? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
Epithelial cell α3β1 integrin links β-catenin
and Smad signaling to promote myofibroblast
formation and pulmonary fibrosis
Kevin K. Kim,1 Ying Wei,1 Charles Szekeres,2 Matthias C. Kugler,1 Paul J. Wolters,1
Marla L. Hill,1 James A. Frank,3 Alexis N. Brumwell,1 Sarah E. Wheeler,1
Jordan A. Kreidberg,2 and Harold A. Chapman1
1Pulmonary and Critical Care Division, Department of Medicine, and Cardiovascular Research Institute, UCSF, San Francisco, California, USA.
2Children’s Hospital and Harvard Medical School, Boston, Massachusetts, USA. 3Pulmonary and Critical Care Division,
Department of Medicine, and San Francisco VA Medical Center, San Francisco, California, USA.
Progressive fibrosis of the lung, especially that of idiopathic pul-
monary fibrosis (IPF), is thought to be a consequence of aberrant
wound healing resulting in progressive scarification (1, 2). Fibro-
blast expansion and activation leading to collagen fibril deposi-
tion is considered to be a final common pathway, and hence much
attention has been directed at mechanisms that lead to fibroblast
proliferation and synthesis of matrix proteins. In addition to
expansion/activation of resident fibroblasts, fibrogenic fibroblasts
may also originate from lung epithelial cells through epithelial-
mesenchymal transition (EMT) (3–6). In this scenario, injurious
stimuli lead to activation of certain key mediators, which cause
some epithelial cells to become reprogrammed as fibroblasts. While
EMT in the lung is not mutually exclusive of endogenous fibroblast
activation, these processes are likely regulated very differently.
EMT is a process involving loss of apical-basal polarity, loss
of cell-cell contacts, detachment from the basement membrane,
cytoskeletal rearrangement, and migration into the provisional
matrix. These vast phenotypic changes are accompanied by sig-
nificant changes in molecular expression within the cell, requir-
ing extensive coordination (7). We recently reported that alveolar
epithelial cells (AECs) undergo EMT in vivo in an animal model
of pulmonary fibrosis by using a murine system in which AECs
are genetically tagged to specifically and permanently express a
reporter protein (8). We further demonstrated that AECs undergo
EMT ex vivo via activation of endogenous TGF-β1.
TGF-β1 signaling has been implicated in many models of EMT
and fibrosis (9–11). TGF-β1 signaling can be regulated at mul-
tiple levels from production and activation of TGF-β1 to for-
mation of a Smad transcriptional complex with various Smad
co-regulators (12, 13). Importantly, TGF-β1 signaling is not an
on/off response, rather cells can respond in a variety of differ-
ent ways to active TGF-β1 depending on convergence with other
signaling pathways. In primary AECs the ECM is an important
determinant to the cellular response to TGF-β1, with provisional
matrix proteins such as fibronectin (Fn) driving EMT and base-
ment membrane proteins such as laminin and type IV collagen
preventing EMT (8). The mechanisms by which the ECM regu-
lates EMT and fibrosis remain to be elucidated but likely involve
signaling through integrin receptors.
Integrins are transmembrane adhesion molecules which bind to
specific ECM ligands. Integrin activation can initiate intracellular
signaling or influence signaling through other receptors (14, 15).
For example, β1 integrins have been shown to regulate signaling
through transmembrane growth factor receptors such as the epi-
dermal growth factor receptor and the platelet-derived growth
factor receptor (16, 17). One report, using a β1 integrin blocking
antibody, demonstrated that β1 integrins are critical for TGF-β1–
mediated transcription and epithelial cell plasticity in vitro (18).
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: AEC, alveolar epithelial cell; EMT, epithelial-
mesenchymal transition; FASC, floxed α3 integrin/SPC-rtTA/tetO-cre (mouse); Fn,
fibronectin; IPF, idiopathic pulmonary fibrosis; mycRI, myc-tagged TGF-β receptor I;
pro-SPC, pro-surfactant protein C.
Citation?for?this?article: J. Clin. Invest. 119:213–224 (2009). doi:10.1172/JCI36940.
Related Commentary, page 7
214? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
α3β1 integrin is a laminin receptor that also localizes to sites of
cell-cell contacts through its interaction with the E-cadherin/
β-catenin complex (19). Signaling through β-catenin has been
implicated in both EMT and pulmonary fibrosis (20), though this
is largely thought to involve the Wnt pathway of stabilization of
β-catenin. Thus, an intriguing hypothesis is that α3β1 integrin is a
key regulator of AEC EMT through its ability to coordinate pheno-
typic changes involving cell-cell and cell-matrix interactions with
To explore the function of α3 integrin in EMT and pulmo-
nary fibrosis, we used triple transgenic mice with lung epithelial
cell–specific loss of α3 integrin. These mice had a normal acute
response to bleomycin injury but failed to progress to fibrosis and
had markedly impaired EMT. These studies also revealed α3β1
integrin as a critical coordinator of prominent EMT signaling
pathways involving β-catenin and pSmad2.
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-spe-
cific 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 α3
integrin mice with mice carrying the human SPC promoter-rtTA and
tetO-CMV-Cre transgenes (8) (Figure 1A). Hereafter, these mice will
be referred to as “floxed α3 integrin/SPC-rtTA/tetO-cre” (FASC).
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
1B). 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 1C). Staining of isolated AECs revealed
that approximately 60%–90% of FASC AECs lacked expression of
α3 integrin (Figure 1, D and E). Collectively, these data confirm
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 archi-
tecture was grossly preserved (Figure 2, 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 2, 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 2G).
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 2, E and F). The appearance of alveolar wall collagen
Lung epithelial cell–specific loss of α3 integrin in
FASC mice. (A) In triple transgenic FASC mice, rtTA is
expressed in lung epithelial cells using the human SPC
promoter. In the presence of doxycycline (dox), rtTA is
an active transcriptional factor leading to expression of
Cre recombinase and removal of the floxed exon 3 of
the α3 integrin gene, resulting in lung epithelial cell–
specific loss of α3 integrin. (B) PCR using primers that
encompass the floxed region of the floxed α3 integrin
gene. 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 non-epithelial
cells of the lung. Littermate control mice and FASC
mice not on doxycycline only demonstrated the 3.5-kb
band. Murine embryonic fibroblasts (MEFs) derived
from a floxed α3 integrin (α3cko) mouse and treated
in vitro with adenovirus expressing Cre (AdCre) was
used as a positive control and only exhibited the 1-kb
band. (C) Immunoblot demonstrating normal levels of
α3 integrin in kidney lysate of a FASC mouse compared
with littermate controls (Ctrl) lacking 1 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. (D and E)
Immunostaining of isolated AECs demonstrates that
about 60%–90% of FASC AECs (E) lack expression of
α3 integrin compared with littermate control AECs (D).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
was not strictly a developmental abnormality because FASC mice
started on doxycycline postnatally also displayed increased col-
lagen 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 3E) 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 base-
ment 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 lam-
inin 5, the main α3β1 integrin ECM ligand, were similar between
FASC and littermate control mice (Figure 2G).
FASC mice also exhibited increased BAL cellularity both at base-
line and 5 days after bleomycin injury compared with littermate
controls that was apparent in lung sections (Figure 3, 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 2, C and D) and no dif-
ferences 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 3).
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 3E).
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, 23). 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 myo-
fibroblasts compared with saline-treated mice (Figure 4, 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 colla-
gen. Immunostaining for collagen I demonstrated thick bands of
fibrosis in control mice, while FASC mice were almost completely
protected from bleomycin-induced fibrosis (Figure 4, D and E).
Collagen I content, quantified by immunoblot from whole lung
lysates, showed no significant increase after bleomycin injury in
FASC mice (Figure 4, F and H). Lung collagen content was further
assessed by hydroxyproline content (Figure 4G). Saline-treated
FASC mice demonstrated a 2-fold increase in hydroxyproline
compared with saline-treated littermate control mice, consis-
tent 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 bleomy-
cin 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).
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 mod-
els of tissue fibrosis (8, 25, 26), but has not yet been demonstrated
Baseline phenotypes of FASC lungs. Littermate con-
trol (A, C, and E) and FASC (B, D, and F) lung sec-
tions. (A and B) Lung sections (original magnification,
×20) stained by H&E demonstrated similar alveolar
architecture. (C and D) Lung sections (original mag-
nification, ×60) immunostained for E-cadherin (E-cad,
green) and pro-SPC (red) demonstrated a similar
E-cadherin staining pattern and increased numbers of
pro-SPC–positive cells in FASC lungs. (E and F) Lung
sections (original magnification, ×60) stained with tri-
chrome demonstrated increased diffuse staining within
the alveolar basement membranes of FASC mice. (G)
Immunoblot showed decreased expression of α3 integ-
rin, increased expression of pro-SPC, a clear increase
in collagen IV (col IV), similar levels of laminin 5, and a
slight increase in collagen I (col I) in FASC lung lysate
compared with littermate control lung lysate.
216? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
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, tetO-CMV-Cre,
and ZEG (floxed lacZ, EGFP) (27) transgenes, resulting in geneti-
cally tagged lung epithelial cells that permanently express GFP.
Mice were given doxycycline throughout gestation, and lung epi-
thelial 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 lack-
ing 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 5, B and C). Epithelial
cell–derived, GFP-positive cells from triple transgenic reporter
mice given saline or bleomycin were analyzed by immunoblot
(Figure 5D) and immunostain for mesenchymal markers (Figure 5,
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 procolla-
gen 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 6, A and B).
These EMT-derived cells were found within the interstitium and
were not observed on the surface of the epithelial lumen.
To study the function of AEC α3 integrin in fibrosis, we estab-
lished quadruple transgenic mice. Saline or bleomycin was intra-
tracheally 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 deter-
Preserved acute lung injury response in FASC mice. (A and B) Five days after intratracheal bleomycin injury, littermate control (A) and FASC
(B) lung sections (original magnification, ×20) were stained with H&E and demonstrated increased inflammation in FASC mice. (C) Cell counts
from BAL of littermate control and FASC mice 5 days after intratracheal saline or bleomycin injury. FASC mice had an increased number of cells
compared with littermate controls (n = 4–6 per group). (D) Lung permeability determined by extravasation of intravascular 125I-albumin into the
lungs and expressed as EVP%. FASC and littermate control mice demonstrated similar permeability 5 days after bleomycin injury (n = 4 per
group). (E) Lung compliance (μl/cm H2O) was determined from anesthetized and paralyzed ventilated mice. FASC and littermate control mice
demonstrated a decrease in compliance 5 days after bleomycin injury. There was a trend toward less compliance in FASC mice after bleomycin
injury compared with littermate control mice after bleomycin injury (P = 0.09; n = 4 per group). (F) Total protein concentration (mg/ml) from BAL
5 days after intratracheal saline or bleomycin injury. FASC and littermate control mice demonstrated a similar increase in BAL protein after
bleomycin injury (n = 4–6 per group). (G) Excess lung water (determined as described in Methods) increased similarly in FASC and littermate
control mice 5 days after bleomycin injury (n = 4 per group).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
mined after 17 days by immunostaining lung sections (Figure 6). In
saline-treated mice, GFP expression was confined to lung epithelial
cells (data not shown). In ZEG-control mice injured with bleomy-
cin (Figure 6, A and B), numerous GFP-positive cells also expressed
α-SMA (7.0%; Figure 6C), indicating epithelial cell–derived myo-
fibroblasts. In contrast, ZEG-FASC mice injured with bleomycin
(data not shown) had much less EMT (1.4%; Figure 6C).
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. Inef-
fective 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 morphol-
ogy (Figure 7A and Supplemental Figure 4A), upregulated clas-
sic mesenchymal markers α-SMA, collagen I, and vimentin, and
completely lost expression of pro-SPC (Figure 7C). FASC AECs
maintained an epithelial morphology (Figure 7B and Supple-
mental 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.
FASC and control AECs had similar levels of Smad2 phosphory-
lation at day 4 after plating on Fn (Figure 7C) 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 produc-
tion, activation, TGF-β1 receptor binding, or Smad2 phosphoryla-
tion. 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 pres-
ence 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
FASC mice have impaired myofibroblast accumulation and
type I collagen response to bleomycin injury. (A and B)
Lung sections (original magnification, ×20) from littermate
control (A; Ctrl Bleo) and FASC (B; FASC Bleo) mice 17
days after intratracheal bleomycin injury immunostained
for α-SMA. (C) Quantification of α-SMA–positive myo-
fibroblasts revealed that FASC mice developed fewer
α-SMA–positive myofibroblasts compared with controls. (D
and E) Lung sections (original magnification, ×20) from lit-
termate control mice lacking at least 1 of the 3 transgenes
(D) and FASC mice (E) were stained for type I collagen 21
days after intratracheal injection with bleomycin. (F) Type I
collagen content from whole lung lysate from littermate
control and FASC mice 21 days after intratracheal injec-
tion with saline was analyzed by immunoblot. (G) Hydroxy-
proline assay from entire left lung of FASC or littermate
control mice 21 days after intratracheal injection of saline
or bleomycin. Uninjured FASC mice had increased levels
of hydroxyproline and a blunted increase in hydroxyproline
after bleomycin injury (n = 7–16 per group). (H) Relative
densitometry of immunoblots for type I collagen from lung
lysate of FASC or littermate control mice 21 days after
intratracheal saline or bleomycin injection. FASC mice had
significantly less type I collagen after bleomycin injury com-
pared with control mice (n = 3 per group).
218? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
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 copre-
cipitated 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, consis-
tent with the formation of a tripartite receptor complex (Figure 7).
Interestingly, in FASC AECs, E-cadherin still associated with
mycRI, indicating that the integrin is not required for this inter-
action. 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 iso-
lated, plated onto Fn for 48 hours to allow for activation of endog-
enous 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 7). 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 7F). 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 coimmunoprecipita-
tion on fresh whole lung lysates from FASC mice and littermate
controls treated with saline and bleomycin (Figure 8A). WT mice
demonstrated increased levels of pSmad2 2 weeks after bleomy-
cin 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 sim-
EMT develops in vivo following intratracheal injection of bleomycin. (A) In triple transgenic mice, rtTA is expressed in lung epithelial cells using
the human SPC promoter. In the presence of doxycycline (dox), rtTA is an active transcriptional factor leading to expression of Cre recom-
binase and the removal of the floxed portion of the ZEG allele, resulting in lung epithelial cell–specific expression of GFP. (B and C) Density
plots obtained during cell sorting for GFP-positive cells from whole lung single-cell suspensions prepared from littermate control (B) and triple
transgenic (C; ZEG/SPC-rtTA/Cre) mice 17 days after intratracheal bleomycin injury. Percentages of GFP-positive cells are indicated. (D)
Seventeen days after intratracheal saline or bleomycin injection, GFP-positive cells were sorted from whole lung single-cell suspensions of
ZEG/SPC-rtTA/tetO-Cre mice. Immunoblot demonstrates de novo expression of α-SMA and downregulation of pro-SPC in epithelium-derived
cells of bleomycin-injured mice. (E–G) Seventeen days after intratracheal saline or bleomycin injury, GFP-positive cells were sorted as described
above and immunostained for GFP and mesenchymal markers vimentin (E), α-SMA (F), and procollagen I (G) and demonstrated expression of
mesenchymal markers in epithelium-derived cells in bleomycin-injured mice, but none in saline-treated mice. The percentages of GFP-positive
cells staining for mesenchymal markers are indicated. Original magnification, ×60.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
ilar 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–depen-
dent 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 (Fig-
ure 8, 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 bleomy-
cin 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.
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 associ-
ated 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 nor-
mal lung lysates (samples N1–N4) (Figure 9A). One of the nonfi-
brotic lung samples (N1) demonstrated high levels of pSmad2 by
Lung epithelial cell α3 integrin regulates EMT in vivo. (A and B) Lung
sections (original magnification, ×60) from a triple transgenic ZEG/SPC-
rtTA/tetO-Cre/α3WT/WT mouse 17 days after intratracheal bleomycin injury,
immunostained for GFP (A) and α-SMA (B). Multiple cells costaining
GFP and α-SMA are indicated by arrows. (C) Percentage of GFP-positive
cells co-expressing α-SMA in ZEG-FASC (ZEG/α3fl/fl/SPC-rtTA/tetO-Cre)
and littermate control (ZEG/α3fl/WT or α3WT/WT/SPC-rtTA/tetO-Cre) mice
treated with saline or bleomycin injection. Control mice demonstrated a
marked increase in cells co-expressing α-SMA and GFP after bleomycin
injury compared with FASC mice (n = 4 per group).
α3 integrin regulates association between β-catenin and
pSmad2 ex vivo. (A and B) Primary AECs from FASC (B) and
littermate control (A) mice were cultured on Fn for 4 days, then
stained for F-actin with phalloidin and counterstained with DAPI
(original magnification, ×60). Control cells demonstrated actin
stress fibers consistent with a mesenchymal morphology, while
FASC AECs demonstrated cortical actin staining consistent with
an epithelial morphology. (C) Primary AECs from FASC or lit-
termate control mice lacking 1 of the 3 transgenes were ana-
lyzed by immunoblot immediately after isolation (day 0) and 4
days after culturing on Fn-coated surfaces (day 4). FASC AECs
had a blunted expression of mesenchymal markers collagen I,
α-SMA, and vimentin. (D) Primary AECs were infected with len-
tivirus expressing mycRI or GFP as a control. Immunoprecipita-
tion of mycRI demonstrated coprecipitation of α3 integrin and
E-cadherin. (E) Coimmunoprecipitation of β-catenin (β-cat) and
pSmad2 was seen with AECs from α3+/+ (control) but not AECs
from FASC mice plated on Fn for 4 days to allow activation of
TGF-β1 (top blot). TGF-β1–dependent tyrosine phosphorylation
of β-catenin at Y654 and pY654–β-catenin/pSmad2 coprecipita-
tion was only seen with AECs from α3+/+ mice (bottom blot). (F)
Primary AECs cultured on Fn for 4 days then stained for α-SMA
and pY654–β-catenin showed nuclear accumulation of β-catenin
(original magnification, ×40).
220? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
immunoblot. Levels of β-catenin were comparable among the sam-
ples. Immunoprecipitation for β-catenin led to robust coprecipita-
tion 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. Further-
more, immunoprecipitation for pY654–β-catenin demonstrated
β-catenin tyrosine phosphorylation only in IPF lung tissues and
not in normal lung (Figure 9A) or in emphysema lung tissue (Sup-
plemental 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.
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 signif-
icant fraction of nuclei of IPF but not normal lungs. Most of
the staining was strikingly localized to subepithelial myofibro-
blasts, and most such myofibroblasts displayed nuclear pY654–
β-catenin accumulation (Figure 9, B and C). In addition, a small
number of AECs in IPF lung showed nuclear accumulation of
The findings reported here add insight into the pathobiology of
tissue fibrosis and demonstrate what we believe is a novel role for
α3β1 integrin. EMT has been demonstrated in vivo in several ani-
mal models of fibrosis (8, 25, 26), but the contribution of this tran-
sition for the fibrotic outcome has remained uncertain. In part,
this is because secreted factors that induce or inhibit EMT are also
important regulators of fibroblast function. Generation of mice
with lung epithelial cell–specific loss of α3 integrin indicates that
this laminin receptor has no essential function in maintaining
normal airway barrier integrity. Instead, α3 integrin is revealed to
be a critical regulator of EMT and tissue remodeling in response
to injury. In a well-described model of lung injury and fibrosis,
lung injury and inflammation were intact and possibly exagger-
ated in α3 integrin–deficient mice, but the degree of in vivo EMT
and fibrosis were dramatically attenuated. To our knowledge, this
is the first report of attenuating fibrosis by epithelial cell–specific
deletion of a protein involved in regulating EMT. Thus, these data
provide the strongest evidence yet that EMT not only occurs dur-
β-catenin and pSmad2 coimmunoprecipitation in murine lungs fol-
lowing bleomycin injury. (A) Two weeks after intratracheal bleomy-
cin or saline injection, FASC and littermate control lungs were lysed
and analyzed by immunoblot and immunoprecipitation for β-catenin.
Control mice injured with bleomycin demonstrated β-catenin/pSmad2
coimmunoprecipitation. (B and C) FASC (C) and littermate control (B)
fresh frozen lung section (original magnification, ×60) 17 days after
bleomycin injury immunostained for α-SMA (green) and pY–β-catenin
(red). Numerous nuclei stained for pY–β-catenin within and around
myofibroblast clusters in littermate control but not FASC mice.
pY654–β-catenin/pSmad2 complexes in IPF lungs. (A) Normal human
lung samples (N1–N4) and IPF lung samples (F1–F5) were lysed and
analyzed by immunoblot and immunoprecipitation for β-catenin or
pY654–β-catenin. All IPF samples demonstrated increased pSmad2
and pY–β-catenin. β-catenin and pY–β-catenin coimmunoprecipitated
with pSmad2 in IPF samples, but not normal lung samples. (B and C)
Fresh frozen normal (B) and IPF (C) lung sections (original magnifi-
cation, ×20) were stained for pY–β-catenin (red) and α-SMA (green).
Numerous nuclei stained for pY–β-catenin in IPF lung but not in normal
lung. Myofibroblasts in IPF lung were frequently pY–β-catenin positive.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
ing injury-induced fibrogenesis but is important to the outcome.
In addition, findings reported here implicate a surprising mech-
anism by which the integrin regulates EMT. Rather than classic
matrix-dependent signaling through integrin clustering, we pos-
tulate that the capacity of α3β1 integrin to engage laminin and
E-cadherin may mainly be a sensing function that regulates epi-
thelial responses to TGF-β1. In this scenario, engagement of the
α3 integrin on the laminin-rich basement membrane or in cell-cell
contact suppresses its role in promoting TGF-β1–induced EMT.
This model clarifies our previous observations that AECs cul-
tured on laminin/collagen IV form tight cell-cell borders and are
resistant to EMT even when stimulated with exogenous TGF-β1
(8). Cells cultured on Fn, which is not an α3β1 integrin ligand,
undergo TGF-β1–mediated EMT. In concurrent parallel studies
of an α3 integrin–deficient kidney cell line reconstituted with WT
α3 integrin or an α3 integrin mutant unable to bind laminin-5,
we observed engagement of laminin-5 to suppress TGF-β1–depen-
dent Y654–β-catenin phosphorylation, β-catenin/pSmad2 com-
plexes, and mesenchymal gene expression. In this context, the pro-
tection of FASC mice from EMT may have resulted in part from
the increased type IV collagen observed in the lungs of these mice
(Figure 2). Type IV collagen has been reported to be a ligand for
α3β1 integrin (31), and kidney epithelial cells cultured on type IV
collagen are reportedly resistant to EMT (32). Further studies into
the mechanism of type IV collagen accumulation in FASC mice
and the role of collagen IV in EMT and lung fibrosis are ongoing.
Synergistic interactions on gene transcription between effectors
of the TGF-β1 and Wnt signaling pathways, Smads and β-catenin
respectively, have been previously reported (33). Although Smads
and β-catenin (34) are each well-described transcriptional effec-
tors of EMT, coordination between these pathways during EMT
has not been defined. α3β1 integrin is identified here as a criti-
cal nidus at which these prominent EMT pathways converge. But
rather than acting through Wnt-dependent inhibition of serine
phosphorylation of β-catenin, in the presence of active TGF-β1,
α3β1 integrin empowers tyrosine phosphorylation of β-catenin
at Y654 and formation of pY–β-catenin/pSmad2 transcriptional
complexes, promoting EMT. Several lines of evidence favor the
conclusion that formation of these complexes is important to
fibrogenesis. (a) pY654–β-catenin/pSmad2 complexes only form
under conditions supporting EMT and fibrosis. Clearly, pSmad2
generation per se is not sufficient for either EMT or lung fibrosis.
Indeed, recent studies of conditionally deleted Smad2 in liver and
skin imply that Smad2 alone may suppress EMT (35, 36). Thus,
the function of pY654–β-catenin is unlikely to be to simply aug-
ment pSmad2 signaling, but rather to redirect or possibly even
to inhibit pSmad2 signaling. Epithelial-specific deficiency of α3
integrin led to loss of pY654–β-catenin phosphorylation both ex
vivo in primary AECs and in vivo in the bleomycin-injured whole
lung. Loss of pY654–β-catenin mirrored attenuation of EMT and
fibrosis. (b) In kidney epithelial cells exposed to active TGF-β1,
stable expression of a dominant-negative β-catenin blocked
α-SMA responses even though no evidence of β-catenin/Lef1 sig-
naling was detectable, consistent with prior studies indicating
that TGF-β1 does not directly activate catenin/Lef1 signaling (37).
(c) Immunostaining of lung tissues obtained from IPF patients
revealed that nuclear pY654–β-catenin was largely confined to
α-SMA–positive fibroblasts and a subset of AECs. No staining
was observed in normal or emphysematous lungs (Figure 9 and
Supplemental Figure 6). Interestingly, the pattern of nuclear
accumulation of pY654–β-catenin was different in injured murine
lungs (Figure 8). A smaller fraction of myofibroblasts exhibited
nuclear staining, possibly consistent with the less well-developed
myofibroblast phenotype and limited nature of the injury in the
bleomycin model as compared with IPF. Although it remains to be
defined exactly how pY654–β-catenin acts along with pSmad2 to
promote myofibroblast formation, and whether persistent nucle-
ar accumulation further drives the process, we speculate that the
appearance of nuclear pY654–β-catenin may be a novel diagnostic
biomarker of in vivo EMT during tissue remodeling.
Although myofibroblast accumulation and fibrosis in injured
murine lungs closely parallels EMT, we cannot exclude the pos-
sibility that epithelial cells contribute to pulmonary fibrosis by
additional α3 integrin–dependent mechanisms. For example, the
2-fold increase in type II AECs and alveolar macrophage numbers
(Figures 3 and 4) could reflect additional unmeasured alterations
in α3 integrin–null AEC function that act concurrently to attenu-
ate fibrogenesis. Indeed it is unlikely that most or all myofibro-
blasts arise from EMT during lung tissue remodeling, though
myofibroblast accumulation is markedly diminished with only
epithelial cell loss of α3 integrin. In the presence of α3 integrin–
deficient AECs, fibroblasts (or myofibroblasts) potentially origi-
nating from other cell types were not able to compensate during
bleomycin-induced fibrosis. It is also possible that epithelial cells
reprogrammed in the lung during EMT may be important in initi-
ating fibrogenesis by driving resident fibroblast proliferation and
activation. If so, the contribution of EMT to fibrogenesis may be
greater than a simple reflection of the fraction of fibroblasts that
are EMT-derived. Fibroblasts from different origins may harbor
unique features rather than blending into a homogeneous pool of
cells. In support of this idea is a recent report indicating fibroblast
strains derived from IPF patients and maintained in culture had
distinct expression profiles, including markers indicative of epi-
thelial origin (38). The ability to permanently delete intracellular
signaling proteins specifically within lung epithelial cells offers the
potential to further define the intricate interconnections between
epithelial and mesenchymal cells important to fibrogenesis.
Finally, our findings may have therapeutic implications.
Although multiple studies have demonstrated the importance
of TGF-β1 to tissue fibrogenesis, a major limitation to the use of
non-specific blocking agents of TGF-β1 signaling as antifibrotic
therapy is the expected toxicity of such inhibition in multiple
organs, especially the likelihood of autoimmunity (39). Dissect-
ing the fibrogenic aspects of TGF-β1 signaling from other TGF-β1
responses is therefore an important next step in potential therapy.
The distinct pathway of the α3β1 integrin–dependent, TGF-β1–
initiated β-catenin/pSmad2 interaction described here must be
active in IPF lung because we are able to demonstrate dramatic
accumulation of nuclear pY654–β-catenin in IPF lung. This inter-
action is absent in normal lung, even when there is evidence for
TGF-β1 activation, and it is absent in at least one other disease
state, emphysema. Targeting physical determinants of E-cadherin/
TGF-β1 receptor association, β-catenin tyrosine phosphorylation,
or pY654–β-catenin/pSmad2 complex formation may offer ther-
apeutic potential for attenuation of interstitial fibrosis without
broadly interrupting the many physiological effects of TGF-β1.
Patient tissue sample accrual and processing. Written informed consent was
obtained from all subjects, and the study was approved by the UCSF Com-
222? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
mittee on Human Research. IPF lung tissues were obtained from diagnos-
tic lung biopsy or explanted lung during lung transplantation. IPF patients
underwent a history, physical examination, high-resolution computed
tomography, pulmonary function testing, and diagnostic lung biopsy. In
all cases the pathologic diagnosis was usual interstitial pneumonia (UIP)
and the consensus clinical diagnosis was IPF. Emphysema tissues were
obtained from explanted lung during lung transplantation. In all cases the
pathologic and clinical diagnosis was emphysema. Normal human lung
tissue was obtained from human lungs not used by the Northern Califor-
nia Transplant Donor Network. Previous studies indicate that these lungs
are physiologically and pathologically normal (40). Lung tissue was either
directly snap-frozen in liquid nitrogen or inflated with PBS and embedded
in OCT prior to snap-freezing. Samples were stored at –80°C until use for
experiments. For immunoblot and immunoprecipitation experiments, fro-
zen lung tissue was pulverized in a stainless steel tissue pulverizer (Fisher
Scientific) pre-cooled in liquid nitrogen. Pulverized frozen lung tissue was
immediately lysed and analyzed as described below.
Reagents. pSmad2 antibody was purchased from Calbiochem. Polyclonal
pro-SPC antibody and plasma Fn were purchased from Chemicon. Mono-
clonal E-cadherin antibody was from Zymed. Matrigel, collagen I, and anti-
bodies to E-cadherin, β-catenin, and α3 integrin were from BD Biosciences —
Transduction Laboratories. Polyclonal myc antibody, α-SMA, vimentin, and
β-actin antibodies were from Sigma-Aldrich. Polyclonal β-catenin antibody
was from Cell Signaling. Collagen I, FITC-conjugated GFP, and GAPDH
antibodies were from Abcam. Smad2/3, Smad7, laminin-5 (γ2), and second-
ary HRP-conjugated antibodies were from Santa Cruz Biotechnology Inc.
Ki67 antibody was from Dako. Polyclonal GFP antibody, pY654–β-catenin,
rhodamine-conjugated phalloidin, and FITC- and PE-labeled secondary anti-
bodies were purchased from Invitrogen. Purified human KGF was purchased
from PeproTech, and purified human TGF-β1 was purchased from R&D
Systems. Small airway basal media and small airway growth media were pur-
chased from Lonzo. Polyclonal collagen IV (α3) antibody was a gift from
Raghu Kalluri (Harvard University, Cambridge, Massachusetts, USA).
Plasmid constructs and virus production. mycRI was a gift from Yoav Henis
(41) (Tel Aviv University, Tel Aviv, Israel) and the Margaret Wheelock and
Keith Johnson laboratory (University of Nebraska Medical Center, Omaha,
Nebraska, USA) and was cloned into a modified version of pSicoR-GFP
enabling lentiviral-mediated expression. pSicoR-GFP (42) was provided
by Michael McManus and was modified by Jonathon Alexander and Chi-
Hui Tang (all from UCSF). Lentivirus was produced by the UCSF Lentivi-
ral Core Facility. Primary AECs were infected with 2 rounds of lentivirus
encoding GFP or mycRI (10 pfu/cell) in the presence of polybrene (6 μg/ml)
on days 1 and 2 after seeding.
Genetically modified mice. To generate conditional α3 integrin–null mice,
a targeting vector was construction using the pDELBOY vector (43), such
that LoxP sites flanked exon 3 of the α3 integrin gene and Frt sites flanked
a NeoR cassette downstream of exon 2. After homologous recombination
in embryonic stem cells and derivation of mice containing this allele, the
Frt NeoR Frt cassette was removed by mating these mice with mice express-
ing FlpE in the germ line (a gift from Susan Dymecki, Harvard University,
Boston, Massachusetts, USA). This left a single Frt site between exon 2 and
the LoxP site upstream of exon 3. The loxP sites flanking exon 3 subject this
portion of the floxed α3 integrin gene to removal by cre, resulting in early
translational termination in exon 4 (Figure 1A). The resulting conditional
α3 integrin–null mice were maintained with a C57BL6/129 background.
PCR was used for genotyping of the floxed α3 integrin allele using primers
5′-TGATGACTATACCAACCGGAC-3′ (forward) and 5′-ACTCCAAGCCA-
CATATCCTC-3′ (reverse), such that the floxed α3 integrin allele yielded
an approximately 620-bp fragment and the WT allele yielded an approxi-
mately 540-bp fragment.
Triple transgenic mice with lung epithelial cell–specific loss of α3 inte-
grin were obtained by breeding and termed FASC mice. In experiments
using FASC mice or cells from FASC mice, littermate controls lacking
at least 1 of the 3 transgenes were used. Conditional ZEG mice, in which
GFP is expressed after cre-mediated recombination, have been previously
described (27). Triple transgenic ZEG/SPC-rtTA/tetO-Cre mice or quadruple
transgenic mice also homozygous for the floxed α3 integrin transgene were
obtained by breeding, resulting in GFP expression in lung epithelial cells
and their derivatives. Pregnant females were maintained on doxycycline
throughout gestation, and resulting litters were genotyped as previously
described (8, 27). All mice were bred and maintained in a specific patho-
gen–free environment. Mouse lung sections (5–7 μm thick) were stained by
H&E and trichrome by the UCSF Research Morphology Core Facility.
Recombination of floxed α3 integrin by PCR. Recombination of the floxed α3
integrin allele was determined by PCR using primers that encompass the
approximately 2.5-kb floxed region: 5′-TGATGACTATACCAACCGGAC-3′
(forward) and 5′-CAGAAGGCATGAATTTGAGAG-3′ (reverse), such that
the recombined floxed α3 integrin produced a 1-kb band while the non-
recombined floxed α3 integrin produced a 3.5-kb band. DNA from murine
embryonic fibroblasts (MEF cells) isolated from floxed α3 integrin mice
and treated in vitro with adenovirus encoding cre (a gift from Lilly Wu,
UCLA, Los Angeles, California, USA) was used as a positive control for the
recombined floxed α3 integrin band.
Mouse type II AEC isolation and culture.?Isolation of primary AECs was
performed as previously described (8) following the method of Corti (44)
with minor modifications. Typical yields were about 106 cells per mouse,
which were more than 95% type II AECs as assessed by cytospin and
immunostaining for pro-SPC. Cells were either analyzed immediately after
isolation or cultured on Fn-coated plates in SAGM supplemented with 5%
CT-FBS and KGF (10 ng/ml) in a 37°C, 5% CO2 incubator as previously
described (8). Whole lung single-cell suspension was obtained from murine
lungs as previously described (8), then immediately FACS sorted for GFP-
expressing cells for further analysis.
Bleomycin lung injury and measurement of fibrosis.?Six-week-old FASC mice
or littermate controls were endotracheally instilled with saline or 1.3 U/kg
of bleomycin (Blenoxane; Bristol-Myers Squibb) dissolved in saline via
surgical tracheotomy (45). Mice were sacrificed 5–21 days after injury.
Hydroxyproline was measured by methods previously described (45).
Briefly, homogenates from the entire left lungs were incubated on ice
in trichloroacetic acid, then baked in 12N HCl. Aliquots of the samples
reconstituted in distilled water were added to 1.4% chloramine T in 10%
isopropanol and 0.5 M sodium acetate for 20 min. Erlich’s solution was
added, and the samples were incubated at 65°C for 15 minutes. Absor-
bance at 550 nm was measured. Type I and type IV collagen was measured
by immunoblot by methods previously described (46, 47).
Measurement of lung permeability and compliance. Lung permeability was
determined by extravasation of intravascular 125I-labeled albumin into the
lungs as previously described (48) and expressed as extravascular plasma
equivalent normalized to plasma volume (EVP%). Total protein concen-
tration (mg/ml) from bronchoalveolar lavage (BAL) 5 days after intratra-
cheal saline or bleomycin injury was measured using the BCA assay (Pierce
Biotechnology) per the manufacturer’s protocol. Lung compliance (μl/cm
H2O) was determined from anesthetized and paralyzed ventilated mice as
previously described (48). Excess lung water was determined as previously
described (48). For each experiment, n = 4–6 mice per group.
Immunofluorescence microscopy. Isolated cells and 5- to 7-μm cryosec-
tions were stained by immunofluorescence as previously described (8).
For immunostaining mouse lung with mouse monoclonal anti–pY654–
β-catenin, the antibody was directly conjugated using Alexa Fluor
Microscale Protein Labeling Kit (Invitrogen) per the manufacturer’s
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 1 January 2009
instructions. Stained sections were visualized on a Nikon fluorescent
microscope and images captured with a SPOT 2.3.1 camera (Diagnostic
Instruments) and analyzed using SPOT 4.0.9 software (Diagnostic Instru-
ments). For cellular quantification of myofibroblasts from immunostained
slides, at least 10 fields (×20) from each mouse were visualized and quan-
tified independently by 2 investigators in a blinded fashion. SimplePCI
(Hamamatsu Corp.) was used for all other cellular quantification of
Statistics. Data are expressed as mean ± SEM. For evaluation of group dif-
ferences, the 2-tailed Student’s t test was used assuming equal variance. A
P value of less than 0.05 was accepted as significant.
Flow cytometry.?Cells were analyzed for expression of GFP by flow cytome-
try using FACSCalibur cytometer (BD Biosciences) and CellQuest Pro soft-
ware or sorted for GFP-expressing cells using a MoFlo Cell Sorter (Dako) at
the UCSF Flow Cytometry Core Facility.
Immunoblot. Tissue and cells were lysed in RIPA buffer (150 mM NaCl,
50 mM Tris, pH 8.0, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%
SDS, supplemented with protease and phosphatase inhibitors) and ana-
lyzed by immunoblot as previously described (49). Scanned immunoblots
are representative of at least 3 separate experiments. Densitometry was
quantified using NIH ImageJ.
Ex vivo and in vivo immunoprecipitation. For primary AEC coimmuno-
precipitation of mycRI and α3 integrin, cells were seeded onto Fn-coated
plates. Cells were infected with lentivirus encoding GFP or mycRI on
days 1 and 2 after seeding then lysed in Triton buffer on day 5. Equal
concentrations of clarified lysates were incubated for 2 hours at 4°C
with monoclonal myc antibody and were then precipitated with pro-
tein A and protein G agarose beads (Invitrogen) overnight at 4°C. Beads
were washed and then analyzed by immunoblot for α3 integrin, E-cad-
herin, and mycRI. For primary AEC immunoprecipitation of β-catenin
and pY–β-catenin, equal numbers of FASC or littermate control AECs
were seeded on Fn-coated plates. Three days after seeding, cells were
rinsed twice with PBS supplemented with sodium orthovanadate and
Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) and then lysed in RIPA
buffer. Clarified lysate was immunoprecipitated for β-catenin and pY–
β-catenin using monoclonal antibodies as described above then analyzed by
immunoblot for β-catenin and pSmad2. For in vivo immunoprecipitation
from murine lungs, FASC and littermate control mice were sacrificed
2 weeks after intratracheal injection with saline or bleomycin. Lungs were
perfused and lavaged with PBS supplemented with sodium orthovana-
date and Phosphatase Inhibitor Cocktail 2 and then immediately lysed in
RIPA buffer. Clarified lysates were further precleared by incubating with
protein A and protein G beads. The precleared supernatants were then
immunoprecipitated for β-catenin and pY–β-catenin as described above.
For in vivo immunoprecipitation of human lung samples, snap-frozen
lungs were pulverized and lysed in RIPA. Clarified lysates were further pre-
cleared with secondary anti-rabbit HRP antibody and protein A and pro-
tein G beads. The precleared supernatants were then immunoprecipitated
for β-catenin and pY–β-catenin as described above.
The authors thank Jonathon Alexander, Chi-Hui Tang, Michael
Galvez, and Liliane Robillard for technical assistance. This work
was supported by the Parker B. Francis Foundation (to K.K. Kim)
and NIH grants K08HL085290 (to K.K. Kim), HL88440 (to J.A.
Frank), and RO1 HL44712 (to H.A. Chapman).
Received for publication July 28, 2008, and accepted in revised
form October 22, 2008.
Address correspondence to: Harold A. Chapman, University of
California, San Francisco, Box 0111, San Francisco, California
94143, USA. Phone: (415) 514-0896; Fax: (415) 502-4995; E-mail:
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