1 Division of Pediatric Pulmonary Medicine, Duke University Medical Center, Durham, North Carolina 27710; and 2 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33101
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ABSTRACT |
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In chronic obstructive pulmonary diseases, the airway epithelium is chronically exposed to neutrophil elastase, an inflammatory protease. The cellular response to neutrophil elastase dictates the balance between epithelial injury and repair. Key regulators of epithelial migration and proliferation are the ErbB receptor tyrosine kinases, including the epidermal growth factor receptor. In this context, we investigated whether neutrophil elastase may regulate expression of MUC4, a membrane-tethered mucin that has recently been identified as a ligand for ErbB2, the major heterodimerization partner of the epidermal growth factor receptor. In normal human bronchial epithelial cells, neutrophil elastase increased MUC4 mRNA levels in both a concentration- and time-dependent manner. RNA stability assays revealed that neutrophil elastase increased MUC4 mRNA levels by prolonging the mRNA half-life from 5 to 21 h. Neutrophil elastase also increased MUC4 glycoprotein levels as determined by Western analysis, using a monoclonal antibody specific for a nontandem repeat MUC4 sequence. Therefore, airway epithelial cells respond to neutrophil elastase exposure by increasing expression of MUC4, a potential activator of epithelial repair mechanisms.
protease; mRNA stability; airway epithelium; mucin glycoprotein
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INTRODUCTION |
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PATIENTS WITH CHRONIC BRONCHITIS (CB) and cystic fibrosis (CF) are plagued by recurrent exacerbations of productive cough, dyspnea, and progressive deterioration of lung function. Although the etiologies of these diseases are different, they share a common pathogenic mechanism characterized by an imbalance between proteolytic injury and antiprotease defenses. Neutrophils dominate the inflammatory response during acute exacerbations of CB (45) and CF (55). Airway obstruction, poor pulmonary function, and chronic expectoration are directly associated with sputum neutrophil concentrations (48). Neutrophils release elastase (NE; EC 3.4.21.37), a serine protease found in high concentrations (µM) in the airways of patients with exacerbations of CB (49) and CF (32). NE exposure injures airway epithelium resulting in ciliary dismotility and injury (1, 34), increased mucin production (6, 10), mucin secretion (13, 24, 28), mucin gene expression (53), and epithelial loss (21). After NE exposure, there is either normal epithelial restitution or secretory and/or squamous metaplasia (21, 45, 55). The mechanisms regulating epithelial proliferation and differentiation following injury are critical for understanding the airway remodeling that occurs in CB and CF but are not yet defined.
Activation of the epidermal growth factor receptor (EGFR), a member of the ErbB receptor tyrosine kinase family, is required for epithelial proliferation and migration (3, 23) following epithelial injury (reviewed in Ref. 27). Three members of the ErbB family have been detected in human airways: EGFR (ErbB1), ErbB2, and ErbB3 (37). When activated, these receptors may homodimerize or heterodimerize (17). ErbB2 is the preferred heterodimerization partner for the other ErbB receptors (16). Importantly, the first ligand specific for ErbB2 activation has recently been identified, the membrane-tethered mucin MUC4 (8).
MUC4 is one member of a family of membrane-tethered mucins that have
protein domains and expression patterns that are very different from
the secreted, gel-forming mucins. MUC4 cDNA encodes a large
domain of tandemly repeating amino acids rich in serine and threonine
that is the site of O-linked glycosylation, characterizing the molecule
as a mucin (33, 38). In addition to the tandem repeat
domain, the carboxyl moiety of human MUC4, MUC4, contains several
interesting protein domains, including two epidermal growth factor
(EGF)-like domains, a non-EGF cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain that contains potential
phosphorylation residues (31). Human MUC4 is
expressed in branching airways during fetal lung development (7,
43). It is also highly expressed in superficial ciliated and
secretory airway epithelial cells from large airways to bronchioles in
adults (2, 7). The protein domains of MUC4 and the
expression of MUC4 during airway development and in
differentiated airway epithelium suggest that MUC4 has important
functions in the airway.
With this background, we sought to determine whether NE regulated the expression of MUC4 mRNA and glycoprotein. We first established that in normal human bronchial epithelial (NHBE) cells both MUC4 mRNA and protein are expressed. We also show that MUC4 glycoprotein is expressed in superficial airway epithelial cells. We then demonstrate that NE increases expression of MUC4 mRNA and glycoprotein. We found that NE regulates MUC4 by enhancing mRNA stability. The increased expression of MUC4 in NHBE cells, following exposure to NE, suggests that MUC4 may have an important role in the epithelial response to injury.
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MATERIALS AND METHODS |
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Reagents.
Dulbecco's modified Eagle's medium, T4 kinase, and 20× SSC were from
GIBCO-BRL/Invitrogen (Carlsbad, CA). NHBE cells, bronchial epithelial
basic medium, and SingleQuot supplements were from Clonetics/BioWhittaker (Walkersville, MD). Six-well plates were purchased from Corning Life Sciences (Cambridge, MA), and 60-mm tissue
culture dishes from BD Biosciences (Bedford, MA). EGF and bovine serum
albumin (BSA) were from Intergen/Serologicals (Norcross, GA). NE (875 U/mg protein) was from Elastin Products (Owensville, MO). Nylon filter
(Nytran SuPerCharged) was from Schleicher and Schuell (Keene, NH).
X-Omat AR film was purchased from Kodak (Rochester, NY). Acrylamide was
from National Diagnostics (Atlanta, GA). Prime-It II random primer
labeling kit was purchased from Stratagene (La Jolla, CA). Enhanced
Chemiluminescence Plus kit, streptavidin-horseradish peroxidase
conjugate, sheep anti-mouse IgG, peroxidase-linked species-specific
whole antibody, microspin G-25 columns, HiTrap Protein G HP column,
[-32P]dCTP, and [
-32P]ATP were from
Amersham Biosciences (Piscataway, NJ). Kaleidoscope prestained protein
standards, 0.45-µm-pore nitrocellulose, DC Protein Assay,
N,N,N',N'-tetramethylethylenediamine, and sodium dodecyl
sulfate (SDS) were purchased from Bio-Rad Laboratories (Hercules, CA),
and cesium chloride was from ICN (Costa Mesa, CA). Pepstatin
was purchased from Roche Molecular Biochemicals (Indianapolis, IN).
Cytotox 96 nonradioactive cytotoxicity assay kit was purchased from
Promega (Madison, WI). Tris, ammonium persulfate, and glycine were from
EM Sciences (Gibbstown, NJ). Ammonium acetate was purchased from
Mallinckrodt Baker (Paris, KY). Biotinylated SDS molecular weight
standards, Ponceau S stain, Tween 20, sodium deoxycholic acid, Triton
X-100, retinoic acid, bovine pancreatic trypsin, guanidine thiocyanate,
methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPV-CMK), and all
other chemicals were from Sigma (St. Louis, MO). Immunohistochemistry
reagents including Retrieval solution, LINK solution, and
diaminobenzidine (DAB) substrate were obtained from Dako (Carpinteria,
CA). NS1 was provided by the Developmental Studies Hybridoma Bank,
Department of Biological Sciences, University of Iowa (Iowa City, IA).
Cell culture.
NHBE were seeded in six-well plates or 60-mm tissue culture dishes in a
serum-free 1:1 mixture of bronchial epithelial cell basic medium, and
Dulbecco's modified Eagle's medium with SingleQuot supplements,
bovine pituitary extract (0.13 mg/ml), EGF (0.5 ng/ml), BSA (1.5 µg/ml), and all trans retinoic acid (5 × 108 M)
in place of SingleQuot retinoic acid and grown to confluency.
Cell stimulation. All studies were carried out on confluent plates of NHBE cells in serum-free growth factor-supplemented medium. Cells were exposed to NE or trypsin at doses and times specified in figure legends. Control conditions included cells treated with 50 µM sodium acetate, pH 5, 100 µM sodium chloride (NE buffer), 1 µM hydrochloric acid (HCl; trypsin buffer), or boiled NE.
RNA isolation and Northern analysis.
RNA was isolated from cell cultures as previously described by the
guanidinium thiocyanate/cesium chloride method (15, 53). Total RNA (10 µg) was separated by 1.2% agarose-formaldehyde gel electrophoresis and transferred by capillary blot to a nylon filter (Nytran SuPerCharged) in 1 M ammonium acetate. After UV
cross-linking, the filters were hybridized at 58°C with
32P-end-labeled oligomer probe (specific activity
>108
counts · min1 · µg
1)
for MUC4 (tandem repeat region)
(5'-GTCGGTGACATGAAGAGGGGTGGTGTCACCTGTGGATGCTGAGGAAGT-3') (38) and at 62°C for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) cDNA probe (53) and 28s rRNA
oligomer probe (GenBank accession no. M11167, nucleotide sequence
4011-4036) (5'-AACGATCAGAGTAGTGGTATTTCACC-3') (18).
Filters were washed twice with 2× SSC and 0.1% SDS at room
temperature for 30 min and then with 0.1× SSC and 0.1% SDS at 58°C
for 5 min. Filters were exposed for autoradiography at
80°C. Band
density on autoradiographs was determined by digitalization with
Fotolook and Photoshop softwares and quantitation using NIH Image software.
RNA stability assay. Transcription in NHBE cells that were resting or stimulated with NE (100 nM, 17 h) was stopped by treatment with actinomycin D (4 µg/ml) for 0, 2, 4, and 8 h after NE treatment (53). For each time point, total cellular RNA was extracted and MUC4 mRNA and 28s rRNA levels were evaluated by Northern analysis and quantitated as described above. Results are plotted as a percentage of starting mRNA levels after the addition of actinomycin D. On the basis of linear regression and plot extrapolation using SigmaPlot software, the half-life is estimated to be at the time point where 50% of the original mRNA level remains.
Western analysis.
The mouse monoclonal antibody 1G8 (J. Zhang and K. L. Carraway, unpublished results) was used to detect MUC4 protein
expression. The antigen for this antibody was purified rat ascites
sialoglycoprotein-2 (rat Muc4; ASGP-2) protein from an ascites tumor
13762 MAT-C1 subline grown in rats. Hybridomas were screened with rat
ASGP-2 from ascites tumor cells (19) and recombinant human
MUC4
/ASGP-2 expressed in Cos7 and HC11 mouse mammary epithelial
cells. 1G8 was selected because of its strong reaction with human
MUC4
/ASGP-2 compared with rat ASGP-2. The antibody was purified
using the HiTrap Protein G HP column.
Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue blocks including normal cartilaginous airway from lung cancer patients (n = 6) were sectioned (5 µm), mounted on slides, and rehydrated. After antigen retrieval with Retrieval solution at 97°C for 20 min and incubation with 3% hydrogen peroxide, slides were incubated with 1G8 (1:100 dilution) at 37°C for 30 min. After two washes in phosphate-buffered saline, LINK solution (biotinylated secondary antibody) was applied to the slides at room temperature for 30 min. After two washes in phosphate-buffered saline, streptavidin peroxidase solution was then applied for 15 min, followed by DAB substrate. After development, slides were counterstained with hematoxylin.
Cytotoxicity assessment. We assessed cytotoxicity by lactate dehydrogenase (LDH) release using a commercially available colorimetric assay for LDH according to the manufacturer's instructions. NHBE cells were exposed to NE (50 or 100 nM NE, 24 h) or control vehicle. Both supernatants and cell lysates were collected and assessed for LDH content. We calculated the percentage of LDH release by taking the ratio of LDH released into the supernatant to the total LDH in the supernatant and the cell lysate.
Statistical analysis. Analysis of data was performed by the Kruskal-Wallis one-way nonparametric analysis of variance and post hoc comparisons by the Wilcoxon/Mann-Whitney rank sum test (47). Differences were considered significant at P < 0.05.
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RESULTS |
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NE increased MUC4 mRNA levels in a dose- and time-dependent manner.
As illustrated in Fig. 1, MUC4 mRNA
expressed in NHBE cells is >9.5 kb. NE treatment (0-100 nM)
increased MUC4 mRNA levels in NHBE cells in a concentration-dependent
manner (Fig. 1A). At a concentration of 100 nM for 24 h, NE increased MUC4 mRNA levels approximately sevenfold compared with
vehicle alone (Fig. 1C). MUC4 expression in NHBE cells was
also regulated by NE in a time-dependent manner (Fig.
2). MUC4 transcript levels were increased
after 1 h of exposure to NE, with significant increases in MUC4
expression exhibited at both 8 and 24 h of continuous NE exposure.
NE treatment under these conditions caused <5% LDH release and was
considered to be noncytotoxic at these concentrations (data not shown).
Heat-inactivated NE (boiled) had no effect on MUC4 expression in NHBE
cells (data not shown).
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MUC4 mRNA expression was differentially regulated by another serine
protease, trypsin.
To determine whether other serine proteases regulated MUC4
expression in a similar manner as NE, we treated NHBE cells with bovine
pancreatic trypsin and examined the regulation of MUC4 expression.
Trypsin stimulated a significant increase in MUC4 mRNA expression in
NHBE cells but required 10-fold higher protease activity (13 U/ml) than
NE (1.3 U/ml 50 nM; Fig. 3). At
equivalent enzymatic activity (1.3 U/ml), trypsin had no significant
effect on MUC4 mRNA expression (Fig. 3). Similar to NE, trypsin did not effect 28s rRNA levels (Fig. 3B).
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NE increased MUC4 mRNA expression by a posttranscriptional
mechanism.
To evaluate whether NE regulated MUC4 gene expression by a
posttranscriptional mechanism, we performed mRNA stability assays. RNA
stability assays revealed that NE treatment prolonged MUC4 mRNA
half-life from 5 h in control cells to 21 h (Fig.
4A). In contrast, NE did not
prolong the half-life of GAPDH mRNA or 28s rRNA, demonstrating that the
effect on MUC4 mRNA stability was specific (Fig. 4, B and
C). These experiments are consistent with the concept that
NE regulates MUC4 expression, at least in part, by enhancing mRNA
stability.
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NE treatment stimulated increased MUC4 glycoprotein production in
NHBE cells.
Western analysis of MUC4 glycoprotein was performed to determine the
effect of NE on MUC4 glycoprotein production. Using the mouse
monoclonal anti-MUC4 antibody 1G8, Western analyses of NHBE cell
lysates revealed a predominant band at ~147 kDa, similar to that
previously observed for the transmembrane subunit ASGP-2/MUC4 of
human MUC4 (Fig. 5) (36) and
six smaller bands between 116 and 45 kDa representing MUC4 glycoprotein
(Fig. 6). Treatment with NE increased
MUC4 glycoprotein levels in NHBE cell lysates compared with control
cell lysates (Fig. 6A). These protein bands were not
detected following incubation with NS1, a control myeloma supernatant
(Fig. 6B), demonstrating the specificity of the
antibody-antigen complex. Ponceau S staining demonstrated equivalent
sample loading for each gel (data not shown).
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MUC4 glycoprotein is expressed in pseudostratified columnar airway
epithelium.
Using the mouse monoclonal anti-MUC4 antibody 1G8, we performed
immunohistochemistry to determine the localization of MUC4 glycoprotein
in vivo. The MUC4 protein was expressed throughout the normal
pseudostratified columnar epithelium from non-CF, non-CB patients (Fig.
7A). Protein detection was
specific because immunostaining was negative with control myeloma
supernatant, NS1 (Fig. 7B). Localization of the protein
agrees with previous reports of MUC4 mRNA expression by in
situ hybridization (2, 7).
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DISCUSSION |
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In this study, we demonstrate that NE increased MUC4 mRNA expression in NHBE cells by a concentration- and time-dependent mechanism. NE proteolytic activity was required for this response, and the concentrations of NE activity used in this study correlate with elastase activity levels found in airway secretions of patients with CF (32) and CB (49). Interestingly, another serine protease, bovine pancreatic trypsin, also increased MUC4 mRNA expression but required higher enzymatic activity compared with NE. Using a monoclonal antibody that detects an MUC4 nontandem repeat protein sequence, we demonstrate that MUC4 glycoprotein is present in superficial airway epithelial cells in control, non-CF, non-CB airways. Importantly, following NE exposure, MUC4 glycoprotein expression increased corresponding to the increase in MUC4 mRNA expression.
Although the functions of MUC4 in the lung are not yet known, its high
level of expression in the lung and its structural features suggest
that the molecule may play important roles in airway homeostasis and
repair. The deduced size of the MUC4 protein backbone is larger than
MUC1 (14, 31). Furthermore, the major protein domain in
MUC4 is a tandemly repeating sequence of 16 amino acids enriched in
serine and threonine that is the major site for O-linked glycosylation
and that shares no homology with other MUC molecules or with the rat
homolog of MUC4, the sialomucin complex (SMC) (19).
Because MUC1 extends 250-500 nm above the apical membrane
(5) and beyond the glycocalyx (50), MUC4 likely extends a unique complex of carbohydrate structures into the
airway lumen beyond other cell-associated glycoproteins
(31). In addition to the tandem repeat domain, the
carboxyl moiety of human MUC4, MUC4, contains several interesting
protein domains, including two EGF-like domains, a non-EGF
cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain
that contains potential phosphorylation residues (31).
These MUC4
protein domains are identical to rat ASGP-2 protein domains.
Most information concerning potential functions of MUC4 protein domains derives from studies with rat Muc4/SMC in cancer tissues. Overexpression of SMC in a human melanoma cell line inhibits killing of tumor cells by masking surface antigens (26) and suppresses tumor cell apoptosis, promoting rapid tumor growth (25). These results suggest Muc4/SMC mediates tumor progression. MUC4 may also regulate cellular growth through two EGF-like domains. In rat mammary adenocarcinoma cells, there is direct interaction between the carboxyl moiety of SMC, ASGP-2, and a member of the EGFR family ErbB2 (8). Furthermore, Muc4/SMC potentiates the receptor tyrosine kinase activity of ErbB2. Moreover, anti-ErbB2 antibody binding to human melanoma cells and human breast carcinoma cells was reduced by overexpression of Muc4/SMC (40). As ErbB2 is a major heterodimerization partner of the EGFR (16, 17), a major growth factor receptor in the lung (11), MUC4 may regulate growth, differentiation, and repair processes in normal airway tissues.
In this report, we used a monoclonal antibody that recognizes a unique
nontandem repeat region of MUC4 to detect MUC4. The epitope used to
develop the monoclonal antibody is a portion of ASGP-2 and has been
used to develop other anti-MUC4 antibodies. By immunohistochemistry,
MUC4
subunit expression is localized to the same cells that express
MUC4 mRNA by in situ hybridization (2, 7).
Anti-MUC4 antibody specificity is further supported by the lack of
staining when the antibody is replaced by control myeloma supernatant.
In an SDS-PAGE system for Western analysis, the MUC4
moiety likely
dissociates from the amino-terminal portion of MUC4, thus resulting in
a smaller size than what would be expected for the full-length
molecule. By Western analysis, we detected the MUC4
glycoprotein
with an approximate size of 147 kDa and several smaller proteins. The
size of MUC4
in NHBE cell lysates is similar to the size of MUC4
detected by Western analysis in human corneal epithelium and tears
(36) and similar to the size of ASGP-2 (120-140 kDa)
in rat epithelial tissues (20, 29). Because our samples
are from cell lysates, the smaller proteins detected by the antibody
may be precursor proteins or products of alternatively spliced
transcripts (30). Importantly, in NHBE cells, increases in
MUC4 glycoprotein expression correlated well with increased MUC4 mRNA levels.
The regulation of MUC4 gene expression is just
beginning to be elucidated. In human pancreatic tumor cells,
transforming growth factor-beta 2 (TGF-2) mediates the upregulation
of MUC4 expression by retinoic acid (9). In the human
pancreatic tumor cell lines CAPAN-1 and CAPAN-2, MUC4 promoter activity
can be increased by protein kinase C activation, EGF, or transforming
growth factor (TGF)-
treatment (35). In addition, in
CAPAN-2 human pancreatic tumor cells, interferon-
in combination
with tumor necrosis factor-
or TGF-
, but not each cytokine or
growth factor independently, increases MUC4 promoter activity
(35). In contrast to transcriptional regulation of MUC4,
in rat mammary epithelial cells, TGF-
treatment decreases Muc4
glycoprotein levels by suppressing Muc4 glycoprotein production without
changing Muc4 transcript levels (39). Furthermore, TGF-
treatment regulates Muc4/SMC expression by a posttranslational mechanism where the processing of the precursor protein is altered (41).
Our study demonstrates that human MUC4 is also
posttranscriptionally regulated. In human bronchial epithelial cells,
NE increased MUC4 expression by enhancing mRNA stability. This
observation adds to a growing number of reports demonstrating
posttranscriptional regulation of mucin genes. We have previously
reported that in A549 cells NE increases the expression of another
major respiratory tract mucin gene, MUC5AC, also by
enhancing mRNA stability (53). Similarly, in NCI-H292
cells, TNF- treatment increases the half-life of MUC5AC
mRNA (4). In HT29 colonic carcinoma cells, in response to
phorbol ester treatment, MUC2 expression is regulated by a posttranscriptional mechanism (52). Collectively, these
studies support the concept that inflammatory mediators amplify the
expression of mucin genes by posttranscriptional mechanisms. These
observations underscore the importance of understanding the molecular
mechanisms required for posttranscriptional regulation of
MUC gene expression.
The specific mechanism(s) of NE-mediated mRNA stability are not yet
known. It is possible that NE interacts with a cell surface receptor
resulting in activation of the receptor and its associated signaling
cascade or that NE releases a secondary mediator that functions in a
paracrine/autocrine manner. On platelets, NE cleaves a surface integrin
important for the potentiation of platelet aggregation
(46). In respiratory epithelial cells, NE upregulates IL-8
expression through a signaling cascade mediated by IL-1
receptor-associated kinase and tumor necrosis factor-associated factor
6 (TRAF6) (54). Signals from TRAF6 can be further relayed
via MAP kinase kinase kinase downstream to activate other members of
the MAP kinase cascades (42) and subsequently may regulate
mRNA stability (22, 56). Similarly, NE treatment can
promote release of growth factors that in turn can mediate
posttranscriptional processes. NE treatment causes the release of
TGF- (51), which has been shown to be important in the
regulation of SMC/Muc4 (20, 41). Similarly, another
protease, thrombin, decreases the half-life of endothelial nitric oxide
synthase through a Rho GTPase-mediated process (12). Stabilization of mammalian gene mRNAs are mediated by many
intracellular signals, including protein kinases, growth factors, ions,
and reactive oxygen species (44). However, the specific
signaling cascades mediating NE-induced posttranscriptional regulation
of MUC4 expression have not been delineated and warrant further investigation.
Our report is one of the first to show that MUC4 expression is regulated in normal human airway epithelium and that expression is increased by a major inflammatory protease. We now have a model system to study the function of MUC4 during the epithelial response to injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jonathan Cohn for critical review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-65611 (J. A. Voynow), CA-52498 (K. L. Carraway), and CA-74072 (K. L. Carraway), grants from the March of Dimes (J. A. Voynow), the American Lung Association (B. M. Fischer), the Duke Children's Miracle Network (B. M. Fischer), and the Florida Biomedical Research Program (K. L. Carraway).
NS1 myeloma supernatant was obtained from the Developmental Studies Hybridoma Bank and is under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
Portions of this work were presented at the annual North American Cystic Fibrosis conferences (Seattle, WA, October 1999 and New Orleans, LA, October 2002).
Address for reprint requests and other correspondence: J. A. Voynow, Div. of Pediatric Pulmonary Medicine, Duke Univ. Medical Center, Box 2994, Durham, NC 27710 (voyno001{at}mc.duke.edu).
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.
First published December 20, 2002;10.1152/ajplung.00220.2002
Received 9 July 2002; accepted in final form 12 December 2002.
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