Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0056
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bronchitis,
asthma, and cystic fibrosis, marked by inflammation and mucus
hypersecretion, can be caused or exacerbated by airway pathogens or
irritants including acrolein, an aldehyde present in tobacco smoke. To
determine whether acrolein and inflammatory mediators alter mucin gene
expression, steady-state mRNA levels of two airway mucins,
MUC5AC and
MUC5B, were measured (by RT-PCR) in
human lung carcinoma cells (NCI-H292).
MUC5AC mRNA levels increased after
0.01 nM acrolein, 10 µM prostaglandin
E2 or 15-hydroxyeicosatetraenoic acid, 1.0 nM tumor necrosis factor-
(TNF-
), or 10 nM phorbol 12-myristate 13-acetate (a protein kinase C activator). In
contrast, MUC5B mRNA levels, although
easily detected, were unaffected by these agonists, suggesting that
irritants and associated inflammatory mediators increase mucin
biosynthesis by inducing MUC5AC
message levels, whereas MUC5B is
constitutively expressed. When transcription was inhibited, TNF-
exposure increased MUC5AC message
half-life compared with control level, suggesting that transcript
stabilization is a major mechanism controlling
increased MUC5AC message levels. Together, these findings imply that irritants like acrolein can directly and indirectly (via inflammatory mediators) increase airway
mucin transcripts in epithelial cells.
aldehyde; chronic obstructive pulmonary disease; cytokine; eicosanoids
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EXCESSIVE MUCUS SECRETION is a hallmark in the
pathogenesis of several airway diseases including chronic bronchitis,
asthma, and cystic fibrosis (34). Patients suffering from these
diseases have pathological abnormalities in both the submucosal glands and surface epithelium, characterized by inflammation, increased mucous
cell number, and excessive airway mucus. Several classes of
inflammatory mediators have been implicated in the process of mucus
hypersecretion based on their ability to stimulate secretion from
cultured cells and tissue explants. These include cytokines [e.g., tumor necrosis factor- (TNF-
)] (12, 31),
proteases (27, 50), reactive oxygen species (2), and arachadonic acid
metabolites (17, 36). The mechanisms controlling mucus secretion are
not completely understood but are likely to involve protein kinase (PK)
C- and calcium-dependent signaling pathways (1).
Mucus glycoproteins (mucins) provide airway secretions with their characteristic adhesiveness, elasticity, and viscosity. Mucins are large heterogeneous proteins (20,000-30,000 kDa) that vary in length from 200 to 4,000 nm (49). As much as 80% of their molecular mass consists of carbohydrate side chains linked by glycosyltransferases to serine and threonine residues of the peptide backbone (52). Mucin peptides are encoded by multiple genes characterized by DNA sequences with variable numbers of tandem repeat domains. These sequences vary in composition and size (>50% of protein) but have the common feature of being rich nucleotide sequences encoding threonine, serine, and proline (18). Nine distinct mucin genes (MUC1-MUC4, MUC5AC, MUC5B, and MUC6-MUC8) have been described in the respiratory, gastrointestinal, and reproductive tracts (reviewed in Ref. 47). Of these, MUC5AC and MUC5B proteins have been isolated from human airway secretions and are considered to be major constituents of the mucous layer (40, 45, 48, 53). The large size and complex repetitive nature of mucin genes have hindered the study of cellular events involved in mucus synthesis and secretion. Control of mucin synthesis likely involves both transcriptional (32, 35) and posttranscriptional mechanisms (56) that regulate mRNA formation and stability, protein translation and glycosylation, and granule formation and exocytosis.
Mucus hypersecretion can also be experimentally induced in response to irritants including tobacco smoke (46). Acrolein (CH2==CHCHO) is a potent irritant aldehyde present in high concentrations [50-70 parts/million (ppm)] in tobacco smoke (5) and is a constituent of wood smoke, diesel exhaust, and photochemical smog (3). Previously, Borchers et al. (7) reported that rats exposed to acrolein develop increased numbers of epithelial mucous cells containing MUC5AC and that mucus hypersecretion in the airways was preceded by increases in steady-state MUC5AC mRNA levels. Exposure to acrolein also results in acute and chronic airway inflammation (30, 33) and increased release of the arachadonic acid metabolites prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) (16, 30). Because these mediators are known agonists of mucus secretion (17, 36), their formation after acrolein exposure represents a possible mechanism by which mucin synthesis is controlled.
The present study was designed to determine whether mucin genes were
regulated at the mRNA level by irritant exposure and inflammatory
mediators. Toward this goal, we investigated the effects of acrolein,
eicosanoids, TNF-, and a PKC activator on MUC5AC and
MUC5B steady-state mRNA levels in
human lung carcinoma cells. Because steady-state mRNA levels are
controlled by transcription rate and mRNA stability, we also examined
the ability of TNF-
to increase message half-life.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental design. To determine
whether acrolein, inflammatory mediators, or a PKC agonist affects
mucin mRNA expression, cultured cells derived from a human lung cancer
(NCI-H292) (10) were exposed to acrolein,
PGE2, 15-HETE, TNF-, phorbol
12-myristate 13-acetate (PMA), or vehicle control for 4 h (37°C, pH
7.4). To determine whether the effects on mucin gene expression were
unique to NCI-H292 cells, another human lung carcinoma cell line (A549) (19) was exposed to TNF-
or PMA. NCI-H292 and A549 cells were selected because increased MUC5AC mRNA
levels are accompanied by increased MUC5AC protein synthesis and mucus
secretion in these cells (26, 58). After exposure, the cells were
lysed, total RNA was isolated, and mucin
(MUC5AC and
MUC5B) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA levels were analyzed by
RT-PCR. In preliminary studies with NCI-H292 cells, maximal
MUC5AC induction by each agonist
occurred between 1 and 4 h. Agonist concentrations were selected based
on previous studies (17, 31, 36) that demonstrated that similar doses
stimulate mucus secretion in epithelial cell explants and cultures. To
examine whether TNF-
regulates MUC5AC mRNA levels by
posttranscriptional mechanisms, NCI-H292 cells were treated with
TNF-
or vehicle control for 2 h, followed by the addition of
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor (59).
Cell culture. NCI-H292 (a human pulmonary mucoepidermoid carcinoma) and A549 cells (human lung carcinoma) were purchased from the American Type Culture Collection. The cells were grown in 75-cm2 plastic tissue culture flasks (Costar, Cambridge, MA). NCI-H292 or A549 cells were maintained in RPMI 1640 medium or Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Life Technologies, Grand Island, NY), respectively, each supplemented with 10% fetal calf serum (BioCell, Rancho Dominguez, CA), penicillin (100 U/ml), and streptomycin (100 µg/ml; both from Sigma, St. Louis, MO) (37°C, pH 7.4). The cells were seeded at a density of 5,000 cells/cm2 and passaged at ~90% confluence.
Acrolein and agonist exposures.
NCI-H292 or A549 cells were seeded (5,000 cells/cm2) into six-well plates
(Corning) and grown to confluence. The cells were incubated (37°C,
pH 7.4) for 20 h in serum-free medium (RPMI 1640 or DMEM) and
subsequently treated with each agent in phosphate-buffered saline (PBS;
GIBCO BRL). The cells were treated for 4 h (37°C, pH 7.4) with
acrolein (0.01-100 nM; Aldrich, Milwaukee, WI), 15-HETE (10 µM;
Sigma), PGE2 (10 µM; Sigma),
TNF- (1 nM; R&D Systems, Minneapolis, MN), or vehicle (0.05%
ethanol) in PBS. After exposure, the medium was removed, and the cells
were lysed in 4 M guanidine thiocyanate (Fisher, Fair Lawn, NJ). The
resulting solution was stored at
70°C until RNA isolation.
MUC5AC mRNA stability.
MUC5AC message half-life and the
effect of TNF- treatment on mRNA stability was examined by exposing NCI-H292 cells to the pharmacological transcription inhibitor DRB (59).
In these experiments, cells were treated for 2 h with TNF-
(1 nM) to
achieve maximum MUC5AC levels. At
time 0, DRB (100 nM) was added, and
the cells were incubated for an additional 0, 1, 2, 4, 8, or 16 h.
After each exposure period, the cells were lysed, total RNA was
isolated, and MUC5AC mRNA levels were determined by RT-PCR analysis. Degradation of
c-myc mRNA (half-life
15 min) was
also examined by RT-PCR to evaluate DRB activity.
RNA isolation and RT-PCR analysis.
Total RNA was isolated from cultured cells by the
guanidinium-phenol-chloroform procedure described by Chomczynski (11).
The purity was estimated by spectrophotometric determination of the
260- to 280-nm absorption ratio. Purified RNA was stored at
70°C until analyzed by RT-PCR. RT-PCR was chosen for these
experiments because smeared signals (continuous bands of multiple
sizes) were produced by Northern analysis of mucin mRNAs, resulting in
difficulties in quantification (55, 57). In contrast, a single band was
generated by RT-PCR by selecting primers in the nonrepeat regions of
the mucin genes.
For RT-PCR, primers were generated from published sequences of MUC5AC (38), MUC5B (15), GAPDH (54), and c-myc (13). All primers except c-myc (Clontech, Palo Alto, CA) were synthesized by the University of Cincinnati (OH) DNA Core Facility. Primer sequences were as follows: MUC5AC lower primer (position 1945), 5'-ACT TGG GCA CTG GTG CTG-3'; MUC5AC upper primer (position 1283), 5'-TCC GGC CTC ATC TTC TCC-3' (product length 680 bp); MUC5B lower primer (position 2688), 5'-CAG TGG CAG AGG CCG TGC AGT A-3'; MUC5B upper primer (position 106), 5'-CAG GGC ATT TGG ACA GTT TTT C-3' (product length 544 bp); GAPDH lower primer, 5'-TGC TGG GGC TGG TGG TC-3'; GAPDH upper primer, 5'-TCA AGT GGG GCG ATG CTG-3'; c-myc lower primer, 5'-TCT TGA CAT TCT CCT CGG TGT CCG AGG ACC T-3'; and c-myc upper primer, 5'-TAC CCT CTC AAC GAC AGC AGC TCG CCC AAC TCC T-3'. RT was carried out in a 10-µl mixture containing total RNA (500 ng of MUC5AC, 50 ng of MUC5B, 250 ng of GAPDH, and 50 ng of c-myc RNAs) and 25 units of Superscript II reverse transcriptase (GIBCO BRL) in buffer containing 10 mM dithiothreitol, 1 mM deoxynucleotide triphosphates (Promega, Madison, WI), 10 units of RNasin (Promega), and 0.2 µM 3' oligonucleotide primer. First-strand synthesis consisted of primer annealing (25°C, 10 min) and template extension (42°C, 45 min) (Biometra Thermocycler, Tampa, FL). Newly synthesized cDNA was amplified by PCR in 50 µl with 0.75 units of Taq polymerase (GIBCO BRL) in Taq buffer (GIBCO BRL) containing 1.5 mM MgCl2 and 0.2 µM 5' primer. The amplification conditions were 20 s at 94°C, 30 s at the annealing temperature (Ta) specific for each primer pair, and 30 s plus 1 s/cycle for n number of cycles (MUC5AC: Ta = 60°C, n = 34; MUC5B: Ta = 59.8°C, n = 30; GAPDH: Ta = 58°C, n = 25; c-myc: Ta = 60°C, n = 30). The specificity of the PCR products was confirmed by dye terminator sequencing with an ABI PRISM cycle sequencing kit (Perkin-Elmer, Foster City, CA).
Quantitation of PCR
products. PCR products were quantitated by densitometry
as previously described (7). DNA (10 µl) was electrophoresed on a 2%
agarose gel containing 0.5 µg/ml of ethidium bromide in 90 mM Tris
phosphate-2 mM EDTA buffer. After electrophoresis, DNA was visualized
by ultraviolet illumination and photographed (type 665 black and white
film, Polaroid). The resulting image was scanned and analyzed by an
image-analysis software program (Mocha, Jandel Scientific), and the
total intensity (average intensity multiplied by total pixels) of each
band was measured. All messages examined amplified exponentially (until
saturation) according to the amount of target mRNA in the sample. The
relationship between mRNA level and band intensity was determined by a
curve-fitting software program (SigmaPlot, Jandel Scientific) and found
to be y = a[1 exp(
bx)] + c, where
a is the amplitude of exponential, b is the rate constant, and
c is the zero intercept. For each RT-PCR, a serial dilution (1,000-32 ng) of total mRNA was
amplified and included on each gel to obtain an internally consistent
reference curve. All samples were analyzed in the linear portion of the curve. The relative amount of mRNA was determined by comparing the
total intensity of each sample against the standard curve. Samples were
analyzed in duplicate, and mucin mRNA levels are expressed as multiple
of increase over control level after normalization to GAPDH.
Data analysis. Mucin mRNA levels were
determined in duplicate from three to six separate experiments and are
presented as means ± SE. Student's
t-test was used to determine
differences between exposed and control groups. Values with
P 0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of acrolein exposure on mucin mRNA
levels. Dose-dependent increases (1.5 ± 0.1-, 2.9 ± 0.6-, and 2.1 ± 0.4-fold) in MUC5AC mRNA levels in NCI-H292 cells
were observed after 4-h exposures to 0.01, 0.1, and 1.0 nM acrolein,
respectively (Fig. 1).
MUC5B mRNA levels were not increased
at any concentration tested. After exposure to 100 nM acrolein, mRNA
levels encoding MUC5AC,
MUC5B, and
GAPDH were significantly less than the
control levels (Fig. 1). Recovery of total RNA was also reduced at 100 nM (data not shown), indicating that decreased message levels may be a
cytotoxic effect of the highest acrolein exposure.
|
Agonist effects on mucin mRNA levels.
Eicosanoid inflammatory mediators associated with acrolein exposures,
PGE2 and 15-HETE significantly
increased steady-state MUC5AC mRNA
levels in NCI-H292 cells (Fig. 2). Levels
of MUC5B mRNA did not change after
exposure to these eicosanoids (Fig. 2). Because TNF- may be an
important mediator of mucus hypersecretion in human diseases (8, 31) and PMA activates a major signaling pathway in mucus secretion (1),
these agonists were also examined for their ability to increase mucin
mRNA synthesis. TNF-
and PMA also increased
MUC5AC but not
MUC5B mRNA levels in NCI-H292 cells
(Fig. 2). The arachadonic acid metabolites
PGE2 or 15-HETE increased
MUC5AC expression (2.1 ± 0.2- and
2.0 ± 0.2-fold, respectively) to a lesser extent than either
TNF-
or PMA (3.0 ± 0.4- and 4.1 ± 1.0-fold, respectively) at
the concentrations tested (Fig. 2). These increases are specific because the control mRNA, GAPDH, was
not altered by any exposure. Differential regulation of
MUC5AC and
MUC5B also occurred in another mucin-secreting cell line (Fig. 3).
MUC5AC mRNA levels increased in A549
cells exposed to TNF-
(2.3 ± 0.2-fold) and PMA (3.1 ± 0.2-fold). Consistent with results from the NCI-H292 cell line, MUC5B mRNA levels were not
significantly changed in A549 cells after these exposures (Fig. 3).
|
|
Under basal conditions, MUC5B could be detected with 10-fold less total RNA and with fewer PCR cycles than those necessary to detect MUC5AC. The abundance of mRNA detected by RT-PCR and the lack of induction by agonists of mucus secretion suggest that MUC5B may be constitutively expressed (Figs. 1-3).
Mechanism of MUC5AC mRNA expression induced by
TNF-. To investigate the mechanism of
MUC5AC mRNA induction, the time course was initially measured. TNF-
stimulated a rapid and transient increase in MUC5AC mRNA levels (Fig.
4). MUC5AC
mRNA levels were significantly increased at 1 h, maximal at 2 h, and
subsequently decreased, returning to control levels at 16 h
posttreatment. MUC5B and
GAPDH mRNA levels were not altered by
TNF-
exposure.
|
To determine whether TNF- affected
MUC5AC mRNA stability, transcription
was pharmacologically inhibited (59) after peak message induction (2 h), and the rate of mRNA decay was determined (Fig.
5). In the absence of TNF-
stimulation,
the half-life (defined as the time at which 50% of mRNA remained) of
MUC5AC in NCI-H292 cells was ~4 h
(Fig. 5). After TNF-
stimulation, the approximate half-life of
MUC5AC mRNA was increased to almost 10 h. This 2.5-fold increase in mRNA half-life could account for most of
the 3-fold increase in MUC5AC
steady-state mRNA levels observed after TNF-
treatment (Fig. 4).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the mediators and mechanisms controlling mucus secretion have
been well studied, less is known regarding the mediators and mechanisms
regulating airway mucin synthesis. In this study, we demonstrate that
in cells derived from human lung cancers, MUC5AC but not
MUC5B is regulated at the mRNA level
by acrolein, inflammatory mediators, and PMA. In addition, we found
that increased message stability is one of the mechanisms involved in
the induction of MUC5AC mRNA after
TNF- exposure.
Exposure to several respiratory tract irritants, including acrolein,
can cause mucus hypersecretion in animals (23, 29, 33). Recently,
Borchers et al. (7) demonstrated that acrolein-induced mucus
hypersecretion is associated with increased
MUC5AC mRNA and protein synthesis. The
present study suggests that acrolein can act directly on epithelial
cells to increase mucin mRNA levels (Fig. 1) or indirectly through
inflammatory mediators (PGE2 or 15-HETE; Fig. 2) released after exposure (16, 22, 30). High concentrations (10-100 µM) of acrolein increase eicosanoid
release from airway epithelial cells (16) or alveolar macrophages (22) and can decrease aldehyde-metabolizing enzymes in the liver (39, 44). A
lower concentration (1 µM) can deplete intracellular glutathione
(40%) in human bronchial epithelial cells (21, 28). The low acrolein
dose (0.01-1.0 nM) inducing
MUC5AC mRNA indicates that this end
point is more sensitive to the direct effects of acrolein exposure than
other end points investigated. At 100 nM acrolein,
MUC5AC mRNA levels were decreased.
Because glutathione depletion and eicosanoid synthesis occur at
doses > 100 nM in other cells and eicosanoid synthesis
was not measured in the present study, whether these mechanisms
are involved in MUC5AC induction in
this experiment are unknown. However, it is possible that small reductions (20%) in glutathione could activate redox-sensitive transcription factors such as nuclear factor-B (43). This
possibility is supported by evidence demonstrating nuclear factor-
B
binding sites within the functional promoter region of
MUC5AC (32).
Acrolein exposure in vivo may nonetheless increase
MUC5AC mRNA levels through the release
of mediators such as eicosanoids. Instillation of
PGE1 induces mucous cell
development and mucin synthesis in mouse airways (41). Additionally,
indomethacin, a prostaglandin synthetase inhibitor, can inhibit mucin
synthesis in rat lung after tobacco smoke exposure (46). Although
eicosanoid levels were not measured in this study,
PGE2 levels increase in the
airways of guinea pigs after 1.3 ppm acrolein exposure (30). Acrolein
exposure of 1.5 ppm increased MUC5AC
mRNA and protein in the rat lung (7). In this study,
PGE2 and 15-HETE increased MUC5AC mRNA levels in human airway
epithelial cells (Fig. 2).
Mucus hypersecretion is a feature of inflammatory airway disorders
associated with increased TNF- production, including asthma (8),
respiratory syncytial virus infection (6), and cystic fibrosis (42). We
therefore examined the regulation of mucin mRNA in response to the
proinflammatory cytokine TNF-
. TNF-
is secreted predominantly by
activated macrophages and monocytes and can increase mucus secretion
and MUC2 mRNA in human airway epithelial cells (31). We found that TNF-
treatment increased MUC5AC but not
MUC5B mRNA levels in both NCI-H292 and
A549 cells (Figs. 2 and 3). In NCI-H292 cells, TNF-
increased
MUC5AC mRNA levels over a period of 8 h, reaching maximal levels by 4 h (Fig. 4). This is consistent with
reports by other investigators who examined TNF-
- and PMA-induced
MUC2 mRNA levels in airway (31) and
colonic (56) epithelial cells. We did not measure mucin protein levels
in this study. However, mucin mRNA induction is relatively short
lasting (
8 h) compared with the duration of mucin secretion after
TNF-
exposure (24-72 h) (31). This suggests that subsequent
translational and posttranslational mechanisms are involved in the
regulation of mucin synthesis and secretion.
One mechanism by which TNF- regulates
MUC5AC mRNA levels is by increasing
the stability of the transcript (Fig. 5). TNF-
can also increase
message stability of a glucose transporter gene, GLUT-1 (37, 51). Message stability of
GLUT-1 involves induction of proteins
that bind to mRNA destabilizing sequences in the 3'-untranslated region (3'-UTR), thus protecting the transcript against
nucleolytic cleavage. Approximately 500 bp of the
MUC5AC 3'-UTR have been sequenced (9, 38), but whether specific elements within this region
control message stabilization remains to be investigated. Although this
is the most studied and best understood mechanism, other determinants
of mRNA stability have also been demonstrated. The formation of
stem-loop structures within the 3'-UTR that serve as protein
binding sites and protein binding within the coding region of
transcripts also result in an increased message half-life (24). Given
that the rate of protein synthesis is directly proportional to the
respective cytoplasmic mRNA levels (24), the ability of a cell to
increase the stability of such a large message (~14 kb) represents a
potentially rapid and efficient means to increase the amount of
templates available for translation. This is especially relevant in the
context of acute irritation or infection reactions when cells deplete
stored mucous granules and must therefore rapidly synthesize nascent
mucin proteins.
MUC5AC has been identified in both submucosal glands and surface epithelial cells (4), whereas MUC5B has been localized primarily within submucosal glands (4, 46). Although this study and others (7, 20, 26, 32) indicate that MUC5AC is regulated at the mRNA level, MUC5B mRNA levels were unchanged in both cell lines examined. The lack of MUC5B induction by agonists of mucin synthesis and secretion suggests that its resulting protein may be constitutively expressed. In normal airways, MUC5B could be responsible for maintaining basal levels of mucin synthesis and secretion within secretory cells. In chronic airway diseases, increases in mucin secretion could thereby be achieved through the marked enlargement of the submucosal glands, accompanied by increases in the number of cells involved in MUC5B synthesis. Having an alternative, inducible mucin like MUC5AC enables rapid responses to irritants. In addition, chronic airway diseases often lead to changes in the biochemical composition of airway mucus (14, 25), possibly provided through the differential regulation of inducible and noninducible forms airway mucins.
In summary, we found that MUC5AC but
not MUC5B mRNA levels are regulated by
acrolein and inflammatory mediators. One mediator, TNF-,
regulates MUC5AC by
posttranscriptional mechanisms. These results suggest that direct
irritant exposure as well as inflammatory mediators contribute to the
pathogenesis of obstructive airway disease by increasing mucin mRNA
synthesis in airway epithelial cells. The details of the regulation of
mucin synthesis now beginning to emerge should increase our
understanding of the relative role of mucin genes in airway
hypersecretion and provide valuable insights into clinical strategies
aimed at alleviating excessive mucus.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Carol Basbaum and Kevin Driscoll for useful advice and suggestions.
![]() |
FOOTNOTES |
---|
This study was supported by National Institute of Environmental Health Sciences Grants R01-ES-06562, R01-ES-06677, and P30-ES-06096 and National Heart, Lung, and Blood Institute Grant R01-HL-58275.
M. T. Borchers is a recipient of a United States Environmental Protection Agency J. Stara Scholarship Award and University of Cincinnati (OH) Graduate Assistantship. This work is in partial fulfillment of the PhD degree requirements at the University of Cincinnati.
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: G. D. Leikauf, Dept. of Environmental Health, Univ. of Cincinnati, PO Box 670056, Cincinnati, OH 45267-0056 (E-mail: Leikaugd{at}ucmail.uc.edu).
Received 23 September 1998; accepted in final form 23 December 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abdullah, L. H.,
J. D. Conway,
J. A. Cohn,
and
C. W. Davis.
Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L201-L210,
1997
2.
Adler, K. B.,
W. J. Holden-Stauffer,
and
J. E. Repine.
Oxygen metabolites stimulate release of high-molecular-weight glycoconjugates by cell and organ cultures of rodent respiratory epithelium via an arachadonic acid-dependent mechanism.
J. Clin. Invest.
85:
75-85,
1990[Medline].
3.
Altshuler, A. P.,
and
S. P. McPherson.
Spectrophotometric analysis of aldehydes in the Los Angeles atmosphere.
J. Air Pollut. Control Assoc.
13:
109-111,
1963.
4.
Audie, J. P.,
A. Janin,
N. Porchet,
M. C. Copin,
B. Gosselin,
and
J. P. Aubert.
Expression of human mucin genes in respiratory digestive and reproductive tracts ascertained by in situ hybridization.
J. Histochem. Cytochem.
41:
1479-1485,
1993
5.
Ayer, H. E.,
and
D. W. Yeager.
Irritants in cigarette smoke plumes.
Am. J. Public Health
72:
1283-1285,
1982[Abstract].
6.
Becker, S. J.,
J. Quay,
and
J. Soucop.
Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophage.
J. Immunol.
147:
4307-4312,
1991
7.
Borchers, M. T.,
S. E. Wert,
and
G. D. Leikauf.
Acrolein-induced MUC5ac expression in rat airways.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L573-L581,
1998
8.
Broide, D. H.,
M. Lotz,
A. J. Cuomo,
D. A. Coburn,
E. C. Federman,
and
S. I. Wasserman.
Cytokines in symptomatic asthma airways.
J. Allergy Clin. Immunol.
89:
958-967,
1992[Medline].
9.
Buisine, M.-P.,
J.-L. Desseyn,
N. Porchet,
P. Degand,
A. Laine,
and
J.-P. Aubert.
Genomic organization of the 3'-region of the human MUC5AC mucin gene: additional evidence for a common ancestral gene for the 11p15.5 mucin gene family.
Biochem. J.
332:
729-738,
1998[Medline].
10.
Carney, D. N.,
A. F. Gazdar,
G. Bepler,
J. G. Guccion,
P. J. Marangos,
T. W. Moody,
M. H. Zweig,
and
J. D. Minna.
Establishment and identification of small cell lung cancer lines having classic and variant features.
Cancer Res.
45:
2913-2923,
1985[Abstract].
11.
Chomczynski, P.
A reagent for the single-step simultaneous isolation of RNA, DNA and protein from cell and tissue samples.
Biotechniques
15:
532-536,
1993[Medline].
12.
Cohan, V. L.,
A. L. Scott,
C. A. Dinarello,
and
R. A. Prendergrast.
Interleukin-1 is a mucus secretagogue.
Cell. Immunol.
136:
425-434,
1991[Medline].
13.
Colby, W. W.,
E. Y. Chen,
D. H. Smith,
and
A. D. Levinson.
Identification and nucleotide sequence of a human locus homologous to the v-myc oncogene of avian myelocytomatosis virus MC29.
Nature
301:
722-725,
1983[Medline].
14.
Davies, J. R.,
H. W. Hovenberg,
C.-J. Linden,
R. Howard,
P. S. Richardson,
J. K. Sheehan,
and
I. Carlstedt.
Mucins in airway secretions from healthy and chronic bronchitis subjects.
Biochem. J.
313:
431-439,
1996[Medline].
15.
Desseyn, J.-L.,
J.-P. Aubert,
I. Van Seuningen,
N. Porchet,
and
A. Laine.
Genomic organization of the 3' region of the human mucin gene MUC5B.
J. Biol. Chem.
272:
16873-16883,
1997
16.
Doupnik, C. A.,
and
G. D. Leikauf.
Acrolein stimulates eicosanoid release from bovine airway epithelial cells.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L222-L229,
1990
17.
Enss, M.-L.,
S. Wagner,
U. Schmidt-Wittig,
H.-K. Heim,
W. Bell,
and
H. J. Hedrich.
Effects of PGE2 on amount and composition of high molecular weight glycoproteins released by human gastric mucous cells in primary culture.
Prostaglandins Leukot. Essent. Fatty Acids
56:
93-98,
1997[Medline].
18.
Gendler, S. J.,
and
A. P. Spicer.
Epithelial cell mucins.
Annu. Rev. Physiol.
57:
607-634,
1995[Medline].
19.
Giard, D. J.,
S. A. Aaronson,
G. J. Todaro,
P. Arnstein,
J. H. Kersey,
H. Dosik,
and
W. P. Parks.
In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors.
J. Natl. Cancer Inst.
51:
1417-1423,
1973[Medline].
20.
Gollub, E. G.,
H. Waksman,
S. Goswami,
and
Z. Marom.
Mucin genes are regulated by estrogen and dexamethasone.
Biochem. Biophys. Res. Commun.
217:
1006-1014,
1995[Medline].
21.
Grafstrom, R. C.,
J. M. Dypbukt,
J. C. Willey,
K. Sundqvist,
C. Edman,
L. Atzori,
and
C. C. Harris.
Pathobiological effects of acrolein in cultured human bronchial epithelial cells.
Cancer Res.
48:
1717-1721,
1988[Abstract].
22.
Grundfest, C. C.,
J. Chang,
and
D. Newcombe.
Acrolein: a potent modulator of lung macrophage arachadonic acid metabolism.
Biochim. Biophys. Acta
713:
149-159,
1982[Medline].
23.
Harkema, J. R.,
C. G. Plopper,
D. M. Hyde,
J. A. St. George,
and
D. L. Dungworth.
Effects of an ambient level of ozone on primate nasal epithelial mucosubstances: quantitative histochemistry.
Am. J. Pathol.
127:
90-96,
1987[Abstract].
24.
Hentze, M. W.
Determinants and regulation of cytoplasmic mRNA stability in eukaryotic cells.
Biochim. Biophys. Acta
1090:
281-292,
1991[Medline].
25.
Jones, R.,
and
L. Reid.
Secretory cell hyperplasia and modification of intracellular glycoprotein in rat airways induced by short periods of exposure to tobacco smoke, and the effect of the antiinflammatory agent phenylmethyloxadiazole.
Lab. Invest.
39:
41-52,
1978[Medline].
26.
Kai, H.,
K. Yoshitake,
A. Hisatsune,
T. Kido,
Y. Isohama,
K. Takahama,
and
T. Miyata.
Dexamethasone suppresses mucus production and MUC-2 and MUC-5AC gene expression by NCI-H292 cells.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L484-L488,
1996
27.
Klingler, J. D.,
B. Tandler,
C. M. Liedtke,
and
T. F. Boat.
Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium.
J. Clin. Invest.
74:
1669-1678,
1984[Medline].
28.
Lam, C.,
M. Casanova,
and
H. d'A. Heck.
Depletion of nasal mucosal glutathione by acrolein and enhancement of formaldehyde-induced DNA-protein cross-linking by simultaneous exposure to acrolein.
Arch. Toxicol.
58:
67-71,
1985[Medline].
29.
Lamb, D.,
and
L. Reid.
Mitotic rates, goblet cell increase and histochemical changes in mucus in rat bronchial epithelium during exposure to sulphur dioxide.
J. Pathol. Bacteriol.
96:
97-111,
1968[Medline].
30.
Leikauf, G. D.,
L. M. Leming,
J. R. O'Donnell,
and
C. A. Doupnik.
Bronchial responsiveness and inflammation in guinea pigs exposed to acrolein.
J. Appl. Physiol.
66:
171-178,
1989
31.
Levine, S. J.,
P. Larivee,
C. Logun,
C. W. Angus,
F. P. Ognibene,
and
J. H. Shelhamer.
Tumor necrosis factor- induces mucin hypersecretion and MUC2 gene expression by human airway epithelial cells.
Am. J. Respir. Cell Mol. Biol.
12:
196-204,
1995[Abstract].
32.
Li, D.,
M. Gallup,
N. Fan,
D. E. Szymkowski,
and
C. B. Basbaum.
Cloning of the amino-terminal and 5'-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts.
J. Biol. Chem.
273:
6812-6820,
1998
33.
Lyon, J.,
L. Jenkins,
R. Jones,
R. Coon,
and
J. Siegel.
Repeated and continuous exposure of laboratory animals to acrolein.
Toxicol. Appl. Pharmacol.
17:
726-732,
1970[Medline].
34.
Lundgren, J. D.,
and
J. H. Shelhamer.
Pathogenesis of airway mucus hypersecretion.
J. Allergy Clin. Immunol.
85:
399-417,
1990[Medline].
35.
Manna, B.,
P. Ashbaugh,
and
S. N. Bhattacharyya.
Retinoic acid-regulated cellular differentiation and mucin gene expression in isolated rabbit tracheal epithelial cells in culture.
Inflammation
19:
489-502,
1995[Medline].
36.
Marom, Z.,
J. H. Shelhamer,
F. Sun,
and
M. Kaliner.
Human airway monohydroxyeicosatetraenoic acid generation and release.
J. Clin. Invest.
72:
122-127,
1983[Medline].
37.
McGowan, K. M.,
S. Police,
J. B. Winslow,
and
P. H. Pekala.
Tumor necrosis factor- regulation of glucose transporter (GLUT1) mRNA turnover.
J. Biol. Chem.
272:
1331-1337,
1997
38.
Meerzaman, D.,
P. Charles,
E. Daskal,
M. H. Polymeropoulos,
B. M. Martin,
and
M. C. Rose.
Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5).
J. Biol. Chem.
269:
12932-12939,
1994
39.
Mitchell, D. Y.,
and
D. R. Peterson.
Inhibition of rat liver aldehyde dehydrogenases by acrolein.
Drug Metab. Dispos.
16:
37-42,
1988[Abstract].
40.
Nguyen, P. L.,
G. A. Niehans,
D. L. Cherwitz,
Y. S. Kim,
and
S. B. Ho.
Membrane bound (MUC1) and secretory (MUC2, MUC3, and MUC4) mucin gene expression in human lung cancer.
Tumour Biol.
17:
176-192,
1996[Medline].
41.
Nygren, H.,
S. Lange,
and
I. Lonnroth.
Development of mucous cells in mouse intrapulmonary airways induced by cholera toxin, dibutyryl cyclic AMP and prostaglandin E1.
Br. J. Exp. Pathol.
65:
549-556,
1984[Medline].
42.
Pfeffer, K. D.,
T. P. Hueckstead,
and
J. R. Hoidal.
Expression and regulation of tumor necrosis factor in macrophages from cystic fibrosis patients.
Am. J. Respir. Cell Mol. Biol.
9:
511-519,
1993[Medline].
43.
Pinkus, R.,
L. M. Weiner,
and
V. Daniel.
Role of oxidants and antioxidants in the induction of AP-1, NF-B, and glutathione-s transferase gene expression.
J. Biol. Chem.
271:
13422-13429,
1996
44.
Pompella, A.,
A. Romani,
A. Benedetti,
and
M. Comporti.
Loss of membrane protein thiols and lipid peroxidation in allyl alcohol hepatotoxicity.
Biochem. Pharmacol.
41:
1255-1259,
1991[Medline].
45.
Reid, C. J.,
S. Gould,
and
A. Harris.
Developmental expression of mucin genes in the human respiratory tract.
Am. J. Respir. Cell Mol. Biol.
17:
592-598,
1997
46.
Rogers, D. F.,
and
P. K. Jeffery.
Inhibition of cigarette smoke-induced airway secretory cell metaplasia by indomethacin, dexamethasone, prednisolone, or hydrocortisone in the rat.
Exp. Lung Res.
10:
285-298,
1986[Medline].
47.
Rose, M. C.,
and
S. J. Gendler.
Airway mucin genes and gene products.
In: Airway Mucus: Basic Mechanisms and Clinical Perspectives, edited by D. F. Rogers,
and M. I Lethem. Boston, MA: Birkhauser Verlag, 1997, p. 41-66.
48.
Rose, M. C.,
B. Kaufman,
and
B. M. Martin.
Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin.
J. Biol. Chem.
264:
8193-8199,
1989
49.
Sheehan, J. K.,
K. Oates,
and
I. Carlstedt.
Electron microscopy of cervical, gastric, and bronchial mucus glycoproteins.
Biochem. J.
239:
147-153,
1986[Medline].
50.
Sommerhoff, C. P.,
J. A. Nadel,
C. B. Basbaum,
and
G. H. Caughey.
Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.
J. Clin. Invest.
85:
682-689,
1990[Medline].
51.
Stephens, J. M.,
B. Z. Carter,
P. H. Pekala,
and
J. S. Malter.
Tumor necrosis factor -induced glucose transporter (GLUT1) mRNA stabilization in 3T3-L1 preadipocytes.
J. Biol. Chem.
267:
8336-8341,
1992
52.
Strous, G. J.,
and
J. Dekker.
Mucin-type glycoproteins.
Crit. Rev. Biochem. Mol. Biol.
27:
57-92,
1992[Abstract].
53.
Thornton, D. J.,
M. Howard,
N. Khan,
and
J. K. Sheehan.
Identification of two isoforms of MUC5B mucin in human respiratory mucus.
J. Biol. Chem.
272:
9561-9566,
1997
54.
Tokunaga, K.,
Y. Nakamura,
K. Sakata,
K. Fujimori,
M. Ohkubo,
K. Sawada,
and
S. Sakiyama.
Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers.
Cancer Res.
47:
5616-5619,
1987[Abstract].
55.
Toribara, N. W.,
A. M. Robertson,
S. B. Ho,
W. Kuo,
E. Gum,
J. W. Hicks,
J. R. Gum,
J. C. Byrd,
B. Siddiki,
and
Y. S. Kim.
Human gastric mucin.
J. Biol. Chem.
268:
5879-5885,
1993
56.
Velcich, A.,
and
L. H. Augenlicht.
Regulated expression of an intestinal mucin gene in HT29 colonic epithelial cells.
J. Biol. Chem.
268:
13956-13961,
1993
57.
Vinall, L. E.,
A. S. Hill,
P. Pigny,
W. S. Pratt,
N. Toribara,
J. R. Gum,
Y. S. Kim,
N. Porchet,
J.-P. Aubert,
and
D. M. Swallow.
Variable number tandem repeat polymorphism of the mucin genes located in the complex on 11p15.5.
Hum. Genet.
102:
357-366,
1998[Medline].
58.
Voynow, J. A.,
T. Horger,
B. M. Fischer,
and
M. C. Rose.
Neutrophil elastase treatment increases MUC5/5ac glycoprotein levels in A549 cell lysates (Abstract).
Am. J. Respir. Crit. Care Med.
157:
A728,
1998.
59.
Zandomeni, R.,
M. C. Zandomeni,
D. Shugar,
and
R. Weinman.
Casein kinase type II is involved in the inhibition by 5,6-dichloro-1--D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription.
J. Biol. Chem.
261:
3414-3419,
1986