolation and Northern analysis.
Western analysis. A549 cells were changed to serum-free medium for 20 h and then treated with 100 nM NE for 22 h. After addition of 1 µM AAPV-CMK, medium was collected, and cells were washed and then lysed in buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1% SDS, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, and 1 mM sodium orthovanadate) at 4°C for 15 min. Cell debris was removed by centrifugation (11,000 g) for 10 min at 4°C. Cell lysates and medium protein concentrations were determined by Pierce bicinchoninic acid protein assay. Cell lysates and medium (25 µg of total protein) were separated on a 1% agarose-Tris-acetate-EDTA-1% SDS gel by electrophoresis, and proteins were transferred under pressure to a polyvinylidene difluoride membrane as previously described (9). The membrane was blocked with milk blocking agent (1:10 dilution) and incubated with a rabbit polyclonal monospecific anti-MUC5AC antibody (1:500 dilution) (9). Membranes were developed with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution) and 10 mg of 4-chloro-1-naphthol-10 mg of 3,3'-diaminobenzidine tetrahydrochloride-0.006% hydrogen peroxide substrate. To determine whether NE digested MUC5AC glycoprotein, conditioned serum-free medium from A549 cells (48-h culture) was treated with 100 nM NE (22 h) or with vehicle control, and 25 µg of total protein from each treatment condition were evaluated for MUC5AC glycoprotein with Western analysis.
Statistical analysis. Analysis of data was performed with the Kruskal-Wallis one-way nonparametric analysis of variance and post hoc comparisons by Mann-Whitney's rank sum test. Differences were considered significant at P < 0.05.
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RESULTS |
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NE increased MUC5AC mRNA levels in a dose- and
time-dependent manner. NE (75 nM, 24 h) increased
MUC5AC mRNA levels in A549 cells
approximately sixfold compared with vehicle alone (Fig. 1A).
NE also upregulated MUC5AC in A549
cells in a time-dependent manner (Fig.
2A).
MUC5AC transcript levels started
increasing after 4 h of exposure to NE and continued to increase up to
24 h. In contrast, NE treatment caused a decrease in -actin mRNA levels over time (Fig. 2B). To
determine whether NE-induced MUC5AC expression occurred in primary airway cells, we examined expression of
MUC5AC in NHBE cells in culture.
Densitometry of Northern analyses showed that 500 nM NE (1 h)
upregulated MUC5AC mRNA levels
approximately two- to fourfold in NHBE cells (Fig.
3A).
There are differences in the kinetics of the NE effect on
MUC5AC expression between NHBE and
A549 cells. The higher concentration of NE required for induction of MUC5AC mRNA expression in
NHBE cells compared with that in A549 cells may be due to the collagen
substratum required for NHBE culture. The shorter duration of treatment
of NHBE cells for NE induction of
MUC5AC expression may be due to
differences in the mechanism of gene regulation or differences in the
survival of NHBE cells after NE treatment. Further studies in NHBE
cells are needed to clarify the etiology of these differences.
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Induction of MUC5AC mRNA expression required
proteolytically active NE. To determine whether NE
enzymatic activity was required for upregulation of
MUC5AC transcript levels, A549 cells
were incubated with inactivated NE (Fig.
4). NE proteolytic activity was completely
ablated by addition of inhibitors
1-antitrypsin or AAPV-CMK or
after boiling for 15 min. NE inactivated by preincubation with
inhibitors or by boiling (Fig. 4) did not increase
MUC5AC mRNA levels in contrast to the
increase induced by active NE (Fig. 4). Treatment with elastase
inhibitors alone did not change MUC5AC mRNA levels compared with resting or vehicle control cells (data not
shown).
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Human NE is prepared from human sputum and therefore may contain lipopolysaccharide. To determine whether the effect of NE on MUC5AC mRNA levels was due to potential lipopolysaccharide contamination, we boiled the NE preparation. Boiling the NE preparation would not affect lipopolysaccharide (29). Treatment with boiled NE did not increase MUC5AC mRNA levels (Fig. 4), suggesting that the effect of NE was due to proteolytic activity and not to potential lipopolysaccharide contamination.
MUC5AC mRNA expression was regulated by serine
protease activity. To determine whether
MUC5AC regulation by NE was
specifically related to serine protease activity, A549 cells were
treated with other classes of proteases as well as a second serine
protease, and the regulation of MUC5AC
expression was examined. Bovine pancreatic trypsin, a serine protease,
stimulated a significant increase in
MUC5AC transcript levels in A549 cells
(Fig.
5A) but
required 100-fold higher protease activity (65 U/ml) than NE (0.65 U/ml; Fig. 5A,
bar
5). At equivalent enzymatic activity
(0.65 U/ml), trypsin had no effect on
MUC5AC mRNA levels (Fig.
5A,
bar
3). In contrast, collagenase, a
metalloprotease, caused only a small change in
MUC5AC mRNA levels at 100-fold higher
activity levels (65 U/ml) than NE (Fig.
5B,
bar
5), and papain, a cysteine protease, caused no increase in MUC5AC mRNA
levels at 10-fold higher activity levels (6.5 U/ml) than NE (Fig.
5C,
bar
4). Importantly, collagenase and
papain caused significantly greater cell dissociation than NE (Table
1). Protease treatments caused no change in
-actin mRNA levels (data not shown) and no change in cell viability
(>95% for all conditions). These data suggest that serine proteases regulated MUC5AC gene expression by a
mechanism distinct from cell dissociation by other proteases.
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NE increased MUC5AC mRNA expression by enhancing mRNA
stability. To evaluate whether NE regulated
MUC5AC gene expression by a
transcriptional or posttranscriptional mechanism, nuclear runoff and
mRNA stability assays were performed. Nuclear runoff studies revealed
that NE treatment did not stimulate new transcription of
32P-labeled
MUC5AC mRNA (Fig.
6). However, RNA stability assays demonstrated an NE-induced increase in
MUC5AC mRNA half-life from 4.5 h in
resting cells to 14.75 h (Fig.
7A). In
contrast to MUC5AC, the half-life of
GAPDH mRNA was similar for resting cells (16.5 h) and NE-stimulated
cells (21 h; Fig. 7B). These
experiments are consistent with the concept that NE regulates
MUC5AC expression by enhancing mRNA
stability.
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NE treatment stimulated increased MUC5AC glycoprotein
production in A549 cells. Western analysis of MUC5AC
glycoprotein was performed to determine the effect of NE on MUC5AC
production. With the use of a rabbit polyclonal monospecific
anti-MUC5AC antibody (9), Western analysis of A549 cell medium revealed
a high-molecular-mass polydisperse band representing fully glycosylated
MUC5AC mucin glycoprotein (Fig. 8). Western
analysis of A549 cell lysate revealed the high-molecular-mass MUC5AC
glycoprotein and two additional discrete bands at approximate molecular
masses of 400 and 500 kDa. These bands probably represent
MUC5AC protein and a partially glycosylated MUC5AC glycoprotein.
Treatment with 100 nM NE (22 h) increased MUC5AC protein and
glycoprotein levels in A549 cell lysates compared with control cell
lysates (Fig. 8, lanes
1 and 2). However, MUC5AC glycoprotein
levels in the medium of NE-treated A549 cells were decreased compared
with those in control cells (Fig. 8,
lanes
3 and
4). Serine proteases have been
reported to digest mucin glycoproteins (24, 34). Therefore, we tested the ability of 100 nM NE (22 h) to digest MUC5AC in cell-free serum-free A549 cell-conditioned medium. MUC5AC glycoprotein was barely
detectable in NE-treated medium, whereas MUC5AC glycoprotein was
present in control vehicle-treated A549 cell-conditioned
medium as shown by Western analysis (Fig.
9). These experiments are consistent with
increased MUC5AC glycoprotein production and MUC5AC degradation in the
medium because of NE treatment.
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DISCUSSION |
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In this study, we demonstrated that NE increased MUC5AC gene expression and glycoprotein production. The concentrations of elastase (25-500 nM) used in this study were within the range found in ASF from CF (10, 20) and asthmatic (18) patients, suggesting that chronic NE exposure to airway epithelium may significantly contribute to mucin overproduction in these diseases.
The effect of NE on MUC5AC regulation
was investigated in two different in vitro models of airway epithelium:
A549 carcinoma cells (reviewed in Ref. 9) and primary respiratory
epithelial cells in culture. In both culture systems, NE treatment
resulted in increased MUC5AC mRNA
levels. The A549 cell line was used in this study because it expresses
both MUC5AC mRNA and glycoprotein (9)
and because it is a well-characterized model for investigating molecular and biochemical processes in airway epithelium, including inducible nitric oxide synthase expression (5), arachidonic acid
metabolism (17), respiratory viral infection (4), and nuclear
factor-B-mediated gene regulation (48). Other cancer cell lines
including NCI-H292 (27-30) and HM3 (28-30) have proven to be
useful models for investigating MUC
gene regulation.
NE has been reported to regulate gene expression of several mammalian genes in addition to MUC5AC including IL-8 (33), secretory leukocyte protease inhibitor (1), elastase-specific inhibitor, elafin (37), and intercellular adhesion molecule-1 (49). However, the mechanism of gene regulation by NE is not well understood. In this report, we demonstrated that inactivation of elastase resulted in abrogation of its effect on MUC5AC gene regulation. Furthermore, MUC5AC gene expression was regulated by serine protease activity (NE and bovine pancreatic trypsin); gene expression was not affected by cysteine protease (papain) or metalloprotease (collagenase) activities. In contrast to the report that NE upregulated IL-8 expression by cell detachment and/or deformation (38), our data suggest that cell detachment alone is not sufficient to regulate MUC5AC expression.
A major finding in this report was that in A549 cells, NE increased MUC5AC mRNA expression by increasing mRNA stability (Fig. 7). To our knowledge, this is the first report of mucin gene regulation by this mechanism. Another secreted mucin, MUC2, has a long half-life in colon cancer cells (22). MUC2 is posttranscriptionally regulated by forskolin and phorbol ester (43); however, the mechanism of posttranscriptional regulation is not due to increased mRNA stability. These reports support the concept that posttranscriptional regulation of mucin genes may be an important regulatory mechanism in disease states.
The molecular mechanisms for mRNA stability have just begun to be elucidated. The half-lives of some mammalian mRNAs are determined by protein binding to 3'-untranslated regions containing instability sequences such as AU-rich sequences or iron response elements (36). There are three short potential AU-rich sequences in the 3'-untranslated region reported for MUC5AC (26, 32) that may be related to mRNA stability. Further studies are needed to explore the role of these sequences in controlling NE enhancement of MUC5AC mRNA stability.
Several studies demonstrated that when elastase is introduced into the trachea of rodents, at first there is increased mucin granule secretion, and then over hours to days, there is an accumulation of granules in secretory cells (14, 15) and secretory cell metaplasia (13). In this report, NE treatment increased the intracellular concentration of MUC5AC glycoprotein compared with that in control vehicle-treated A549 cells. The digestion of MUC5AC glycoprotein in A549 cell medium is consistent with previous reports of mucin degradation by elastase in hamster airway cells (24) and CF mucins (34). MUC5AC was detectable in NE-treated A549 cell medium (Fig. 8) but not in NE-treated cell-free A549 cell-conditioned medium (Fig. 9). These experiments suggest that there is replacement of digested MUC5AC in the medium by cell secretion and/or that A549 cells rapidly inactivate NE by producing an anti-protease (37, 44).
There is a growing body of evidence that inflammatory mediators
increase expression of mucin genes. Tumor necrosis factor- upregulates expression of MUC2 in
NCI-H292 cells, a pulmonary mucoepidermoid carcinoma cell line (27).
Pseudomonas aeruginosa exoproducts
increase expression of MUC2 and
MUC5AC in two cancer cell lines, HM3
and NCI-H292, by transcriptional regulation (28-30). MUC5AC mRNA expression is increased in
transgenic mice overexpressing interleukin-4 (41). Both MUC5AC mRNA and
glycoprotein are induced in rat airways by exposure to acrolein, an
aldehyde found in cigarette smoke (11). In inflammatory airway
diseases, a combination of NE and these mediators may be present.
Together, these mediators may act synergistically to upregulate
expression of several mucin genes, resulting in increased mucin production.
In summary, NE treatment resulted in increased stability of MUC5AC mRNA expression in airway epithelial cells by a mechanism requiring serine proteolytic activity. Furthermore, NE treatment increased production of MUC5AC glycoprotein in A549 cell lysates. This study provides an important link in understanding the pathogenesis of NE-induced mucin production.
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ACKNOWLEDGEMENTS |
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We thank S. Erzurum, S. Chu, and J. Cohn for critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants R29-HL-50694 (to J. A. Voynow) and R01-HL-33152 (to M. C. Rose) and a Cystic Fibrosis Foundation New Investigator Award (to J. A. Voynow).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. A. Voynow, Div. of Pediatric Pulmonary Diseases, Box 2994, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: voyno001{at}mc.duke.edu).
Received 24 November 1998; accepted in final form 28 January 1999.
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