The purpose of this study was to examine the consequences of epithelial-specific stabilization and accumulation of β-catenin on lung morphogenesis and cell differentiation. To this end, we used the cre-loxP system to permanently remove the floxed exon 3 of β-catenin thus resulting in accumulation of CatnbΔex3 protein, which lacks the N-terminal phosphorylation sites required for β-catenin degradation (Harada et al., 1999
). We generated CatnbEx3(cko
double transgenic mice by mating between Catnb[
mice and Nkx2.1-cre
mice (Xing et al., 2008
; Xu et al., 2008
). Accumulation of CatnbΔex3 in the lung epithelial cells was accompanied by increased β-catenin mRNA. Morphologically, accumulation of CatnbΔex3 led to dilation of airways in the lung and formation of polyp-like structures in the proximal airways including trachea and main-stem bronchi. Functionally, accumulation of CatnbΔex3 led to cell fate changes as evidenced by, 1) inhibition of ciliated, Clara and basal cell differentiation, and 2) activation of UCHL1, a marker of pulmonary neuroendocrine cells. Cell differentiation was also inhibited in epithelial cells that lacked, but were adjacent to those with CatnbΔex3 accumulation. As the sites of CatnbΔex3 accumulation overlap with high level expression of Bmp4, we propose that inhibition of cell differentiation in these sites occurs via paracrine Bmp4 signaling.
To generate epithelial-specific gain-of-function mutation for β-catenin we used a novel cre mouse line in which expression of the recombinase is under the control of the Nkx2.1 regulatory elements. Nkx2.1 is a homeodomain transcription factor and the earliest known marker of lung endodermal determination. Activity of Nkx2.1-cre was detected in epithelial airways of embryonic lungs from as early as E10.5 (Tiozzo et al., Manuscript submitted). Thus, Nkx2.1-cre provides the opportunity for investigating the role of early activation of β-catenin in lung morphogenesis and cell lineage determination. A surprising finding of the current study is that stabilization of β-Catenin in epithelial cells occurred in a selective, rather than uniform manner in CatnbEx3(cko
lungs. For example, CatnbΔex3 protein accumulated in epithelial cells of the outer wall, but not the inner wall of the main-stem bronchi. It is unlikely that the selective pattern of β-catenin accumulation observed in the mutant lungs is due to Nkx2.1-cre selectivity (). Whether the cells permissive to accumulation possess mechanisms that’s lacking in those without accumulation remains unknown. Alternatively, the inner wall epithelial cells may have a mechanism
that degrades truncated β-catenin via an exon3-independent pathway. Cell-type selective stabilization of β-catenin was also observed in early embryogenesis. Using a zona pellucida3-cre (Zp3-cre)
, Kemler et al expressed the CatnbΔex3 in developing oocytes (Kemler et al., 2004
). CatnbΔex3 was not stabilized in pre-implantation embryos, but stabilized in early postimplantation embryos and led to premature cell fate changes in the embryonic ectoderm (Kemler et al., 2004
). Detailed mechanism underlying cell specificity is unknown. Several GSK3β-independent β-catenin degradation mechanisms have been reported. For example, ubiquitin ligase Siah-1 and Siah-2, the mammalian homolog of Drosophila seven in absentia
, degrade β-catenin independently of the GSK3β pathway (Liu et al., 2001
; Matsuzawa and Reed, 2001
; Topol et al., 2003
). Also, the Gq pathway triggers calpain-mediated proteolysis of β-catenin in the human colon cancer cell line, SW480, independent of GSK3β (Li and Iyengar, 2002
). Finally, although its relationship to GSK3β pathway remains unknown, Smad7, an antagonist for TGFβsignaling also induces β-catenin degradation through Smurf2 in keratinocytes (Han et al., 2006
Another finding was increased mRNA for β-catenin in CatnbEx3(cko
lungs. Gain-of-function mutation generated by removal of exon 3 from β-catenin is thought to result in accumulation of available β-catenin simply by stabilization of the protein. In CatnbEx3(cko
lungs, we found not only a robust increase in β-catenin protein, but also mRNA (). The mechanism for increased mRNA remains unknown, but as β-catenin is a target of Wnt signaling, it may potentially represent auto-regulation of β-catenin transcription by the stabilized protein. In support of this possibility, increased β-catenin mRNA has been observed in several intestinal-type gastric cancers with APC mutations (Ebert et al., 2002
). Therefore, robust accumulation of β-catenin in the CatnbEx3(cko
lungs may be established by combined effects of stabilization of β-catenin protein as well as increased steady state level of β-catenin mRNA.
Stabilization and accumulation of β-catenin in lung epithelial cells caused both structural and cell fate changes in CatnbEx3(cko
lungs. Structural changes consisted of abnormal cartilage formation in the trachea and large, dilated airways especially in the anterior tip of the left lobe (Figures & ). The latter dilated airway phenotype was also found in gain-of-function mutations generated by Dox-regulated CCSP-rtTA;(tetO)7
-CMV-cre (Mucenski et al., 2005
). However, the Nkx2.1-cre generated CatnbEx3(cko
lungs, also included structures resembling polyps which were not reported by previous studies (Mucenski et al., 2005
; Okubo and Hogan, 2004
; Reynolds et al., 2008
). Intestinal polyposis is commonplace and thought to be precursor of colorectal cancer (Harada et al., 1999
; Morin et al., 1997
; Morson, 1974
). Mutations of Wnt signaling mediators including APC, β-catenin and Axin are found in colorectal cancers (Fearnhead et al., 2005
; Kolligs et al., 2002
). Epithelial-specific stabilization of β-catenin in the intestine of transgenic Catnb[
mice causes intestinal adenomas (Harada et al., 1999
). In contrast, the formation of polyps in the lung is uncommon and a novel finding of the present study. The tracheal and bronchial “polyps” differ from intestinal polyps in the following aspects. First, unlike their intestinal counterparts which are composed solely of epithelial cells, the tracheal and bronchial polyps in CatnbEx3(cko
lungs consist of a mesenchymal core covered by an epithelium that is contiguous with the bronchial epithelial wall. Second, cell proliferation is not increased in tracheal and bronchial “polyps”, but is increased in intestinal polyps (Harada et al., 1999
). This suggests
that the lung polyps are unlikely to advance to tumors. However, as the CatnbEx3(cko
mice do not survive postnatally, this issue requires further experimentation. In several tissues such as liver, hyperactive Wnt signaling alone is not sufficient for tumorigenesis and may require additional genetic or epigenetic changes (Harada et al., 2002
). Clinical report on tracheal polyposis is limited and its mechanism remains unknown (Fein et al., 1982
). The CatnbEx3(cko
lung provides a useful in vivo model for studying tracheal and bronchial polyposis as well as the function of Wnt/β-catenin signaling in epithelial cell lineage specification.
In the mutant lungs, CatnbΔex3 accumulation is associated with ectopic Bmp4 expression in the apical epithelium of the polyps. It was reported previously that expression of Bmp4 is activated by Wnt/β-catenin signaling (Haegele et al., 2003
). Whether Bmp4 has a role in polyp formation in the CatnbEx3(cko
lungs remains to be investigated. Several previous studies have demonstrated that BMP4 inhibits epithelial cell proliferation. For example, over-expression of BMP4 by SpC promoter results in reduced epithelial cell proliferation during lung morphogenesis (Bellusci et al., 1996
). Similarly, recombinant BMP4 protein inhibits proliferation of cultured epithelial explants (Hyatt et al., 2002
; Weaver et al., 2000
). Therefore, high levels of BMP4 in epithelial cells with accumulated β-catenin may be the reason for a lack of proliferation in these epithelial cells.
Stabilization and accumulation of β-catenin in epithelial cells of CatnbEx3(cko/+) lungs also caused abnormalities in cell fate and differentiation. Careful characterization of cells particularly in regions of high CatnbΔex3 accumulation revealed that the impact of excess β-catenin on epithelial cell differentiation and cell fate can occur via at least two mechanisms.
First, stabilization of β-catenin and subsequent activation of Wnt signaling () may lead to cell fate changes in embryonic lung epithelial cells. This conclusion is based on the observation that expression of CatnbΔex3 in the lung epithelium resulted in inhibition of Clara, ciliated and basal cell differentiation (Figures & ). In addition, UCHL1, a marker of PNEC was found to be ectopically expressed in the epithelial cells where CatnbΔex3 accumulated. We propose that the latter changes are either direct consequences of excess intracellular β-catenin or secondary to its paracrine effects (please see below). In support of this proposal, level of β-catenin in PNECs of wild-type lungs is indeed higher than other epithelial cells along the airway (). However, unlike a previous report (Okubo and Hogan, 2004
) we found that robust accumulation of CatnbΔex3 had no impact on Nkx2.1, a transcription factor that is expressed in epithelial progenitor cells early in lung development, suggesting maintenance of lung epithelial cell identity (). Furthermore, TTF3, a marker for intestinal cells, was clearly absent from either wild-type or CatnbEx3(cko
lungs (data not shown). These inconsistent results may simply reflect the inherent functional differences between CatnbΔex3 and β-catenin-Lef1 that was used in Okubo and Hogan’s report.
Second, we found that differentiation of Clara and ciliated cells was also blocked in the epithelium that lacked robust expression of CatnbΔex3 but was adjacent to the polyps. The robust accumulation of CatnbΔex3 overlaps high level expression of Bmp4 (). The function of BMP4 in inhibiting proximal airway epithelial cell differentiation has been demonstrated by both in vivo and in vitro studies. Bmp4 signaling negatively regulates proximal epithelial cell differentiation in transgenic animals that over-express BMP4 inhibitors (Lu et al., 2001
; Weaver et al., 1999
). Tracheal epithelial cells cultured in Matrigel differentiate and express CCSP (CC10) and HFH4 (Foxj1) in presence of FGF1. BMP4 inhibits the latter process and the BMP4 inhibitor, Noggin, blocks the inhibitory effect of BMP4 (Hyatt et al., 2002
). Expression of Bmp4 is activated by Wnt/β-catenin signaling (Haegele et al., 2003
). Thus, it is possible that accumulation of stabilized β-catenin stimulates Bmp4 expression thereby inhibiting Clara and ciliated cell differentiation in the adjacent epithelium. This is the paracrine mechanism of excess β-catenin effect on cell differentiation (). It is noteworthy that basal cell differentiation, as assessed by p63 expression was not blocked by high level of Bmp4 signaling in the adjacent cells (), whereas it was blocked in cells with direct accumulation of CatnbΔex3. Therefore, differentiation of ciliated, Clara and basal cells may be differentially regulated by mechanisms involving Wnt/β-catenin and Bmp4 signaling.
Figure 11 A simplistic schematic model of mechanisms related to β-catenin accumulation. In apical epithelial cells of the polyps, accumulation of β-catenin results in cell fate changes associated with increased expression of Bmp4 and β-catenin. (more ...)
In summary, stabilization of β-catenin in the lung epithelium occurs in a non-uniform pattern suggesting potential differences amongst epithelial cells that hitherto had been thought to be of similar or identical developmental history. Excess β-catenin has both direct and paracrine effects on cell fate determination and differentiation. Finally, β-catenin gain-of-function using the cre-loxP system reflects both increased protein as well as mRNA. These observations should help elucidate the functional role of Wnt/β-catenin signaling during mammalian development.