Reactivation of Wnt signaling reinduces Fgf10 expression in PSMCs 3 days after naphthalene-mediated Clara cell injury.
We previously showed that a large proportion of PSMCs in the lung are derived from Fgf10
-expressing cells in the distal mesenchyme during early lung development (10
) and that treatment of embryonic lung explants with Dickkopf1 (Dkk1), a canonical Wnt/β-catenin inhibitor, impairs PSMC differentiation (12
). In addition, we have shown that amplification of these PSMC progenitors, as well as their Fgf10
expression, is regulated by mesenchymal β-catenin signaling and that deletion of β-catenin leads to their premature differentiation into PSMCs (9
). During lung development, Fgf10 signaling is critical in the maintenance of lung epithelial progenitors (5
), in part through direct activation of epithelial β-catenin signaling. Although the Fgf/β-catenin signaling axis in lung development is relatively well described, it is unclear whether this signaling pathway is recapitulated in adult lungs after lung epithelial injury as part of the repair process.
BrdU labeling of lungs 3 days after naphthalene-mediated epithelial injury indicated a robust proliferation of the PSMCs compared with a lack of proliferation in corn oil–treated lungs (Figure , A and B; 0% ± 0% vs. 6.6% ± 0.7% SMA+
≥ 3; P
= 0.00004). This proliferation was abrogated in mice induced to overexpress Dkk1
3 days prior to injury (Figure C; 0.3% ± 0.3% vs. 6.6% ± 0.7% SMA+
= 7; P
= 0.00002), demonstrating the importance of this pathway for PSMC proliferation. To visualize which cells were undergoing active β-catenin signaling in the adult lung during homeostasis as well as after naphthalene-mediated epithelial injury, we used the TOPGAL reporter line for Wnt signaling (14
). During normal homeostasis in 2-month-old mice, we showed that numerous Clara cells were TOPGAL positive (Figure D and Supplemental Figure 1G; supplemental material available online with this article; doi:
), demonstrating active β-catenin signaling. Interestingly, when TOPGAL mice were injured with naphthalene, we noticed that the majority of Clara cells were lost at 3 days after challenge, but detected robust TOPGAL activity in the surviving variant Clara cells at the BADJs (Figure E). Variant Clara cells adjacent to NEBs were also TOPGAL positive (see below). Strikingly, we could also detect TOPGAL activation in the PSMCs surrounding the injured airways (Figure E, arrow, and Supplemental Figure 1H), indicative of Wnt pathway reactivation in these cells. TOPGAL activity was not detected in the PSMCs during normal homeostasis in adult lungs (Supplemental Figure 1G).
Wnt7b expressed by surviving ciliated cells induces Fgf10 expression in PSMCs 3 days after naphthalene-mediated Clara cell injury.
To demonstrate that TOPGAL activity in PSMCs, as well as in variant Clara cells, is Wnt ligand dependent, we crossed TOPGAL mice with Rosa-rtTA;Tet-Dkk1
mice. Ubiquitous overexpression of Dkk1
starting 3 days before injury prevented the induction of TOPGAL activity in PSMCs as well as in variant Clara cells 3 days after naphthalene injury (Figure F). We then used Fgf10LacZ
reporter mice (10
), which express LacZ
under control of the Fgf10
promoter, to analyze Fgf10
expression in 2-month-old adult lungs during normal homeostasis, 3 days after naphthalene injury, and 3 days after naphthalene injury while overexpressing Dkk1
. PSMCs, unlike their progenitors during development, did not express Fgf10
during normal homeostasis (Figure G and Supplemental Figure 1, A and E), but showed strong induction of Fgf10
expression 3 and 7 days after injury (Figure H and Supplemental Figure 1, B and F) that was inhibited by Dkk1
overexpression (Figure I and Supplemental Figure 1, C and N). Note that we have identified the Fgf10
-expressing cells in the alveolar compartment (Figure , G–I) as lipofibroblasts (S.P. De Langhe, unpublished observations).
Dkk1 acts by inducing degradation of the LRP5/6 coreceptor, thereby preventing the binding of a Wnt ligand to the Frizzled receptor (16
). We therefore compared the expression levels of different Wnt ligands in WT lungs 3 days after corn oil or naphthalene treatment by quantitative real-time PCR (qPCR) analysis using a Wnt signaling pathway qPCR array. Only Wnt7b
expression levels were significantly changed, showing approximately 3-fold upregulation 3 days after injury (Figure L and Supplemental Figure 2). Interestingly, a recent paper by Cohen et al. demonstrated that Wnt7b secreted by the airway epithelium plays an important role in PSMC development and in activation of the canonical Wnt pathway in PSMCs (18
). The authors also demonstrated that the Wnt pathway becomes reactivated in the PSMCs in a mouse model for asthma, as well as in VSMCs in patients with pulmonary arterial hypertension (PAH) (18
). Here we found that 3 days after naphthalene injury, surviving airway ciliated cells showed robust induction of Wnt7b expression (Figure , J and K), possibly activating surrounding PSMCs in a paracrine fashion. Interestingly, Fgf10
expression was also induced in PSMCs after ozone- or bleomycin-mediated epithelial injury (Supplemental Figure 3, A–C).
Wnt-induced Fgf10 secreted by PSMCs is essential for epithelial repair after naphthalene injury.
Next, we investigated the importance of Wnt-induced Fgf10, secreted by the PSMCs for epithelial regeneration after naphthalene injury. We generated 5 different mouse models, which we injured at 2 months of age with naphthalene. Rosa26-rtTA;Tet-sFgfr2b mice, which overexpress a dominant-negative soluble secreted Fgfr2b receptor (sFgfr2b), were used to block Fgf10 signaling by sequestering the Fgf10 ligand. Rosa26-rtTA;Tet-Fgf10 mice were used to overexpress Fgf10. Rosa26-rtTA;Tet-Dkk1 mice were generated to overexpress Dkk1 and to suppress Fgf10 expression in the PSMCs. We generated Rosa26-rtTa;Tet-Dkk1;Tet-Fgf10 mice to conditionally overexpress both Dkk1 and Fgf10. Finally, we generated Myh11-cre+;Fgf10fl/fl mice to conditionally delete Fgf10 specifically in the SMCs (note that Myh11 is also known as smooth muscle myosin heavy chain [smMHC]).
Immunofluorescence and qPCR for Scgb1a1 were used to assess and quantify the expansion of the Clara cells at different time points after injury. Inhibition of Fgf10 signaling by overexpression of the dominant-negative Fgfr2b receptor significantly impaired Clara cell regeneration after naphthalene injury (Figure , A, B, and I). On the other hand, overexpression of Fgf10 significantly accelerated airway epithelial regeneration (Figure , C and I). Mice overexpressing Dkk1 showed a significant impairment in regeneration 7 days after naphthalene injury (Figure I), and the majority did not survive up to 2 weeks after injury. The latter effect was not due to general overexpression of Dkk1, as noninjured doxycycline-induced (dox-induced) Rosa26-rtTA;Tet-Dkk1 mice survived readily up to 6 months of age, after 4 months of continuous induction. However, the impaired regeneration was rescued by coexpressing Dkk1 and Fgf10: Rosa26-rtTA;Tet-Dkk1;Tet-Fgf10 mice showed accelerated epithelial repair, similar to that of mice overexpressing only Fgf10 (Figure , G–I). Together, these data suggest that the main mechanism by which Dkk1 overexpression impairs epithelial regeneration is inhibition of Wnt7b-induced Fgf10 expression in the PSMCs. Fgf10 affected the amplification of variant Clara cells at the BADJs as well as near the NEBs (Figure , A–K).
Wnt-induced Fgf10 secreted by PSMCs is essential for epithelial repair after naphthalene injury.
To confirm that PSMCs are indeed the source of Fgf10 important for regeneration, we performed naphthalene injury on Myh11-Cre+;Fgf10fl/fl mice. Clara cell regeneration was severely impaired in mice with SMC-specific deletion of Fgf10 (Figure , L–P). Moreover, qPCR analysis revealed a dramatic decrease in Fgf10 expression in lungs from Myh11-Cre;Fgf10fl/fl versus control lungs 14 days after naphthalene injury (Supplemental Figure 1N). Because Myh11-Cre;Fgf10fl/fl mice were very susceptible to injury, they were therefore injured suboptimally to allow for survival so that we could address the role of PSMC-derived Fgf10 in airway epithelial regeneration.
Fgf10 mediates airway epithelial repair independent of Wnt ligands, in part by directly activating/enhancing β-catenin signaling in the epithelium.
Epithelial Wnt signaling has previously been shown to accelerate Clara cell regeneration after naphthalene injury and is known to amplify the variant Clara cells at the BADJs (19
). Our previous work has suggested that Fgf10 can activate β-catenin signaling directly in the epithelium (8
). To test whether Fgf10 directly activates β-catenin signaling during repair, we crossed the TOPGAL Wnt signaling reporter allele into the different mouse lines described above, performed naphthalene injury, and monitored epithelial β-catenin signaling 7 days after injury. At 7 days after naphthalene injury, lungs from control TOPGAL mice were partially regenerated and showed TOPGAL activity in the regenerating Clara cells at the BADJs as well as near the NEBs (Figure , A, F, K, and P). Mice overexpressing Fgf10
showed a robust increase in Clara cell regeneration, as described above, and showed a strong increase in epithelial TOPGAL activity in the Clara cells at both the BADJs and the NEBs (Figure , B, G, L, and Q). Mice overexpressing sFgfr2b
showed a profound decrease in Clara cell regeneration and had limited TOPGAL activity in the Clara cells at both BADJs and NEBs. This indicates that inhibition of Fgf10 signaling through either sequestering of the Fgf10 ligand (Figure , C, H, M, and R) or suppressing Fgf10
expression (Figure , D, I, N, and S) by overexpressing sFgfr2b
, respectively, inhibits not only Clara cell regeneration, but also epithelial β-catenin signaling.
Fgf10 mediates epithelial repair independent of Wnt ligands in part by activating/enhancing β-catenin signaling in the epithelium.
Finally, we demonstrated that naphthalene-injured lungs from mice overexpressing both Dkk1 and Fgf10 showed a rescue in Clara cell regeneration as well as an increase in β-catenin signaling at the BADJs and NEBs (Figure , E, J, O, and T), which indicates that Fgf10 signaling can induce repair independent of Wnt ligands and activate the β-catenin signaling pathway directly.
Fgf10 signaling induces Akt-mediated phosphorylation of β-catenin and maintenance/amplification of variant Clara cells.
The main mechanism through which Fgf10 is thought to activate β-catenin signaling is by activation of the PI3K-Akt pathway. Akt acts to inhibit GSK3β, therefore preventing the degradation of β-catenin, as well as to phosphorylate β-catenin directly on Ser552 to drive it to the nucleus (21
). To establish the mechanism by which Fgf10 activates β-catenin signaling, we performed immunostaining for phosphorylated Akt (p-Akt) and p–β-catenin–Ser552 on lung samples from control, Rosa26-rtTa;Tet-Fgf10
, and Rosa26-rtTa-Tet-sFgr2b
mice 3 days after naphthalene injury. Modest phosphorylation of Akt and β-catenin was observed in control lungs (Figure , A and D). This phosphorylation was increased in lungs overexpressing Fgf10
(Figure , B and E) and dramatically decreased in lungs overexpressing sFgfr2b
(Figure , C and F). We also observed phosphorylation of Akt and β-catenin–Ser552 in Clara cells at the BADJ after prolonged overexpression of Fgf10
in noninjured lungs (Supplemental Figure 4, I–L).
Fgf10 signaling induces Akt-mediated phosphorylation of β-catenin and maintenance/amplification of variant Clara cells.
The putative stem cell population of variant Clara cells at the BADJ has been shown to be double positive for Sftpc and Scgb1a1 (19
); such cells have also been called bronchoalveolar stem cells (BASCs). Sftpc is a marker of not only alveolar epithelial type II cells, but also distal epithelial progenitor cells in the developing lung, where its expression is controlled by Fgf10. Therefore, reexpression of Sftpc in variant Clara cells could reflect an epithelial progenitor–like state. To analyze the effect of Fgf10 on the amplification and maintenance of variant Clara cells, we performed double immunostaining for Scgb1a1 and Sftpc on control and Fgf10
-overexpressing lungs 21 days after naphthalene injury (Figure , I and J). Whereas an increase in variant Clara cells (Scgb1a1-Sftpc double-positive cells) is normally seen shortly after naphthalene injury, these double-positive cells usually did not persist by 21 days after injury (Figure I). Interestingly, overexpression of Fgf10
not only amplified the number of BASCs after injury, but also maintained them as long as Fgf10
expression was induced (Figure J). These findings indicate that Fgf10, possibly (at least in part) by directly activating/enhancing epithelial β-catenin signaling, is important in generating variant Clara cells at the BADJs. Importantly, Fgf10 was also able to generate these Scgb1a1-Sftpc double-positive airway epithelial stem cells near the NEBs. Figure K shows a cluster of Sftpc-positive Clara cells adjacent to a NEB in an Fgf10
-overexpressing lung 21 days after naphthalene injury, which indicates that reexpression of Sftpc in variant Clara cells may be more universal in nature and possibly reflects partial dedifferentiation into a lung epithelial progenitor–like state. We also identified the Fgf10 receptor Fgfr2b as an extracellular marker for these Scgb1a1-Sftpc double-positive airway epithelial stem/progenitor cells (Figure , G and H, and see below). It is important to note that Fgfr2b is also a marker for distal embryonic lung epithelial progenitors and is downstream of Fgf10 as well as β-catenin signaling (9
). Amplification of Fgfr2b-expressing variant Clara cells at the BADJ was also observed after prolonged overexpression of Fgf10
in noninjured lungs and after ozone- and bleomycin-induced lung injury (Supplemental Figure 4, A–D).
Flow cytometry analysis of Scgb1a1-Sftpc or Scgb1a1-Fgfr2 double-positive airway epithelial stem cells.
To quantify the effect of naphthalene injury and Fgf10 expression on the generation of Scgb1a1-Sftpc or Scgb1a1-Fgfr2 double-positive cells, we performed flow cytometry analysis for these markers on permeabilized fixed single cell whole lung digests from noninjured control lungs as well as control and Fgf10-overexpressing lungs 7 days after naphthalene injury (Figure ). In noninjured control lungs, a small fraction of the cells were Scgb1a1-Sftpc and Scgb1a1-Fgfr2 double-positive (0.18% and 0.54%, respectively; Figure , A and B). Injured control lungs showed an approximately 14-fold increase in Scgb1a1-Sftpc and Scgb1a1-Fgfr2 double-positive airway epithelial stem cells 7 days after injury relative to noninjured lungs (Figure , C and D). Finally, Fgf10-overexpressing lungs showed 22- and 33-fold increases, respectively, in Scgb1a1-Sftpc and Scgb1a1-Fgfr2 double-positive airway epithelial stem cells 7 days after injury (Figure , E and F). The forward scatter/side scatter plots in Figure also illustrate that the same population of airway epithelial stem cells was isolated, by sorting for either the Scgb1a1 and Sftpc markers or the Scgb1a1 and Fgfr2 markers. This identifies Fgfr2b as a marker for airway epithelial stem cells in the adult lung after naphthalene injury, a finding we believe to be novel.
Flow cytometry analysis of airway epithelial stem cells.
Fgf10 signaling induces Fgfr2b expression, Notch activation, and subsequent Snail1 induction in activated variant Clara cells.
Because of the importance of Fgf10 during lung development in maintaining the distal epithelial lung progenitors and preventing them from differentiating into more proximal cell types, we next sought to unravel the mechanism by which Fgf10 generates airway epithelial stem cells. A recent paper by Mani and colleagues (24
) established that in the mammary gland (another organ in which Fgf10 plays an important role; refs. 25
), the EMT generates mammary gland epithelial stem cells.
Our cell sorting data indicated that airway epithelial stem cells are positive for Fgfr2b. Immunostaining 3 days after naphthalene injury demonstrated that regenerating Clara cells adjacent to NEBs were Fgfr2b positive in control and Fgf10
-overexpressing lungs, but not in sFgfr2b
- or Dkk1
-overexpressing lungs (Figure , A–D). Fgfr2b expression in Dkk1
-overexpressing lungs was rescued by simultaneous overexpression of Fgf10
(Figure E). The most robust inducer of EMT is Snail1 (encoded by Snai1
), which is tightly regulated on a transcriptional as well as a posttranslational level. Wnt, Fgf, and Notch pathways often act together to regulate Snail1 and induce an EMT program. Fgf and Notch have been previously shown to directly regulate Snai1
expression, whereas Fgf and Wnt, through inhibition of GSK3β, act to stabilize the Snail1 protein and protect it from degradation (reviewed in refs. 27
Fgf10 signaling induces Fgfr2b expression, Notch activation, and subsequent Snail induction in the activated variant Clara cells.
Immunostaining for the cleaved Notch1 intracellular domain (NotchICD) demonstrated that Notch signaling was activated in regenerating Clara cells/airway epithelial stem cells 3 days after injury at both BADJs and NEBs (Figure , F and K). Additionally, Notch activation was increased in lungs overexpressing Fgf10 and inhibited in lungs overexpressing sFgfr2b or Dkk1, but was rescued upon overexpression of both Dkk1 and Fgf10 (Figure , F–O). Moreover, we found an induction in Snail1 expression in the regenerating Clara cells adjacent to NEBs and at BADJs in WT control lungs 3 days after naphthalene injury (Figure P and Supplemental Figure 1I). This induction in Snail1 was dramatically increased in lungs overexpressing Fgf10, inhibited in lungs overexpressing sFgfr2b or Dkk1, and rescued upon overexpression of both Dkk1 and Fgf10 (Figure , P–T, and Supplemental Figure 1, K and L).
Variant Clara cells undergo a transient EMT in response to Fgf10-induced Notch activation and subsequent Snail1 induction.
Finally, we sought to determine whether regenerating (naphthalene-resistant) Clara cells are indeed undergoing an EMT to generate airway epithelial stem cells. We crossed Myh11-Cre mice, which express the Cre recombinase under control of the smMHC promoter, with Rosa26R-eYFP or Rosa26R-LacZ reporter mice. During normal homeostasis, Myh11-Cre;Rosa26R mice only labeled airway and VSMCs in the lung, never lung epithelial cells. However, we observed labeled epithelial cells around 3 days after naphthalene injury (Figure A), which indicates that Clara cells undergo a transient EMT to generate airway epithelial stem cells or adopt epithelial stem cell properties. The labeled clusters expanded to eventually give rise to regenerated Clara cells (Figure , B and C). Rosa26R-labeled Clara cells were detected near both NEBs and BADJs (Figure , D and E). We further checked for expression of other markers consistent with EMT; we found loss of E-cadherin and increased smMHC and vimentin in Clara cells 3 days after naphthalene injury (Supplemental Figure 4, M–O). Additionally, we performed bleomycin injury on Myh11-Cre+;Rosa26R-LacZ mice, which demonstrated that bleomycin injury also induced a transient EMT in a nonfibrotic area of airway epithelium 21 days after bleomycin injury (Supplemental Figure 4P).
Variant Clara cells undergo transient EMT in response to Fgf10-induced Notch activation and subsequent Snail induction.
To determine whether Fgf10, via induction of Notch signaling, induces Snai1 expression in Clara cells and therefore induces this transient EMT, we overexpressed Fgf10 in 2-month-old noninjured lungs for 14 and 21 days and found that prolonged Fgf10 exposure induced Snai1 expression and Notch activation in Clara cells at the BADJ in the absence of injury (Figure , G and H, Supplemental Figure 1, K–M, and Supplemental Figure 4, E and F). Furthermore, Snail1 was induced in Clara cells at BADJs after ozone or bleomycin injury (Supplemental Figure 4, G and H). To determine the role of Notch signaling directly in the generation of lung airway epithelial stem cells, we generated Scgb1a1-rtTa;Tet-O-Cre;Rosa26-NotchICD mice, allowing us to inducibly overexpress the NotchICD activator domain in Clara cells. Interestingly, noninjured lungs from Scgb1a1-rtTa;Tet-O-Cre;Rosa26-NotchICD mice looked normal, but showed robust Snail1 expression in Clara cells (Figure I). We then tested whether airway epithelial regeneration after naphthalene injury is increased in Scgb1a1-rtTa;Tet-O-Cre;Rosa26-NotchICD mice induced 3 days prior to injury. We found that activation of Notch signaling in Clara cells indeed accelerated regeneration of the airway epithelium after naphthalene injury (Figure F). To investigate the importance of Snail1 in airway epithelium regeneration after naphthalene injury, we generated Shh-Cre+;Snai1fl/fl mice, in which Snai1 is conditionally deleted from all lung epithelial cells, and found that Snail1 in Clara cells undergoing repair was important for the proper restoration and function of the airway epithelium after injury (Figure , J–L, and Supplemental Figure 1J). Finally, we crossed Myh11-Cre;Rosa26R-LacZ mice with mice overexpressing sFgfr2b or Dkk1 and found that inhibition of Fgf10 signaling resulted in a lack of labeled epithelial cells (Figure , M–O). We therefore conclude that Fgf10 signaling is necessary for the generation of airway epithelial stem cells through the process of transient EMT.