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Logo of ajrccmIssue Featuring ArticlePublisher's Version of ArticleSubmissionsAmerican Thoracic SocietyAmerican Thoracic SocietyAmerican Journal of Respiratory and Critical Care Medicine
 
Am J Respir Crit Care Med. 2009 October 15; 180(8): 701–712.
Published online 2009 July 2. doi:  10.1164/rccm.200901-0100OC
PMCID: PMC2778148

Deletion of Pten Expands Lung Epithelial Progenitor Pools and Confers Resistance to Airway Injury

Abstract

Rationale: Pten is a tumor-suppressor gene involved in stem cell homeostasis and tumorigenesis. In mouse, Pten expression is ubiquitous and begins as early as 7 days of gestation. Pten−/− mouse embryos die early during gestation indicating a critical role for Pten in embryonic development.

Objectives: To test the role of Pten in lung development and injury.

Methods: We conditionally deleted Pten throughout the lung epithelium by crossing Ptenflox/flox with Nkx2.1-cre driver mice. The resulting PtenNkx2.1-cre mutants were analyzed for lung defects and response to injury.

Measurements and Main Results: PtenNkx2.1-cre embryonic lungs showed airway epithelial hyperplasia with no branching abnormalities. In adult mice, PtenNkx2.1-cre lungs exhibit increased progenitor cell pools composed of basal cells in the trachea, CGRP/CC10 double-positive neuroendocrine cells in the bronchi, and CC10/SPC double-positive cells at the bronchioalveolar duct junctions. Pten deletion affected differentiation of various lung epithelial cell lineages, with a decreased number of terminally differentiated cells. Over time, PtenNxk2.1-cre epithelial cells residing in the bronchioalveolar duct junctions underwent proliferation and formed uniform masses, supporting the concept that the cells residing in this distal niche may also be the source of procarcinogenic stem cells. Finally, increased progenitor cells in all the lung compartments conferred an overall selective advantage to naphthalene injury compared with wild-type control mice.

Conclusions: Pten has a pivotal role in lung stem cell homeostasis, cell differentiation, and consequently resistance to lung injury.

Keywords: Pten, lung progenitor cells, injury

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

PTEN is a well-known tumor suppressor that plays a key role in stem cell homeostasis in multiple organs, including the lung. The role of PTEN in lung injury and repair has not been studied yet.

What This Study Adds to the Field

This study demonstrates that lung epithelial-specific deletion of Pten leads to the expansion of epithelial progenitor cells and allows increased protection as well as regeneration of the airways after injury.

Cell renewal is critical for maintenance of tissue homeostasis, aging, and repair after injury. It is currently believed that this ability is derived from resident progenitor cells that have long-term self-renewal capacity and the potential to regenerate highly specialized differentiated cell types (1, 2). Most of our understanding of the repair process has been obtained in mice. In particular, this model system has been used to study lung regeneration. The lung epithelium is a major target of insults and is organized into functional compartments along its proximal-distal axis. The proximal lung includes ciliated cells (β-tubulinpos), Clara cells (CC10pos), and a small number of innervated neuroendocrine (NE) cells (calcitonin gene related peptide, CGRPpos). The cartilaginous airways (bronchi) include a relatively unspecialized basal cell type that expresses P63 and keratins 14 and 5. In the more distal bronchi and bronchioles, the epithelium consists mostly of Clara cells. Respiratory alveoli, the most distal compartments of the lung, are composed of alveolar type I (T1-α pos) and type II (SPCpos) cells.

The lung contains both multipotent and lineage-restricted progenitor cells (3). Repair of tissue after injury or during normal aging entails different strategies and progenitor cells in each of the various lung compartments. In the proximal lung, the basal cells meet the criteria for “stemness” (46). A subpopulation of NE cells expressing both CGRP and CC10 may also have progenitor cell properties (7). In the airways, a variant type of Clara cells that lacks detectable cytochrome P450 2F2 isozyme (CYP2F2) proteins is known to restore the epithelium after naphthalene injury (8). In the distal lung, differentiated alveolar type II epithelial cells are likely facultative progenitor cells (9, 10). Recently, a rare population of progenitor cells referred to as the bronchioalveolar stem cells or BASC have been identified within the transition region between the terminal bronchioles and the alveoli, the bronchioalveolar duct junction (BADJ) (11). Rarity of progenitor cells represents a major technical block to the badly needed characterization of their functional properties.

Pten (phosphatase tensin homolog) was initially identified as a tumor-suppressor gene because of its link to Cowden's disease. Pten encodes a lipid phosphatase responsible for the degradation of phosphatidyl-inositol triphosphate. Through this action, PTEN counterbalances the activity of the phosphatidyl-inositol-3-kinase, a central pathway of growth factor signaling, providing a sensitive and critical counterbalance to growth factor stimulation. Through the inhibition of the phosphatidyl-inositol-3-kinase signaling pathway, PTEN controls cell growth, cell cycle and apoptosis, glucose oxidation, and cell migration (12). Pten is also expressed at high levels in embryonic stem cells and regulates their proliferation (13). Pten deletion in mouse causes early embryonic lethality (14, 15). Tissue-specific deletion of Pten in the brain causes a phenotype similar to macrocephaly in humans due to increased number of progenitor cells (16). Loss of Pten in the intestinal progenitor cells initiates polyposis, a condition characterized by precancerous neoplastic increase in the number of crypts, which contain intestinal progenitor cells (17). In the hematopoietic system, PTEN is required to maintain hematopoietic stem cells (HSCs) in a quiescent state and absence of PTEN drives the entry of HSC into cell cycle generating leukemic stem cells (18). PTEN, therefore, has an important role in self-renewal and stem cell homeostasis in several organs.

In the current study, we examined the consequences of epithelial-specific early deletion of Pten in lung development using a novel Nkx2.1-cre driver line. Deletion of Pten caused an expansion of all known progenitor/stem cell populations in the lung: in the proximal epithelial cells, Pten deletion increased the P63/K14 double-positive cells; in the progeny of Pten-deleted distal epithelial cells, both CC10/SPC double-positive cells in the BADJ and the CGRP/CC10 double-positive NE cells were more abundant.

Our data indicate that such increase of the progenitor cells in the lung leads to an arrest in cell differentiation in the proximal and distal compartments. Epithelial cells in the mutant lungs were more resistant to injury and recovered faster than the control lungs. Therefore, Pten deletion in the airway epithelium confers relative resistance to airway injury.

Some of the results of these studies have been previously reported in the form of abstracts (19, 20).

METHODS

Additional details on the methods are provided in the online data supplement.

Generation of Nkx2.1-cre

A novel transgenic mouse strain carrying the genomic integration of a modified bacterial artificial chromosome (BAC) in which the second exon of Nkx2.1 is replaced by the cre recombinase was recently published (21). The Nkx2.1-cre transgenic mice are fertile and show no obvious abnormalities.

Generation of PtenNkx2.1cre Mice

Ptenflox/flox females (BALBc background) were mated with Nkx2.1-cre male mice (C57BL6 background). We backcrossed the mice for five generations to obtain mice carrying Nkx2.1-cre; Ptenflox/flox (henceforth referred to as PtenNkx2.1-cre) in a pure BALBc background. Ptenflox/flox mice were used as control.

Genotyping of the Nkx2.1-cre mice (21) and of Ptenflox, PtenΔ, and Ptenwt alleles was performed as previously described (22).

All animal experiments were approved by the University of Southern California Animal use and care committee.

Histological Analysis

Embryonic lungs from control and mutant embryos were collected at E15.5 and E18.5. Adult lungs were dissected, inflated at 20 cm water pressure with 4% paraformaldehyde, and fixed overnight. The lungs were then dehydrated through increasing ethanol gradient concentration and embedded in paraffin. Sections (5 μm) were mounted on slides for histological analysis.

After performing antigen retrieval and blocking, the lung tissues were incubated overnight with the primary antibodies at different concentration (see online supplement for more details). Signals were visualized with the Histostatin Rabbit or Mouse Primary Kit (Zymed-Invitrogen, Carlsbad, CA) or with secondary antibodies from Jackson Immunoresearch (West Grove, PA).

Naphthalene Treatments

Naphthalene treatments were performed as previously described (9).

Cell Proliferation Analysis

Cell proliferation was assessed using Ki67 staining on 2-month-old control and mutant lungs (n = 3).

Protein Extraction and Western Blot

Total protein extracts were prepared from 3-week-old Ptenflox/flox and PtenNkx2.1-cre lungs with radio-immunoprecipitation assay buffer (Sigma, St. Louis, MO), separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then blotted to polyvinylidene diflouride membrane (Millipore, Billerica, MA). p-AKT was detected with antibodies purchased from Cell Signaling (Danvers, MA) (p-AKT) at the concentration suggested by the manufacturer.

RNA Extractions and Quantitative Reverse Transcriptase–Polymerase Chain Reaction

Total RNA was isolated from lungs of transgenic mice and wild-type littermate control animals using a Qiagen (Carlsbad, CA) RNAeasy kit and cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen). An ABI PRISM 7700 Sequence Detection System was used to detect the studied genes using pre-developed TaqMan assay reagents (Applied Biosystems, Foster City, CA). Data were normalized to β-actin (ACTB) mRNA levels as described previously (23).

Fluorescence-Activated Cell Sorting

Single lung cells were prepared from control and mutant lungs (n = 3) as described previously with some modifications (11). Sca-1/Lys6A, CD45, CD34, and CD31 antibodies were purchased from Pharmingen (San Jose, CA). Cell sorting was performed in a FACSAria Cytometer (BD Bioscience, San Jose, CA) and the data were analyzed by FACSDiva software version.

Data Presentation and Statistical Analysis

Data were presented as mean ± SEM unless otherwise stated. Statistical analyses were performed on the data with Student t test for comparison of two groups. P values 0.05 or less were considered as significant.

RESULTS

Nkx2.1-cre Recombinase Driver Mouse Line

Nkx2.1 encodes a key transcriptional regulator of lung morphogenesis whose onset of expression in the mouse occurs at around embryonic Day E9.5 concomitant with the specification of the lung primordium (24). The murine Nkx2.1 gene consists of three exons and a highly complex cis-active DNA region that controls its expression in the lung, brain, and thyroid (25). A novel transgenic cre mouse line was generated by inserting a modified BAC in which the second exon of Nkx2.1 is replaced by the cre recombinase (21, 26). The pattern and efficiency of the Nkx2.1-cre line in mediating LoxP-dependent DNA excision in the lung epithelium was determined using ROSA26R-LacZ reporter mice. LacZ activity was virtually absent in the wild-type lungs (Figure 1C). In E10.5 ROSA26R-LacZ Nkx2.1-cre embryos, LacZ activity was limited to the primordial lung and brain (Figures 1A and 1B, arrows). At E13.5, it was possible to detect Lac-Z activity in the lung epithelium, brain, and thyroid (Figures 1D and 1E, arrows) in the ROSA26R-LacZNkx2.1-cre embryos. In E13.5 lungs, the pattern of LacZ activity was nearly homogeneous throughout the tracheal lung epithelium, with the exception of some random peripheral tips (Figures 1E–1G). In E15.5 and adult lungs (Figures 1H–1K), homogeneous epithelial staining was present in all epithelial cells, with the strongest expression proximally.

Figure 1.
Nkx2.1-cre expression during lung development. (AK) Detection of cre-induced β-galactosidase activity at different embryonic stages. (A, B) E10.5 whole mount β-galactosidase staining of Rosa26RNkx2.1-cre detecting (A) activity ...

Thus, Nkx2.1-cre mice represent a highly useful tool for conditional deletion of epithelial genes very early in the course of lung development.

Epithelial-Specific Deletion of Pten by Nkx2.1-cre

To determine the potential role of Pten in lung morphogenesis, we used the Nkx2.1-cre mouse line to delete Pten in the lung epithelium. Homozygous deletion of Pten via Nkx2.1-cre was postnatally viable with a frequency consistent with expected mendelian ratios. Immunohistochemistry (IHC) analysis in PtenNkx2.1-cre lungs at E15.5 showed absence of PTEN protein in nearly 100% of epithelial cells with only rare positive staining in the mutant lungs (Figures 2E and 2F, arrows). PTEN-negative epithelial cells in the mutant lungs were positive for Nkx2.1, indicating their lung epithelial cell identity (Figures 2G and 2H). At the trachea level the hyperproliferation of the epithelia was already present early during development (Figures 2I and 2J) in the PtenNkx2.1-cre mice. IHC for PTEN showed a homogenous deletion of the gene in approximately all the cells (Figure 2, compare L to K). We confirmed the deletion of Pten by polymerase chain reaction (PCR) using genomic DNA from lung tissue and two different sets of primers. Our results indicate the presence of the Δ5 allele that confirms Pten deletion (22) (Figure 2I). At E15.5 there were no detectable abnormalities in branching morphogenesis of the embryonic mutant versus control (Ptenflox/flox) lungs (Figure 2, compare A and C to B and D). However, quantification of the number of double-positive cells for E-cadherin (marker for epithelial cells) and phosphohistone H3 (marker of proliferation) in E15.5 lungs (n = 3 for each group) showed an increase in the proliferation rate of the epithelial cells in the mutant compared with the control (1.51 ± 0.14% vs. 0.7 ± 0.1%, P ≤ 0.01, data not shown).

Figure 2.
Deletion of Pten does not affect branching morphogenesis during lung development. (AD) Hematoxylin and eosin (H&E) staining of lung sections of control animals (n = 4, A and C) and mutant PtenNkx2.1-cre (n = 4, B and ...

In the proximal lung epithelium, progressive epithelial hyperplasia extending from the trachea to the small bronchioles (Figures 3A–3F) was detected in the mutant embryos of all embryonic stages examined. In the adult stages, the epithelial cells positive for E-cadherin displayed a papillae-like structure with the apical side of the cells facing the airway lumen (Figures 3E and 3F). The hyperplastic epithelium showed evidence of increased cell proliferation, as documented by Ki67 immunostaining (Figures 3G–3M). The numbers of Ki67-positive cells in the mutant lungs exceeded by threefold the numbers found in the control lungs (11 ± 1.3% vs. 4 ± 0.4%, respectively; n = 3, P ≤ 0.01). In addition, analysis by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) revealed decreased apoptosis in the mutant lungs when compared with control lungs (Figures 3K and 3L). Further quantification of apoptosis (Figure 3N) confirmed the statistically significant decrease of the number of apoptotic cells in the mutants (n = 3) versus wild-type lungs (n = 3) (0.3 ± 0.06% vs. 1 ± 0.07%, P ≤ 0.01). Therefore, early epithelial deletion of Pten causes airway hyperplasia that is detectable from early stages of lung development and in adult mice, due to increased cell proliferation and decrease of apoptosis.

Figure 3.
Absence of Pten leads to bronchiolar hyperplasia secondary to an increase in proliferation rate and to a decrease in apoptosis. (AD) Histological analysis through hematoxylin and eosin staining of lungs from wild type and mutants at E15.5 and ...

Deletion of Pten Results in Expansion of Epithelial Cell Populations in Multiple Progenitor Cell Niches

When compared with control lungs, PtenNkx2.1-cre lungs showed expansion of cells within a number of previously defined progenitor cell niches. In the proximal lung the tracheal basal cells, defined by expression of P63 (Figures 4A and 4B) and keratin14 (Figures 4C and 4D), were significantly more abundant (Figures 4G and 4H, low magnification; Figures 4I and 4J, high magnification). Quantification confirmed the results obtained by immunofluorescence (IF) (29 ± 0.4% vs. 51 ± 1.1%, n = 3, P ≤ 0.01; Figure 4K). More distally, in the bronchi, the neuroepithelial bodies (NEB), identifiable by CGRP/CC10 overlapping expression, were also increased in number in the PtenNkx2.1-cre versus control lungs (38 ± 2.04 vs. 12.5 ± 0.95, n = 3, P ≤ 0.01). Of note, although IF is not a quantitative technique, the NEB clusters were not only more numerous (Figure 5I) but also showed stronger immunoreactivity (Figures 5G and 5H). Real-time PCR data confirmed our IF data, showing a nearly 80-fold increase in CGRP expression in mutant compared with control lungs (Figure 5J). This observation suggests that either cells within the NEB clusters express higher levels of the two markers or that each cluster contains a larger number of cells.

Figure 4.
Deletion of Pten increases number of basal cells in the PtenNkx2.1-cre trachea. (AJ) Lung sections from mutant PtenNkx2.1-cre and control littermates at 2 months of age were stained (A and B) for P63 and (C and D) for keratin14. Increased number ...
Figure 5.
Inactivation of Pten leads to an increase of the neuroepithelial bodies in the bronchi. (AH) Immunofluorescence for CC10 (A and B) and CGRP (C and D) in 8-week-old mutant and control lung sections. Increased size and brightness in the neuroepithelial ...

When compared with control lungs, PtenNkx2.1-cre lungs also showed an expansion of progenitor cells occupying the BADJ region (Figure 6, compare A and C to B and D, respectively). Many of the PtenNkx2.1-cre cells were distinctly larger in size (Figures 6C and 6D, arrows). We used IF to determine whether any of the overexpanded cells in the BADJ were double positive for CC10 and SPC, a characteristic previously associated with putative progenitor cells in this region (11). Although in the control lungs these cells are extremely rare (Figures 6E and 6H), double staining for anti-CC10 and anti-SPC antibodies detected an increased number of CC10/SPC double-positive cells in the mutant lungs (1.8 ± 0.57% vs. 0.3 ± 0%, n = 3, P ≤ 0.01; Figure 6, compare I and J to H; quantification analysis, Figure 6O). The CC10/SPC-positive cells were more convincingly revealed by confocal microscopy (Figures 6K–6N). Using fluorescence-activated cell sorter to further confirm this observation, we gated the BACs, defined as Sca1+CD45CD31CD34+ cells, in the mutant and in the control lungs (n = 3 for each). The number of Sca1+ cells in the CD45CD31CD34+ cell population was more than threefold increased in PtenNkx2.1-cre compared with the control lungs (9.5 vs. 2.8%) (Figures 6P and 6Q). Thus, early epithelial deletion of Pten by Nkx2.1-cre expands several putative epithelial progenitor cell populations throughout the proximal-distal axis of the lung.

Figure 6.
Deletion of Pten increases the double-positive cells CC10/SPC in the BADJ. (A, D) Hematoxylin and eosin staining showed in the mutant an increase of cells at the BADJ level (compare A to B, lower magnification ×20; C to D, higher magnification, ...

PtenNkx2.1-cre Cells Form Putative Progenitor Cell Masses in the BADJ

We examined the behavior of the PtenNkx2.1-cre cells residing within the BADJ in the mutant lungs over time. PtenNkx2.1-cre progenitor cells undergo proliferation as a function of time and within approximately 8 weeks of postnatal life, a mass consisting of PtenNkx2.1-cre epithelial cells around the BADJ area is detected in some, but not all, mutant lungs (Figures 7A and 7B). These masses are slow growing and do not interfere with viability or respiratory status of the animals (data not shown). Importantly, the cells within the mass express SPC (at higher level) and CC10 (at lower level) (Figures 7D and 7E). E-cadherin immunostaining showed that within the mass, the cells are organized into ductlike structures reminiscent of the pseudoglandular stage of early lung development and a distinct property of lung endodermal progenitor cells (Figure 7C). Interestingly, N-myc, a downstream target of activated β-catenin–dependent WNT signaling, was also highly expressed in the nuclei of the cells within the PtenNkx2.1-cre mass (Figure 7F).

Figure 7.
The BADJ cells proliferate inside the parenchyma and give rise to masses. (A, B) Hematoxylin and eosin staining on PN180 PtenNkx2.1-cre lung showing that over time the cells in the BADJ grow in the parenchyma and give rise to a tumorlike mass. (C) E-cadherin ...

Proliferation analysis inside the masses (Figure 7G) showed very few cells positive for Ki67: the quantification confirmed the slow proliferation rate inside the masses, not statistically significant compared with the parenchyma (8.3 ± 1.55% vs. 12.3 ± 1.85%, P > 0.05) (Figure 7H). These data indicate that the physiological role of Pten during normal lung development may be to constrain epithelial progenitor cell proliferation within the BADJ, an important lung progenitor cell niche.

Activation of AKT and β-Catenin in PtenNkx2.1-cre Cells

Deletion or inactivation of Pten is expected to cause increased activity (phosphorylation) of AKT. In addition, increased β-catenin activation has been associated with progenitor cell homeostasis and tumorigenesis in a number of tissues, including the intestine (17). Under normal physiological conditions, PTEN inhibits the stabilization of β-catenin by increasing the activity of GSK3. Recently, He and colleagues showed that the PTEN-AKT pathway phosphorylates β-catenin at the level of serine (Ser) 552, resulting in nuclear localization of this phosphorylated form of β-catenin in the intestinal stem cells. It is proposed that this nuclear form is acting as a transcription factor controlling stem cell homeostasis (17). Considering that the lung and the gastrointestinal tract share the same embryological origin, we therefore examined whether lung epithelial-specific deletion of Pten leads to increased phospho-AKT and nuclear β-cateninSer 552 localization. Immunohistochemical staining of PtenNkx2.1-cre lungs showed increased level of phospho-AKT (Figure 8, compare C to D). Increased expression of P-AKT in mutant versus wild-type lungs was confirmed by Western blot analysis (Figure 8F). Double staining for E-cadherin and phosphorylated β-cateninSer 552 was performed (Figures 8A and 8B). Quantification of nuclear phosphorylated β-cateninSer 552/E-cadherin double-positive cells over the total number of E-cadherin– positive cells (n = 3 for each group) showed an increase of β-cateninSer 552 nuclear localization in the epithelial cells in the PtenNkx2.1-cre compared with the Ptenflox/flox control lungs (3.99 ± 0.8% vs. 1.13 ± 0.34%, P ≤ 0.01, Figure 8E).

Figure 8.
Deletion of Pten increases p-AKT and β-catenin activation. (A, B) Immunofluorescence for β-cateninSer522 and E-cadherin in E15.5 lung sections. Double-positive cells are increased in the mutant (B) compared with the control (A). (C, D ...

Finally, nuclear localized N-myc was increased in Pten-depleted epithelial cells compared with the surrounding parenchyma, which can be considered as an internal control (Figure 7F). Overall, these data indicate that β-catenin signaling has been functionally activated in PtenNkx2.1-cre lungs.

Impact of Pten Deletion on Epithelial Cell Lineage Determination

Expression of a number of cell markers was examined by IF and real-time PCR in PtenNkx2.1-cre versus Ptenflox/flox (control) lungs to determine the impact of epithelial Pten deletion on the emergence and differentiation of various lung epithelial cell lineages localized in the proximal and distal lung compartments. In the Clara cell lineage, IF for CC10 revealed a markedly increased number of Clara cells in the mutant lungs (Figures 9A and 9B). The increase in CC10-positive cells was associated with a decrease in the number of ciliated cells, believed to be their terminally differentiated progeny, as revealed by β-tubulin staining (Figures 9C and 9D, arrows). In the distal compartments, SPC, a Type II cell marker, was increased (Figures 9E and 9F), whereas T1-α and Aqp 5, two Type I cell markers, were decreased in PtenNkx2.1-cre lungs (Figures 9G and 9H), indicating a block in transition from precursor to terminally differentiated cell types. The IHC results were validated by real-time PCR analysis of mRNA for the latter cell lineage markers. This analysis showed statistically significant increases of CGRP, SPC, and CC10 and a decrease of β-tubulin IV (marker for ciliated cells) in the mutant versus control lungs. To better understand the mechanism underlying this phenomenon, we examined hairy and enhancer of split 1 (HES1), known to be involved in cell determination in lung, particularly in the Clara cell lineage (27). Both IHC and real-time PCR showed increased HES-1 in the mutant compared with the control lungs (Figures 9I and 9J). Therefore, Pten appears to play a necessary function in normal epithelial cell fate determination.

Figure 9.
Absence of Pten impairs cell fate. (AH) Immunofluorescence for (A, B) CC10, (C, D) β-tubulin, (E, F) SPC, and (G, H) T-1α from 2-month-old control and mutant lungs. (A, B) In the mutant, an increase of Clara cells (CC10 positive) ...

Impact of Pten Deletion on Airway Epithelial Cell injury

Conditional deletion of Pten causes airway epithelial hyperplasia in the trachea and in the bronchi. In the bronchi, these cells are CC10 positive. However, in the trachea, these cells are negative for CC10 as well as for all the known lung epithelial markers (data not shown). Based on this observation, we hypothesized that these cells are arrested during their differentiation process and, thus, may display a selective advantage in coping with airway injury. We therefore examined the response of the tracheal and bronchial airway epithelium of PtenNkx2.1-cre and control mice to naphthalene, a simple and well-defined model of lung injury. Corn oil was used as control.

In Ptenflox/flox control mice, naphthalene injury was detected in the trachea (Figures 10A and 10D) and in the distal compartments (Figures 10G and 10J) when compared with the corn oil control animals. In the wild-type animals, intraperitoneally administered naphthalene denuded entirely the tracheal epithelium after 3 days (Figure 10B) with a partial reepithelization after 7 days (Figure 10C). In the distal airway, naphthalene caused epithelial cell death within 72 hours (Figure 10H) followed by reepithelization of the airways by presumably the P450neg variant of Clara cells (28). After 7 days of injury, the epithelium was in part restored (Figure 10I). In contrast, after naphthalene administration, the proximal airway epithelium of PtenNkx2.1-cre lungs at 3 and 7 days post injury appeared to remain intact with no signs of injury (Figures 10E and 10F). At the bronchial level, injury was reduced (Figure 10, compare K to H) and repair enhanced (Figure 10, compare L to I) in the PtenNkx2.1-cre lungs, in comparison with the control group. Morphometric analysis of epithelial damage was carried out by measuring after CC10 IF staining, the ratio of nude surface (damaged surface) versus total epithelial surface in control compared with mutant lungs 1, 3, and 7 days after injury (Figure 10M). One day after injury, less epithelial damage is observed in mutant versus control lungs (21 ± 0.4% vs. 79 ± 0.4%, P ≤ 0.01). However, epithelial damage in mutant lungs increases at Day 3 after injury (from 21 ± 0.4% at Day 1 to 70 ± 4.4% at Day 3), whereas in the control, the extent of the epithelial damages remained similar (from 79 ± 0.4% at Day 1 to 88 ± 3% at Day 3). At 7 days after injury, epithelial damage was lower in mutant compared with control lungs (37 ± 4.7% vs. 54 ± 7%, P ≤ 0.05%). The ratio of epithelial damage (Day 7 vs. Day 3) can be considered as a measure of the efficiency of the repair process during this time period. Our results indicate indeed a better repair process in mutant versus wild-type lungs (0.53 vs. 0.61). The observed overall resistance to the injury observed at Day 1 correlates with the increased number of SPC/CC10 double-positive progenitor cells in the mutant compared with control lungs at this stage (3.3 ± 1% vs. 0.2 ± 0.06%, P ≤ 0.05) (Figure 10N). The improved repair process between Day 3 and Day 7 in mutant versus wild-type lungs correlates with the increased proliferation of CC10-positive cells (2.9 ± 0.8% vs. 0.4 ± 0.35%, P ≤ 0.05) (Figure 10O).

Figure 10.
Absence of Pten protects the airways from naphthalene injury. (AF) Hematoxylin and eosin staining of a control and mutant trachea after corn oil or naphthalene injection. (A, D) Corn oil administration did not affect the tracheal structure in ...

These results suggest that the PtenNkx2.1-cre airway epithelium has a significantly increased level of resistance to naphthalene and confers a better capacity to recover after injury.

DISCUSSION

Early Pten deletion in the lung does not affect branching morphogenesis but leads to conducting airway hyperplasia.

The results of our work confirm that Pten does not affect lung branching morphogenesis, but affects cell differentiation and blocks cells in a less differentiated status.

As this study was underway two reports outlined the results of epithelial-specific deletion of Pten on lung morphogenesis (29, 30). In both previous studies Pten deletion was achieved using the SPC-rtTA;Tet(O)-cre line.

Our findings are partially consistent with both reports: Yanagi and colleagues (2007), who induced Pten deletion in the distal lung epithelium from E10 to E16, found delayed lung branching along with impaired epithelial cell differentiation and neonatal lethality in 90% of mice, due likely to respiratory insufficiency (29). By contrast, Davé and coworkers (2007), used the same inducible cre model to effect epithelial Pten deletion within a different time frame, from E0.5 to E14.5, and reported airway hyperplasia without impact on lung development or epithelial cell differentiation (30).

The differences in phenotype may simply be related to the different time points at which cre activation was effected or to the mixed genetic background of the mice; the latter has been well documented by observations that link onset and severity of tumorigenesis to the genetic background in Pten knockout mice (15). This dependence on the genetic background may well apply to the role of Pten in organogenesis and could provide another potential explanation for the differences in lung phenotype observed in various studies.

In the current work, a homogeneous BALBc background was used to avoid the possible bias created by a mixed genetic background. Nkx2.1-cre, moreover, is not an inducible cre system and follows, with few exceptions, the pattern of endogenous Nkx2.1 gene expression in the lung (Figure 1). In our hands, Pten deletion did not affect lung branching morphogenesis but caused epithelial airway hyperplasia. Moreover, none of the PtenNkx2.1-cre neonates experienced any respiratory distress, and any sporadic death within the first 2 weeks of life was always associated with enlarged thyroid and obstruction of the trachea.

Thus, our results confirm a major role for Pten in proximal compared with distal lung morphogenesis. These data are supported by the fact that proliferation is affected only in the proximal airways of the mutant lungs, whereas there is no effect on the distal compartment.

Pten deletion through Nkx2.1-cre, therefore, represents a mixed phenotype between the two recent reports, without branching defects, but with airway epithelial hyperplasia and impaired cell fate.

PTEN Controls Epithelial Progenitor Cell Pool Size in the Lung

Progenitor cells are localized along the proximal-distal axis of the lung, notably in specialized environments known as niches in the conducting airways and the BADJ. In PtenNkx2.1-cre lungs, a significant increase in K14/P63-positive cells localized in the trachea was observed (Figure 4). Other putative progenitor cells, including the CC10/CGRP double-positive NEB and SPC/CC10 double-positive cells in the BADJ were also increased (Figures 5 and and6).6). Yanagi and colleagues and Davé and colleagues also described an increased of NEB and BADJ cells, but an increase in progenitor cells in the PtenNkx2.1cre trachea (area not affected by SPC-rtTA;Tet(O)cre driver line) reveals an additional role for Pten that had gone unnoticed by the previous studies (29, 30).

The increase in progenitor cells was also linked to impaired cell differentiation: in the proximal lung, the CC10-positive cells (called Clara cells) were present in higher numbers at the expense of ciliated cells, (recognized by β-tubulin staining). More distally we observed an increase in alveolar type II cells (SPC positive) at the expense of type I cells (T1α positive). Both Clara cells and type II cells are considered to be progenitor cells, respectively, of the ciliated and the type I cells.

The increase in Clara cells is correlated with the increase of Hes-1, a transcriptional factor controlling the balance between endocrine and nonendocrine epithelial cell fate (27). Interestingly, in our study we did not observe a decrease in the neuroepithelial bodies, which is inconsistent with previous reports in which Hes-1 inhibited neuroendocrine differentiation. Further studies are necessary to clarify the mechanisms underlying the impact of Pten in lung epithelial cell determination.

PTEN May Control Cell Fate and Progenitor Cell Homeostasis through β-Catenin

Absence of Pten in the cells leads to phosphatidyl-inositol triphosphate accumulation, which in turn leads to overactivation of several key signaling molecules, including AKT/PKB, mTOR, and S6 KINASE. AKT is the most characterized of these molecules. Numerous substrates for AKT have been identified that participate in control of cell metabolism, cell death, cell cycle progression, and cell differentiation (12). A primary target of AKT is GSK3, which destabilizes β-catenin and causes its degradation. Thus, deletion of Pten can activate β-catenin–dependent WNT signaling, a known regulator of progenitor cell behavior.

In addition, constitutive expression of a stable form of β-catenin in the lung epithelium leads to proximal airway hyperplasia similar to the one present in the PtenNkx2.1-cre lungs (C. Li, personal communication, 2008). Deletion of Pten increases β-catenin expression; thus it is possible to hypothesize that these cells may be arrested in a less differentiated state. Finally, deletion of Pten leads to an expansion of the progenitor cells and prevents the cells from undergoing terminal differentiation.

Absence of Pten at the BADJ Leads to Generation of Masses

Transformed cells, in which pathways related to self-renewal or stem cell homeostasis are activated, may be the source for tumor initiation, survival, and progression (31). Cancer may also arise from a selected number of progenitor cells that have in common the activation of selected pathways. This concept of “tumor stem cells” is already known in the hematopoietic system, where a rare group of stem cells (called leukemic stem cells), with an extensive capacity of self-renewal, can give rise to the majority of the leukemic cells (32).

Different candidate genes are suggested as regulator for the proliferative capacity of these cells. One of these is Pten, which has a role in restricting the activation of hematopoietic stem cells as well as preventing leukemogenesis (33).

In our model it appears that, in the absence of PTEN, the CC10/SPC double-positive cells, considered as progenitor cells in the lung, in time give rise to slow-growing masses that do not interfere with respiratory mechanism. These cells proliferate inside the parenchyma, and at some point lose CC10 expression while retaining the more undifferentiated marker SPC. Over time, the cells form structures resembling the branching ductlike processes that are formed during early lung development, again indicating the less differentiated nature of these cells that may act as progenitors. A more detailed characterization of these cells is currently underway.

PtenNkx2.1-cre Airway Epithelium Exhibits Relative Resistance to Naphthalene Injury

Because mutant lungs showed an increase in the number of progenitor cells, we examined whether they may also demonstrate altered resistance to experimentally induced airway injury by naphthalene. In animal models of airway injury, exposure to naphthalene kills most Clara cells within the first 72 hours. Naphthalene is converted by the P450 (CytP4502F2) enzyme into its toxic derivatives, 1, 2-epoxide (34), a diepoxide (35), and quinines (36). A rare population of variant Clara cells (Clarav cells) is believed to lack CytP4502F2 enzyme activity and hence is resistant to naphthalene killing. Clarav cells are believed to act as progenitors, undergoing proliferation and subsequently repopulating the airway epithelium and reestablishing its cellular composition. In the absence of commercially available reagents for detecting CytP4502F2, an alternative, but functional assay for a putative progenitor cells in the airway may be their relative resistance to naphthalene killing. If expansion of the cells in the airway epithelium of PtenNkx2.1-cre lungs includes a larger number of such “progenitors,” then their presence can be indirectly examined by assaying their relative resistance to naphthalene. Indeed, our results indirectly suggest the presence of an expanded population of progenitor Clara cells in the Pten mutant lungs as evidenced by their relative resistance to naphthalene injury and improved repair.

In summary, the current study, which was performed in a pure BALB genetic background, shows that epithelial deletion of Pten during early lung development does not affect lung branching morphogenesis. Deletion of Pten increased several progenitor pools in both proximal and distal lung compartments. In addition, absence of Pten inhibited cell differentiation of specialized epithelial cell types. The current study also shows that Pten has an important role in tracheal epithelial progenitor cell homeostasis. Finally, to our knowledge, our work has uncovered for the first time an impact of Pten deletion on lung epithelial airway injury.

Supplementary Material

[Online Supplement]

Acknowledgments

The authors thank Dr. Linghen Li for providing anti-β cateninSer522 antibodies. They also thank Andre Nagy for his precious help with the immunohistochemistry; Mia Brockop, Clarence Wigfall, and Denise Al Alam for critical reading of the manuscript; and Benjamin Lopez, Maria Lavaredda-Pearce, Laura Perin, and Stefano Da Sacco for their spontaneous technical assistance.

Notes

Supported by NIH PO1 HL060231 (P.M.) and R01HL086322 (S.B.), Hastings Foundation (P.M.), CIRM Clinical Fellowship (C.T.), and the “Young Investigator Award,” European Society of Pediatrics (C.T.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200901-0100OC on July 2, 2009

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

References

1. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell 2004;116:769–778. [PubMed]
2. Lanza R. Essentials of stem cells biology. New York: Academic Press; 2006.
3. Engelhardt JF, Schlossberg H, Yankaskas JR, Dudus L. Progenitor cells of the adult human airway involved in submucosal gland development. Development 1995;121:2031–2046. [PubMed]
4. Schoch KG, Lori A, Burns KA, Eldred T, Olsen JC, Randell SH. A subset of mouse tracheal epithelial basal cells generates large colonies in vitro. Am J Physiol Lung Cell Mol Physiol 2004;286:L631–L642. [PubMed]
5. Avril-Delplanque A, Casal I, Castillon N, Hinnrasky J, Puchelle E, Péault B. Aquaporin-3 expression in human fetal airway epithelial progenitor cells. Stem Cells 2005;23:992–1001. [PubMed]
6. Liu JY, Nettesheim P, Randell SH. Growth and differentiation of tracheal epithelial progenitor cells. Am J Physiol 1994;266:L296–L307. [PubMed]
7. Hong KU, Reynolds SD, Giangreco A, Hurley CM, Stripp BR. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Am J Respir Cell Mol Biol 2001;24:671–681. [PubMed]
8. Reynolds SD, Hong KU, Giangreco A, Mango GW, Guron C, Morimoto Y, Stripp BR. Conditional clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells. Am J Physiol Lung Cell Mol Physiol 2000;278:L1256–L1263. [PubMed]
9. Aso Y, Yoneda K, Kikkawa Y. Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab Invest 1976;35:558–568. [PubMed]
10. Isakson BE, Lubman RL, Seedorf GJ, Boitano S. Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF. Am J Physiol Cell Physiol 2001;281:C1291–C1299. [PubMed]
11. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121:823–835. [PubMed]
12. Stiles B, Groszer M, Wang S, Jiao J, Wu H. PTENless means more. Dev Biol 2004;273:175–184. [PubMed]
13. Takahashi K, Murakami M, Yamanaka S. Role of the phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells. Biochem Soc Trans 2005;33:1522–1525. [PubMed]
14. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi PP. Pten is essential for embryonic development and tumor suppression. Nat Genet 1998;19:348–355. [PubMed]
15. Freeman D, Lesche R, Kertesz N, Wang S, Li G, Gao J, Groszer M, Martinez-Diaz H, Rozengurt N, Thomas G, et al. Genetic background controls tumor development in PTEN-deficient mice. Cancer Res 2006;66:6492–6496. [PubMed]
16. Groszer M, Erickson R, Scripture-Adams DD, Dougherty JD, Le Belle J, Zack JA, Geschwind DH, Liu X, Kornblum HI, Wu H. PTEN negatively regulates neural stem cell self-renewal by modulating G0–G1 cell cycle entry. Proc Natl Acad Sci USA 2006;103:111–116. [PubMed]
17. He XC, Yin T, Grindley JC, Tian Q, Sato T, Tao WA, Dirisina R, Porter-Westpfahl KS, Hembree M, Johnson T, et al. PTEN-deficient intestinal stem cells initiate intestinal polyposis. Nat Genet 2007;39:189–198. [PubMed]
18. Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006;441:475–482. [PubMed]
19. Tiozzo C, Yu M, Xing Y, Li M, Li C, DeLanghe S, Bellusci S, Anderson S, Minoo P, Lung-specific early deletion of Pten alters the pool of progenitor/stem cells in the lung [abstract]. Presented at the Keystone Symposia, February 24–29, 2008, Vancouver, BC.
20. Tiozzo C, Yu M, Xing Y, Li M, Li C, DeLanghe S, Bellusci S, Anderson S, Minoo P. Lung-specific early deletion of Pten alters the pool of progenitor/stem cells in the lung [abstract]. Presented at the PAS Annual Meeting, May 3–6, 2008, Honolulu, HI.
21. Xu Q, Tam M, Anderson SA. Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. J Comp Neurol 2008;506:16–29. [PubMed]
22. Lesche R, Groszer M, Gao J, Wang Y, Messing A, Sun H, Liu X, Wu H. Cre/loxP-mediated inactivation of the murine Pten tumor suppressor gene. Genesis 2002;32:148–149. [PubMed]
23. Londhe VA, Belperio JA, Keane MP, Burdick MD, Xue YY, Strieter RM. CXCR2/CXCR2 ligand biological axis impairs alveologenesis during dsRNA-induced lung inflammation in mice. Pediatr Res 2005;58:919–926. [PubMed]
24. Minoo P, Su G, Drum H, Bringas P, Kimura S. Defects in tracheoesophageal and lung morphogenesis in Nkx2.1(−/−) mouse embryos. Dev Biol 1999;209:60–71. [PubMed]
25. Pan Q, Li C, Xiao J, Kimura S, Rubenstein J, Puelles L, Minoo P. In vivo characterization of the Nkx2.1 promoter/enhancer elements in transgenic mice. Gene 2004;331:73–82. [PubMed]
26. Xing Y, Li C, Hu L, Tiozzo C, Li M, Chai Y, Bellusci S, Anderson S, Minoo P. Mechanisms of TGFbeta inhibition of LUNG endodermal morphogenesis: the role of TbetaRII, Smads, Nkx2.1 and Pten. Dev Biol 2008;320:340–350. [PMC free article] [PubMed]
27. Ito T, Udaka N, Ikeda M, Yazawa T, Kageyama R, Kitamura H. Significance of proneural basic helix-loop-helix transcription factors in neuroendocrine differentiation of fetal lung epithelial cells and lung carcinoma cells. Histol Histopathol 2001;16:335–343. [PubMed]
28. Stripp BR, Maxson K, Mera R, Singh G. Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am J Physiol 1995;269:L791–L799. [PubMed]
29. Yanagi S, Kishimoto H, Kawahara K, Sasaki T, Sasaki M, Nishio M, Yajima N, Hamada K, Horie Y, Kubo H, et al. Pten controls lung morphogenesis, bronchioalveolar stem cells, and onset of lung adenocarcinomas in mice. J Clin Invest 2007;117:2929–2940. [PMC free article] [PubMed]
30. Davé V, Wert SE, Tanner T, Thitoff AR, Loudy DE, Whitsett JA. Conditional deletion of Pten causes bronchiolar hyperplasia. Am J Respir Cell Mol Biol 2008;38:337–345. [PMC free article] [PubMed]
31. Lahad JP, Mills GB, Coombes KR. Stem cell-ness: a “magic marker” for cancer. J Clin Invest 2005;115:1463–1467. [PMC free article] [PubMed]
32. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730–737. [PubMed]
33. Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, Haug JS, Rupp D, Porter-Westpfahl KS, Wiedemann LM, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 2006;441:518–522. [PubMed]
34. Jerina DM, Daly JW, Jeffrey AM, Gibson DT. Cis-1,2-dihydroxy-1,2-dihydronaphthalene: a bacterial metabolite from naphthalene. Arch Biochem Biophys 1971;142:394–396. [PubMed]
35. Stillwell WG, Horning MG, Griffin GW, Tsang WS. Identification and synthesis of the isomeric tetrahydroxytetrahydronaphthalene metabolites excreted in rat urine. Drug Metab Dispos 1982;10:11–14. [PubMed]
36. Hesse S, Mezger M. Involvement of phenolic metabolites in the irreversible protein-binding of aromatic hydrocarbons: reactive metabolites of [14C]naphthalene and [14C]1-naphthol formed by rat liver microsomes. Mol Pharmacol 1979;16:667–675. [PubMed]

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