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Abstract |
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Abstract
Introduction
References
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Lung carcinoma cell lines are being used in many laboratories to study various airway epithelial functions, including mucin gene expression. To identify model systems for investigating regulation of MUC5/5AC gene expression and secretion of MUC5/5AC mucins in airway epithelial cells, we evaluated the expression of several mucin genes in six carcinoma cell lines of respiratory tract origin. RNA was extracted from A549, Calu-3, NCI H292, Calu-6, RPMI 2650, and A-427 cells; MUC1, MUC2, MUC4, MUC5/5AC, and MUC5B messenger RNA (mRNA) expression was determined. By Northern analyses, all cell lines expressed MUC1 mRNA, whereas MUC2 mRNA was not detectable in any of the cell lines. RPMI 2650 cell lines expressed only MUC1 mRNA. NCI-H292 cells expressed MUC4 and low levels of MUC5/5AC mRNA. Calu-3 and A549 cells expressed MUC5/5AC mRNA; A549 cells also expressed MUC5B mRNA. Glycoconjugates secreted by lung carcinoma cells were also examined. By wheat germ lectin analysis, Calu-3, H292, and A549 cells secreted high molecular weight glycoproteins having N-acetylglucosamine and/or sialic acid moieties. Western blot analyses with an anti-MUC5:TR-3A antibody demonstrated that Calu-3 and A549 cells secreted MUC5/5AC mucins. All six carcinoma cell lines secreted large, radiolabeled, sulfated macromolecules; the majority were proteoglycans that were digested by hyaluronidase. However, Calu-3 cells also secreted sulfated high molecular-weight glycoproteins that were immunoprecipitated by anti-MUC5:TR-3A antibody. These studies demonstrated that Calu-3 and A549 cell lines expressed high and moderate amounts of MUC5/5AC mRNA and MUC5/5AC mucins, whereas H292 cells expressed lesser amounts. These cell lines should prove useful for studies of MUC5/5AC gene expression and MUC5/5AC biosynthesis, trafficking, and secretions in airway epithelial cells.
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Introduction |
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Abstract
Introduction
References
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Epithelial cells in the upper and lower respiratory tract are exposed in vivo to a variety of airborne noxious substances including microorganisms, toxins, and abrasive particulates. They are protected against this constant assault by the viscoelastic mucosal layer (mucus) that coats their cell surfaces. Mucus is hypersecreted in response to inflammatory and environmental insults (1). Persistence of hypersecretion can result in airway obstruction in diseases such as asthma, cystic fibrosis (CF), and chronic obstructive pulmonary diseases, and thus alter airway physiology and/or clearance defenses. Because mucin glycoproteins, highly O-glycosylated proteins that impart viscous and elastic properties to airway mucus (2, 3), are the major macromolecular components of mucus (4, 5), it is likely that increased expression of mucin genes and biosynthesis of mucins accompanies mucus hypersecretion.
The protein backbone of human mucins is encoded
by a recently identified family of MUC genes, seven of
which
MUC1, MUC2, MUC4, MUC5,1 MUC5B, MUC7,
and MUC8are normally expressed as messenger RNA (mRNA) in adult respiratory epithelium (reviewed in Reference 6). The post-translationally modified protein products of these seven MUC genes are presumably secreted
into the mucosal layer, although correlation of specific
MUC gene expression with MUC mucin secretion has not
yet been reported. Elucidation of the roles of MUC genes
and their gene products in healthy and diseased human
airways will require both human cell and animal models. Primary airway epithelial cell cultures that mimic airway
epithelium have been established, but cell populations are
heterogeneous, culture conditions are rigorous, components are expensive, and cells can be passaged only a few
times (7, 8). However, cell lines derived from carcinomas
of epithelial origin grow quickly, are readily passaged, and
have provided model systems for studying epithelial-specific diseases such as CF (9) and inflammatory processes
(10, 11). Identification of lung carcinoma cell lines that express and secrete specific mucins would prove useful for
studies of MUC gene expression/regulation and MUC mucin secretion. Thus, we investigated several carcinoma cell
lines derived from respiratory tract epithelial tissue to
identify cell lines that expressed some MUC genes known
to be normally expressed in upper and lower respiratory
tract tissues (6). We also investigated whether the glycoconjugates secreted by these cells were predominantly glycoproteins or proteoglycans. Finally, we determined whether cell lines that expressed MUC5 mRNA also secreted
MUC5 mucins, to identify cell lines for investigating the
role of MUC5 gene and gene products in airway health
and diseases.
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Materials and Methods |
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Cell Lines
Carcinoma cell lines (Table 1), obtained from the American Type Culture Collection (ATCC, Rockville, MD), were established as cultures according to ATCC instructions. Cell media were supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin (100 µ/ml), and streptomycin (100 µg/ml). All cells were grown to confluency at 37°C in a humidified 5% CO2 atmosphere.
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Northern Analysis
Total cellular RNA was extracted from confluent respiratory cell cultures by a modified single-step guanidine isothiocyanate-phenol-chloroform procedure (12) using TRIzol
reagent (GIBCO BRL, Gaithersburg, MD). Ten micrograms of total RNA from each sample were electrophoresed on 1.2% agarose/formaldehyde gels and transferred
to Nytran membranes (Nytran Plus; Schleicher & Schuell,
Keene, NH) by capillary blotting in 1 M ammonium acetate as previously described (13). After ultraviolet crosslinking (Stratalinker; Stratagene, La Jolla, CA), membranes
were prehybridized in a buffer containing 7% sodium dodecyl sulfate (SDS)/0.01 mM ethylenediaminetetraacetic acid (EDTA)/0.5% bovine serum albumin/0.5 M sodium phosphate buffer, pH 7.6, for a minimum of 2 h at 62°C and hybridized overnight at 62°C with 10 to 20 million cpm of
32P-labeled complementary DNA (cDNA) probes. Blots
for MUC5B and MUC4 analyses were prehybridized and
hybridized at 58°C. Membranes were washed twice with
2× standard saline citrate (SSC)/0.1% SDS for 30 min at
room temperature; and then with 0.1× SSC/0.1% SDS for
15 min at the same temperature used for hybridization, before being exposed to Kodak X-OMAT AR film (Kodak,
Rochester, NY) at 
70°C for 1 to 4 d. In some experiments, gels were treated with 50 mM NaOH/1.5 M NaCl
for 15 min after electrophoresis to promote transfer of
high molecular-weight (MW) transcripts (14), washed with
1.5 M NaCl/0.5 M Tris, pH 7.4, and transferred in 20× SSC. Membranes were then prehybridized for a minimum of 2 h
at 65°C and hybridized overnight at 65°C with a 32P-labeled
541-base pair (bp) EcoR1/BamH1 fragment of the MUC5 cDNA clone NP3a (15).
Nucleotide Probes
Inserts from previously described cDNA clones encoding nontandem repeats in MUC1, MUC2, and MUC5 (13); cDNA clone HGBM2-3 encoding the 3' end of MUC5B (16) (kindly provided by Dr. Gwenyth Offner, Boston University School of Medicine, Boston, MA); a cDNA clone encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (13); and the MUC5 541-bp EcoR1/BamH1 fragment of cDNA clone NP3a (15) were labeled with [32P]deoxycytidine triphosphate, 3,000 Ci/mmol (NEN, Boston, MA) using a random primer labeling kit (Stratagene) as previously described (13, 15) to a specific activity of 20 × 108 cpm/µg. Synthetic 48-mer oligonucleotide probes for MUC4, MUC5, and MUC5B with sequences complementary to tandem repeat regions (17) were synthesized and labeled to a specific activity of 20 × 108 cpm/µg with [32P]deoxyadenosine triphosphate using T4-polynucleotide kinase (Promega, Madison, WI).
Metabolic Labeling
Cells grown to 90 to 95% confluency in 75-cm2 Corning tissue-culture flasks were incubated in medium (5 ml) containing 200 µCi of [35S]sulfate (NEN) for 20 to 24 h at 37°C.
Agarose Electrophoresis and Transfer to PVDF Membranes
Electrophoresis of secretions was carried out on 1.0% (wt/
vol) agarose (molecular biology grade; GIBCO BRL) gels
(11 × 14 cm) prepared in electrophoresis buffer (0.1% [wt/
vol] SDS/1 mM EDTA/40 mM Tris acetate, pH 8.0) at
room temperature for 30 min at 40 V, then for 18 to 20 h
at 15 or 30 V. Samples were solubilized in electrophoresis
sample buffer (2.5% [wt/vol] SDS/4.5 M urea/5% [vol/vol]
-mercaptoethanol/25% [vol/vol] glycerol/0.005% [wt/vol] bromophenol blue/0.08 M Tris HCl, pH 7.5) and loaded
into sample wells (20-well comb, 2 mM thick) in a horizontal H5 gel apparatus (GIBCO BRL). After electrophoresis, samples were transferred to PVDF membranes (Millipore, Bedford, MA) by positive pressure transfer (18) in
0.6 M NaCl/0.06 M sodium citrate buffer, pH 7.5, in a Possiblot apparatus (Stratagene) at 75 mm Hg for 70 to 90 min. Complete transfer of high-MW macromolecules was
demonstrated by fluorography of gels after transfer of radiolabeled secretions and lysates. Prestained (Bio-Rad,
Hercules, CA) and [14C]-high-MW protein standards (Amersham, Arlington Heights, IL), laminin (Becton Dickinson
Labware, Franklin Lakes, NJ), 
2-macroglobulin and [14C]-
myosin (Sigma, St. Louis) were used as MW standards.
Samples and standards, except for laminin, were dissolved
in electrophoresis sample buffer. Laminin was solubilized in
3% SDS/17% glycerol/0.008% bromophenol blue/134 mM
dithiothreitol/77.5 mM Tris HCl, pH 7.5.
Immunochemical Analyses of Cell Secretions
Monospecific anti-MUC5 antibodies were used for immunostaining and immunoprecipitation. The TBM:TR-3A peptide sequence WFDVDFPSPGPHGGDKETYNNI (15, 19) was used to prepare MUC5 antigenic peptide derivatives. Five milligrams of peptide cys-TR-3A, in which cysteine replaced the amino terminal tryptophan of the TR-3A peptide, was reduced and coupled to keyhole limpet hemocyanin by standard techniques (20) and used to raise antibodies in rabbits. Monospecific antibodies, anti-MUC5:TR3A (21), were isolated from polyclonal antisera by affinity chromatography on a Sulfolink column (Pierce, Rockford, IL) to which cys-TR-3A peptide had been attached according to the manufacturer's instructions.
For Western blot analysis, PVDF membranes were blocked with 40 ml of milk-blocking agent (Kirkegaard & Perry Labs, Inc.) diluted 1:10 in water for 1 h at room temperature, and then transferred to a seal-a-meal bag containing 40 ml of milk-blocking agent (diluted 1:20) and anti-MUC5 monospecific antibodies (diluted 1:200) (21). Membranes were incubated overnight by rocking at 4°C and washed three times for 5 min with phosphate-buffered saline (PBS) containing 0.05% (vol/vol) Tween 20 (Sigma), and twice with PBS. Membranes were incubated with horseradish peroxidase-conjugated goat antirabbit antibody (Kirkegaard & Perry Labs, Inc., Gaithersburg, MD) at a dilution of 1:5,000 for 2.5 h, washed three times with PBS/0.05% (vol/vol) Tween 20 and three times with PBS, and then developed for 2 min in freshly made developer (40 ml of PBS added to 10 ml of methanol containing 30 mg of 4-chloro-1-naphthol [Sigma] and 10 mg of 3,3'diaminobenzidine tetrahydrochloride [Sigma]; 100 µl of 3% hydrogen peroxide was added after mixing).
Immunoprecipitation reactions with anti-MUC5 monospecific antibodies were carried out as previously described
(21) using prewashed protein A-agarose beads (Boehringer
Mannheim, Indianapolis, IN). For peptide-inhibition experiments, 30 µl of monospecific antibody was incubated
overnight at 4°C with 50 µg of cys-TR-3A peptide that had
been precoupled to albumin. Prior to fluorography, gels
were soaked for 30 min in 40% methanol/10% acetic acid
and then for 15 to 30 min in Enlightening (NEN), dried,
and exposed to Kodak X-Omat AR films at 70°C.
Lectin Analyses
Secretions were electrophoresed on 1% agarose gels and transferred to PVDF membranes as described previously. Lectin staining with wheat germ agglutinin (WGA) was carried out using the Vectastain horseradish peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions.
Enzymatic Digestions
Radiolabeled secretions were dialyzed for two successive
24-h periods and divided in half. One aliquot was digested
with bovine testes hyaluronidase (Calbiochem, San Diego,
CA) at 37°C for 16 h in PBS, adjusted to pH 6.0 by the addition of 0.1 M citric acid, as described by Kim and colleagues (22). The second aliquot was incubated in a buffer
that lacked hyaluronidase. Reactions were quenched by
boiling for 2 min. Samples were chromatographed on Sepharose CL-4B (30 × 0.5 cm) in 0.15 M ammonium acetate/
0.1% SDS/0.01 M -mercaptoethanol as previously described (23).
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Results |
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Expression of MUC Genes in Respiratory Epithelial Cell Lines
To identify cell lines that express MUC genes normally expressed in respiratory epithelium, we evaluated mRNA expression of MUC1, MUC2, MUC4, MUC5, and MUC5B in six respiratory carcinoma cell lines. Figure 1 shows representative Northern analyses; results are summarized in Table 1. Hybridization of blots with GAPDH cDNA probe (Figure 1E) demonstrated similar loading and transfer of RNA to membranes.
The 4-kb transcript of MUC1 was observed by Northern analysis in the six carcinoma cell lines studied (Figure
1A); expression by Calu-6 (lane 1) and A549 (lane 6) cell
lines was minimal at 24 h and required a 4-d exposure.
Larger transcripts (
9 kb), likely reflecting allelic variation in the MUC1 gene sequence (24, 25), were moderately expressed by Calu-3 (lane 2) and H292 (lane 5) cell
lines and minimally by RPMI and A-427 cell lines (lanes 3 and 4). A 7-kb transcript was also observed in the H292
cell line (lane 5).
MUC2 transcripts were not detected by Northern analysis in any of the six respiratory carcinoma cell lines even after 4 d exposure, although MUC2 mRNA (> 9 kb) was well expressed on the same blot by LS174T human colon adenocarcinoma cells (data not shown) as previously reported (13, 26). MUC4 (Figure 1B), MUC5 (Figure 1C), and MUC5B (Figure 1D) mRNA presented as > 9.5-kb transcripts in some respiratory carcinoma cell lines. MUC4 mRNA was well expressed only by NCI-H292 cells (Figure 1B, lane 5). MUC5 was well expressed by Calu-3 cells, moderately expressed by A549 cells, and minimally expressed by H292 cells, as shown in Figure 1C, lanes 2, 6, and 5, respectively. Identical results were obtained with other MUC5 probes: cDNA clones NP3a (15) and MUC5/ TH46 (13), and tandem repeat oligonucleotides (17) (data not shown). MUC5B was expressed by A549 cells (Figure 1D, lane 6). Identical results were obtained when hybridization was carried out with cDNA clone HGBM 2-3 (16) (data not shown).
Respiratory Carcinoma Cell Line Secretions
Primary respiratory epithelial cells have been shown to
secrete two major classes of high-MW glycoconjugates
mucin glycoproteins and proteoglycansdepending on cell
culture conditions (27). Carcinoma cell lines can also secrete proteoglycans and/or high-MW glycoproteins (28, 29).
Thus, to assess the type of glycoconjugates secreted by respiratory carcinoma cell lines, we examined their large-MW secretory products.
Secretions were initially analyzed by WGA lectin staining after electrophoresis on 1% agarose gels and transfer to PVDF membranes (Figure 2). WGA recognizes both N-acetylglucosamine and sialic acid moieties in glycoproteins and thus is a useful marker for both O-glycosides and N-glycosides (30). Several high-MW (> 450 kD) components in Calu-3 (lane 2), H292 (lane 5), and A549 (lane 6) secretions reacted strongly with WGA. High-MW components (> 450 kD) that reacted with WGA were either minimally detected or not detected in secretions from Calu-6, RPMI 2650, or A-427 cell lines.
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To determine whether these respiratory carcinoma cell lines secreted proteoglycans, cells were radiolabeled with [35S]SO4 and their secretions were analyzed. Electrophoresis on 1% agarose showed that sulfated high-MW components were secreted by Calu-6, Calu-3, RPMI 2650, and NCI-H292 cell lines (Figure 3, lanes 1-4); by A549 cells (Figure 4, lane 1); and by A427 cells (data not shown). At least two very high-MW (> 450 kD) radiosulfated components (Figure 3, lane 2; Figure 4, lane 3) with electrophoretic mobilities similar to WGA-stained macromolecules (Figure 2, lane 2) were secreted by Calu-3 cells.
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The radiosulfated secretions were treated with hyaluronidase (which enzymatically fragments glycosaminoglycans but not O-glycans) and analyzed by molecular sieving chromatography. Before hyaluronidase treatment, a moderate amount of radiosulfated components from all respiratory carcinoma cell secretions eluted in the void volume of Sepharose CL-4B, as shown for A549 and Calu-3 secretions (Figures 5a and 5c). After hyaluronidase treatment, the radiolabeled void volume peak in Calu-6, A549, A-427, H292, and RPMI 2650 secretions disappeared, as shown for A549 radiosulfated secretions (Figure 5b). However, the amount of radiolabeled sulfate in the void volume of Calu-3 secretions decreased approximately 50% after treatment with hyaluronidase (Figures 5c and 5d), suggesting that Calu-3 cells secreted high-MW sulfated glycoproteins. This interpretation was validated by comparing the electrophoretic properties of the radiosulfated secretions before and after hyaluronidase treatment. No high-MW radiosulfated band was observed in A549 secretions after hyaluronidase treatment (Figure 4, lane 2), whereas the hyaluronidase-resistant fraction in the Calu-3 secretions (Figure 4, lane 4) migrated with an electrophoretic mobility similar to that of the highest MW fraction that reacted with a WGA band (Figure 2, lane 2).
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Identification of MUC5 Mucins Secreted by Respiratory Tract Carcinoma Cell Lines
To begin examining the correlation between MUC mRNA expression and MUC mucin secretion, we investigated whether respiratory tract carcinoma cell lines that expressed MUC5 mRNA also secreted MUC5 mucins. Northern and Western analyses were carried out on RNA samples and secretions isolated from the same flask of cultured cells. In agreement with the MUC5 mRNA expression data (Figure 1C), Western blot analyses demonstrated that Calu-3 and A549 cells secreted detectable amounts of MUC5 mucins (Figure 6, lanes 2 and 6) whereas secretions from H292 cells, which expressed low but detectable levels of MUC5 by Northern analysis (Figure 1C), secreted barely detectable amounts of MUC5 mucin (Figure 6, lane 5). The immunoreactive bands did not stain when MUC5 antibodies were inhibited by preincubation with peptide MUC5:TR-3A (data not shown). The electrophoretic mobility of the immunoreactive mucins correlated with the largest of the WGA-reactive bands in Calu-3, A549, and H292 secretions (Figure 2), thus identifying these bands as MUC5 mucins of different electrophoretic mobilities, likely reflecting differences in O-glycosylation. No detectable immunostaining of secretions from Calu-6, A-427, and RPMI 2650 cell lines with the anti-MUC5 antibody was observed, in keeping with the observation that these cell lines did not express detectable amounts of MUC5 mRNA (Figure 1C).
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Immunoprecipitation analyses of radiosulfated secretions of the three cell lines that expressed and secreted MUC5 mucins (Figure 7) were carried out. In agreement with the hyaluronidase digestion experiments (Figures 5b and 4), sulfated MUC5 mucins were not immunoprecipitated from A549 (Figure 7, lane 2) or H292 (data not shown) secretions. However, Calu-3 cells secreted sulfated mucins, some of which were immunoprecipitated by anti-MUC5 antibody (Figure 7, lane 5). Incubation of anti-MUC5 antisera with cys-TR3A peptide inhibited immunoprecipitation (Figure 7, lane 6).
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Discussion |
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Epithelial cell lines derived from carcinomas are frequently
used as in vitro model systems to study gene regulation
and signal transduction. We initiated this study to identify
epithelial cell lines of respiratory tract origin that could be
useful in elucidating the role of mucins in airway health
and diseases. Our results demonstrate that several carcinoma cell lines, as discussed subsequently, express profiles
for MUC1, MUC2, MUC4, MUC5, and/or MUC5B mRNA that are similar to those reported for normal and cancer
lung tissues. We also examined the high-MW glycoconjugates secreted by these cells and demonstrated that whereas
all six carcinoma cell lines secreted proteoglycans, only three
cell linesCalu-3, A549, and NCI H292secreted high-MW glycoproteins. We further showed that Calu-3, A549,
and NCI H292 cell lines expressed differing amounts of
MUC5 mRNA and secreted variable, but detectable,
amounts of MUC5 mucins.
Mucin mRNA Expression in Respiratory Tract Epithelial Cells
MUC1. The prevailing concept that MUC1 is a panepithelial gene expressed in different normal tissues, primary cells, and cancer cell lines (31) was supported by this study of cell lines derived from primary respiratory tract cancer tissues. MUC1 was detected in all carcinoma cell lines, although expression was low in Calu-6 and A549 cells (Figure 1A and Table 1). Healthy human lung tissues normally express MUC1 mRNA with transcript sizes ranging from 4.4 to 7.4 kb, reflecting allelic variations (24, 25). Variable levels of expression of different size transcripts have been reported in primary bronchial (32) and nasal (32, 33) epithelial cells from normal individuals and CF patients.
MUC1 has been shown both by Northern analysis and reverse transcription-polymerase chain reaction (RT-PCR) to be expressed by lung cancers of various histopathologic types encompassing squamous, adenocarcinoma, large cell, and poorly differentiated cells (34, 35), including the Calu-3, Calu-6, A-427, and A549 cell lines studied here (36). That two or three transcripts are expressed in Calu-3 or H292 cell lines, respectively, likely reflects different length MUC1 alleles, as previously observed in other epithelial cancer cell lines (37) as well as in primary bronchial (32) and nasal (32, 33) epithelial cells. Lung adenocarcinomas have higher levels of MUC1 expression than do squamous or adenosquamous carcinomas (34), and well-differentiated lung adenocarcinomas have higher levels of expression than do poorly differentiated adenocarcinoma tumors (38). Thus, MUC1 may be a useful marker of well-differentiated epithelial cells.MUC2.
MUC2, originally cloned from a small intestinal cDNA library, is well expressed in intestinal tissue and
colon cancer cell lines (26). In contrast to MUC1, MUC2
mRNA is expressed at low levels in normal respiratory epithelial cells and usually requires RT-PCR (39, 40), in situ
hybridization (41, 42), or RNA slot blot (33) for detection.
MUC2 mRNA was shown by in situ hybridization to be
present at lower levels than MUC5 and MUC4 in normal
nasal turbinate epithelium (41). By quantitative RNA slot-blot analysis, MUC2 mRNA was expressed at lower levels
than MUC5 in freshly isolated nasal epithelial cells from
normal individuals and subjects with rhinitis or CF (33)
and in nasal polyps (13). Although one study, using a
competitive RT-PCR method, demonstrated that MUC2
mRNA was expressed at greater levels than MUC5 in normal human bronchial epithelial cells in culture (43), the
majority of studies to date support the concept that MUC2
is expressed at lower levels than MUC5 in normal respiratory tract tissue. However, inflammation appears to increase MUC2 mRNA expression. It is highly expressed in
bronchial cells of smokers and patients with chronic bronchitis (44, 45). Both a proinflammatory marker, tumor necrosis factor- (TNF-), and a bacterial product, Pseudomonas aeruginosa lipopolysaccharide, have recently been shown
to stimulate upregulation of MUC2 expression in NCI-H292
cells (46, 47).
MUC4. This cDNA was originally cloned from a cDNA library generated from respiratory tract tissues (48) and has been shown by in situ hybridization analysis to be moderately expressed in both the upper (41) and lower (17) respiratory tract airways. MUC4 mRNA expression, relative to normal lung tissue, is increased in adenocarcinomas and squamous, adenosquamous, and large-cell carcinomas (34, 35). In the present study, MUC4 mRNA was strongly expressed by NCI-H292 cells, minimally expressed by Calu-3 cells, and not expressed by the other four respiratory tract carcinoma cell lines investigated (Figure 1B and Table 1).
MUC5. cDNA clones for MUC5 have been isolated from cDNA libraries generated from respiratory tract tissues (15, 49) as well as from gastric tissues (50). MUC5 has been shown by in situ hybridization analysis to be moderately expressed in both the upper (41) and lower (17) respiratory tract airways. MUC5 is expressed at greater levels than MUC2 or MUC1 in the respiratory tract (see previous discussion on MUC2) and is generally localized to goblet cells in normal respiratory tissues (17, 41). MUC5 is also highly expressed in non-small-cell lung cancer associated with metastases (38, 51). In the present study, we observed high levels of expression of MUC5 in Calu-3 cells, moderate expression in A549 cells, and minimal expression in NCI-H292 cells (Figure 1C and Table 1).
MUC5B. MUC5B was originally cloned from a cDNA library generated from respiratory tract tissues (52) and has been shown by in situ hybridization analysis to be well expressed in normal respiratory tract tissue, predominantly in the submucosal glands (17, 41). MUC5B expression is increased in non-small-cell lung cancer tumors, particularly those which metastasize (51). In the present study, MUC5B was well expressed by A549 cells and minimally expressed by NCI-H292 cells (Figure 1D and Table 1).
In summary, these studies show that three carcinoma cell lines derived from respiratory tract tissuesA549,
Calu-3, and NCI-H292expressed combinations of MUC1,
MUC4, MUC5, and MUC5B genes (Table 1). This is in
agreement with the consensus of current opinion that MUC1,
MUC4, MUC5, and MUC5B are moderately well expressed
in both normal respiratory tract and lung cancer tumor tissues, with MUC2 being expressed at lower levels, and suggests that the profile of mucin gene expression in these cell
lines is similar to that in both normal and tumor respiratory epithelium.
Airway Epithelial Carcinoma Cell Secretions
Our intent was to identify airway epithelial cells that
would provide useful model systems for studying MUC
mRNA expression and mucin secretion. Analysis of mucins secreted by cell lines is complicated by the fact that
both primary (27) and carcinoma cell lines (28, 29) derived
from epithelial tissues secrete two major classes of high-MW glycoconjugates: mucin glycoproteins and proteoglycans. In this study we showed that all six carcinoma cell
lines secreted sulfated proteoglycans on the basis of their sensitivity to hyaluronidase (Figure 5). Furthermore, three
cell linesCalu-3, H292, and A549secreted large glycoproteins (> 450 kD) that reacted strongly with WGA
lectin, a marker for N-acetylglucosamine and N-acetylneuraminic acid moieties in both N-linked and O-linked glycoconjugates (30). The largest of the WGA-staining
bands in Calu-3, A549, and H292 secretions (Figure 2)
exhibited electrophoretic mobility similar to MUC5 mucins (Figure 6) . WGA staining of high-MW glycoproteins
was minimally or not detected in secretions from Calu-6,
RPMI 2650, or A-427 cells (Figure 2), consistent with the
observation that these cell lines did not express detectable amounts of secretory mucin gene MUC2, MUC4, MUC5
and MUC5B mRNA (Table 1).
Cells may express a specific MUC mRNA yet not synthesize and secrete its mature gene product; mucin protein synthesis, maturation, and secretion are post-transcriptionally
regulated events that are not yet well studied. Some genes
that are expressed in the respiratory tract1-antitrypsin,
secretory leukocyte proteinase inhibitor, and elastase-specific inhibitorhave been shown to exhibit increased gene
expression without a corresponding increase in protein
levels (11, 53). The present study demonstrates correlation
of MUC5 mRNA expression (Figure 1C) and MUC5 mucin secretion (Figure 6) in Calu-3, A549, and NCI-H292 cells
and, as far as we know, is the first study to show correlation of a specific MUC mRNA and protein expression.
Calu-3, A549, and H292 Cells: Model Systems for Investigating MUC5 Expression and Secretion
The A549 cell line, initiated through explant culture of lung carcinomatous tissue from a 58-yr-old Caucasian male (54), was originally reported to retain features of type II alveolar cells, including cytoplasmic multilamellar inclusion bodies and an ability to synthesize surfactant (54, 55). The tumor from which the cell line is derived showed cuboidal epithelial cells in clumps and acini on histologic examination, and its acinar-like spaces were filled with a mucinous material (54). Characterization of A549 cells at passage 68, the stock culture from which ATCC provides starter cells, did not reveal prominent inclusion bodies or increased incorporation of 14C-choline into phosphatidylcholine relative to biosynthesis in lung fibroblasts. These observations precluded the A549 cell line from being definitively categorized as being of type II origin or function (ATCC catalog). Furthermore, it has been reported that A549 cells do not express surfactant protein B (56), a marker for alveolar cells.
A549 cells have previously been used as a respiratory epithelial cell model for various studies including (1) gene regulation of elastase-specific inhibitor/elafin and secretory leukoprotease inhibitor (11), inducible nitric oxide synthase (57), and interleukin-8 (58); (2) arachidonic acid metabolism (59); (3) production of extracellular matrix (60); (4) gene therapy (10); (5) respiratory syncytial virus infection (61, 62); and (6) signal transduction (63). Thus, A549 cells have provided a useful model for molecular and biologic studies of airway epithelium. In the present study, we have further characterized this cell line and shown that it expresses MUC1, MUC5B, and MUC5 mRNA and secretes MUC5 mucins. Thus, these well-characterized cells may be a useful system for studying MUC5, and probably MUC5B, gene regulation and permit investigation into the relationships between mucin regulation and inflammation, infection, or signal transduction.
Calu-3 cells expressed higher levels of MUC5 mRNA and MUC5 mucins than did A549 cells, although the latter grow considerably more quickly. Calu-3 cells have the advantage of expressing the CF transmembrane conductance regulator (CFTR) (64), which may permit studies to determine the relationship between CFTR and mucin regulation, biochemistry, and function, especially with regard to altered sulfation of CF mucins (65, 66). Calu-3 MUC5 mucins were shown to be sulfated, in contrast to MUC5 mucins secreted by A549 cells (Figure 7). Thus, the MUC5 mucins secreted by Calu-3 may be analogous to airway mucins secreted in vivo, which contain low to moderate amounts of sulfate (67). The MUC5 mucins secreted by Calu-3 migrated more rapidly than those secreted by A549 cells, suggesting that O-glycosylation of A549 and of Calu-3 MUC5 mucins are not identical.
The NCI-H292 cell line, a mucoepidermoid cell line, is
currently being used as a model system for studying regulation of MUC gene expression. Recently, it has been used
to demonstrate increased expression of MUC2 mRNA by
TNF- (46), transcriptional regulation of MUC2 by P. aeruginosa lipopolysaccharide (47), and downregulation of MUC2
and MUC5 by dexamethasone (68).
In summary, we have identified and partially characterized several cell lines derived from respiratory tract tissue that should prove useful in understanding regulation of MUC mucin secretion and gene expression in lung disease. These data indicate that Calu-3, A549, and NCI H292 cells will be useful for studying at least three of the MUC genes and gene products normally expressed in airway cells and secretions. In particular, A549 and Calu-3 cell lines, as well as the NCI-H292 cell line, have major advantages because regulation of both MUC5 mRNA and MUC5 glycoprotein can now be studied in the same model.
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Footnotes |
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Address correspondence to: Dr. M. C. Rose, Center for Molecular Mechanisms of Diseases, Rm. 5700, Children's Research Institute, 111 Michigan Ave., Washington, DC 20010-2970. E-mail: MRose{at}CNMC.org
(Received in original form March 27, 1998 and in revised form July 16, 1998).
* Current address: Department of Cell Biology & Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.Abbreviations: complementary DNA, cDNA; cystic fibrosis, CF; messenger RNA, mRNA; molecular weight, MW; phosphate-buffered saline, PBS; reverse transcription-polymerase chain reaction, RT-PCR; sodium dodecyl sulfate, SDS; wheat germ agglutinin, WGA.
1 The MUC5/5AC gene is referred to as MUC5, MUC5AC, and MUC5/ 5AC. For simplicity, we have used the MUC5 notation, except in the Abstract.
Acknowledgments: This work was funded by NIH HL33152 (M.C.R.), NIH R29 HL50694 (J.A.V.), and a Children's Research Institute award (J.T.B.). The authors thank Teresa Horger for excellent technical assistance.
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Abstract
Introduction
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