Generation of floxed α3 integrin/SPC-rtTA/tetO-cre mice (lung epithelial cell–specific loss of α3 integrin).
To explore the in vivo significance of AEC α3 integrin, we used the cre-lox system, in which tissue-specific expression of cre-recombinase results in permanent removal of sequences of DNA flanked by loxP sites (floxed) within those tissues. We generated triple transgenic mice by crossing floxed
mice with mice carrying the human SPC promoter-rtTA
) (Figure A). Hereafter, these mice will be referred to as “f
). Lung epithelial cell–specific recombination was verified by several techniques. PCR primers encompassing the floxed region were designed. DNA from lungs of FASC mice fed doxycycline revealed a 1-kb band consistent with the recombined floxed α3 integrin and a 3.5-kb band corresponding to non-recombined floxed α3 integrin in nonepithelial cells of the lung. Littermate control mice and FASC mice not on doxycycline only demonstrated the 3.5-kb band (Figure B). FASC mice had normal levels of α3 integrin in kidney lysate compared with littermate controls lacking one of the transgenes but an approximately 50% reduction of α3 integrin in whole lung lysate and an approximately 80% reduction of α3 integrin in lysates of isolated AECs (Figure C). Staining of isolated AECs revealed that approximately 60%–90% of FASC AECs lacked expression of α3 integrin (Figure , D and E). Collectively, these data confirm lung epithelial cell–specific loss of α3 integrin in FASC mice.
Lung epithelial cell–specific loss of α3 integrin in FASC mice.
FASC mice have a normal acute lung injury response to bleomycin injury.
In contrast to α3 integrin–null mice, which die perinatally due to renal failure (21
), FASC mice have a normal lifespan and body weight compared with their WT littermates. Alveolar architecture was grossly preserved (Figure , A and B), and there were no differences in total lung capacity and airway resistance (data not shown). Interestingly, FASC mice demonstrated a mild type II AEC hyperplasia demonstrated by increased numbers of pro-surfactant protein C–positive (pro-SPC–positive) cells (Figure , C and D) and increased numbers of Ki67-positive cells (0.89% vs. 0.41%) from immunofluorescence staining of lung sections (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI36940). This approximately 2-fold increase in type II AECs was not progressive but was maintained over the lifespan of the mouse. As expected, an increase in pro-SPC was confirmed by immunoblot of whole lung lysates of FASC mice (Figure G).
Baseline phenotypes of FASC lungs.
Surprisingly, FASC lung sections stained by trichrome revealed increased collagen staining diffusely within the alveolar septa with otherwise preserved architecture compared with littermate control mice (Figure , E and F). The appearance of alveolar wall collagen was not strictly a developmental abnormality because FASC mice started on doxycycline postnatally also displayed increased collagen after several weeks (data not shown). While apparent from tissue staining, the degree of collagen deposition did not result in significant differences in baseline compliance between normal and FASC mice (Figure E) and did not have an impact on lifespan. Because trichrome staining does not distinguish among different collagen subtypes, we analyzed FASC and control mouse lungs for type IV collagen, a normal major component of the alveolar basement membrane, and type I collagen, prominent in pulmonary fibrosis. Indeed, FASC mice demonstrated a clear increase in type IV collagen by immunoblotting and immunostaining consistent with the increased trichrome staining. Levels of type I collagen and laminin 5, the main α3β1 integrin ECM ligand, were similar between FASC and littermate control mice (Figure G).
Preserved acute lung injury response in FASC mice.
FASC mice also exhibited increased BAL cellularity both at baseline and 5 days after bleomycin injury compared with littermate controls that was apparent in lung sections (Figure , A and B) and by BAL cell counts. BAL cell differential was similar between FASC and littermate control mice, both at baseline and after bleomycin injury (data not shown). Importantly, FASC lungs demonstrated normal staining for E-cadherin (Figure , C and D) and no differences in lung permeability at rest or acutely after bleomycin injury as measured by I125-albumin uptake in the lungs, BAL total protein, and excess lung water, all reflecting a normal vascular response to injury and intact epithelial barrier function (Figure ). Despite this normal permeability response, FASC mice trended toward lower compliance acutely after bleomycin injury (P = 0.09), likely due to the exaggerated inflammation (Figure E).
α3 integrin is critical for pulmonary fibrosis.
Bleomycin injury can initiate lung fibrosis in addition to acute lung injury, and recent evidence suggests that these pathways are distinct (22
). To explore the role of α3 integrin in pulmonary fibrosis, 6-week-old FASC mice or littermate controls were injected intratracheally with saline or bleomycin. We first looked at the accumulation of myofibroblasts, which are thought to be important fibrogenic effector cells (24
). As expected, control mice developed robust myofibroblast accumulation 17 days after bleomycin injury, as indicated by staining lung sections for α-SMA. In contrast, FASC mice demonstrated a minimal increase in the number of myofibroblasts compared with saline-treated mice (Figure , A–C). Three weeks after bleomycin injury, the extent of fibrosis was determined by several methods. Lung fibrosis is characterized by the accumulation of fibrillar collagens, such as type I collagen. Immunostaining for collagen I demonstrated thick bands of fibrosis in control mice, while FASC mice were almost completely protected from bleomycin-induced fibrosis (Figure , D and E). Collagen I content, quantified by immunoblot from whole lung lysates, showed no significant increase after bleomycin injury in FASC mice (Figure , F and H). Lung collagen content was further assessed by hydroxyproline content (Figure G). Saline-treated FASC mice demonstrated a 2-fold increase in hydroxyproline compared with saline-treated littermate control mice, consistent with the increased levels of type IV collagen in FASC mice. Completely uninjured FASC mice exhibited a similar increase in hydroxyproline, suggesting that this increase in hydroxyproline was not due to a response to saline injection (data not shown). Again, FASC mice demonstrated a blunted response to bleomycin with no significant increase in hydroxyproline after bleomycin injury compared with a 2-fold increase in injured control mice. Six-month-old FASC and control mice assessed by hydroxyproline had a similar pattern, with saline-treated FASC mice having an approximately 3-fold increase in hydroxyproline compared with control mice at baseline and a blunted increase in hydroxyproline after bleomycin injury (Supplemental Figure 2).
FASC mice have impaired myofibroblast accumulation and type I collagen response to bleomycin injury.
α3 integrin regulates EMT in vivo.
Because loss of α3 integrin in FASC mice is limited to lung epithelial cells, we hypothesized that protection from myofibroblast accumulation and fibrosis could be due to ineffective EMT. EMT has been reported in several models of tissue fibrosis (8
), but has not yet been demonstrated in the bleomycin model of lung fibrosis. To determine whether α3 integrin regulates EMT in vivo, we first assessed whether EMT occurs in this model. We developed triple transgenic reporter mice containing at least 1 copy of the human SPC-rtTA
, and ZEG
(floxed lacZ, EGFP) (27
) transgenes, resulting in genetically tagged lung epithelial cells that permanently express GFP. Mice were given doxycycline throughout gestation, and lung epithelial cell–specific expression of GFP was confirmed by several techniques. Frozen lung sections stained for GFP revealed robust GFP expression within lung epithelial cells, predominantly pro-SPC–positive AECs, while mesenchymal structures within the lung (smooth muscle layers within vessels and airways) were completely GFP negative (Supplemental Figure 3). Several extrapulmonary organs were examined by immunostaining and immunoblot and revealed no expression of GFP. Primary AECs isolated from triple transgenic mice and littermate control mice were analyzed by flow cytometry, demonstrating that approximately 10%–40% of isolated type II AEC were GFP positive. Thus, GFP expression is specific for lung epithelial cells, but with a low recombination efficiency in ZEG/SPC-rtTA/tetO-CMV-Cre
mice. Littermate control mice lacking either the SPC-rtTA
or the tetO-CMV-Cre
transgenes revealed no expression of GFP by these techniques (data not shown).
EMT during fibrogenesis was observed in the bleomycin model using these reporter mice by several methods. Triple transgenic mice were treated with bleomycin or saline, then analyzed after 17 days. Whole lung, single-cell suspensions were prepared and sorted for GFP-positive cells. As expected, cells from littermate control mice lacking any one of the transgenes did not express GFP even after bleomycin injury (Figure , B and C). Epithelial cell–derived, GFP-positive cells from triple transgenic reporter mice given saline or bleomycin were analyzed by immunoblot (Figure D) and immunostain for mesenchymal markers (Figure , E–G). Immunoblot revealed marked de novo expression of α-SMA and loss of pro-SPC within GFP-positive cells from bleomycin-injured mice compared with saline-treated mice. By immunostain, GFP-positive cells from saline-treated mice revealed no staining for mesenchymal markers, while in the bleomycin-injured mice, a surprisingly high percentage of epithelium-derived cells expressed classic mesenchymal markers (α-SMA, vimentin, and procollagen I). Finally, lung sections from bleomycin-injured and saline-treated mice were stained for GFP and α-SMA. Again, numerous cells costaining for GFP and α-SMA were identified in bleomycin-injured mice, but none in saline-treated mice (Figure , A and B). These EMT-derived cells were found within the interstitium and were not observed on the surface of the epithelial lumen.
EMT develops in vivo following intratracheal injection of bleomycin.
Lung epithelial cell α3 integrin regulates EMT in vivo.
To study the function of AEC α3 integrin in fibrosis, we established quadruple transgenic mice. Saline or bleomycin was intratracheally injected into ZEG/SPC-rtTA/tetO-Cre/α3fl/fl (ZEG-FASC) and littermate ZEG/SPC-rtTA/tetO-Cre/α3fl/WT or ZEG/SPC-rtTA/tetO-Cre/α3WT/WT (ZEG-control), and the EMT response was determined after 17 days by immunostaining lung sections (Figure ). In saline-treated mice, GFP expression was confined to lung epithelial cells (data not shown). In ZEG-control mice injured with bleomycin (Figure , A and B), numerous GFP-positive cells also expressed α-SMA (7.0%; Figure C), indicating epithelial cell–derived myofibroblasts. In contrast, ZEG-FASC mice injured with bleomycin (data not shown) had much less EMT (1.4%; Figure C).
Overall, these findings indicate that lung epithelial cell α3β1 integrin is not required for normal lung epithelial cell barrier function but is required for patterned responses of the lung to at least one well-defined injurious stimulus, bleomycin. Although it is unlikely that all myofibroblasts are derived from epithelial cells, the data indicate that myofibroblast development occurs in an epithelial α3 integrin–dependent manner and that EMT-derived myofibroblasts clearly appear following bleomycin injury, accounting for a significant fraction of these myofibroblasts. Ineffective myofibroblast development in the absence of α3 integrin likely accounts for the greatly attenuated fibrotic response. We next considered a mechanism by which α3 integrin could regulate EMT and fibrogenesis.
α3 integrin is required for tyrosine phosphorylation of β-catenin and β-catenin/pSmad2 complex formation.
We have previously shown that primary AECs cultured on provisional matrix proteins, such as Fn, undergo EMT via activation of endogenous TGF-β1 (8
). Primary AECs from control and FASC mice were analyzed immediately after isolation and 4 days after culturing cells on Fn-coated plates. As expected, control AECs demonstrated a mesenchymal morphology (Figure A and Supplemental Figure 4A), upregulated classic mesenchymal markers α-SMA, collagen I, and vimentin, and completely lost expression of pro-SPC (Figure C). FASC AECs maintained an epithelial morphology (Figure B and Supplemental Figure 4B) and had a dramatically limited EMT with weak upregulation of several mesenchymal markers even though pro-SPC expression was largely lost over time in culture.
α3 integrin regulates association between β-catenin and pSmad2 ex vivo.
FASC and control AECs had similar levels of Smad2 phosphorylation at day 4 after plating on Fn (Figure C) and at all other time points examined from day 2 to day 7 (data not shown), indicating that α3β1 integrin does not affect endogenous TGF-β1 production, activation, TGF-β1 receptor binding, or Smad2 phosphorylation. Levels of inhibitory Smad7 were also similar between FASC and control AECs (Supplemental Figure 5), indicating that α3 integrin regulation of EMT is distinct from a recently described mechanism in which α3 integrin regulates levels of Smad7 and pSmad2 in keratinocytes (28
We recently observed that an immortalized α3 integrin–null kidney epithelial cell line also had defective responses to TGF-β1 compared with WT cells (29
). Levels of pSmad2 following TGF-β1 stimulation were similar between a3 integrin–null and WT kidney epithelial cells. Smad7 levels were also independent of the presence or absence of α3 integrin in these kidney epithelial cells. The α3β1 integrin was also found to physically associate with TGF-β1 receptors as well as E-cadherin in a tripartite complex and to be required for TGF-β1–induced phosphorylation of β-catenin at Y654. Phosphorylation of Y654–β-catenin has previously been reported to be important for both its release from E-cadherin and stabilization from proteosomal turnover (30
). We found that pY654–β-catenin formed a complex with pSmad2 shortly after TGF-β1 stimulation and was required for development of EMT in this kidney epithelial cell line (29
). We therefore asked whether α3 integrin has a similar role in primary AECs and, if so, whether this mechanism operates in vivo.
As expected, immunoprecipitation of E-cadherin robustly coprecipitated α3 integrin in WT primary AECs (data not shown). To confirm that α3 integrin associates with TGF-β1 receptors, we used lentiviral-mediated transduction to express a myc-tagged TGF-β receptor I (mycRI). Lentiviral infections led to equal expression of mycRI in control and FASC AECs. Immunoprecipitation of mycRI led to coprecipitation of both α3 integrin and E-cadherin, consistent with the formation of a tripartite receptor complex (Figure ). Interestingly, in FASC AECs, E-cadherin still associated with mycRI, indicating that the integrin is not required for this interaction. Specificity of these immunoprecipitations was confirmed by lack of precipitation of these proteins in cells infected with a control lentivirus expressing only GFP. These findings confirm the physical associations of α3 integrin, E-cadherin, and the TGF-β1 receptor in primary AECs and raise the possibility that α3 integrin in these cells could also regulate assembly of β-catenin/pSmad2 complexes, as we recently observed in a kidney cell line.
Primary AECs from FASC and littermate control mice were isolated, plated onto Fn for 48 hours to allow for activation of endogenous TGF-β1, and further stimulated with exogenous TGF-β1 for 1 hour. Control AECs demonstrated formation of β-catenin/pSmad2 complexes by coimmunoprecipitation, while FASC AECs lacking α3 integrin failed to form this complex. Further, only WT AECs developed Y654 phosphorylation of β-catenin in response to TGF-β1 (Figure ). Indeed, coimmunoprecipitation between pY654–β-catenin and pSmad2 was as robust as the total β-catenin/pSmad2 coimmunoprecipitation, indicating that pY654–β-catenin is likely the principal form of β-catenin in complex with pSmad2. Thus the α3 integrin is critical to formation of pY654–β-catenin/pSmad2 complexes in AECs. Finally, primary WT AECs cultured on Fn for 4 days demonstrated nuclear accumulation of pY654–β-catenin by immunostaining (Figure F). Many pY654–β-catenin–positive cells had also formed α-SMA stress fibers consistent with EMT.
pY654–β-catenin/pSmad2 complexes in mouse and human lung tissues.
To extend this analysis in vivo, we performed coimmunoprecipitation on fresh whole lung lysates from FASC mice and littermate controls treated with saline and bleomycin (Figure A). WT mice demonstrated increased levels of pSmad2 2 weeks after bleomycin injury compared with saline-treated WT mice, consistent with activation of TGF-β1 signaling during fibrogenesis. FASC mice treated with bleomycin also demonstrated levels of pSmad2 similar to WT mice treated with bleomycin. Of note, saline-treated (and completely uninjured) FASC mice demonstrated relatively high levels of baseline pSmad2. However, despite similar levels of pSmad2 after bleomycin injury, immunoprecipitation of β-catenin only coprecipitated pSmad2 in WT whole lung lysates but not in FASC whole lung lysates, confirming an α3 integrin–dependent β-catenin/pSmad2 complex formation in vivo during fibrogenesis. Fresh frozen lungs from FASC and littermate control mice 17 days after bleomycin injury were sectioned and immunostained for pY654–β-catenin and α-SMA (Figure , B and C). Nuclear pY654–β-catenin staining was seen within and around clusters of myofibroblasts in α3+/+ control mice (mice that have normal levels of α3 integrin) injured with bleomycin but not in FASC mice. Although α3 integrin is deficient only in lung epithelial cells in the FASC mouse, this selective deficiency virtually completely abrogates formation of β-catenin/pSmad2 complexes in the whole lung in this model, implying that these transcriptional complexes are largely initiated in epithelial cells in response to TGF-β1.
β-catenin and pSmad2 coimmunoprecipitation in murine lungs following bleomycin injury.
To assess whether β-catenin/pSmad2 complexes accumulate in IPF, we used flash-frozen lung tissue obtained by biopsy at the time of diagnosis from 5 patients with IPF and compared them with flash-frozen normal human lung tissues and lung tissues from 6 patients with emphysema, a common lung disease associated with subepithelial fibrosis. Tissues were lysed and analyzed by immunoblot and immunoprecipitation. In general, levels of pSmad2 were consistently higher in IPF (samples F1–F5) than normal lung lysates (samples N1–N4) (Figure A). One of the nonfibrotic lung samples (N1) demonstrated high levels of pSmad2 by immunoblot. Levels of β-catenin were comparable among the samples. Immunoprecipitation for β-catenin led to robust coprecipitation of pSmad2 in all IPF samples but in none of the normal lung samples. Interestingly, the 1 nonfibrotic lung sample (N1) that demonstrated elevated levels of pSmad2 within the lung lysate did not show β-catenin/pSmad2 coimmunoprecipitation, suggesting that this association is specific to fibrotic lung disease. Furthermore, immunoprecipitation for pY654–β-catenin demonstrated β-catenin tyrosine phosphorylation only in IPF lung tissues and not in normal lung (Figure A) or in emphysema lung tissue (Supplemental Figure 6). Accordingly, pSmad2 coimmunoprecipitated with pY654–β-catenin in IPF samples but not in either normal or emphysema lung samples. Thus, tyrosine phosphorylation of β-catenin at Y654 and its association with pSmad2 are prominent in IPF, but seemingly absent in normal or emphysematous lungs.
pY654–β-catenin/pSmad2 complexes in IPF lungs.
Finally, we explored where in IPF lungs the pY654–β-catenin accumulated. Flash-frozen normal or IPF lung tissues were also immunostained for pY654–β-catenin and α-SMA. Again, distinct nuclear pY654–β-catenin staining was observed within a significant fraction of nuclei of IPF but not normal lungs. Most of the staining was strikingly localized to subepithelial myofibroblasts, and most such myofibroblasts displayed nuclear pY654–β-catenin accumulation (Figure , B and C). In addition, a small number of AECs in IPF lung showed nuclear accumulation of pY654–β-catenin.