β-Catenin Dosage Is a Critical Determinant of Tracheal Basal Cell Fate Determination
2011; Elsevier BV; Volume: 179; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2011.03.016
ISSN1525-2191
AutoresHeather M. Brechbuhl, Moumita Ghosh, Mary Smith, Russell Smith, Bilan Li, Douglas A. Hicks, Brook B. Cole, Paul R. Reynolds, Susan D. Reynolds,
Tópico(s)Cancer-related gene regulation
ResumoThe purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells. The purpose of this study was to determine whether β-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/β-catenin pathway gene expression suggested a role for β-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by β-catenin. This issue was overcome by analysis of β-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for β-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for β-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of β-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent β-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells. The human tracheobronchial region is characterized by a pseudostratified epithelium and the presence of smooth muscle and cartilage.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar This anatomy extends from the trachea through the first six intrapulmonary generations. Thus, the mouse trachea serves as a model for identification of pathways that regulate repair of the human tracheobronchial epithelium (TBE). Pulse-chase and lineage tracing analyses have demonstrated that the mouse basal cell,1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 2Evans M.J. Shami S.G. Cabral-Anderson L.J. Dekker N.P. Role of nonciliated cells in renewal of the bronchial epithelium of rats exposed to NO2.Am J Pathol. 1986; 123: 126-133PubMed Google Scholar, 3Hong K.U. Reynolds S.D. Watkins S. Fuchs E. Stripp B.R. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium.Am J Pathol. 2004; 164: 577-588Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 4Hong K.U. Reynolds S.D. Watkins S. Fuchs E. Stripp B.R. In vivo differentiation potential of tracheal basal cells: evidence for multipotent and unipotent subpopulations.Am J Physiol Lung Cell Mol Physiol. 2004; 286: L643-L649Crossref PubMed Scopus (266) Google Scholar similar to its human counterpart,5Engelhardt J.F. Allen E.D. Wilson J.M. Reconstitution of tracheal grafts with a genetically modified epithelium.Proc Natl Acad Sci U S A. 1991; 88: 11192-11196Crossref PubMed Scopus (54) Google Scholar, 6Engelhardt J.F. Schlossberg H. Yankaskas J.R. Dudus L. Progenitor cells of the adult human airway involved in submucosal gland development.Development. 1995; 121: 2031-2046PubMed Google Scholar serves as a progenitor for all differentiated cell types in the mouse tracheal epithelium. In the mouse trachea, parenteral naphthalene (NA) exposure depleted the secretory progenitor cell pool (termed Clara-like cells) and the ciliated cell population within 3 days.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Basal cells, defined by the expression of keratin (K) 5, proliferated on recovery days 3 to 9. Nascent Clara-like cells, which were defined by the expression of Clara cell secretory protein (CCSP) and nascent ciliated cells, that expressed forkhead box protein J1 (FoxJ1) or acetylated tubulin (ACT) were detected between recovery days 6 and 13. The basal cell–mediated reparative process was uniform along the proximal to distal axis of the trachea, suggesting that the basal cell progenitors were uniformly distributed. The signals that may regulate the reparative process include developmentally important pathways such as Notch, Sonic hedgehog, and Wnt/β-catenin.7Stripp B.R. Reynolds S.D. Maintenance and repair of the bronchiolar epithelium.Proc Am Thorac Soc. 2008; 5: 328-333Crossref PubMed Scopus (112) Google Scholar Wnt/β-catenin signaling waxes and wanes during lung development, suggesting that this signaling pathway regulates similar processes over time or that it mediates multiple but distinct components of organ formation.8Reynolds S.D. Zemke A.C. Giangreco A. Brockway B.L. Teisanu R.M. Drake J.A. Mariani T. Di P.Y. Taketo M.M. Stripp B.R. Conditional stabilization of β-catenin expands the pool of lung stem cells.Stem Cells. 2008; 26: 1337-1346Crossref PubMed Scopus (113) Google Scholar, 9Dean C.H. Miller L.A. Smith A.N. Dufort D. Lang R.A. Niswander L.A. Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland.Dev Biol. 2005; 286: 270-286Crossref PubMed Scopus (87) Google Scholar, 10Okubo T. Hogan B.L. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm.J Biol. 2004; 3: 11Crossref PubMed Google Scholar, 11De Langhe S.P. Reynolds S.D. Wnt signaling in lung organogenesis.Organogenesis. 2008; 4: 100-108Crossref PubMed Scopus (53) Google Scholar Loss- and gain-of-function studies have demonstrated that β-catenin is necessary and sufficient to alter lung branching morphogenesis. Okubo and Hogan10Okubo T. Hogan B.L. Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm.J Biol. 2004; 3: 11Crossref PubMed Google Scholar demonstrated distalization of the foregut endoderm using a Lef1–β-catenin fusion protein and suggested that excess β-catenin signaling altered specification of proximal endodermal lineages. Li et al12Li C. Li A. Li M. Xing Y. Chen H. Hu L. Tiozzo C. Anderson S. Taketo M.M. Minoo P. Stabilized β-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis.Dev Biol. 2009; 334: 97-108Crossref PubMed Scopus (36) Google Scholar stabilized β-catenin early in lung epithelial development (approximate embryonic day 9.5) using the Nkx2.1-cre transgene and the floxed exon 3 β-catenin allele.13Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. Intestinal polyposis in mice with a dominant stable mutation of the β-catenin gene.EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (968) Google Scholar Cre recombinase–mediated excision of exon 3 resulted in generation of a transcriptionally active β-catenin protein that lacked the GSK3β phosphorylation sites. This β-catenin mutant is "stabilized." The study by Li et al12Li C. Li A. Li M. Xing Y. Chen H. Hu L. Tiozzo C. Anderson S. Taketo M.M. Minoo P. Stabilized β-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis.Dev Biol. 2009; 334: 97-108Crossref PubMed Scopus (36) Google Scholar demonstrated polyp formation in the trachea and upper airways. These polyps were devoid of ciliated and Clara-like cells, suggesting that excess β-catenin blocked generation of the tracheal secretory/ciliated lineage. In contrast to the tracheal phenotype, stabilization of β-catenin during the pseudoglandular phase of lung development (approximate embryonic day 15.5) using the CCSP-cre transgene and the floxed exon 3 β-catenin allele8Reynolds S.D. Zemke A.C. Giangreco A. Brockway B.L. Teisanu R.M. Drake J.A. Mariani T. Di P.Y. Taketo M.M. Stripp B.R. Conditional stabilization of β-catenin expands the pool of lung stem cells.Stem Cells. 2008; 26: 1337-1346Crossref PubMed Scopus (113) Google Scholar attenuated postnatal maturation of bronchiolar Clara cells. β-Catenin stabilization did not alter Clara cell proliferation in response to NA injury but did block Clara to ciliated cell differentiation. These studies indicated that β-catenin did not drive Clara cell proliferation. However, β-catenin did play an important role in Clara cell fate determination. Clara cell–specific knockout of β-catenin demonstrated that β-catenin was not necessary for embryonic development after the pseudoglandular stage, for postnatal maturation of bronchiolar Clara cells, or for repair of the NA-injured bronchiolar airways.14Zemke A.C. Teisanu R.M. Giangreco A. Drake J.A. Brockway B.L. Reynolds S.D. Stripp B.R. β-Catenin is not necessary for maintenance or repair of the bronchiolar epithelium.Am J Respir Cell Mol Biol. 2009; 41: 535-543Crossref PubMed Scopus (50) Google Scholar Collectively, the gain- and loss-of-function studies indicated that a threshold level of β-catenin signaling was important for Clara and ciliated cell differentiation through the pseudoglandular stage and that an overabundance of β-catenin signaling altered Clara cell fate in the adult. Analysis of β-catenin signaling in basal cells and its effect on basal cell fate has not been reported. We demonstrate that NA-mediated tracheobronchial injury results in transient β-catenin stabilization during the period of basal cell proliferation/differentiation. This observation led us to hypothesize that β-catenin serves as a signal that toggles the basal cell life cycle between cell division and generation of differentiated ciliated and Clara-like cells. Analysis of the β-catenin reporter transgene TOPGal15DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar demonstrated that β-catenin stabilization was associated with basal cell proliferation and differentiation in tracheal epithelial cell cultures. Genetic stabilization of β-catenin demonstrated that β-catenin did not promote basal cell proliferation. However, β-catenin down-regulation was required for generation of appropriate numbers of ciliated cells and for basal to secretory cell differentiation. We conclude that β-catenin is a critical determinant of basal cell fate determination in the tracheal epithelium. All the animals were cared for and treated according to procedures approved by the National Jewish Health Institutional Animal Care and Use Committee. All the experiments used adult mice 6 to 8 weeks old. TOPGal-C57Bl/6 congenic (TOPGal-B6) mice were generated by backcrossing CD1-TopGal15DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar mice with C57Bl/6 mice for 10 generations. C57Bl/6 mice were used for in vivo gene expression. Air-liquid interface (ALI) cultures were established from TOPGal-B6 mice for X-Gal reporting experiments. Fvb/n mice were used for in vitro gene expression analysis. K14/rtTA/TRE-cre/DE3 mice were generated by breeding transgenic mice harboring the K14–reverse tetracycline (T) transactivator (K14-rtTA),16Xie W. Chow L.T. Paterson A.J. Chin E. Kudlow J.E. Conditional expression of the ErbB2 oncogene elicits reversible hyperplasia in stratified epithelia and up-regulation of TGFα expression in transgenic mice.Oncogene. 1999; 18: 3593-3607Crossref PubMed Scopus (122) Google Scholar T response element-cre recombinase trangenic mice (TRE-cre),17Perl A.K. Wert S.E. Nagy A. Lobe C.G. Whitsett J.A. Early restriction of peripheral and proximal cell lineages during formation of the lung.Proc Natl Acad Sci U S A. 2002; 99: 10482-10487Crossref PubMed Scopus (411) Google Scholar to mice homozygous for the floxed β-catenin exon 3 allele (DE3).13Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. Intestinal polyposis in mice with a dominant stable mutation of the β-catenin gene.EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (968) Google Scholar Bitransgenic mice that were homozygous for the floxed allele were used to establish the ALI cultures used for β-catenin stabilization experiments. Dose-response experiments were used to determine the dose of NA needed to cause 95% depletion of Clara cells on posttreatment day 3 in TOPGal-B6 mice. This dose was 275 mg/kg. NA was delivered i.p. as previously reported.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Mice were weighed daily. Survival was >95%. Tissue was recovered for histologic and gene expression analyses on recovery days 3, 6, 9, and 13. ALI cultures were established based on the method of You et al18You Y. Richer E.J. Huang T. Brody S.L. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.Am J Physiol Lung Cell Mol Physiol. 2002; 283: L1315-L1321PubMed Google Scholar using tracheae from 6- to 8-week-old mice. Tracheae were carefully cleaned of excess tissue and glands by blunt dissection. Cells were recovered by digestion with 0.15% pronase (from Streptomyces griseus) in Ham's F-12 supplemented with l-glutamine and 5% penicillin/streptomycin. Digestion was overnight at 4°C. The protease was inactivated by the addition of 10% fetal bovine serum. Cells were removed from the trachea by gentle agitation, were pelleted at 300 × g, and were plated in Dulbecco's modified Eagle's medium/5% penicillin/streptomycin/10% fetal bovine serum for 3 hours at 37°C with 5% CO2 to remove macrophages and fibroblasts. The cells were pelleted at 300 × g and were plated in collagen I–coated Transwells (Corning Inc., Corning, NY) at a density of 1.0 × 105 cells/cm2. The proliferation phase of ALI cultures is defined as the period when cells are proliferating and creating a polarized epithelium. During the proliferation phase, cultures were grown in MTEC+, Dulbecco's modified Eagle's medium/F-12 (1:1) (Gibco, Grand Island, NY) supplemented with 2 mmol/L l-glutamine (Mediatech Inc., Manassas, VA), 0.25 μg/mL of amphotericin B (Sigma-Aldrich Corp., St. Louis, MO), 5% penicillin/streptomycin, 7.5% NaHCO3, 5% fetal bovine serum (HyClone, Logan, UT), insulin-transferrin-selenium (Gibco), 10 μg/mL of insulin, 5 μg/mL of transferrin, 5 μg/mL of selenite, 0.1 mg/mL of cholera toxin (Sigma-Aldrich Corp.), 0.1 μg/mL epithelial growth factor (BD Biosciences, Franklin Lakes, NJ), 25 ng/mL of bovine pituitary extract (Gibco), 0.03 mg/mL of hydrocortisone (MP Biomedicals, Solon, OH), and 50 nmol/L retinoic acid (Sigma-Aldrich Corp.). Proliferation day 0 is defined as the day the cells are seeded. Transepithelial resistance is measured beginning on proliferation day 4. Cultures that reach >330 Ω-cm2 are fed proliferation media and are cultured for 1 additional day. At this time, transepithelial resistance averaged 2000 Ω-cm2. Tracheal RNA was isolated for gene expression analysis according to previously described methods.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Tracheae were isolated as indicated previously herein. Tissue was stored in RNAlater (Ambion, Foster City, CA) at 4°C overnight and at −80°C until RNA isolation. RNA from ALI cultures was isolated for gene expression using the Absolutely RNA microprep kit (Agilent Technologies, La Jolla, CA) according to manufacturer protocols. Pools of RNA from normal tracheae were used as a calibrator for tracheal and ALI gene expression. Real-time analysis of gene expression was completed as previously reported.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Assays on demand used in this study were purchased from Applied Biosystems (Carlsbad, CA) and included CCSP (Mm00442046_m1), β-glucuronidase (Mm00446953_m1), FoxJ1 (Mm00807215_m1), Lef1 (Mm00550265_m1), Myc (alias c-myc) (Mm00487803_m1), Tcf7 (Mm00493445_m1), K5 (Mm00503549_m1), Axin 2 (Mm00443610_m1), and Plunc (Mm00465064_m1). Standard reagents and protocols from Applied Biosystems were used. Relative gene expression was calculated using the delta-delta cycle threshold method.19Heid C.A. Stevens J. Livak K.J. Williams P.M. Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5002) Google Scholar Tissue staining was as previously reported.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar ALI cultures were fixed for 20 minutes at 4°C with 3.0% sucrose/3.2% paraformaldehyde/1X PBS solution. Membranes were rinsed with 1X PBS, removed from the plastic insert, placed in a 24-well plate, and stained using standard methods and previously validated antibodies.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 8Reynolds S.D. Zemke A.C. Giangreco A. Brockway B.L. Teisanu R.M. Drake J.A. Mariani T. Di P.Y. Taketo M.M. Stripp B.R. Conditional stabilization of β-catenin expands the pool of lung stem cells.Stem Cells. 2008; 26: 1337-1346Crossref PubMed Scopus (113) Google Scholar Images were captured using a Zeiss AxioVision microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) using 11 steps on the Z-axis and the extended focus function. Regions of interest were identified in the DAPI channel followed by imaging in the red or green channels. At least three images were acquired at ×200 or ×400. Cells were defined by the presence of a DAPI-stained nucleus. The number of nuclei per field was determined and was set as the denominator. The number of cells expressing the marker of interest in each field was determined and was set as the numerator. Experiments were repeated up to three times and included a minimum of three replicates. Data are presented as mean ± SEM. Tracheae were collected into ice-cold radioimmunoprecipitation assay buffer [1% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), 0.35 mol/L SDS, 0.15 mol/L NaCl, and 0.05 mol/L Tris (pH 8)] with 1X SigmaFast protease inhibitor tablets (#S8820; Sigma-Aldrich, St Louis, MO), 1X Halt phosphatase inhibitor cocktail (#78420; Thermo Scientific, Waltham, MA), and 1 mmol/L phenylmethanesulfonyl fluoride (#78830; Sigma-Aldrich). Tracheae were homogenized by alternating bead-beating for 60 seconds at 4000 rpm (Tomy Micro Smash MS-100, CS Bio Co, Menlo Park, CA) and incubating on ice for 5 minutes. Homogenates were centrifuged at 2000 rpm at 4°C for 5 minutes, and supernatants were recovered for protein analysis. Twenty micrograms of sample was added to each lane of BioRad Criterion XT precast 4% to 12% Bis-Tris gels (BioRad Laboratories, Hercules, CA), and electrophoresis and transfer (polyvinylidene difluoride membranes) were completed on BioRad's Criterion gel box system according to manufacturer protocols. Membranes were blocked overnight at 4°C in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE). Primary antibodies were added overnight at 4°C at 1:1000 in Odyssey blocking buffer with 0.2% Tween 20 (Roche Diagnostics GmbH). Membranes were washed three times for 10 minutes at room temperature in PBS/0.1% Tween 20. Secondary antibodies were applied 1:20,000 in Odyssey blocking buffer with 0.2% Tween 20 and 0.01% SDS for 1 hour in the dark. Membranes were washed as described previously and then were scanned using the Li-Cor Odyssey imager. The primary antibodies used in this study were mouse monoclonal anti-actin (C-2) (#sc-8432; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–β-catenin (amino-terminal) (#9581; Cell Signaling Technology, Danvers, MA), and mouse anti–β-catenin (C-terminal) (#610154; BD Biosciences Pharmingen, San Diego, CA). The secondary antibodies used were goat anti-mouse IR 800 (#926-32210; Li-Cor) and goat anti-rabbit IR 680 (#926-3222; Li-Cor). Analysis of variance with Tukey comparisons and 2-way analysis of variance with Bonferroni comparisons were calculated using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA). A P < 0.05 was considered statistically significant. The goal of this study was to evaluate β-catenin–dependent gene expression in steady state and in response to NA-mediated injury. To benchmark this analysis to previous studies, the NA dose response and injury and repair patterns were documented. A previous analysis using female Fvb/n mice demonstrated that NA exposure resulted in depletion of tracheal Clara-like and ciliated cells.1Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal basal cells: a facultative progenitor cell pool.Am J Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar The abundance of CCSP mRNA, a Clara-like cell marker, was 5% of control levels on recovery day 3 and returned to 80% of control levels on recovery day 13. Preliminary analysis demonstrated that the original TOPGal-CD1 strain15DasGupta R. Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation.Development. 1999; 126: 4557-4568Crossref PubMed Google Scholar was insensitive to NA relative to the Fvb/n strain mice. Minimal injury was observed at an NA dose of 325 to 350 mg/kg body weight, and the lung epithelial injury pattern was inconsistent (data not shown). In contrast, an NA dose-response analysis in TOPGal (C567Bl/6 congenic, TOPGal-B6) mice demonstrated that 275 mg/kg of NA resulted in a 97% decrease in CCSP mRNA abundance on recovery day 3 and a return to 85% of control on recovery day 9 (see Supplemental Figure S1A at http://ajp.amjpathol.org). This analysis detected more variation in CCSP mRNA abundance on recovery days 6 and 9 than previously reported for Fvb/n mice. Body weight change paralleled changes in CCSP mRNA abundance (see Supplemental Figure S1B at http://ajp.amjpathol.org). A sexually dimorphic response to NA treatment was not observed for the C57Bl/6 background (see Supplemental Figure S1C at http://ajp.amjpathol.org). The number and distribution of basal and Clara-like cells in the tracheae of steady state and NA-injured TOPGal-B6 mice was assessed by histologic analysis. A single layer of K5+ basal cells was positioned adjacent to the basement membrane (Figure 1, A and B). Rare K5+ cells co-expressed K14 (data not shown). Basal cell shape varied from tall/pyramidal to short/squamous. This variation did not differ between wild-type and transgenic positive (data not shown). Clara-like cells expressed CCSP and exhibited an apical surface that projected into the airway lumen (Figure 1, F and G). Columnar and cuboidal Clara-like cells were noted, and the frequency of these morphologic variants did not vary between wild type and transgenic positive. Three days after NA administration, the epithelium was severely disrupted. Most regions had an intact basal cell layer containing squamated K5+ cells (Figure 1C). Clara-like cells were absent (Figure 1H), as were ciliated cells (data not shown). In most animals, epithelial repair was evident by recovery day 6, but three subpatterns were noted. An intermediate repair pattern was characterized by increased density of basal cells (Figure 1D), with some areas of basal cell hyperplasia. Moderate restoration of Clara-like cells was detected (Figure 1I). A slower repair process was noted in ∼25% of animals. This "slow repair" was typified by a broken line of basal cells and some regions that were devoid of basal cells (Figure 1K). Clara-like cells were not detected on recovery day 6 in the slow repair group (Figure 1M). The remaining 25% of animals underwent a "rapid repair" process in which numerous Clara-like cells were detected on recovery day 6 (Figure 1N). These differences in repair kinetics could not be attributed to the level of injury because depletion of CCSP mRNA and body weight loss on recovery day 3 was similar for all groups (see Supplemental Figure S1 at http://ajp.amjpathol.org; data not shown). Regardless of the repair pattern, the epithelium regained much of its pseudostratified appearance by recovery day 13 and was populated by basal (Figure 1E), Clara-like (Figure 1J), and ciliated (data not shown) cells. Analysis of transgene-negative C57Bl/6 mice demonstrated a high level of endogenous (eukaryotic) β-galactosidase (β-gal) activity in the normal tracheal epithelium (Figure 1, A and F). This β-gal activity persisted despite careful adjustment of the buffer pH.20Tenu J.P. Viratelle O.M. Garnier J. Yon J. pH dependence of the activity of β-galactosidase from Escherichia coli.Eur J Biochem. 1971; 20: 363-370Crossref PubMed Scopus (101) Google Scholar The eukaryotic β-gal activity was limited to a "belt-like" staining pattern in CCSP+ Clara-like cells (compare Figure 1, A and F). An independent flow cytometry analysis used a fluorescent β-gal reporter and demonstrated that cells from control C57Bl/6 mice contained a high level of β-gal activity. This activity could not be inhibited by chloroquine diphosphate and suggested that the enzyme was lysosomal. Other strains, such as Fvb/n, also demonstrated this high level of endogenous β-gal activity (data not shown). Therefore, it is important to evaluate individual strains for endogenous β-gal activity when using a β-gal reporter. Control TOPGal-B6 transgene-positive mice exhibited a β-gal pattern that was indistinguishable from that observed in control C57Bl/6 wild-type mice (compare Figure 1, A and F, with Figure 1, B and G). Consequently, we were unable to use the TOPGal reporter to evaluate β-catenin–dependent gene expression in the tracheal epithelium under steady state conditions. NA treatment resulted in ablation of Clara-like cells on recovery day 3 and complete loss of the steady state β-gal staining pattern (Figure 1, C and H). The absence of β-gal activity on recovery day 3 indicated that any subsequent changes in β-gal activity could be attributed to reexpression of the β-gal reporter in previously negative cells. On recovery day 6, intense β-gal activity was detected in mice exhibiting intermediate repair kinetics (Figure 1, D and I). This activity was pH dependent (data not shown), indicating that it was due to expression of the prokaryotic β-gal reporter. On recovery day 6, β-gal activity was detected throughout the epithelial layer and was present in the previously negative K5+ basal cells. β-Gal activity was also detected in nascent CCSP+ cells. Transgene activity was not detected in glandular structures (data not shown) or in nonepithelial tissues. Mice exhibiting slow repair were negative for β-gal activity on recovery day 6 (Figure 1, K and M), and mice undergoing rapid repair exhibited a β-gal pattern that was similar to that of control on recovery day 6 (Figure 1, L and N). On recovery day 13, all the mice exhibited the control β-gal activity pattern (Figure 1, E and J). This analysis indicated that β-catenin–dependent gene expression was transiently activated in basal cells after NA-mediated Clara-like and ciliated cell depletion. TOPGal transgene activation implied expression of β-catenin target genes. Genes in this analysis were selected on the basis of a preliminary survey of 86 WNT/β-catenin pathway genes (SuperArray; data not shown), which demonstrated differential expression of Lef1, TCF7, Myc, and Axin 2 after NA treatment. Transcript abundance in total tracheal RNA from control C57Bl/6 mice and those exposed to 275 mg/kg of NA and recovered for 5 to 9 days was quantified by real-time RT-PCR. Lef1 mRNA decreased fourfold between control and recovery days 5 and 6 and returned to control levels on recovery days 7 to 9 (F
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