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
). In both previous studies Pten
deletion was achieved using the SPC-rtTA;Tet(O)-cre
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 (). 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 (). Other putative progenitor cells, including the CC10/CGRP double-positive NEB and SPC/CC10 double-positive cells in the BADJ were also increased ( and ). 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
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.