Rapamycin Prevents Transforming Growth
Factor-a–Induced Pulmonary Fibrosis
Thomas R. Korfhagen1, Timothy D. Le Cras1, Cynthia R. Davidson2, Stephanie M. Schmidt2, Machiko Ikegami1,
Jeffrey A. Whitsett1, and William D. Hardie2
1Divisions of Pulmonary Biology and2Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
Transforming growth factor (TGF)-a is a ligand for the epidermal
growth factor receptor (EGFR). EGFR activation is associated with
fibroproliferative processesin human lungdiseaseand animalmod-
els of pulmonary fibrosis. Overexpression of TGF-a in transgenic
response are unknown. Using a doxycycline-regulatable transgenic
mouse model of lung-specific TGF-a expression, we observed in-
creased PCNA protein and phosphorylation of Akt and p70S6K in
whole lung homogenates in association with induction of TGF-a.
over a 7-week period. Daily administration of rapamycin prevented
accumulation of total lung collagen, weight loss, and changes in
pulmonary mechanics. Treatment of mice with rapamycin 4 weeks
after the induction of TGF-a prevented additional weight loss, in-
creases in total collagen, and changes in pulmonary mechanics.
Rapamycin prevented further increases in established pulmonary
fibrosis induced by EGFR activation. This study demonstrates that
determine the role of mTOR in the pathogenesis and treatment of
Keywords: epidermal growth factor receptor; PI3K; Akt; mTOR
Pulmonary fibrosis is characterized by mesenchymal cell proli-
feration in the lung, expansion of the extracellular matrix (ECM),
and extensive remodeling of the lung parenchyma (1). Clinical
diseases causing pulmonary fibrosis are heterogeneous and in-
clude connective tissue disorders, occupational and environmen-
tal exposures, and interstitial lung diseases (ILD) (2). Currently
there are no proven therapies that prevent or reverse pulmonary
fibrosis, emphasizing the need to identify new molecular targets.
The molecular pathways leading to pulmonary fibrosis are
the cytokines TNF-a, GM-CSF, IL-11, IL-13, and IL-1b develop
varying degrees of pulmonary fibrosis associated with inflamma-
tion. In addition, several growth factors regulate matrix deposi-
tion and fibroblast proliferation in the lung, including connective
tissue growth factor, platelet-derived growth factor (PDGF),
basic fibroblast growth factor, insulin-like growth factor, and
transforming growth factor (TGF)-b1 (3, 4). TGF-a, along with
epidermal growth factor (EGF) and amphiregulin are ligands for
the epidermal growth factor receptor (EGFR). We previously
generated doxycycline (Dox)-regulatable transgenic mice where-
and extensive vascular adventitial, peribronchial, interstitial, and
pleural fibrosis that was independent of inflammatory or de-
velopmental influences (5). Gene expression profiles observed
found in pulmonary fibrotic disease in humans (6).
The EGFR is a membrane-bound receptor tyrosine kinase
that belongs to a subfamily of four closely related receptors:
HER1/EGFR/ERBB1, HER2/NEU/ERBB2, HER3/ERBB3,
and HER4/ERBB4. After ligand binding, these receptors form
homo- and heterodimers leading to autophosphorylation of tyro-
sine residues in the cytosolic domains of the proteins. The phos-
phorylated tyrosine residues become docking sites for signaling
molecules that activate multiple downstream effector pathways
including MAPK, Src kinases, STAT, and the phosphatidylino-
sitol 3-kinase pathway (PI3K) (7, 8). The mammalian target of
rapamycin (mTOR) a is highly conserved intracellular serine/
threonine kinase and a major downstream component of PI3K
(9). Activation of mTOR, in complex with raptor (mTORC1),
leads to phosphorylation of ribosomal P70S6 kinase (S6K) and
eukaryotic initiation factor-4E–binding protein-1 (4E-BP1). Both
S6K and 4E-BP1 control the translation of specific mRNAs and
protein synthesis involved in cell cycle regulation (9). Inhibitors
of mTOR, such as rapamycin bind to an intracellular cytoplas-
mic receptor, the FK506-binding protein-12 (9). This complex
interacts and inhibits mTOR function leading to cell cycle arrest
in the G1 phase. In addition to blocking cell proliferation,
mTOR inhibitors have been identified with anti-inflammatory,
anti-tumor, and anti-fibrotic properties that implicate mTOR
signaling in a wide range of cellular functions.
In this study we first evaluated whether PI3K/Akt-mTOR
pathway is activated by expression of TGF-a in the lungs of
transgenic mice. We then determined the role of mTOR in the
initiation and propagation of pulmonary fibrosis by administer-
ing rapamycin at the time of TGF-a induction. Finally, we
determined the effectiveness of rapamycin as a late treatment
for established and progressive fibrosis in the TGF-a model.
Expression of epidermal growth factor receptor (EGFR)
and EGFR ligands have been identified in animal models
of pulmonary fibrosis and human disease. Using a transgenic
mouse model of pulmonary fibrosis caused by lung-specific
a, the present study demonstrates that administration of
rapamycin prevents both the initiation and the progression
of established pulmonary fibrosis and associated alterations
in lung mechanics. These findings support the need for fur-
ther studies to carefully determine the role of mammalian
target of rapamycin activation in the pathogenesis of pul-
monary fibrosis and to assess the efficacy of therapies de-
signed to inhibit its activity.
(Received in original form October 6, 2008 and in final form December 19, 2008)
This work was supported by National Institutes of Health grants: HL086598
(W.D.H.), HL061646 (M.I.), HL058795 (T.R.K.), HL90156 (J.A.W.) and HL61646
(J.A.W.), and American Heart Association grant 0740069N (T.D.L.).
Correspondence and requests for reprints should be addressed to William D.
Hardie, M.D., Department of Pulmonary Medicine, Cincinnati Children’s Hospital
Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: william.
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2008-0377OC on February 24, 2009
Internet address: www.atsjournals.org
Vol 41. pp 562–572, 2009
MATERIALS AND METHODS
CCSP-rtTA activator mice expressing the reverse tetracycline-responsive
transactivator (rtTA) under control of the 2.3-kb rat Clara cell secretory
protein (CCSP) (a.k.a. secretoglobin, family 1A, member 1 [Scgb1a1])
mice containing the human TGF-a cDNA under the control of seven
copies of the tetracycline operon ((TetO)7-cmv TGFa) plus a minimal
CCSP-rtTA1/2/(TetO)7-cmv TGF-a1/2mice (herein called CCSP/
TGF-a mice) were produced within the same litter by mating homozy-
gous CCSP-rtTA1/1mice to hemizygous (TetO)7-cmv TGFa1/2mice.
Mice were genotyped as previously described (5). All mice were derived
from the FVB/NJ inbred strain. Mice were maintained in virus-free
containment and protocols were approved by the Institutional Animal
induces phosphorylation of the Akt, P70S6K, and
S6. Epithelial cell TGF-a expression increased
phosphatidylinositol 3-kinase (PI3K)/mammalian
target of rapamycin (mTOR) activity as assessed
by increased phosphorylation of Akt, P70S6K, and
S6. CCSP/TGF-a mice were treated with doxycy-
cline (Dox) to induce TGF-a expression for 1 or 4
days and compared with littermate control single
transgene CCSP/- mice treated with 1 day of Dox.
CCSP/TGF-a mice demonstrated (A) increased
phosphorylation of Akt at Ser473 and (B) phos-
phorylation of P70S6K at Thr389. Immunostain-
ing for phosphorylated Akt and phosphorylated
S6 demonstrated increased staining in the airway
epithelium in CCSP/TGF-a mice after 4 days of
Dox compared with CCSP/TGF-a mice not re-
ceiving Dox (C, arrows). Mean 6 SE, n 5 3–6 mice
of each genotype; * P , 0.05.
Transforming growth factor (TGF)-a
Korfhagen, Le Cras, Davidson, et al.: Rapamycin Inhibits Pulmonary Fibrosis563
old)were placedonDox-containingdrinkingwater (0.5mg/ml)andfood
(62.5 mg/kg). Dox-containing water was replaced three times per week.
Administration of Erlotinib and Rapamycin
Erlotinib powder (100 mg/kg; OSI Pharmaceuticals Melville, NY) was
suspended in 0.5% methyl cellulose (0.015 mg/ml; 378C; Colorcon, West
Point, PA). Three hours before administration, food and water were
removed from cages. Mice were then anesthetized (Isoflurane; Abbott
gavage using a 20-gauge feeding catheter (Harvard Apparatus, Hollis-
a 30 mg/ml stock solution by dissolving 2 mg in 67 mL 100% EtOH.
Rapamycin was diluted to a 0.6 mg/ml concentration in 0.25% PEG400,
and intraperitoneal rapamycin (4 mg/kg) or vehicle was administered
using a 1-ml syringe with a 27G1/2 needle (BD Syringe, Franklin Lakes,
NJ). Drug dosage was based upon previous studies in mice using
rapamycin and was not adjusted for changes in body weight during the
study period (11). Mice were weighed at the beginning of the study and
at weekly intervals.
Western blot analysis was performed on lung homogenates of CCSP/
TGF-a mice treated with either 1 or 4 days of Dox. Controls were lit-
on a 4 to 20% Tris/glycine SDS-PAGE (Invitrogen, Carlsbad, CA) and
electroblotted to PVDF membranes (0.45 mm; Bio-Rad, Hercules, CA).
150 mM NaCl, 0.1% Tween 20) and incubated with antibodies against
total and phosphorylated Akt (Ser 473; Cell Signaling Technology,
Beverly, MA), total and phosphorylated p70 S6 kinase (Thr 389; Cell
Signaling Technology), phosphorylated EGFR (pY1086; Epitonic,
Burlingame, CA), total EGFR (rabbit polyclonal; kind gift from
Dr. Brad Warner, Washington University) and proliferatingcell nuclear
antigen (PCNA) (Cell Signaling Technology). Blots were washed in
TBST and incubated with goat anti-rabbit horseradish peroxidase–
conjugated (Calbiochem/EMD Biosciences, Madison, WI) secondary
antibodies and developed on film by chemiluminescence using the
ECL Plus system (Amersham Biosciences, Piscataway, NJ). Densitom-
Imagersoftware Imagequant5.2 (Molecular Dynamics,Sunnyvale, CA)
after scanning the films.
Rapamycin Prevention and Late Treatment Studies
To determine if rapamycin prevented the fibrotic effects of TGF-a
expression, CCSP/TGF-a mice were administered 7 weeks of Dox, and
treated with either rapamycin (4 mg/kg once daily 6 d/wk) or vehicle.
Controls were littermate single transgene CCSP/- mice administered
Dox and treated with rapamycin.
For the late treatment studies, CCSP/TGFa mice were administered
4 wk of Dox. A subgroup of mice were killed at 4 weeks to measure
endpoints as a ‘‘fibrotic baseline’’ to determine the effectiveness of
rapamycin for reversing or preventing progression of established
fibrosis. At the beginning of the fifth week, remaining mice were then
treated with either vehicle or rapamycin while continuing on Dox. Mice
in both groups were killed after 3 or 7 weeks of treatment (7 or 11 wk
of total Dox, respectively). Control mice were littermate single trans-
gene CCSP/- mice administered Dox for 11 weeks and treated with
7 weeks of rapamycin beginning on the fifth week of Dox. Endpoints
for the prevention and late treatment studies were changes in lung
histology, body weights, total lung collagen, and lung mechanics.
Lung Histology and Immunohistochemistry
Mice were killed with pentobarbital sodium (65 mg/ml) euthanasia
solution (Fort Dodge Animal Health, Fort Dodge, IA), and lungs were
inflation fixed using 4% paraformaldehyde at 25 cm H2O of pressure,
and then allowed to fix overnight at 48C. Fixed lungs were then washed
with phosphate-buffered saline (PBS), dehydrated through a graded
series of ethanols, and processed for paraffin embedding. Sections
(5 mm) were loaded onto polysine slides for immunostaining, hematoxylin
and eosin (H&E) staining as previously described (12). Immunostain-
ing was performed on CCSP/TGF-a mice treated with 4 days of Dox
and compared with CCSP/TGF-a mice not receiving Dox. Immunos-
taining for phosphorylated Akt (Ser 473; Cell Signaling) used citrate/
TBST antigen unmasking with antibody diluted 1:50. Immunostaining
for phosphorylated S6 (Ser 235/236; Cell Signaling) used citrate/TBST
antigen unmasking with antibody diluted 1:1,000. Secondary antibodies
and DAB detection were preformed as previously described (5).
Figure 1. (continued).
564 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 412009
Total Lung Collagen
Total lung collagen was determined by measuring total soluble collagen
in 5 ml 0.5 M acetic acid containing pepsin (1 mg/10 mg tissue; Sigma-
rpm for 12 min), and the pellet was suspended (1 ml 0.5 M NaOH). The
optical density measured with a spectrophotometer (540 nm).
Lung mechanics were assessed on anesthetized mice using a computeri-
zed Flexi Vent system (SCIREQ, Montreal, PQ, Canada), as previously
described (13, 14). Briefly, mice were anesthetized with ketamine and
xylazine, tracheostomized, and then ventilated with a tidal volume of
8 ml/kg at a rate of 450 breaths/minute and positive end-expiratory
pressure (PEEP) of 2 cm H2O computerized by the SCIREQ system,
thereby permitting analysis of dynamic lung compliance. The ventila-
tion mode was changed to forced oscillatory signal (0.5–19.6 Hz), and
respiratory impedance was measured. Tissue elastance was obtained
for mice at 2 cm H2O PEEP by fitting a model to each impedance spec-
trum. With this system, the calibration procedure removed the im-
pedance of the equipment and tracheal tube.
Using normal plots and tests for normality (Shapiro-Wilk and
Kolmogorov-Smirnov), all response variables showed a significant de-
Figure 2. Erlotinib and rapamycin inhibit TGF-a–induced phosphor-
ylation of P70S6K and PCNA. (A) Pretreatment of CCSP/TGF-a mice
with erlotinib (100 mg/kg) prevented increased phosphorylation of
Akt and P70S6K and increased levels of PCNA after 1 day of Dox.
Pretreatment of CCSP/TGF-a mice with rapamycin (Rapa, 4 mg/kg)
did not alter phosphorylation of Akt, but prevented increased
phosphorylation of P70S6K and increased levels of PCNA after
1 day of Dox. (B) Rapamycin did not effect phosphorylation of EGFR
(Y1086) in lung homogenates after 1 day of Dox. Mean 6 SE, *P ,
0.05 compared with vehicle (veh)-treated (the rapamycin vehicle or
0.25% PEG400, 0.25% Tween 20) CCSP/- controls.
Korfhagen, Le Cras, Davidson, et al.: Rapamycin Inhibits Pulmonary Fibrosis565
a–dependent pulmonary fibrosis. CCSP/
TGF-a mice were administered 7 weeks of
Dox, and treated with either rapamycin
(4 mg/kg once daily 6 d/wk) or vehicle.
Controls were littermate single transgene
CCSP/- mice administered Dox and trea-
ted with rapamycin. The treatment pro-
tocol is represented schematically in A. (B)
toxylin and eosin. (C) CCSP/TGF-a mice
Dox caused a marked attenuation of pul-
monary fibrosis compared with CCSP/
TGF-a vehicle-treated mice. (D) Rapamy-
cinpreventedTGF-a–induced increases in
total lung collagen. Photomicrographs
shown are representative of lungs from
graphs are taken at the same magnifica-
tion, and bar is 200 mm. *P , 0.05
CCSP/TGF-a vehicle-treated mice.
Rapamycin prevents TGF-
566 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 412009
parture from the normality assumption. Therefore, log transformations
of the above response variables were used to compare group means in
a one-way ANOVA. Differences in group means were calculated and
tested using a simulation-based adjustment for multiple comparisons.
variance/covariance structure was used for each Group. Differences in
selected (a priori) Group*Week means were calculated and tested using
a simulation-based adjustment for multiple comparisons.
TGF-a Induces Phosphorylation of the Akt/mTOR Pathway
CCSP/TGF-a mice were treated with Dox to induce TGF-a ex-
pression for 1 and 4 days. Induction of TGF-a caused increased
1B). Increased p-Akt and phosphorylated S6 were identified by
immunohistochemistry primarily in epithelial cells (Figure 1C).
Less prominent staining was also present in mesenchymal cells
along the lung pleura and perivascular and peribronchial adven-
titia (not shown).
Erlotinib and Rapamycin Inhibit TGF-a–Induced
CCSP/TGF-a mice pretreated with erlotinib (100 mg/kg) then
administered Dox for 24 hours demonstrated reduced p-Akt, p-
P70S6K, and total PCNA in whole lung homogenates compared
with vehicle-treated CCSP/TGF-a mice (Figure 2A). CCSP/
TGF-a mice pretreated with rapamycin (4 mg/kg) then admin-
istered Dox for 24 hours demonstrated reduced p-P70S6K and
PCNA in whole lung homogenates compared with vehicle-treated
CCSP/TGF-a mice with no change in p-Akt (Figure 2A), indi-
cating that TGF-a–induced proliferation was mediated through
the mTOR pathway. Administration of Dox and rapamycin did
not alter increased phosphorylation of EGFR in lung homoge-
nates of CCSP/TGF-a mice (Figure 2B).
Rapamycin Prevents TGF-a–Induced Pulmonary Fibrosis
CCSP/TGF-a mice were treated with Dox to induce TGF-a ex-
pression and concomitantly treated daily with either vehicle or
rapamycin (4 mg/kg 6 d/wk) for 7 weeks (Figure 3A). Control
mice were CCSP/- mice administered 7 weeks of Dox and
rapamycin. Body weights of CCSP/TGF-a mice treated with 7
weeks of Dox and administered vehicle decreased 14.7 6 0.2%
from baseline, while CCSP/TGF-a mice treated with 7 weeks of
Dox and administered rapamycin increased 4.0 6 0.1% from
baseline, similar to that of CCSP/- mice (14.9 6 0.1%, P ,
0.001) (Figure 3B). Induction of TGF-a caused extensive fibro-
sis localized to the pleural surfaces and to the perivascular and
peribronchial adventitia. Rapamycin reduced pulmonary fibro-
sis with minimal residual disease, represented by scattered
areas of perivascular pulmonary fibrosis and pleural thickening
(Figure 3C). Increases in lung collagen content (Figure 3D) and
altered lung mechanics (Figures 4A–4D) in CCSP/TGF-a
vehicle-treated mice were all prevented in the rapamycin-
Rapamycin Prevents Progression of Established
TGF-a–Induced Pulmonary Fibrosis
To determine whether rapamycin influences the progression of
established fibrosis, after 4 weeks of Dox treatment, CCSP/
TGF-a mice were administered either daily rapamycin or
vehicle while remaining on Dox (Figure 5A). Body weights of
CCSP/TGF-a mice treated with vehicle decreased 25% from
baseline after 8 weeks of Dox (Figure 5B). Between Weeks
8 and 11, body weight loss stabilized but was likely influenced
mechanics. Rapamycin administered daily to CCSP/TGF-a mice at the
time of Dox treatment prevented TGF-a–mediated (A–C) increases in
airway resistance, and airway and tissue elastance, and (D) decreases in
compliance compared with CCSP/TGF-a receiving vehicle. *P , 0.05
compared with CCSP/- control and CCSP/TGF-a vehicle-treated mice.
Rapamycin prevents TGF-a–dependent changes in lung
Korfhagen, Le Cras, Davidson, et al.: Rapamycin Inhibits Pulmonary Fibrosis567
sion of TGF-a–dependent pulmonary fi-
brosis. After 4 weeks of Dox, CCSP/TGF-a
mice were administered either daily rapa-
mice were evaluated after 3 or 7 weeks
of treatment. Vehicle-treated CCSP/TGF-
a mice were recovered at 4, 7, and 11
weeks of Dox for comparison. Control
mice were CCSP/- mice treated with
rapamycin after 4 weeks of Dox. The
treatment protocol is represented sche-
matically in A. (B) Dox-induced expres-
sion of TGF-a caused progressive weight
loss. Body weight stabilized in mice trea-
ted with rapamycin 4 weeks after treat-
ment with Dox. Sections of lungs were
stained with hematoxylin and eosin. (C)
Expression of TGF-a caused progressive
adventitial fibrosis. CCSP/TGF-a mice ad-
ministered rapamycin after 4 weeks of
Dox demonstrated reduced (C) adventi-
tial fibrosis and (D) total lung collagen
compared with CCSP/TGF-a vehicle-treated
mice receiving 7 or 11 weeks of Dox.
*P , 0.05 compared with rapamycin-
treated controls. 1P , 0.05 compared
with 7 and 11 week vehicle-treated
CCSP/TGF-a mice. Small images of dead
mice in B denote mouse death during
Rapamycin prevents progres-
568AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 41 2009
by the deaths of three severely affected mice. Rapamycin
administered at the beginning of Week 5 prevented further
body weight loss compared with vehicle-treated mice, but body
weights remained less than those of control mice. Lung fibrosis
as assessed by histology, total lung collagen, and lung mechan-
ics was improved compared with that of vehicle-treated mice
at 7 weeks and 11 weeks, but was unchanged compared with
vehicle-treated mice after 4 weeks of Dox (Figures 5C–5D and
Using a transgenic mouse model of pulmonary fibrosis caused
by lung specific expression of the EGFR ligand, TGF-a, the
Figure 5. (continued).
gression of TGF-a–dependent changes
in lung mechanics. CCSP/TGF-a trans-
genic mice administered rapamycin
onstrated (A–C) reduced increases in
elastance, and (D) decreases in compli-
ance compared with vehicle-treated
CCSP/TGF-a mice receiving 7 and 11
weeks of Dox. *P , 0.05 compared
with CCSP/- control mice; 1 P , 0.05
compared with 7 and 11 week vehicle-
treated CCSP/TGF-a transgenic mice.
Rapamycin prevents pro-
Korfhagen, Le Cras, Davidson, et al.: Rapamycin Inhibits Pulmonary Fibrosis569
present study demonstrates that administration of rapamycin
prevents both the initiation and the progression of established
pulmonary fibrosis and associated alterations in lung mechanics.
Increased expression of EGFR ligands and activation of EGFR
have been implicated in the pathogenesis of pulmonary fibrosis
in a number of animal models including bleomycin, naphtha-
lene, asbestosis, and ovalbumin-induced lung injury (15–18).
Signaling pathways downstream of EGFR have not been
identified in these models. Our findings demonstrate the
activation of the PI3K-Akt-mTOR pathway in association with
the induction of EGFR-mediated pulmonary fibrosis. The
efficacy of rapamycin in preventing TGF-a–induced pulmonary
fibrosis was similar to a previous study in which the EGFR
inhibitors gefitinib and erlotinib also prevented fibrosis in the
CCSP/TGF-a transgenic model (19). Together, these data sup-
port mTOR as a major effector of EGFR-mediated pulmonary
The antifibrotic effects of mTOR inhibition have recently
been reported in several rat models of chronic kidney disease,
including diabetic nephropathy, chronic glomerulosclerosis, and
tubulointerstitial fibrosis (20–22). Likewise, rapamycin pre-
vented extracellular matrix deposition in CCL4-induced liver
fibrosis in rats (23). In rat models of established liver cirrhosis,
rapamycin reduced fibrosis and attenuated disease progression
(24). In pulmonary fibrosis models there are limited data on
activation of mTOR and the effectiveness of rapamycin treat-
ment. The rapamycin analog SDZ RAD prevented bleomycin-
induced pulmonary fibrosis, although it was unclear whether
changes in lung inflammation may have contributed to these
improvements (25). CCSP/TGF-a transgenic mice develop fibro-
sis independent of inflammation (5, 6). Therefore, the efficacy of
rapamycin in this study is unlikely to be attributed to the anti-
inflammatory properties of rapamycin.
Fibrosis in the CCSP/TGF-a transgenic model is progressive,
allowing assessment of rapamycin in reversing established and
accumulating fibrosis. The effectiveness of mTOR inhibition in
established and ongoing lung fibrosis has not been previously
studied. Mice treated with 3 weeks of rapamycin after 4 weeks
of Dox had significantly reduced cachexia, lung collagen, and
changes in lung mechanics compared with mice receiving Dox
and vehicle for 7 and 11 weeks (Figures 5 and 6). However, lung
collagen and lung mechanics in these rapamycin-treated mice
of Dox. In our previous study, lung collagen accumulation and
abnormalities in lung mechanics were partially reversed by
removing dox treatment without any additional pharmacologic
intervention, showing that TGF-a–induced lesions are at least
partially reversible (6). Since the rapamycin treatment did not
completely reverse fibrotic changes, which were present in mice
treated with Dox for 4 weeks, we cannot determine if rapamycin
changes. Mice that received an additional 4 weeks of rapamycin
(7 wk total) did not show further benefit. Together, these results
demonstrate that rapamycin was effective in preventing the
progression of fibrosis and improvements were maintained. An
extended rapamycin treatment period or increased rapamycin
doses may be necessary to further reduce the extent of fibrosis.
However, in CCSP/TGF-a mice treated with the EGFR inhib-
itor gefitinib in a late treatment study, fibrosis was not com-
pletely reversed (19). Thus, some degree of pulmonary fibrosis in
the CCSP/TGF-a model may be irreversible. Alternatively, addi-
tional profibrotic pathways activated after the establishment of
Figure 6. (continued).
570AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 412009
fibrosis may remain active after removal of EGFR or mTOR
protein synthesis in a number of cell lines, including fibroblasts,
vascular smooth muscle, and epithelial cells (22, 26). Pulmonary
fibrosis in the TGF-a model is characterized by epithelial and
mesenchymal proliferation and increased extracellular matrix
deposition (6, 27), and both processes were effectively inhibited
with rapamycin administration in the prevention studies. Al-
Akt, there is increasing evidence that mTOR signals indepen-
28). Both S6K and 4E-BP1 possess several phosphorylation sites
and are activated not only by mTOR, but also by PI3K and ERK
(9, 29). Additional studies using specific pathway component
inhibitors, phosphorylation assays, and specific rescue experi-
ments in vitro will be needed to precisely identify the signaling
pathways leading to mTOR activation. In addition, studies of
gene targets of mTOR will be needed to further identify other
fibrotic components causing fibrosis in this model.
In addition to EGFR, the mTOR pathway may be regulated
by multiple fibrotic activators. Although the Smad pathway is
believed to be the primary conduit for signals from the TGF-b1
receptors, emerging evidence supports the importance of non-
SMAD pathways. Fibroblasts stimulated with TGF-b1 dem-
onstrate Smad-independent proliferation and matrix protein
production which are regulated by MAPK and PI3K pathways
(23, 30, 31). Pulmonary fibrosis caused by TGF-b1 was atten-
uated when mice were treated with an Akt inhibitor (32).
Platelet-derived growth factors are profibrotic cytokines that
have also been implicated in inflammatory models of lung
fibrosis (33, 34). Platelet-derived growth factors act via two
receptors which, like EGFR, are receptor tyrosine kinases and
activate PI3K and MAPK (35). Future studies targeting mTOR,
S6K, and 4E-BP1 will be valuable in determining whether the
mTOR pathway influences diverse fibrotic processes in the lung.
Rapamycin and related analogs are currently used clinically
as an immunomodulating agent for kidney transplant (9). Ab-
normal activation of signaling pathways of mTOR appears to
occur frequently in human cancer, which led to the evaluation
of the antiproliferative effects of rapamycin in malignant neo-
plasms. EGFR inhibitors combined with rapamycin analogs are
in trials in lung cancer, with the rationale that mTOR inhibition
may rescue proliferation pathways that remain activated in tu-
mors resistant to EGFR-specific tyrosine kinase inhibitors (36).
The utility of mTOR inhibition in clinical pulmonary disease is
currently under investigation. Rapamycin was used to success-
fully treat a patient with idiopathic pulmonary fibrosis (IPF) (37).
The effectiveness of rapamycin for IPF is currently under in-
vestigation in a randomized clinical trial (38). Lymphangioleio-
myomatosis (LAM) is a progressive lung disease characterized
by infiltration of abnormal smooth muscle–like cells and for-
mation of parenchymal cysts. Inhibition of the mTOR pathway
with Sirolimus improved spirometric measurements and gas
trapping in LAM patients that persisted after cessation of
The predominant pathways involved in experimental fibrosis
and the response to pharmacologic agents varies widely with the
specific strain of mice used and with the specific pro-fibrotic
challenge. Results from this study using FVB/N mice need to be
tested in additional mouse strains and other models of pulmo-
nary fibrosis before extrapolating results form this study directly
to human disease.
In summary, the present study demonstrates that rapamycin
prevents and inhibits progression of ongoing pulmonary fibrosis
caused by expression of TGF-a and increased EGFR signaling.
These findings support the need for further studies to carefully
determine the role of mTOR activation in the pathogenesis of
pulmonary fibrosis and to assess the efficacy of therapies de-
signed to inhibit its activity.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Matt Fenchel, who provided assistance in
statistical analysis of data, and technical assistance in measuring lung mechanics
from Angelica Falcone.
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