Am J Physiol Lung Cell Mol Physiol 289: L971–L979, 2005.
First published July 22, 2005; doi:10.1152/ajplung.00172.2005.
␤-Catenin regulates differentiation of respiratory epithelial cells in vivo
Michael L. Mucenski,1,2 Jennifer M. Nation,1 Angela R. Thitoff,1
Valérie Besnard,1 Yan Xu,1,2 Susan E. Wert,1,2 Naomoto Harada,3
Makoto M. Taketo,4 Mildred T. Stahlman,5 and Jeffrey A. Whitsett1,2
1
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati; 2Department
of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; 3Banyu Tsukubu Research Institute
(Merck), Tsukuba, Japan; 4Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Kyoto, Japan; and 5Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee
Submitted 15 April 2005; accepted in final form 14 July 2005
Mucenski, Michael L., Jennifer M. Nation, Angela R. Thitoff,
Valérie Besnard, Yan Xu, Susan E. Wert, Naomoto Harada,
Makoto M. Taketo, Mildred T. Stahlman, and Jeffrey A. Whitsett. ␤-Catenin regulates differentiation of respiratory epithelial cells
in vivo. Am J Physiol Lung Cell Mol Physiol 289: L971–L979, 2005.
First published July 22, 2005; doi:10.1152/ajplung.00172.2005.—An
activated form of ␤-catenin [Catnb⌬(ex3)] was expressed in respiratory
epithelial cells of the developing lung. Although morphogenesis was
not altered at birth, air space enlargement and epithelial cell dysplasia
were observed in the early postnatal period and persisted into adulthood. The Catnb⌬(ex3) protein caused squamous, cuboidal, and goblet
cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had
morphological characteristics of alveolar type II cells were observed
in bronchioles of the transgenic mice. Catnb⌬(ex3) inhibited expression
of Foxa2 and caused goblet cell hyperplasia associated with increased
staining for mucins and the MUC5A/C protein. In vitro, both wild
type and activated ␤-catenin negatively regulated the expression of
the Foxa2 promoter. Catnb⌬(ex3) also caused pulmonary tumors in
adult mice. Activation of ␤-catenin caused ectopic differentiation of
alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the
Wnt/␤-catenin signal transduction pathway in the differentiation of
the respiratory epithelium in the postnatal lung.
lung morphogenesis; tumorigenesis; goblet cell hyperplasia; air space
enlargement
THE RESPIRATORY TRACT IS LINED by distinct subsets of respiratory
epithelial cells in the conducting and peripheral airways. The
numbers of epithelial cell types and their gene expression
patterns are precisely regulated along the cephalo-caudal axis,
as well as during development in the mammalian lung (40).
Differentiation of various epithelial cell types is influenced by
a variety of developmental, humoral, and environmental factors. Lung morphogenesis and homeostasis are dependent upon
precise autocrine-paracrine signaling events that control transcriptional programs that influence cell proliferation, differentiation, and/or function (4, 7). Cytodifferentiation of respiratory epithelial cells occurs progressively in late gestation and in
the postnatal period, as conducting airways become lined by an
increasing complexity of airway epithelial cell types, including
basal, ciliated, goblet, and nonciliated bronchiolar cells and
cuboidal (type II) and squamous (type I) cells in the alveoli.
Alveolarization and differentiation of the conducting airways
Address for reprint requests and other correspondence: M. L. Mucenski,
Div. of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center,
3333 Burnet Ave., Cincinnati, OH 45229-3039 (e-mail: michael.mucenski
@cchmc.org).
http://www.ajplung.org
are highly active processes in the postnatal period, which are
substantially completed 21–28 days after birth in the mouse.
Thereafter, environmental, infectious, and other inflammatory
conditions influence airway differentiation and function.
Marked changes in respiratory epithelial cell differentiation,
including goblet cell hyperplasia and squamous cell metaplasia, are associated with common medical conditions, including
asthma, smoking, chronic obstructive pulmonary disease, and
cystic fibrosis.
The Wnt/␤-catenin pathway regulates intracellular signaling
and gene transcription, which in turn, alter proliferation and/or
differentiation of many cell lineages. ␤-Catenin interacts with
the cytoplasmic tail of E-cadherin and ␣-catenin, a protein
linked to actin filaments, to maintain cell adhesion (10, 24). In
the Wnt/␤-catenin signal transduction pathway, a subset of
secreted Wnt glycoproteins interacts with their Frizzled (Fzd)
receptors, thereby inhibiting ␤-catenin phosphorylation by glycogen synthase kinase-3␤ and its degradation. Hypophosphorylated ␤-catenin accumulates in the cytoplasm, translocates to
the nucleus, and interacts with high mobility group domaincontaining, DNA-binding proteins [including T cell factor
lymphoid enhancer factor (LEF) and Sox family members] to
regulate the expression of downstream genes (17, 33). ␤-Catenin pathways influence many biological processes, including
cell fate decisions, stem cell proliferation, and axis specification (22, 42). A number of Wnt and Fzd family members are
expressed in the developing lung (9, 18, 20, 29). The importance of the Wnt/␤-catenin pathway in lung morphogenesis
was supported recently by studies in which ␤-catenin was
deleted in the developing respiratory epithelium, using a doxycycline-inducible conditional system to express Cre recombinase, which resulted in homologous recombination between
loxP sites to remove exons 3–7 of the ␤-catenin gene (22).
␤-Catenin expression in respiratory epithelial cells was shown
to be required for normal lung morphogenesis. Formation and
differentiation of peripheral lung tubules, including the specification of type I and type II epithelial cells in the alveolus,
were disrupted by the lack of ␤-catenin expression in respiratory epithelial cells of the mouse (22). Thus ␤-catenin influenced specification of peripheral versus proximal airway epithelial cells in vivo. When a ␤-catenin-LEF1 fusion protein
was expressed under control of the human surfactant protein C
(SFTPC) promoter early in lung morphogenesis, lung formation was disrupted and genes typically expressed in the gut tube
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were aberrantly expressed (25). Taken together, these studies
support a potential role for Wnt/␤-catenin signal transduction
pathway in cell fate specification during foregut and respiratory
epithelial differentiation.
In the present study, an activated ␤-catenin isoform,
Catnb⌬(ex3), was expressed after Cre-mediated recombination
in respiratory epithelial cells of the fetal and postnatal lung
under control of the rat Clara cell secretory protein (CCSP)
promoter.
MATERIALS AND METHODS
Transgenic mice. Catnb⫹/lox(ex3) mice were generated as previously described (14). The generation of triple transgenic mice, doxycycline regiments, conditions for genotyping CCSP-reverse tetracycline transactivator gene (rtTA) and (tetO)7-CMV-Cre transgenic
mice, processing of fetal tissue, and inflation fixation of postnatal
lungs were described previously (19, 22, 36). The Catnblox(ex3)
transgene was identified with PCR primers for neomycin forward
(5⬘-CAC ACC AGA CAA TCG GCT GCT-3⬘) and reverse (ACA
GTT CGG CTG GCG CGA G-3⬘). PCR parameters used were: 95°C
for 5 min, followed by 30 cycles at 95°C for 30 s, 60°C for 30 s, and
72°C for 45 s, followed by a 7-min extension at 72°C. Mice used in
this study were housed and maintained according to protocols approved by the Institutional Animal Care and Use Committee at
Cincinnati Children’s Hospital Research Foundation. In these mice,
conditional expression of Cre recombinase caused deletion of exon 3
of the mouse ␤-catenin gene, producing Catnb⌬(ex3), an activated
form of the ␤-catenin protein.
Histology, immunohistochemistry, and electron microscopy. A
minimum of three controls and three compound mutants were analyzed at each time point, with the exception of postnatal day (PN) 21,
in which two controls were analyzed. Antibodies used were generated
to: ␤-catenin (1:2,000 –1:4,000, goat polyclonal, sc-1496; Santa Cruz
Biotechnology), MUC5AC [1:6,000, chicken polyclonal, designated
H08, kindly provided by Dr. Samuel Ho, University of Minnesota
(38)], CCSP (1:2,000, rabbit polyclonal generated in this laboratory),
surfactant pro protein C (pro-SP-C, 1:4,000, rabbit polyclonal generated in this laboratory), thyroid transcription factor (TTF-1, 1:2,000,
rabbit polyclonal generated in this laboratory), Foxa2 (1:2,000, rabbit
polyclonal generated in this laboratory), cyclin D1 (1:60, mouse
monoclonal, 72-13G, sc-450; Santa Cruz Biotechnology), ␤-tubulin
IV (1:400, mouse monoclonal, MU178-UC; Biogenex), and phosphohistone H3 (1:500, rabbit polyclonal, H5110-14B; US Biological).
Biotinylated secondary antibodies and a streptavidin-biotin-peroxidase detection system (Vector Laboratories) were used to localize the
antibody-antigen complexes in the tissues, as previously described
(43). A mouse-on-mouse blocking kit (Vector Laboratories) was used
with the mouse monoclonal antibodies. Antigen detection was enhanced with nickel-diaminobenzidine and Tris-cobalt, followed by
counterstaining with nuclear fast red. To detect acidic or neutral
mucins, we stained lungs with Alcian blue or periodic acid Schiff
(PAS) following manufacturer’s instructions (Poly Scientific R&D).
The Hart’s method was used to stain for elastin fibers. Vascular
patterning was assessed using a biotinylated, endothelium-specific
isolectin Griffonia simplicifolia B4 as previously described (1). Mice
used in the ovalbumin challenge model for goblet cell hyperplasia
were treated as previously described (39).
Tissue for ultrastructural analysis was fixed in modified Karnovsky’s fixative containing 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed in 1% osmium
tetroxide in 0.1 M sodium cacodylate buffer (reduced with 1.5%
potassium ferrocyanide), stained en bloc with aqueous 4% uranyl
acetate, dehydrated, and embedded in Embed 812 (Electron Microscopy Sciences). Sections were analyzed on a Morgangni electron
microscope and the images stored digitally.
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Western blot and ELISA analyses. Bronchoalveolar lavage (BAL)
was done as previously described (30). BAL fluid was concentrated
with a Centricon YM-10 (Millipore) following manufacturer’s specifications. Protein concentrations in BAL supernatants were determined by the Lowry protein assay. BAL supernatants were stored at
⫺20°C. Equal protein amounts were resolved on 10 –20% SDS-Trisglycine-polyacrylamide gels (Invitrogen Life Technologies) and
transferred to a nitrocellulose membrane. The membrane was blocked
in Tris-buffered saline containing 0.1% Tween (TBST) and 5% wt/vol
powdered milk for 30 min at room temperature. The blot was
incubated overnight at 4°C in TBST containing rabbit anti-human
matrix metalloproteinase (MMP)-12 (BioMol International) at a
1:1,000 dilution. The blot was washed in TBST and incubated with
peroxidase-conjugated goat anti-rabbit IgG antibody (Calbiochem) at
1:5,000 dilution in TBST for 2 h at room temperature. After being
washed in TBST, blot was developed with ECL Western blotting
detection reagents (Amersham Biosciences) following manufacturer’s
specifications. Lung lavage fluid from a surfactant protein D null
mutant (Sftpd⫺/⫺) mouse served as a positive control for activated
MMP-12 (41).
RT-PCR analysis. Total RNA was isolated from the lungs of two
adult controls and three compound mutant animals and reverse transcribed by standard methodologies. RNA was also isolated from adult
intestine to serve as a positive control. Primer pairs used for three
intestinal genes were the following: Atoh1 forward (5⬘-GCT TCC
TCT GGG GGT TAC TC-3⬘) and reverse (5⬘-ACA ACG ATC ACC
ACA GAC CA-3⬘), Cryptdin 6 forward (5⬘-TTC CAG GTC CAG
GCT GAT CCT ATC-3⬘) and reverse (5⬘-CCA TTC ATG CGT TCT
CTT CCT TTG-3⬘), and Tff3 forward (5⬘-TCT GGC TAA TGC TGT
TGG TGG-3⬘) and reverse (5⬘-GTT GTT ACA CTG CTC CGA TGT
GAC-3⬘). PCR parameters used were: 95°C for 5 min, followed by 30
cycles at 95°C for 30 s, 62°C for 30 s, 72°C for 30 s, followed by a
7-min extension at 72°C.
Microarray analysis. Lung RNAs from PN8 Catnb⌬(ex3) and littermate controls (n ⫽ 3 per group) were prepared with Trizol reagent
(Invitrogen Life Technologies) following the manufacturer’s specifications. RNA was treated with DNase at 37°C for 30 min. Lung
cRNA was hybridized to the murine genome MOE430 chips (Affymetrix). Affymetrix MicroArray Suite version 5.0 was used to scan
and quantitate the gene chips on default scan settings. Normalization
was performed with the Robust Multichip Average model, which
consists of three steps: background adjustment, quartile normalization, and summarization (15, 16). Detection of differential expression
was performed with Welch’s approximate t-test for mutant and control groups at P ⱕ 0.05. Additional filters for selection of candidate
genes included a minimum of a 1.5-fold change in absolute ratio and
a minimum of two present calls by the Affymetrix algorithm in three
samples.
Cytospins. Lung lavage and cytospins were done on three control
and three Catnb⌬(ex3)-expressing adult mice by standard methodologies. Cells were stained using the Diff-Quik staining kit (Dade
Behring). Total cell counts were determined from cytospin slides. The
Student’s t-test was used to determine significant changes at P ⬍ 0.05.
Morphometric analysis. Morphometric measurements were made
on inflation-fixed adult lungs (n ⫽ 315 fields per group, n ⫽ 7 mice
per group) to determine fractional air space area as previously described (19, 41). Values are expressed as means ⫾ SE, and the P
values for significance were ⬍0.05.
Cell culture, plasmid constructs, and transfection assay. HeLa cells
were grown in Dulbecco’s modified Eagle’s medium and H292
pulmonary adenocarcinoma cells in RPMI containing 4.5g/l D-glucose
and 10 mM HEPES, both media supplemented with 50 units of
penicillin/ml, 50 ␮g of streptomycin/ml, and 10% fetal calf serum.
Cells were cultured in 5% CO2-95% air atmosphere at 37°C. The
mouse ␤-catenin expression plasmids pEGFP-C1-␤CAT and pEGFPC1-⌬GSK-␤CAT were kindly provided by Dr. Angela Barth (Stanford University). SacII/BamHI fragments encoding ␤-catenin (2) or
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ACTIVATED ␤-CATENIN AND LUNG CELL DIFFERENTIATION
⌬GSK-␤-catenin (3) were removed from pUDH1O-3 and subcloned
into pEGFP-C1 (BD Biosciences). The 1.6-kb mouse Foxa2 promoter
was generated by PCR with the following primers: Foxa2 promoter
5⬘-primer, 5⬘-ATG TCA GCT AGC CTT GAT ATC GAA T-3⬘;
3⬘-primer, 5⬘-GTC GTC AAG CTT CTC AGT CCT CT-3⬘; and
cloned into a pGL3-basic vector, a luciferase reporter plasmid (Promega) at the NheI/HindIII sites. Cells were transfected with the
Effectene transfection reagent (QIAGEN) according to the manufacturer’s protocol. Forty-eight hours after transfection, luciferase activity was assessed and normalized for cotransfection efficiency by
␤-galactosidase activity. All transfections were performed in triplicate. ANOVA was used to determine the differences between groups.
All values were expressed as means ⫾ SE with P values for significance ⬍0.05.
RESULTS
Conditional activation of ␤-catenin in the lung. CCSPrtTA⫹/tg or tg/tg, (tetO)7CMV-Cre⫹/tg or tg/tg, Catnb⫹/⌬(ex3) mice
were generated by breeding CCSP-rtTA⫹/tg or tg/tg, (tetO)7CMVCre⫹/tg or tg/tg mice with Catnb⫹/lox(ex3) mice. The Catnb alleles
were either wild type (Catnb⫹/⫹) or heterozygous [Catnb⫹/lox(ex3)].
The doxycycline-inducible Cre/loxP system is outlined in Fig.
1. The 2.3-kb rat CCSP promoter selectively expresses the rtTA
gene in the respiratory epithelium. This promoter is active in
epithelial cells of the developing lung tubules, causing recombination at approximately embryonic day (E) 14.5 and thereafter in subsets of conducting and peripheral respiratory epithelial cells (26). In the presence of doxycycline, the rtTA
protein binds to the (tetO)7-CMV promoter construct, activating expression of Cre recombinase. Cre recombinase recognizes loxP sites, causing homologous recombination and permanent deletion of exon 3 of the ␤-catenin gene. In-frame
deletion of exon 3 results in the synthesis of an amino terminally truncated ␤-catenin protein that constitutively activates
the Wnt/␤-catenin signal transduction pathway. When pregnant females were placed on doxycycline from identification of
a vaginal plug at E0.5, mutant mice were born at the expected
Mendelian frequency.
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␤-Catenin staining after conditional recombination. ␤-Catenin immunostaining was detected in respiratory epithelial cells
in conducting airways of the normal mouse lung (Fig. 2, A and
C). ␤-Catenin was localized primarily to cellular membranes
and was infrequently detected in the nuclei in the normal
postnatal lung. ␤-Catenin staining was not observed in alveolar
type II cells (Fig. 2E). ␤-Catenin staining was increased in
epithelial cells throughout the conducting airways and in subsets of alveolar type II epithelial cells after exposure of CCSPrtTA compound mutant mice to doxycycline (Fig. 2, B, D, and
F). In mice expressing the Catnb⌬(ex3) protein, increased
␤-catenin staining was observed primarily in the cytoplasm and
plasma membranes and less frequently in the nuclei of bronchiolar and alveolar epithelial cells. ␤-Catenin staining was
decreased or absent in conducting and peripheral airways of
mice in which ␤-catenin was deleted by the conditional rtTA
system (22), confirming the specificity of the ␤-catenin immunostaining. Although lung morphology was not perturbed before birth, expression of activated ␤-catenin protein caused
abnormalities in lung morphology that were detected as early
as PN8 and thereafter to adulthood. Focal regions of epithelial
cell hyperplasia and squamous cell dysplasia were noted predominantly in the more proximal regions of intraparenchymal
conducting airways (Fig. 3, B, D, and F). Subsets of cells
stained for both acidic and neutral mucins as assessed by
Alcian blue and PAS, respectively (Fig. 3, B and D).
MUC5A/C immunostaining was also detected in bronchiolar
epithelial cells of Catnb⌬(ex3)-expressing mice (Fig. 3F) but
was lacking in lungs of littermate control mice (Fig. 3E).
Although generalized inflammation was not readily apparent in
the CCSP-rtTA compound mutant mice, focal areas of inflammatory cell infiltrates consisting primarily of macrophages
were observed as early as PN8 (Supplemental Fig. 1; for all
supplemental material see: http://ajplung.physiology.org/cgi/
content/full/00172.2005/DC1). Although total cell counts in
lung lavage fluid were not different, the fraction of neutrophils
was increased (16.6 ⫾ 4.2% vs. 2.9 ⫾ 1.5%, P ⬍ 0.05 by
Fig. 1. Doxycycline-inducible activation of
␤-catenin in triple transgenic mice. Triple
transgenic mice were generated that express
the reverse tetracycline transactivator (rtTA)
protein specifically in epithelial cells of the
lung, using the rat Clara cell secretory protein (rCCSP) promoter. In the presence of
doxycycline, rtTA activates the expression
of the (tetO)7-CMV-Cre recombinase transgene. The Cre protein recognizes the loxP
sites flanking exon 3, causing homologous
recombination and deletion of exon 3 in the
Catnb locus producing Catnb⌬(ex3) transgenic mice expressing the truncated ␤-catenin protein.
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nin on the activity of a murine Foxa2 promoter was assessed in
vitro. Cotransfection of a 1.6-kb mouse Foxa2 promoter construct with plasmids expressing either a wild-type ␤-catenin or
an activated ␤-catenin protein caused a dose-dependent decrease in luciferase activity in the HeLa and H292 cell lines in
vitro, supporting the concept that ␤-catenin may inhibit Foxa2
gene expression at the transcriptional level (Fig. 4). These
results suggest that epithelial cell dysplasia and goblet cell
differentiation caused by the expression of Catnb⌬(ex3) was
mediated, at least in part, by its inhibitory effects on Foxa2
gene expression in airway epithelial cells.
␤-Catenin causes ectopic expression of pro-SP-C in conducting airways. Because the loss of ␤-catenin blocked peripheral epithelial cell specification in the lung (22), we tested
whether enhanced ␤-catenin might induce ectopic production
of peripheral lung cells in conducting airways. Squamous and
cuboidal cell differentiation was observed in the conducting
airways of the Catnb⌬(ex3)-expressing mice, sites which normally are lined entirely by columnar cells (Figs. 3F and 5, A
and B). Decreased CCSP immunostaining was observed in
epithelial cells lining the conducting airway of compound
mutant lungs compared with littermate controls (Fig. 5, A and
B), consistent with changes in Clara cell differentiation. Expression of ␤-tubulin, a marker for ciliated cells, was not
observed in the atypical squamous and cuboidal airway cells
Fig. 2. ␤-Catenin immunostaining in respiratory epithelial cells in Catnb⌬(ex3)
mice. ␤-Catenin was localized primarily to epithelial cell membranes in the
lungs of control mice at embryonic day (E) 18.5 (A). Immunostaining of
␤-catenin was increased in epithelial cells of the conducting airways in
Catnb⌬(ex3) mice at E18.5 (B). ␤-Catenin staining was less intense in cell
membranes of epithelial cells in conducting airways of adult controls (C) and
was increased in the conducting airways of adult mutant mice (D). ␤-Catenin
was not detected in alveolar type II cells of adult controls (E) but was observed
in a subset of alveolar type II cells (arrows) in Catnb⌬(ex3)-expressing mice (F).
Scale bar ⫽ 50 ␮m.
Student’s t-test) in adult Catnb⌬(ex3) mice. There was no
histological or serological evidence of infection, and sentinel
mice did not harbor viral or bacterial pathogens.
Decreased Foxa2 at sites of goblet cell hyperplasia. Foxa2
is required for the maintenance of airway epithelial cell differentiation in the postnatal lung. Deletion of the Foxa2 gene
caused goblet cell hyperplasia and increased expression of
MUC5A/C (39). Expression of the T helper 2 cell cytokines
IL-4 and IL-13 induces goblet cell hyperplasia associated with
the loss of Foxa2 immunostaining in conducting airway cells
(39). To assess whether Catnb⌬(ex3) influenced goblet cell
hyperplasia via Foxa2-dependent pathways, we performed
Foxa2 immunostaining. In Catnb⌬(ex3)-expressing mice, Foxa2
staining was absent at sites of goblet cell differentiation,
supporting a potential cell autonomous effect of ␤-catenin on
Foxa2 expression (Fig. 3, A and B). ␤-Catenin staining was not
altered, however, by allergen challenge with ovalbumin, indicating that goblet cell hyperplasia and inflammation in this
model are not likely mediated by the ␤-catenin-dependent
pathways (data not shown).
␤-Catenin inhibits Foxa2 promoter activity in vitro. Because
Foxa2 immunostaining was decreased at sites of goblet cell
hyperplasia in mice expressing Catnb⌬ex3, the effects of ␤-cateAJP-Lung Cell Mol Physiol • VOL
Fig. 3. Activation of ␤-catenin causes goblet cell hyperplasia. Alcian blue
staining was observed in goblet cells in the conducting airways of Catnb⌬(ex3)
mice (B) but not in littermate controls (A) at postnatal day (PN) 8. Whereas
Foxa2 immunostaining was readily detected in epithelial cells lining the
conducting airway of controls (A), Foxa2 staining was decreased or absent in
goblet cells of Catnb⌬(ex3) mice (B, arrows). PAS and MUC5AC staining were
observed in Alcian blue-positive goblet cells of Catnb⌬(ex3) lungs (D and F)
compared with controls (C and E) at PN21. Mice were exposed to doxycycline
from E0.5 until killed. Scale bar ⫽ 100 ␮m.
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ACTIVATED ␤-CATENIN AND LUNG CELL DIFFERENTIATION
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Fig. 4. ␤-Catenin inhibits Foxa2 gene transcription. Effect of ␤-catenin or activated ␤-catenin
on Foxa2 promoter activity was assessed after
cotransfection of a plasmid containing 1.6 kb of
the mouse Foxa2 promoter (pGL3-Foxa2-luciferase) with increasing amount of plasmid containing either wild type (A, B) or activated
␤-catenin (C, D). Expression plasmids were
used at 0, 1, and 2 ␮g per well (x-axis) in HeLa
(A, C) or H292 cells (B, D). Luciferase activity
was normalized to ␤-galactosidase activity. Values were obtained from 3 separate experiments
carried out in triplicate, means ⫾ SE; n ⫽ 9.
*P ⬍ 0.05 vs. control (0 ␮g).
induced by activated ␤-catenin (data not shown). Subsets of the
abnormal epithelial cells lining conducting airways of the
compound mutant mice stained intensely for pro-SP-C, an
alveolar type II epithelial cell marker (Fig. 5D) in comparison to controls (Fig. 5C). Thus the heterogeneous populations of columnar, squamous, and cuboidal cells, the latter
with characteristics of alveolar rather than conducting airway epithelium, lined the lungs of Catnb⌬(ex3)-expressing
mice (Fig. 5).
At the ultrastructural level, regions of the bronchiolar epithelium of Catnb⌬(ex3)-expressing mice were lined by atypical
heterogeneous populations of columnar, cuboidal, and squamous cells. The atypical cuboidal cells lacked cilia and/or
Clara cell characteristics and some contained lamellar body-
like organelles, short microvilli, and multivesicular bodies,
characteristics of alveolar type II cells (Fig. 6). Because the
ectopic expression of an activated ␤-catenin/LEF1 protein in
early fetal lungs under control of the SFTPC promoter induced
expression of markers normally restricted to the gastrointestinal tract (25), expression of three intestinal genes (Atoh1,
Defcr6, and Tff3) was examined in the lungs of adult CCSPrtTA compound mutants. These mRNAs were readily detected
in mouse intestinal mRNA but were not detected in the lungs
of Catnb⌬(ex3) transgenic mice (data not shown). Because the
rat CCSP promoter is active later in development than the
Fig. 5. Catnb⌬(ex3) alters epithelial cell morphology and induces ectopic
expression of pro-SPC in conducting airways. Decreased CCSP immunostaining was observed in conducting airways of Catnb⌬(ex3) mice (B) compared with
littermate controls (A). The proportion of columnar cells was decreased and
that of cuboidal and squamous cells increased in the conducting airways of
mutant mice. Pro-SP-C immunostaining was detected in a subset of cells in the
conducting airway of Catnb⌬(ex3) mutant mice (D, arrows) but was never
detected in the conducting airway of controls (C). Pro-SP-C staining was
detected in alveolar type II cells of both mutants and controls (arrowheads).
Scale bar ⫽ 100 ␮m.
Fig. 6. Ultrastructure of the bronchiolar epithelium of Catnb⌬(ex3) mice.
Electron microscopy was performed on adult lung tissue from Catnb⌬(ex3) (B,
C, and D) and control (A) mice. Atypical squamous cells (B) and cuboidal
cells, lacking ciliated or a Clara cell morphology (C), were observed in the
bronchioles of Catnb⌬(ex3) transgenic mice. These cuboidal cells contained
organelles similar to lamellar bodies (D, arrow) and had short apical microvilli
and multivesicular bodies (D, arrowheads) typical of alveolar type II cells. In
contrast, the bronchioles of control littermates were lined by characteristic
Clara cells (nonciliated respiratory epithelial cells with large, dense mitochondria) and ciliated cells (A). Magnification ⫽ ⫻3,000 in A–C and ⫻8,000 in D.
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SFTPC promoter used previously (25), the lack of expression
of intestinal genes in the present study likely represents a
difference in the timing or sites of ␤-catenin activation.
Activated ␤-catenin causes air space enlargement in the
postnatal period. Although lung morphology was not perturbed at E18.5, air space abnormalities were observed as early
as PN8. In spite of marked changes in alveolar septum formation, there was no evidence of inflammation or infection in the
perinatal period. However, focal regions of inflammatory cells
were observed in CCSP-rtTA compound mutant mice as early
as PN8 (Supplemental Fig. 1). Peripheral air space enlargement
was observed as early as PN8 and progressed with advancing
age. The extent of air space enlargement was variable but
observed in all compound mutant mice (Fig. 7, D and F–H). In
adult mice expressing activated ␤-catenin, the relative percent
fractional air space was significantly increased compared with
controls (69.0 ⫾ 0.56 vs. 57.7 ⫾ 0.28%, respectively; P ⬍ 0.05
by paired t-test). Staining for vascular endothelium, using G.
simplicifolia B4 isolectin, indicated that alveoli were well
vascularized (data not shown). In young mice (PN8 –PN21),
there was no evidence of elastin fragmentation, which is
normally associated with emphysema caused by inflammation,
protease activation, and remodeling (Supplemental Fig. 2).
However, in the lungs of adult CCSP-rtTA compound mutant
mice, focal regions of elastin fiber thickening and blunting of
secondary septa were observed (Supplemental Fig. 2). Although microarray analysis indicated that the expression of
MMP-2 and MMP-9 was similar, MMP-12 mRNA increased
approximately sixfold at PN8 (data not shown), and the activated form of MMP-12 was detected by Western blot in lung
lavage fluid from adult Catnb⌬(ex3), but not from control
littermates (Supplemental Fig. 3). Thus air space enlargement
in Catnb⌬(ex3)-expressing mice may be related to developmental processes as well as remodeling of the tissue. No changes in
cell proliferation, as assessed by phosphohistone H3 immunostaining, were observed in the lungs of control and CCSP-rtTA
compound mutant mice at any of the time points examined
(data not shown).
Lung tumors in compound mutant mice. Activation of
␤-catenin-dependent pathways has been associated with tumorigenesis in the gastrointestinal tract and other organs (6, 8, 28).
Pulmonary tumors of epithelial cell origin were observed in
three out of 27 compound mutant mice analyzed (54 –225 days
of age) and were not detected in control mice. Tumors were
solid and consisted of sheets of epithelial cells that lacked
staining for both pro-SP-C and CCSP, but expressed TTF-1
and Foxa2 (Fig. 8, A–D), indicating that the tumors were not
highly differentiated. Increased ␤-catenin (Fig. 8E) immunostaining was observed in the nuclei and cytoplasm of the tumor
cells. Although Catnb⌬(ex3) inhibited Foxa2 gene expression at
sites of goblet cell hyperplasia, Foxa2 expression was not
decreased in the tumors. Increased immunostaining for cyclin
D1, a known transcriptional target of ␤-catenin, was observed
in the nuclei of the tumor cells (Fig. 8F).
DISCUSSION
Fig. 7. Catnb⌬(ex3) causes air space enlargement. Hematoxylin and eosin
staining of control (A, C, and E) and Catnb⌬(ex3) (B, D, F, G, and H) lungs. At
E18.5, mutant lungs (B) are morphologically indistinguishable from control
littermates (A). By PN8, enlarged air spaces were observed in lungs of
Catnb⌬(ex3)-expressing mice (D) compared with littermate control mice (C).
Air space enlargement varied in severity (F, G, and H) in adult mice compared
with controls (E). Scale bar ⫽ 200 ␮m.
AJP-Lung Cell Mol Physiol • VOL
Activation of ␤-catenin in respiratory epithelial cells of the
conducting and peripheral airways perturbed epithelial cell
differentiation and caused goblet cell hyperplasia, air space
enlargement, and pulmonary tumors in transgenic mice. Goblet
cell hyperplasia was associated with inhibition of Foxa2 expression in the conducting airways. Consistent with the known
requirement for ␤-catenin in the specification of peripheral
airway epithelial cells (22), activated ␤-catenin induced expression of an alveolar marker, pro-SP-C, and caused metaplasia of epithelial cells lining conducting airways. Catnb⌬(ex3)
caused ectopic differentiation of alveolar type II-like cells and
squamous metaplasia lining the conducting airways. Enhanced
activity of ␤-catenin also caused pulmonary tumors in a subset
of adult mice. ␤-Catenin influences epithelial cell differentiation in the lung and is required for the normal differentiation of
the bronchiolar and alveolar epithelium.
Lung architecture was not altered by the expression of
Catnb⌬(ex3) under control of the CCSP promoter before birth.
In contrast, peripheral air space abnormalities were readily
apparent in the early postnatal period of alveolarization and
preceded significant inflammatory changes within the lung.
The timing of the observed abnormalities are likely deter-
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ACTIVATED ␤-CATENIN AND LUNG CELL DIFFERENTIATION
Fig. 8. Catnb⌬(ex3) causes pulmonary adenomas. Adenomatous tumors were
observed in Catnb⌬(ex3)-expressing mice. Pro-SP-C (A) and CCSP (B) staining
was lacking, whereas TTF-1 (C), Foxa2 (D), and nuclear ␤-catenin (E) staining
was detected in tumor cells. Cyclin D1 was observed in the nuclei of epithelial
cells in the tumors (F). Mice were exposed to doxycycline from E0.5 until
death. Scale bars ⫽ 100 ␮m.
mined, at least in part, by the timing and sites of expression of
the CCSP-rtTA transgene used to delete exon 3 of the ␤-catenin gene. Increased staining of ␤-catenin was detected at E18.5
but was more extensive thereafter, occurring in both conducting and peripheral airway epithelial cells. The rat CCSP promoter used in the CCSP-rtTA construct is first expressed in
conducting airways at approximately E14.5 in the fetal lung, a
time at which branching of the major airways is complete (26),
and in the presumptive alveolar epithelium by E18.5 (Wert S,
unpublished observation). Although increased ␤-catenin staining was observed as early as E18.5, lung morphology was not
altered by expression of ␤-catenin until the postnatal alveolar
period of development. Previous studies with this CCSP-rtTA
transgenic line also demonstrated expression of target genes in
the peripheral lung in the postnatal period. Because the floxed
allele is permanently deleted following expression of Cre
recombinase, epithelial cells in both conducting airway and the
alveoli are targeted by the CCSP-rtTA, (tetO)7CMV-Cre transgenic mouse system (26). These findings are supported by
present immunohistochemical data demonstrating the sites of
increased ␤-catenin staining in the lung. Thus the postnatal
phenotype and the survival of these mice are likely related to
the timing of gene activation, which occurs relatively late in
lung morphogenesis. Consistent with this notion is expression
of an activated ␤-catenin/LEF1 fusion protein or activated
␤-catenin under control of the human SFTPC promoter early in
lung development (E9 –10), that markedly disturbed lung formation, resulting in respiratory failure at birth (Ref. 25 and
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Mucenski ML, unpublished data, respectively). In the present
model, air space enlargement occurred within several days
after birth in the absence of significant inflammation or perturbation of elastin fibers, supporting the concept that air space
abnormalities are caused, at least in part, by changes in morphogenetic processes, rather than by inflammation alone. However, increased expression of MMP-12 and the loss of Foxa2
seen in Catnb⌬(ex3) mice may play a role in air space abnormalities in this model.
␤-Catenin plays important roles in cell fate determination
and differentiation in various organ systems. Deletion of
␤-catenin during lung morphogenesis blocked peripheral (alveolar) epithelial cell differentiation, resulting in lungs composed primarily of conducting airways, thus demonstrating a
critical requirement for ␤-catenin for the formation of alveoli
(22). Activation of ␤-catenin in the present study caused
ectopic expression of pro-SP-C, whose expression is normally
restricted to alveolar epithelial cells in the postnatal lung, to
sites along the conducting airways. Cuboidal cells with features typical of alveolar type II cells, including lamellar bodylike organelles, microvilli, and multivesicular bodies, were
found in bronchioles of the Catnb⌬(ex3)-expressing mice. Because it is not known whether Catnb⌬(ex3) altered the differentiation of progenitor cells or of differentiated conducting airway cells, it remains unclear whether the process should be
termed transdetermination or transdifferentiation, respectively.
Thus ␤-catenin influenced peripheral epithelial cell differentiation in the bronchiolar epithelium consistent with its proposed
role in establishing epithelial cell type specification along the
cephalo-caudal axis of the lung (22, 25). In the present study,
Catnb⌬(ex3) altered the differentiation of airway epithelial cells
in the conducting airways. An even more dramatic change in
cell differentiation was observed when the SFTPC promoter
was used to express a constitutively activated ␤-catenin-LEF1
fusion protein (25), wherein expression of genes normally
restricted to the gastrointestinal tract were detected. Differences in these observations and the present study are likely
influenced by the sites and timing of expression of the transgenes in the two models, the CCSP promoter being active
much later in lung development, likely at a time in which cell
fate of endodermal progenitors was restricted from differentiation to gut epithelium.
Activation of ␤-catenin caused goblet cell hyperplasia that
was evident as early as PN8. Goblet cell hyperplasia and
associated mucin expression are induced during injury by
activation of various signals, including cytokines, epidermal
growth factor, and lipopolysaccharide (5, 35, 37). Pathological
findings seen in the Catnb⌬(ex3)-expressing mice are similar in
many respects to those associated with chronic obstructive lung
disease related to smoking and other forms of lung injury.
Foxa2 is required for the suppression of mucin expression and
for the maintenance of airway epithelial cell differentiation in
the normal postnatal lung. Foxa2 was markedly decreased or
absent at sites of ␤-catenin activation of goblet cell hyperplasia
in Catnb⌬(ex3)-expressing mice. The finding that the Foxa2
promoter was inhibited by cotransfection with ␤-catenin in
vitro provides a potential mechanism by which ␤-catenin
influences Foxa2-mediated goblet cell differentiation. Thus the
loss of Foxa2 induced by activated ␤-catenin is likely sufficient
to cause goblet cell hyperplasia in conducting airways. Because Foxa2 is also required for normal alveolarization (39),
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ACTIVATED ␤-CATENIN AND LUNG CELL DIFFERENTIATION
air space enlargement in Catnb⌬(ex3) mice may be influenced
by changes in Foxa2 expression in alveolar cells.
The expression of an activated ␤-catenin in epithelial cells of
the lung caused pulmonary adenomatous tumors in a subset of
mice. Increased nuclear ␤-catenin staining was observed in
tumors associated with mutations in ␤-catenin including gastric cancers (6), hepatocellular carcinomas (8), and pulmonary
adenocarcinomas (23). Similar activating mutations, involving
the serine and/or threonine residues found in exon 3, are
infrequently detected in human adenocarcinomas of the lung
(23, 31, 34). The incidence of tumors in the present model was
low, indicating that other epigenetic or genetic changes must
promote tumor initiation and/or progression in the Catnb⌬(ex3)
mice. Increased cyclin D1 staining was observed in the nuclei
of tumor cells in the Catnb⌬(ex3) mice but was not observed in
nontumorous regions of the lung. The low incidence of tumors
seen in the present model is consistent with previous studies
demonstrating the lack of tumorigenicity in mice expressing
Catnb⌬(ex3) in the liver, wherein addition of activated H-ras
markedly increased the formation of hepatocellular carcinomas
(12, 13). The relatively low frequency of tumors presently
observed supports the concept that tumor progression likely
requires subsequent genetic alterations, in addition to the
expression of the activated ␤-catenin protein. Activated ␤-catenin also caused transdifferentiation in mammary epithelial cells
without adenocarcinoma formation (21). In contrast, expression of activated ␤-catenin in secretory epithelia using a
MMTV-Cre construct caused high-grade prostatic neoplasia,
whereas in other organs, terminal squamous transdifferentiation but not neoplastic transformation was observed (11). Thus
the effects of ␤-catenin on epithelial cell differentiation and
tumorigenesis are likely influenced by the expression of other
cell-specific cofactors.
ACKNOWLEDGMENTS
We thank Jason Murray, Stephanie Thompson, David Loudy, Haiming Xu,
and Ann Maher for their contributions.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-56387 (J. A. Whitsett, S. E. Wert, M. T. Stahlman) and HL-75770
(J. A. Whitsett), as well as American Lung Association Research Grant
RG-175-N (M. L. Mucenski) and American Cancer Society Institutional Grant
#92-126-09 (M. L. Mucenski).
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