Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.
glycoprotein; binding; cystic fibrosis; airway; infection
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
MUCINS ARE HIGH MOLECULAR MASS glycoproteins constituting the major protein component of mucus and are thought to be encoded by 12 separate genes (15, 32, 39). Although the majority of mucin genes encode secretory glycoproteins, four of them, namely, MUC1 (4, 14), MUC3 (40), MUC4 (21), and MUC12 (39), contain transmembrane domains indicating that they most likely encode cell surface mucins. MUC1 (Muc1 in nonhuman species) mucins are abundantly expressed on most carcinoma cells and have been shown to reduce intercellular (16, 36) as well as matrix adhesion (38) through their extended and rigid structure (9). These cell surface mucins are also expressed by normal epithelial cells in various glandular tissues of the respiratory, gastrointestinal, and female reproductive organs (7, 10, 25) and have been shown to be associated with cell differentiation in mammary (23), uterine epithelial (7), and airway epithelial (22) cells. Similar to cancer cells, Muc1 mucins in uterine epithelial cells have been shown to exhibit an antiadhesive property, suggesting that they play an important role in maintaining the prereceptive phase in the uterus (8). The role of MUC1 mucin in the airway, however, is unknown.
Previously, Kim et al. (12) reported that mucins released by neutrophil elastase via its proteolytic action were found on the surface of primary hamster tracheal surface epithelial (HTSE) cells. These cell surface mucins were composed of two major types: one similar to secreted mucins both in size and charge and the other resembling human MUC1 mucins based on its immunoreactivity (24). Expression of Muc1 mRNA in HTSE cells was confirmed through cDNA cloning of its gene and shown to be closely associated with goblet cell differentiation (22). Pseudomonas aeruginosa, a gram-negative bacterium almost invariably associated with airway mucus in cystic fibrosis patients, is known to bind to secreted mucins purified from airway mucus (29). Because Muc1 mucins constitute only a minor, if any, component of airway secreted mucins, it was of interest to determine whether P. aeruginosa could also adhere to these glycoproteins present on the cell surface. We approached this question by stably transfecting the hamster Muc1 gene into a hamster epithelial cell line that normally does not produce Muc1 mucins followed by monitoring P. aeruginosa adhesion to the transfected cells.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. All materials used in this study were purchased from Sigma (St. Louis, MO) unless otherwise stated.
P. aeruginosa Two P. aeruginosa strains were used: CF3, a mucoid isolated from a cystic fibrosis patient, and PAK, a nonmucoid laboratory strain (11) (a generous gift from Dr. Alice Prince, Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY). Both strains were grown in Luria-Bertani broth for 7 h at 37°C and metabolically radiolabeled in sulfate-free M9 medium containing 10 µCi/ml of Na235SO4 (1,175 Ci/mmol, 100 mCi/ml, carrier-free; American Radiolabeled Chemicals, St. Louis, MO) for 16 h at 37°C. The radioactive bacteria were washed twice and resuspended to the appropriate concentrations in PBS containing 2 mg/ml of glucose for use in the binding assay.
Establishment of Chinese hamster ovary-Muc1 and Chinese hamster ovary-X cell lines. The hamster Muc1 expression plasmid pcDNA/Muc1 was constructed by inserting a full-length hamster Muc1 cDNA (22) into the pcDNA3 vector (Invitrogen, San Diego, CA). The pcDNA/Muc1 plasmid and pcDNA3 vector alone were separately transfected into Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA) with a cationic liposome, LIPOFECTIN (Life Technologies, Manassas, VA), according to the manufacturer's protocol. Clones were selected and passaged 10 times to identify stable cell lines. On the basis of Northern blot analysis, a stable clone expressing hamster Muc1 mRNA is referred to as CHO-Muc1 and a clone derived from the vector alone as CHO-X. The latter was used as a negative control in the adhesion assay. Both cell lines were maintained in Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 200 µg/ml of G-418, 5% (vol/vol) fetal bovine serum, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (all from Life Technologies).
Construction and expression of Muc1 mucin deletion mutants. Two Muc1 mucin deletion mutants were constructed: pcDNA/Muc1-NTR, lacking the tandem repeat region (NTR), and pcDNA/Muc1-NEC, lacking the entire extracellular (EC) domain (NEC). For both, a polymerase chain reaction (PCR) was used to amplify pcDNA3/Muc1 outside of the region to be deleted, and the amplicon was gel purified, digested with restriction endonucleases, ligated, and cloned into pcDNA3. PCR primer pairs were as follows, with the underlined nucleotides corresponding to the restriction sites used for joining amplicons and plasmid insertion: pcDNA/Muc1-NTR NH2-terminal region: MucTR-1, 5'-GCATGGATCCGGCACGAGCACAGCCACAGC-3', and MucTR-2, 5'-CGTACCCGGGGTTCAAGGTTGAGGAACTGC-3'; pcDNA/Muc1-NTR COOH-terminal region: MucTR-3, 5'-GCATCCCGGGTCCTCCATGCAAACCACCGA-3', and MucTR-4, 5'-CGATCTCGAGGCGCGTGTCAGGAAGGCTGG-3'; pcDNA/Muc1-NEC NH2-terminal region: MucTR-1, 5'-GCATGGATCCGGCACGAGCACAGCCACAGC-3', and MucTR-5, 5'-CGATGTCGACACTGTTTGGATCTGTTAC-3'; and pcDNA/Muc1-NEC COOH-terminal region: MucTR-6, 5'-GATCCTCGAGATGCAGTTTCCTTCCTCTG-3', and MucTR-7, 5'-GATCTCTAGAACTCTGGCTCACCAGCCC-3'. PCR was performed for 1 cycle at 94°C for 3 min followed by 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and final extension at 72°C for 10 min. Stably transfected CHO-NTR cells were isolated as described in Establishment of Chinese hamster ovary-Muc1 and Chinese hamster ovary-X cell lines. Transiently transfected CHO-NEC cells were prepared by incubating 2 × 105 CHO cells/well in 24-well culture plates for 24 h at 37°C in 5% CO2, adding 0.2 µg of pcDNA/Muc1-NEC in serum-free Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 1 µl of SuperFect reagent (QIAGEN, Valencia, CA), incubating for 2.5 h, replacing the medium with Ham's F-12 medium-Dulbecco's modified Eagle's medium containing 200 µg/ml of G-418 and 5% fetal bovine serum and incubating the cells for 48 h at 37°C.
Northern blot analysis.
Total RNA was extracted from CHO-X, CHO-Muc1, and HTSE cells as
previously described (22). Details of the primary HTSE
cell culture were reported elsewhere (37). RNA was
electrophoresed in 1% agarose-formaldehyde gels at 5 V/cm, transferred
to nitrocellulose membrane (Bio-Rad, Richmond, CA), prehybridized for
2 h, and hybridized overnight at 42°C in a solution containing
the 1.5-kb fragment of the hamster Muc1 cDNA (22), 50%
formamide, and 6× saline-sodium citrate (SSC). The probe was labeled
with a random-primer DNA labeling kit (Boehringer Mannheim,
Indianapolis, IN). The membrane was washed in 0.1× SSC containing
0.1% sodium dodecyl sulfate (SDS) at room temperature and exposed to
X-ray film with intensifying screens at 80°C for 2 days.
Metabolic radiolabeling, immunoprecipitation, and SDS-PAGE. In an experiment designed to radiolabel the EC domain of Muc1 mucins, CHO-X and CHO-Muc1 cells were incubated in Ham's F-12 medium-Dulbecco's modified Eagle's medium containing a "low" level of glucose (0.4 mg/ml) and D-[6-3H]-glucosamine (100 µCi/ml, 20 Ci/mmol; American Radiolabeled Chemicals) for 24 h and lysed as previously described (24). The lysis buffer contained 40 mM sodium phosphate, pH 7.2, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 10 mM benzamidine, 25 µg/ml of leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml of aprotinin. Lysates were immunoprecipitated overnight at 4°C as described by Boshell et al. (1) using a polyclonal antibody (CT-1) raised against a synthetic peptide (SSLSYTNPAVAATSANL) corresponding to the COOH-terminal region of the human MUC1 cytoplasmic domain (25). Both the CT-1 antibody and its blocking peptide (the synthetic peptide used to prepare it) were generous gifts from Dr. Sandra Gendler (Mayo Clinic, Scottsdale, AZ). Although produced against the human sequence, this antibody cross-reacts with hamster Muc1 mucin (24, 25). Immune complexes were precipitated with protein A agarose, and isolated proteins were resolved by SDS-PAGE on a 5% acrylamide gel and visualized by fluorography.
Immunoblot analysis. CHO-X and CHO-Muc1 cell lysates were immunoprecipitated with CT-1 antibody, resolved on a 15% SDS-acrylamide gel, and transblotted to nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in Tris · HCl, pH 7.2, containing 0.15 M NaCl and 0.1% Tween 20 (TBS-T), reacted overnight with the CT-1 antibody (1:2,000 dilution), and washed with TBS-T, and the bound antibody was reacted with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5,000 dilution; Pierce, Rockford, IL). After the membrane was washed with TBS-T, immunoreactive bands were visualized with enhanced chemiluminescence reagent (Amersham Pharmacia, Piscataway, NJ).
Bacterial adherence assay.
Adhesion of P. aeruginosa to transfected CHO cells was
performed according to the method described by Rostand and Esko
(33) with slight modification. Briefly, the cells were
plated in 24-well dishes at 2 × 105 cells/well and
grown for 24 h to confluence. The monolayer was washed twice with
PBS and fixed for 10 min with 2.5% (vol/vol) glutaraldehyde in PBS at
room temperature. The fixed monolayer was washed three times with PBS
and incubated with 35S-radiolabeled bacteria [2 × 107 colony-forming units (cfu) · 0.5 ml1 · well
1] for 40 min at 37°C.
After incubation, the monolayer was extensively washed three times with
PBS to remove the unbound bacteria. Adhered cells were lysed with 1.0 ml of 2% SDS, and radioactivity was measured with a liquid
scintillation counter. The degree of adhesion is expressed as number of
bacteria bound per cell.
Treatment with human neutrophil elastase. Cells were incubated with human neutrophil elastase (HNE; 5 µg/ml) (12) for 30 min at 37°C in serum-free Ham's F-12 medium-Dulbecco's modified Eagle's medium and washed twice with PBS before being fixed with glutaraldehyde for the bacterial adherence assay.
Statistical analyses. The number of bacteria adhered to epithelial cells was calculated in the following order: 1) determination of the number of bacteria adhered to each well by converting disintegrations per minute to actual number of bacteria, 2) determination of the number of epithelial cells at the end of the assay using a separate parallel group not exposed to bacteria, and 3) assessment of the significance of the difference between groups with Student's t-test for unpaired samples or analysis of variance.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the functional role of Muc1 mucins, we sought to identify
a nonexpressing hamster epithelial cell line to serve as a suitable
host for transfection of our previously cloned hamster Muc1 cDNA.
During initial screening of various cell lines, we observed that CHO
cells did not express Muc1 mRNA (Fig. 1).
Furthermore, a recent report by Rostand and Esko (33)
showed that P. aeruginosa adhesion to CHO cells was fully
accounted for by the presence of cholesterol and cholesterol esters on
the epithelial cell surface, whereas glycoconjugates such as
glycoproteins, glycolipids, or proteoglycans were not involved.
Therefore, CHO cells turned out to be an ideal system for transfecting
the hamster Muc1 gene in the present study.
|
After two stable clones (CHO-Muc1 and CHO-X cells) were obtained (Fig. 1) as described in METHODS, we examined whether Muc1-encoded proteins are expressed on the cell surface based on the following structural information available for human MUC1 mucin: 1) MUC1 mucins are made up of two subunits noncovalently bound via a hydrophobic interaction: a larger subunit containing the EC domain where all the O-linked glycosylation sites are present and a smaller subunit containing the transmembrane region and cytoplasmic domain (9, 17); 2) the COOH-terminal amino acid sequence of hamster Muc1 mucin is virtually identical to human and mouse sequences, rendering it immunoreactive with rabbit CT-1 antibody against the 17 COOH-terminal residues (22); and 3) hydrophobic binding between the two subunits can be disrupted by high concentrations of SDS (17). We also exploited the previous observation that HNE released Muc1 mucins from the surface of airway goblet cells (37). In the present experiment, cells were metabolically radiolabeled with [3H]glucosamine to identify the glycosylated EC domain and immediately treated with HNE to cleave the EC domain, the cells were then lysed in detergent solution, and the lysates were immunoprecipitated with CT-1 antibody (25). After immunoprecipitation, the cell lysates were subjected to SDS-PAGE, and the larger subunit containing most of the radiolabeled sugar moieties was identified by 3H fluorography, whereas the smaller subunit containing the cytoplasmic tail was identified by immunoblotting with the CT-1 antibody.
Figure 2A shows the presence
of a glycoprotein with an apparent molecular mass of 180 kDa in
CHO-Muc1 cells (lane 2) but in not CHO-X cells (lane
1). This protein was degraded by HNE (Fig. 2A,
lane 3), thus identifying it as the larger subunit of Muc1 mucins. Because the concentration of HNE used in this experiment did
not cause lactate dehydrogenase release from the cells (data not
shown), we concluded that the reduced signal of the
[3H]glucosamine-labeled EC domain after enzyme treatment
was not due to nonspecific disruption of the plasma membrane.
Immunoblot analysis of these samples with the CT-1 antibody (Fig.
2B) showed the presence of a 26-kDa protein in CHO-Muc1
cells regardless of HNE treatment (lanes 2 and
3), thus identifying this protein as the smaller subunit of
Muc1 mucins. When the immunoprecipitation reaction was blocked with the
synthetic peptide used to prepare the CT-1 antibody, appearance of the
26-kDa band was completely abolished (data not shown). Collectively,
the results presented in Fig. 2 indicate that CHO-Muc1 cells express
Muc1 mucins on the cell surface, whereas CHO-X cells do not.
|
Having characterized the transfected cells for Muc1 mucin expression,
we next compared the ability of CHO-X and CHO-Muc1 cells to bind to
P. aeruginosa. In preliminary experiments, we tried both
live and fixed cells for the P. aeruginosa adhesion and
found no significant differences between them, with the exception that live cells tended to give greater variation in bacterial adhesion compared with that in fixed cells (data not shown). Moreover, a recent
report (26) that live human airway epithelial cells may
internalize P. aeruginosa led us to use fixed cells for the P. aeruginosa adhesion experiments. Results from kinetic
studies with CHO-X cells (data not shown) were practically
identical to those previously reported for nontransfected CHO cells
(33). P. aeruginosa adhesion to CHO-X cells
reached a plateau after 40 min, with approximately five bound bacteria
per CHO-X cell. Based on these results, we chose adhesion assay
conditions utilizing 2 × 107 cfu of radioactive
P. aeruginosa incubated with 2 × 105 cells
for 40 min at 37°C and quantified the number of bound bacteria. Figure 3 shows that although P. aeruginosa adhered to both CHO-X and CHO-Muc1 cells in a
concentration-dependent fashion, adhesion to CHO-Muc1 cells was
consistently about twofold greater than that to CHO-X cells regardless
of the number of P. aeruginosa added.
|
Several previous studies found that mucoid P. aeruginosa
strains adhered to epithelial cells better than nonmucoid variants (18-20), but others found no difference (2,
31) or that the mucoid forms bound less strongly (30,
41). These discrepancies could be due to differences in the
bacterial isolates and/or cell types used. To investigate whether the
increased adhesion of the CF3 strain of P. aeruginosa to
CHO-Muc1 cells that we observed was due to its mucoid nature, we next
tested the nonmucoid PAK strain that has been used extensively for
studying adhesion to the airway epithelial cell (3).
Figure 4 shows the results of CF3 and PAK
adhesion to CHO-X and CHO-Muc1 cells. Although the former P. aeruginosa strain displayed substantially greater adhesion to both
CHO cell transfectants, the degree of adhesion to CHO-Muc1 cells was
significantly greater than to CHO-X cells with both bacterial strains.
Thus the binding of CF3 and PAK to CHO-Muc1 cells was 194 ± 6 and
242 ± 8% greater, respectively, than to CHO-X cells. The
addition of a 100-fold excess of "cold" P. aeruginosa
(2 × 109 cfu) displaced >90% of
35S-labeled P. aeruginosa adhesion to CHO-Muc1
cells by both strains, suggesting the presence of specific binding to
the cells (data not shown).
|
Because the only difference between CHO-Muc1 and CHO-X cells is
most likely the presence of the Muc1 mucin on the surface of the former
cells, the results presented above strongly suggested that increased
P. aeruginosa adhesion to CHO-Muc1 cells was mediated by
Muc1 mucins. However, these results do not rule out the alternative, although unlikely, possibility that transfection with the Muc1 mucin
cDNA induced expression of a nonmucin protein responsible for bacterial
adhesion. To more directly address the role of this glycoprotein in
P. aeruginosa adhesion, two experimental approaches were
taken: proteolytic digestion of mature mucins on the cell surface with
HNE and analysis of EC domain deletion mutants. HNE has previously been
shown to specifically and efficiently release Muc1 mucin from primary
HTSE cells (12) and CHO-Muc1 cells (Fig. 2). As shown in
Fig. 5, the addition of HNE to the
binding assay completely abolished the increased binding of the CF3 and
PAK strains to CHO-Muc1 cells to the basal levels seen with CHO-X cells. Reduced adhesion of P. aeruginosa to HNE-treated
CHO-Muc1 cells was significant (P < 0.01).
|
Finally, CHO cells transfected with mutants of the Muc1 cDNA that were
either partially (CHO-NTR) or completely (CHO-NEC) deleted of the EC
domain were analyzed for P. aeruginosa adhesion. Construction of the NTR deletion mutant generated a molecule devoid of
O-linked sugar units while still retaining the majority of the protein
core and all N-linked glycosylation sites. The complete NEC deletion
mutant, on the other hand, was constructed to produce a Muc1 mucin
molecule containing only the transmembrane and cytoplasmic domains of
the protein. Expression of both mutants in transfected CHO cells was
confirmed by immunoblot analysis with the CT-1 antibody (data not
shown). As shown in Fig. 6, CHO cells
expressing these mutants displayed significantly altered binding to
P. aeruginosa compared with the nonmutant Muc1 mucin. Thus
although PAK binding to CHO-Muc1 cells was 280 ± 24% greater
than to CHO-X cells, CHO-NTR and CHO-NEC cells showed 163 ± 7 and
80 ± 4% P. aeruginosa binding, respectively,
compared with CHO-X cells. In other words, deletion of the tandem
repeat region led to a 60% reduction in P. aeruginosa adhesion, whereas complete deletion of the EC domain resulted in a
total loss of the increased P. aeruginosa adhesion in
CHO-Muc1 cells. Collectively, these results strongly suggested that
Muc1 mucin is the cellular receptor for P. aeruginosa
adherence.
|
The exact glycosylation patterns of MUC1 mucins, human and nonhuman, are not known. Previous reports (28, 34, 35) on P. aeruginosa binding to epithelial cells, however, implicated sugar residues in the primary binding domains. Most of the sugar moieties of human MUC1 and hamster Muc1 mucins are O-linked, are found in the tandem repeat region, and are composed of 20-amino acid repeats rich in serine, threonine, and proline (5). However, the number of repeating units varies greatly between these two species such that the hamster molecule contains 12 repeats (22) and the human 25-125 repeats (5). Thus MUC1 mucin may possess more binding sites and higher affinity for P. aeruginosa. Interspecies differences in glycosylation between MUC1 and Muc1 mucin molecules may result in heterogeneous patterns of P. aeruginosa adhesion, potentially limiting our study. On the other hand, such alterations in glycosylation may be crucially important for P. aeruginosa adhesion to epithelial cells. It is interesting that cells expressing the deletion mutant devoid of tandem repeats (CHO-NTR) showed significantly increased (~40%) P. aeruginosa adhesion compared with CHO-NEC cells, suggesting the presence of at least two binding subdomains. Current experiments are underway to further define the molecular nature of these subdomains with regard to the involvement of N-linked sugars and/or the protein core of Muc1 mucin.
The physiological significance of P. aeruginosa adhesion to
MUC1 mucins remains unknown. These molecules may serve as a secondary defense barrier in the lung against airborne bacteria that manage to
escape the gel phase of airway mucus, the primary physical barrier.
Bacteria "trapped" by MUC1 mucins might be cleared from the airway
by some as yet unidentified process, possibly involving proteolytic
cleavage and release of the mucin-bacteria complex from the cell
surface for subsequent removal by the mucociliary escalator.
Alternatively, although not mutually exclusive, MUC1 mucins may serve
as signal transduction receptors for microorganisms that penetrate the
primary mucous layer, bind to epithelial cells, and induce an
inflammatory response (13). For example, it was recently
shown that adhesion of P. aeruginosa to glycolipids on airway epithelial cells resulted in nuclear translocation of nuclear factor-B and interleukin-8 expression (3), indicating
an important role for these molecules in initiating an epithelial
proinflammatory response to P. aeruginosa adhesion.
Important in this regard, MUC1/Muc1 mucins from human, mouse, hamster,
and rabbit have virtually identical amino acid sequences in their
cytoplasmic domains, with conserved consensus motifs for tyrosine
phosphorylation (4, 6, 14, 19). Recent reports by
Zrihan-Licht et al. (42) and Quin and McGuckin
(27) that MUC1 mucins from mammary and ovarian carcinoma
cell lines can be phosphorylated on both tyrosine and serine residues
in the cytoplasmic domain strongly implicate activation of
intracellular signal transduction pathway(s) mediated by this cell
surface mucin. We are currently investigating downstream signaling
mechanisms and cellular inflammatory responses in the P. aeruginosa/CHO-Muc1 model system.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-47125 and research grants from the Cystic Fibrosis Foundation.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Rm. 446, Baltimore, MD 21201.
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. Section 1734 solely to indicate this fact.
Received 17 January 2000; accepted in final form 16 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Boshell, M,
Lalani EN,
Pemberton L,
Burchell J,
Gendler S,
and
Taylor-Papadimitriou J.
The product of the human MUC1 gene when secreted by mouse cells transfected with the full-length cDNA lacks the cytoplasmic tail.
Biochem Biophys Res Commun
185:
1-8,
1992[ISI][Medline].
2.
Boyd, RL,
Ramphal R,
Rice R,
and
Mangos JA.
Chronic colonization of rat airways with Pseudomonas aeruginosa.
Infect Immun
39:
1403-1410,
1983[ISI][Medline].
3.
DiMango, E,
Ratner AJ,
Bryan R,
Tabibi S,
and
Prince A.
Activation of NF-kappaB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells.
J Clin Invest
101:
2598-2605,
1998
4.
Gendler, SJ,
Lancaster CA,
Taylor-Papadimitriou J,
Duhig T,
Peat N,
Burchell J,
Pemberton L,
Lalani EN,
and
Wilson D.
Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin.
J Biol Chem
265:
15286-15293,
1990
5.
Gendler, SJ,
and
Spicer AP.
Epithelial mucin genes.
Annu Rev Physiol
57:
607-634,
1995[ISI][Medline].
6.
Hewetson, A,
and
Chilton BS.
Molecular cloning and hormone-dependent expression of rabbit Muc1 in the cervix and uterus.
Biol Reprod
57:
468-477,
1997[Abstract].
7.
Hey, NA,
Graham RA,
Weif MW,
and
Aplin JD.
The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase.
J Clin Endocrinol Metab
78:
337-342,
1994[Abstract].
8.
Hild-Petito, S,
Fazleabas AT,
Julian J,
and
Carson DD.
Mucin (Muc-1) expression is differentially regulated in uterine luminal and glandular epithelia of the baboon (Papio anubis).
Biol Reprod
54:
939-947,
1996[Abstract].
9.
Hilkens, J,
Ligtenberg MJL,
Vos HL,
and
Litvinov SV.
Cell membrane-associated mucins and their adhesion-modulating property.
Trends Biochem Sci
17:
359-363,
1992[ISI][Medline].
10.
Hollingsworth, MA,
Batra SK,
Qi WN,
and
Yankaskas JP.
MUC1 mucin mRNA expression in cultured human nasal and bronchial epithelial cells.
Am J Respir Cell Mol Biol
6:
516-520,
1992[ISI][Medline].
11.
Johnson, K,
Parker ML,
and
Lory S.
Nucleotide sequence and transcriptional initiation sites of two Pseudomonas aeruginosa pilin genes.
J Biol Chem
261:
15703-15708,
1986
12.
Kim, KC,
Wasano K,
Niles RM,
Schuster JE,
Stone PJ,
and
Brody JS.
Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.
Proc Natl Acad Sci USA
84:
9304-9308,
1987[Abstract].
13.
Krivan, HC,
Roberts DD,
and
Ginsburg V.
Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc1-4Gal found in some glycolipids.
Proc Natl Acad Sci USA
85:
6157-6161,
1988[Abstract].
14.
Lan, MS,
Batra SK,
Qi WN,
Metzgar RS,
and
Hollingsworth MA.
Cloning and sequencing of a human pancreatic tumor mucin cDNA.
J Biol Chem
265:
15294-15299,
1990
15.
Lapensee, L,
Paquette Y,
and
Bleau G.
Allelic polymorphism and chromosomal localization of the human oviductin gene (MUC9).
Fertil Steril
68:
702-708,
1997[ISI][Medline].
16.
Ligtenberg, MJ,
Buijs F,
Vos HL,
and
Hilkens J.
Suppression of cellular aggregation by high levels of episialin.
Cancer Res
52:
2318-2324,
1992[Abstract].
17.
Ligtenberg, MJ,
Kruijshaar L,
Buijs F,
van Meijer M,
Litvinov SV,
and
Hilkens J.
Cell-associated episialin is a complex containing two proteins derived from a common precursor.
J Biol Chem
267:
6171-6177,
1992
18.
Mai, GT,
McCormack JG,
Seow WK,
Pier GB,
Jackson LA,
and
Thong YH.
Inhibition of adherence of mucoid Pseudomonas aeruginosa by alginase, specific monoclonal antibodies, and antibiotics.
Infect Immun
61:
4338-4343,
1993[Abstract].
19.
Marcus, H,
Austria A,
and
Baker NR.
Adherence of Pseudomonas aeruginosa to tracheal epithelium.
Infect Immun
57:
1050-1053,
1989[ISI][Medline].
20.
Marcus, H,
and
Baker NR.
Quantitation of adherence of mucoid and non-mucoid Pseudomonas aeruginosa to hamster tracheal epithelium.
Infect Immun
47:
723-729,
1985[ISI][Medline].
21.
Moniaux, N,
Nollet S,
Porchet N,
Degand P,
Laine A,
and
Aubert JP.
Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.
Biochem J
338:
325-333,
1999[ISI][Medline].
22.
Park, HR,
Hyun SW,
and
Kim KC.
Expression of MUC1 mucin gene by hamster tracheal surface epithelial cells in primary culture.
Am J Respir Cell Mol Biol
15:
237-244,
1996[Abstract].
23.
Parry, G,
John Stubbs JL,
Bissell MJ,
Schmidhauser C,
Spicer AP,
and
Gendler SJ.
Studies of Muc-1 mucin expression and polarity in the mouse mammary gland demonstrate developmental regulation of Muc-1 glycosylation and establish the hormonal basis of mRNA expression.
J Cell Sci
101:
191-199,
1992[Abstract].
24.
Paul, E,
Lee DI,
Hyun SW,
Gendler SJ,
and
Kim KC.
Identification and characterization of high molecular-mass mucin-like glycoproteins in the plasma membrane of airway epithelial cells.
Am J Respir Cell Mol Biol
19:
681-690,
1998
25.
Pemberton, L,
Taylor-Papadimitriou J,
and
Gendler SJ.
Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals.
Biochem Biophys Res Commun
185:
167-175,
1992[ISI][Medline].
26.
Pier, GB,
Grout M,
Zaidi TS,
Olsen JC,
Johnson LG,
Yankaskas JR,
and
Goldberg JB.
Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections.
Science
271:
64-67,
1996[Abstract].
27.
Quin, RJ,
and
McGuckin MA.
Phosphorylation of the cytoplasmic domain of the MUC1 mucin correlates with changes in cell-cell adhesion.
Int J Cancer
87:
499-506,
2000[ISI][Medline].
28.
Ramphal, R,
Carnoy C,
Fievre S,
Michalski J-C,
Houdret N,
Lamblin G,
Strecker G,
and
Roussel P.
Pseudomonas aeruginosa recognizes carbohydrate chains type 1 (Gal1-3GlcNAc) or type 2 (Gal
1-4GlcNA) disaccharide units.
Infect Immun
59:
700-704,
1991[ISI][Medline].
29.
Ramphal, R,
Guay C,
and
Pier GB.
Pseudomonas aeruginosa adhesins for tracheobronchial mucin.
Infect Immun
55:
600-603,
1987[ISI][Medline].
30.
Ramphal, R,
Koo L,
Ishimoto KS,
Totten PA,
Lara JC,
and
Lory S.
Adhesion of Pseudomonas aeruginosa pilin-deficient mutants to mucin.
Infect Immun
59:
1307-1311,
1991[ISI][Medline].
31.
Ramphal, R,
and
Pyle M.
Adherence of mucoid and nonmucoid Pseudomonas aeruginosa to acid-injured tracheal epithelium.
Infect Immun
41:
345-351,
1983[ISI][Medline].
32.
Rose, M,
and
Gendler S.
Airway mucin genes and gene products.
In: Airway Mucus: Basic Mechanisms and Clinical Perspectives, edited by Rogers DF,
and Lethem MI.. Basel: Birkhauser Verlag, 1997, p. 41-66.
33.
Rostand, KS,
and
Esko JD.
Cholesterol and cholesterol esters: host receptors for Pseudomonas aeruginosa adherence.
J Biol Chem
268:
24053-24059,
1993
34.
Saiman, L,
and
Prince A.
Pseudomonas aeruginosa pili bind to asialo GM1 which is increased on the surface of cystic fibrosis epithelial cells.
J Clin Invest
92:
1875-1890,
1993[ISI][Medline].
35.
Sajjan, U,
Reisman J,
Doig P,
Irvin RT,
Forstner G,
and
Forstner J.
Binding of nonmucoid Pseudomonas aeruginosa to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.
J Clin Invest
89:
657-665,
1992[ISI][Medline].
36.
Van de Wiel-van Kemenade, E,
Ligtenberg MJL,
de Boer AJ,
Buijs F,
Vos HL,
Melief CJM,
Hilkens J,
and
Figdor CG.
Episialin (MUC1) inhibits cytotoxic lymphocyte-target cell interaction.
J Immunol
151:
767-776,
1993
37.
Wasano, K,
Kim KC,
Niles RM,
and
Brody JS.
Membrane differentiation markers of airway epithelial secretory cells.
J Histochem Cytochem
36:
167-178,
1988[Abstract].
38.
Wesseling, J,
van der Valk SW,
Vos HL,
Sonnenberg A,
and
Hilkens J.
Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components.
J Cell Biol
129:
255-265,
1995[Abstract].
39.
Williams, SJ,
McGuckin MA,
Gotley DC,
Eyre HJ,
Sutherland GR,
and
Antalis TM.
Two novel mucin genes down-regulated in colorectal cancer identified by differential display.
Cancer Res
59:
4083-4089,
1999
40.
Williams, SJ,
Munster DJ,
Quin RJ,
Gotley DC,
and
McGuckin MA.
The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.
Biochem Biophys Res Commun
261:
83-89,
1999[ISI][Medline].
41.
Woods, DE,
Bass JA,
Johanson WG,
and
Straus DC.
Role of adherence in the pathogenesis of Pseudomonas aeruginosa lung infection in cystic fibrosis patients.
Infect Immun
30:
694-699,
1980[ISI][Medline].
42.
Zrihan-Licht, S,
Baruch A,
Elroy-Stein O,
Keydar I,
and
Wreschner DH.
Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine receptor-like molecules.
FEBS Lett
356:
130-136,
1994[ISI][Medline].