An activated form of β-catenin [CatnbΔ(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 CatnbΔ(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. CatnbΔ(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 β-catenin negatively regulated the expression of the Foxa2 promoter. CatnbΔ(ex3) also caused pulmonary tumors in adult mice. Activation of β-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/β-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.
- lung morphogenesis
- 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 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/β-catenin pathway regulates intracellular signaling and gene transcription, which in turn, alter proliferation and/or differentiation of many cell lineages. β-Catenin interacts with the cytoplasmic tail of E-cadherin and α-catenin, a protein linked to actin filaments, to maintain cell adhesion (10, 24). In the Wnt/β-catenin signal transduction pathway, a subset of secreted Wnt glycoproteins interacts with their Frizzled (Fzd) receptors, thereby inhibiting β-catenin phosphorylation by glycogen synthase kinase-3β and its degradation. Hypophosphorylated β-catenin accumulates in the cytoplasm, translocates to the nucleus, and interacts with high mobility group domain-containing, DNA-binding proteins [including T cell factor lymphoid enhancer factor (LEF) and Sox family members] to regulate the expression of downstream genes (17, 33). β-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/β-catenin pathway in lung morphogenesis was supported recently by studies in which β-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 β-catenin gene (22). β-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 β-catenin expression in respiratory epithelial cells of the mouse (22). Thus β-catenin influenced specification of peripheral versus proximal airway epithelial cells in vivo. When a β-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 were aberrantly expressed (25). Taken together, these studies support a potential role for Wnt/β-catenin signal transduction pathway in cell fate specification during foregut and respiratory epithelial differentiation.
In the present study, an activated β-catenin isoform,CatnbΔ(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
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 β-catenin gene, producing CatnbΔ(ex3), an activated form of the β-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: β-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), β-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.
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-Tris-glycine-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).
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.
Lung RNAs from PN8 CatnbΔ(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 ≤ 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.
Lung lavage and cytospins were done on three control and three CatnbΔ(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 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 μg of streptomycin/ml, and 10% fetal calf serum. Cells were cultured in 5% CO2-95% air atmosphere at 37°C. The mouse β-catenin expression plasmids pEGFP-C1-βCAT and pEGFP-C1-ΔGSK-βCAT were kindly provided by Dr. Angela Barth (Stanford University). SacII/BamHI fragments encoding β-catenin (2) or ΔGSK-β-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 β-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.
Conditional activation of β-catenin in the lung.
CCSP-rtTA+/tg or tg/tg, (tetO)7CMV-Cre+/tg or tg/tg, Catnb+/Δ(ex3) mice were generated by breeding CCSP-rtTA+/tg or tg/tg, (tetO)7CMV-Cre+/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 β-catenin gene. In-frame deletion of exon 3 results in the synthesis of an amino terminally truncated β-catenin protein that constitutively activates the Wnt/β-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.
β-Catenin staining after conditional recombination.
β-Catenin immunostaining was detected in respiratory epithelial cells in conducting airways of the normal mouse lung (Fig. 2, A and C). β-Catenin was localized primarily to cellular membranes and was infrequently detected in the nuclei in the normal postnatal lung. β-Catenin staining was not observed in alveolar type II cells (Fig. 2E). β-Catenin staining was increased in epithelial cells throughout the conducting airways and in subsets of alveolar type II epithelial cells after exposure of CCSP-rtTA compound mutant mice to doxycycline (Fig. 2, B, D, and F). In mice expressing the CatnbΔ(ex3) protein, increased β-catenin staining was observed primarily in the cytoplasm and plasma membranes and less frequently in the nuclei of bronchiolar and alveolar epithelial cells. β-Catenin staining was decreased or absent in conducting and peripheral airways of mice in which β-catenin was deleted by the conditional rtTA system (22), confirming the specificity of the β-catenin immunostaining. Although lung morphology was not perturbed before birth, expression of activated β-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 CatnbΔ(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 Student's t-test) in adult CatnbΔ(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 CatnbΔ(ex3) influenced goblet cell hyperplasia via Foxa2-dependent pathways, we performed Foxa2 immunostaining. In CatnbΔ(ex3)-expressing mice, Foxa2 staining was absent at sites of goblet cell differentiation, supporting a potential cell autonomous effect of β-catenin on Foxa2 expression (Fig. 3, A and B). β-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 β-catenin-dependent pathways (data not shown).
β-Catenin inhibits Foxa2 promoter activity in vitro.
Because Foxa2 immunostaining was decreased at sites of goblet cell hyperplasia in mice expressing CatnbΔex3, the effects of β-catenin 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 β-catenin or an activated β-catenin protein caused a dose-dependent decrease in luciferase activity in the HeLa and H292 cell lines in vitro, supporting the concept that β-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 CatnbΔ(ex3) was mediated, at least in part, by its inhibitory effects on Foxa2 gene expression in airway epithelial cells.
β-Catenin causes ectopic expression of pro-SP-C in conducting airways.
Because the loss of β-catenin blocked peripheral epithelial cell specification in the lung (22), we tested whether enhanced β-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 CatnbΔ(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 β-tubulin, a marker for ciliated cells, was not observed in the atypical squamous and cuboidal airway cells induced by activated β-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 CatnbΔ(ex3)-expressing mice (Fig. 5).
At the ultrastructural level, regions of the bronchiolar epithelium of CatnbΔ(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 β-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 CCSP-rtTA compound mutants. These mRNAs were readily detected in mouse intestinal mRNA but were not detected in the lungs of CatnbΔ(ex3) transgenic mice (data not shown). Because the rat CCSP promoter is active later in development than the 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 β-catenin activation.
Activated β-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 β-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 CatnbΔ(ex3), but not from control littermates (Supplemental Fig. 3). Thus air space enlargement in CatnbΔ(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 β-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 β-catenin (Fig. 8E) immunostaining was observed in the nuclei and cytoplasm of the tumor cells. Although CatnbΔ(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 β-catenin, was observed in the nuclei of the tumor cells (Fig. 8F).
Activation of β-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 β-catenin in the specification of peripheral airway epithelial cells (22), activated β-catenin induced expression of an alveolar marker, pro-SP-C, and caused metaplasia of epithelial cells lining conducting airways. CatnbΔ(ex3) caused ectopic differentiation of alveolar type II-like cells and squamous metaplasia lining the conducting airways. Enhanced activity of β-catenin also caused pulmonary tumors in a subset of adult mice. β-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 CatnbΔ(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 determined, at least in part, by the timing and sites of expression of the CCSP-rtTA transgene used to delete exon 3 of the β-catenin gene. Increased staining of β-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 β-catenin staining was observed as early as E18.5, lung morphology was not altered by expression of β-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 β-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 β-catenin/LEF1 fusion protein or activated β-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 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 CatnbΔ(ex3) mice may play a role in air space abnormalities in this model.
β-Catenin plays important roles in cell fate determination and differentiation in various organ systems. Deletion of β-catenin during lung morphogenesis blocked peripheral (alveolar) epithelial cell differentiation, resulting in lungs composed primarily of conducting airways, thus demonstrating a critical requirement for β-catenin for the formation of alveoli (22). Activation of β-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 body-like organelles, microvilli, and multivesicular bodies, were found in bronchioles of the CatnbΔ(ex3)-expressing mice. Because it is not known whether CatnbΔ(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 β-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, CatnbΔ(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 β-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 β-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 CatnbΔ(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 β-catenin activation of goblet cell hyperplasia in CatnbΔ(ex3)-expressing mice. The finding that the Foxa2 promoter was inhibited by cotransfection with β-catenin in vitro provides a potential mechanism by which β-catenin influences Foxa2-mediated goblet cell differentiation. Thus the loss of Foxa2 induced by activated β-catenin is likely sufficient to cause goblet cell hyperplasia in conducting airways. Because Foxa2 is also required for normal alveolarization (39), air space enlargement in CatnbΔ(ex3) mice may be influenced by changes in Foxa2 expression in alveolar cells.
The expression of an activated β-catenin in epithelial cells of the lung caused pulmonary adenomatous tumors in a subset of mice. Increased nuclear β-catenin staining was observed in tumors associated with mutations in β-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 CatnbΔ(ex3) mice. Increased cyclin D1 staining was observed in the nuclei of tumor cells in the CatnbΔ(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 CatnbΔ(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 β-catenin protein. Activated β-catenin also caused transdifferentiation in mammary epithelial cells without adenocarcinoma formation (21). In contrast, expression of activated β-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 β-catenin on epithelial cell differentiation and tumorigenesis are likely influenced by the expression of other cell-specific cofactors.
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).
We thank Jason Murray, Stephanie Thompson, David Loudy, Haiming Xu, and Ann Maher for their contributions.
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.
- Copyright © 2005 the American Physiological Society