From the Division of Pulmonary and Critical Care
Medicine, Department of Internal Medicine, University of Michigan
Medical Center, Ann Arbor, Michigan 48109 and ¶ Pulmonary and
Critical Care Division, Department of Medicine, Tufts-New England
Medical Center, Boston, Massachusetts 02111
Received for publication, August 20, 2002, and in revised form, January 15, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myofibroblast differentiation and activation by
transforming growth factor- Cell differentiation and phenotypic plasticity of cells, including
that of adult stem cells, is critically dependent on the nature of the
cellular microenvironment. Rapid and dynamic alterations of this
microenvironment occur in the setting of tissue injury, inflammation,
and repair. Fibroblasts are active participants in such processes and
are characterized by their exceptional ability to undergo various
interconversions between related but distinctly different cell types.
This phenotypic plasticity of fibroblasts allows them to play an active
role in the tissue repair and remodeling process.
Fibroblast phenotype heterogeneity has been described in normal
physiologic and diverse pathologic conditions (1, 2). Gabbiani et
al. (3) first described the transient appearance and disappearance
of so-called myofibroblasts in the granulation tissue of
healing cutaneous wounds. Myofibroblasts possess ultrastructural features intermediate between fibroblasts and smooth muscle cells and
have been defined by their ability to express contractile proteins,
particularly Transforming growth factor- Integrins are the major cell adhesion receptors for ECM ligands that
mediate adhesion-dependent signaling in diverse biological processes including cell proliferation, migration, and apoptosis (18).
Adhesion-dependent integrin activation recruits focal adhesion kinase (FAK) and activates it at focal adhesions (19, 20). FAK
activation is mediated primarily by autophosphorylation of tyrosine 397 that creates a binding site for the Src homology 2 domain of Src (21)
and phosphatidylinositol 3-kinase (22). Recent studies (23, 24) suggest
reciprocal catalytic activation of FAK and Src kinases at focal
adhesions, specifically in adhesion-dependent integrin aggregation.
Cellular proliferation and differentiation, in general, activate
distinct and often opposing signaling pathways (25, 26). Yet both
processes are critically dependent on cellular adhesion to the ECM
(27), suggesting that at least some components of adhesion-dependent signaling might be common to both cell
proliferation and differentiation. This study was undertaken to examine
the role of adhesion-generated signal(s) in myofibroblast
differentiation of normal, untransformed human lung fibroblasts by
TGF- Cell Culture and Reagents--
All experiments were performed on
early passage normal human fetal lung fibroblasts (IMR-90; Institute
for Medical Research, Camden, NJ). The cells were maintained in medium
consisting of Dulbecco's modified Eagle's medium (Invitrogen)
supplemented with 10% fetal calf serum (Sigma), 100 units/ml
penicillin/streptomycin (Sigma), and fungizone (Invitrogen); medium was
changed every 3 days. Cells were plated on 35- or 100-mm dishes at a
density of 106 cells/dish and incubated in 5%
CO2, 95% air. For immunofluorescence studies, IMR-90 cells
were plated on glass coverslips placed in 35-mm Petri dishes. In some
experiments, cells were plated on dishes pre-coated with various ECM
proteins (BD Biocoat cellware, BD Biosciences). Cells were
growth-arrested by reducing the concentration of fetal calf serum in
the medium to 0.01% for 48 h prior to stimulation with
TGF- Antibodies--
Mouse monoclonal antibodies against Immunoprecipitation and Western Blotting--
Cells grown on
tissue culture plates were gently washed with 5 ml of
phosphate-buffered saline and lysed in 0.5 ml of cold RIPA lysis buffer
(1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M NaH2PO4,
2 mM EDTA, 0.5 mM NaF) containing 2 mM sodium orthovanadate and 1:100 dilution of protease
inhibitor mixture III (Calbiochem). Protein concentrations were
determined using the BCA protein assay (Pierce). For each
immunoprecipitation, equal amounts of protein were incubated for 4 h in 1:100 dilution of antibody and protein A-agarose beads (15 µl/500-µl sample) (Santa Cruz Biotechnology, Santa Cruz, CA).
Immunoprecipitated protein samples or whole cell lysates were mixed
with a 1:5 v/v ratio of 6× electrophoresis sample buffer (0.2 M EDTA, 40 mM dithiothreitol, 6% SDS, 0.06 mg/ml pyronin, pH 6.8) and boiled at 95 °C for 5 min to denature
protein. Sample mixtures were then loaded and subjected to
electrophoresis in a 4-20% polyacrylamide gradient gel. Proteins were
electrophoretically transferred to polyvinylidene difluoride membrane
(Immobilon-P, Millipore Inc., Bedford, MA) and incubated in blocking
buffer containing 75 mM sodium phosphate, 70 mM
sodium chloride, and 0.1% Tween 20 (pH 7.4) with 5% bovine serum
albumin for 1 h at room temperature. The blot was treated with a
1:1000 dilution of primary antibody in blocking buffer overnight at
4 °C. Three washes with a buffer containing 10 mM Tris,
100 mM NaCl, and 0.1% Tween 20 were then performed prior
to incubation with a secondary antibody conjugated to horseradish
peroxidase. The washes were repeated, and membrane was incubated
with SuperSignal Substrate Western blotting reagent (Pierce) for 10 min. The blot was then exposed to chemiluminescent-sensitive Kodak
X-Omat AR film (Eastman Kodak). Image analysis was performed using the
public domain NIH Image program available on the internet at
rsb.info.nih.gov/nih-image.
Expression Plasmids and Transient Transfections--
The
mammalian expression plasmid pKH3 encoding mutant FAK (substitution of
Phe for Tyr-397; Y397F-FAK) was a gift from Dr. Hong-Chen Chen
(Institute for Biochemistry, National Chung Hsing University, Taiwan)
and has been described previously (22). Plasmid encoding green
fluorescent protein (GFP) was from Clontech, Palo
Alto, CA. Co-expression of FAK plasmid constructs with GFP was by
transient transfections of IMR-90 cells using the cationic lipid
reagent, LipofectAMINE (Invitrogen) according to manufacturer's instructions. Optimal ratio of DNA (µg) to LipofectAMINE (µl) was
determined to be ~1:5 for IMR-90 cells. Cells were incubated with
DNA-lipid complexes in serum-free RPMI medium for 4-5 h prior to introducing serum (10%) for 16-20 h. The next day, transfection medium was removed, and cells were again placed in serum-free medium
for 4 h prior to treatment with TGF- Immunofluorescence Staining--
Cells grown on glass coverslips
were initially rinsed with Dulbecco's modified Eagle's medium
(Invitrogen) for 30 s at ambient temperature and then fixed in 4%
formaldehyde for 5 min. Cells were then washed three times in
phosphate-buffered saline prior to permeabilization and after each
subsequent step. Permeabilization was performed in buffer consisting of
0.1% Triton in 50 mM PIPES (pH 7.0), 90 mM
HEPES (pH 7.0), 0.5 mM MgCl2, 0.5 mM EGTA, and 75 mM KCl for 30 s at room
temperature. Coverslips were sequentially incubated with mouse
monoclonal anti- Assay for Plasminogen Activator Inhibitor-1 Promoter
Activity--
Mink lung epithelial cells (MLECs-clone 32) stably
transfected with an 800-bp fragment ( TGF- Pharmacologic Inhibition of FAK/Src Kinase(s) Activity
Blocks TGF- TGF- Early TGF- FAK Autophosphorylation Is Required for TGF- TGF- Regulation of Myofibroblast Differentiation on Different ECM-coated
Surfaces--
Integrins bind to ECM ligands with varying and,
sometimes, overlapping specificities (29). To determine whether ECM
proteins differentially regulate myofibroblast differentiation, cells
were plated on tissue culture-treated dishes known to promote cell adhesion by integrin binding, dishes coated with ECMs (collagen I,
fibronectin, laminin and collagen IV), and dishes coated with a
non-integrin-binding polypeptide, poly-D-lysine. Fig.
7A demonstrates that the basal
expression of Myofibroblasts are active participants in normal wound repair, but
persistence of these cells in injured tissues prevents normal healing
and promotes a dysregulated repair process characterized by progressive
connective tissue remodeling and fibrosis (30). Transformation of
fibroblasts to the myofibroblast phenotype is primarily mediated by the
pro-fibrotic cytokine, TGF- Combinatorial signalings involving integrins and mitogenic growth
factor receptors have been well characterized, and synergistic activations of downstream signals such as mitogen-activated protein kinase activation are essential for anchorage-dependent
cell cycle progression (31). For example, growth factor-induced
activation of MEK, an activator of mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase, is dependent on
cell adhesion (32). The mechanisms by which cell adhesion/integrin
signals regulate cellular differentiation are less clear, although a
critical role for the ECM is well recognized (27, 33). Our data
strongly support a role for adhesion/integrin-dependent FAK
activation in the differentiation response to TGF-1 (TGF-
1) is a critical event in the
pathogenesis of human fibrotic diseases, but regulatory mechanisms for
this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-
1 and adhesion-dependent signals. TGF-
1-induced
myofibroblast differentiation of lung fibroblasts is blocked in
non-adherent cells despite the preservation of TGF-
receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-
1
induces tyrosine phosphorylation of focal adhesion kinase (FAK)
including that of its autophosphorylation site, Tyr-397, an effect that
is dependent on cell adhesion and is delayed relative to early Smad
signaling. Pharmacologic inhibition of FAK or expression of
kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit
TGF-
1-induced
-smooth muscle actin expression, stress fiber
formation, and cellular hypertrophy. Basal expression of
-smooth
muscle actin is elevated in cells grown on fibronectin-coated dishes
but is decreased on laminin and poly-D-lysine, a
non-integrin binding polypeptide. TGF-
1 up-regulates expression of
integrins and fibronectin, an effect that is associated with
autophosphorylation/activation of FAK. Thus, a safer and more effective
therapeutic strategy for fibrotic diseases characterized by persistent
myofibroblast activation may be to target this integrin/FAK pathway
while not interfering with tumor-suppressive functions of TGF-
1/Smad signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-smooth muscle actin
(
-SMA)1 protein (3, 4).
This "contractile phenotype" is functionally important for the
closure of cutaneous wounds (4). In addition, these cells represent an
"activated" fibroblast phenotype with high synthetic capacity for
extracellular matrix (ECM) proteins (5, 6), growth factors/cytokines
(7), growth factor receptors (8), integrins (9), and oxidants (10). The
presence/activation of myofibroblasts appears to be a consistent
finding in the pathology of human fibrotic diseases involving diverse
organ systems such as the lung, liver, and kidney (11). Thus,
persistent myofibroblast proliferation and/or survival represent a
pathologic repair process that can result in aberrant architectural
remodeling of tissues associated with end-stage fibrosis and organ failure.
1 (TGF-
1) has been linked to most of
the fibrotic diseases in humans (11). TGF-
1 has been shown to induce
myofibroblast differentiation both in vitro (12) and
in vivo (13), but regulatory mechanisms for this effect are
unclear. TGF-
receptor(s) activation results in the rapid recruitment and phosphorylation of the Smad proteins (14). We have
shown previously (10, 15) that TGF-
1 induces delayed protein
tyrosine phosphorylation in human lung fibroblasts, an effect that is
associated with extracellular H2O2 production
by a cell surface oxidase. ECM signals, either chemical or physical, can modulate TGF-
1-induced myofibroblast differentiation (16, 17),
suggesting a role for cell adhesion receptors in mediating/promoting myofibroblast differentiation.
1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. Porcine platelet-derived TGF-
1 was obtained from R & D
Systems, Minneapolis, MN. PP2
(4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and its inactive analog, PP3, were from Calbiochem. LipofectAMINE transfection reagent was from Invitrogen. All other reagents were from Sigma.
-SMA and
fibronectin were from Sigma. Phosphorylation-specific antibody to
Tyr-397-FAK was from BIOSOURCE International,
Carmillo, CA. Antibodies against the integrin subunits,
4 (sc-14008),
5 (sc-10729), and
1 (sc-8978) and fibronectin were from Santa Cruz
Biotechnology, Santa Cruz, CA.
1 (2 ng/ml) for 24 h.
Cells were then analyzed by immunofluorescence staining.
-SM antibody (Dako Corp., Carpenteria, CA) and
rhodamine-labeled anti-mouse antibody (Jackson ImmunoResearch, West
Grove, PA), each for 60 min at room temperature. Cells were then
visualized and photographed using a Zeiss fluorescence microscope.
700 to +71) of the 5' end of the human plasminogen activator inhibitor-1 (PAI-1) gene fused to the
firefly luciferase reporter gene in a p19LUC-based vector containing
the neomycin resistance gene from pMAMneo was a gift from Dr. Dan
Rifkin (Department of Cell Biology, New York University Medical Center,
New York). Assays were performed as described previously (28) with
minor modifications.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-induced Myofibroblast Differentiation Is Dependent on Cell
Adhesion--
In cultured human lung fibroblasts (IMR-90), TGF-
1 (2 ng/ml) induces a time-dependent steady increase in
-SMA
protein expression (Fig. 1A).
The up-regulation of
-SMA was dose-dependent up to 10 ng/ml and remained elevated for up to 5 days without repeat dosing
(data not shown), consistent with a stably differentiated state. This
effect of TGF-
1 was associated with morphological changes of
cellular hypertrophy and well formed actin stress fibers (Fig.
2A, middle panels),
characteristic of myofibroblasts. To examine the role of cell adhesive
events to modulate TGF-
1-induced myofibroblast differentiation, the
effect of TGF-
1 on cells stimulated under non-adherent conditions
was examined. Cells were detached by EDTA treatment, dispersed by
pipetting, and washed with Ca2+- and
Mg2+-containing Hanks' balanced salt solution. Cells were
then stimulated with TGF-
1 (2 ng/ml) in suspension, and the effect
on
-SMA protein expression was assessed at 24 h. TGF-
1 was
unable to induce
-SMA expression in suspended cells (Fig.
1B). The inability to up-regulate
-SMA expression in
non-adherent cells following TGF-
1 stimulation is not explained by
loss of cell viability because these cells are capable of re-attaching
and growing when re-plated on culture dishes; moreover, up-regulation
of
-SMA expression was partially restored when cells treated with
TGF-
1 were returned to an adherent state (data not shown). This
suggests that adhesion-dependent signal(s) mediate and/or
maintain the myofibroblast differentiated state induced by
TGF-
1.
View larger version (54K):
[in a new window]
Fig. 1.
A, time course of the induction of
-SMA expression by TGF-
1 in human lung fibroblasts.
Quiescent, adherent human lung fibroblasts (IMR-90 cells) were
stimulated with TGF-
1 (2 ng/ml) under serum-free conditions, and
cell lysates were obtained at the times indicated. Cell lysates were
then subjected to SDS-PAGE followed by immunoblotting with antibodies
against
-SMA and
-actin. B, cell adhesion is required
for the TGF-
1-induced up-regulation of
-SMA expression. Quiescent
IMR-90 cells were stimulated with TGF-
1 (2 ng/ml for 24 h in
serum-free medium) under conditions where cells were adherent or
non-adherent (suspended). Cell lysates were obtained and
subjected to SDS-PAGE followed by immunoblotting with antibodies
against
-SMA and
-actin. Control, lane C.
View larger version (83K):
[in a new window]
Fig. 2.
A, pharmacologic inhibition
of FAK/Src kinases with PP2 blocks TGF- 1-induced cell hypertrophy
and formation of actin stress fibers in human lung fibroblasts.
Quiescent, adherent human lung fibroblasts (IMR-90 cells) were
stimulated with TGF-
1 (2 ng/ml) in the absence/presence of PP2 (10 µM) for 24 h. Cell morphology was photographed by
phase contrast microcopy (upper panels). Immunofluorescent
cell staining was performed with monoclonal
-SMA antibody
(bottom panel). B, PP2, but not PP3, inhibits
TGF-
1-induced
-SMA expression in lung fibroblasts. Quiescent,
adherent IMR-90 cells were stimulated with TGF-
1 (2 ng/ml) in the
absence and presence of PP2 (1, 5, and 10 µM), PP3
(inactive analog of PP2; 10 µM), and vehicle control
(DMSO) for 24 h. Cell lysates were then obtained and
subjected to SDS-PAGE followed by immunoblotting with antibodies
against
-SMA and
-actin.
1-induced Myofibroblast
Differentiation--
Adhesion-dependent signal(s) may
be transmitted by integrins, and these adhesion receptors activate FAK
and Src kinases at focal adhesion complexes (23, 24). PP2 is an
inhibitor of both Src kinases and FAK (23). To determine whether
FAK/Src kinase(s) may be required for TGF-
1-induced myofibroblast
differentiation, the effect of PP2 on the formation of actin stress
fibers was examined. Fig. 2A demonstrates that PP2 (10 µM) is able to inhibit completely the formation of stress
fibers as well as the associated cellular hypertrophy induced by
TGF-
1. The effect of varying doses of PP2 and its inactive analog,
PP3, on the expression of
-SMA was also examined. Fig. 2B
shows a dose-dependent inhibition of TGF-
1-induced
-SMA expression by PP2, with mild inhibition at 1 µM,
greater than 50% inhibition at 5 µM and complete
inhibition to basal levels at 10 µM. PP3 (10 µM) and vehicle control (Me2SO) had no effect
on TGF-
1-induced
-SMA expression. These results suggest that
FAK/Src kinase(s) activity may be required for differentiation of
adherent lung fibroblasts to myofibroblasts in response to TGF-
1.
1 Induces Delayed, Adhesion-dependent FAK
Autophosphorylation--
Based on the finding that FAK/Src kinase(s)
may be involved in TGF-
1-induced myofibroblast differentiation, the
ability of TGF-
1 to activate this pathway in lung fibroblasts was
examined. Activation of Src kinases, measured by in vitro
kinase assays of Src, Yes, and Fyn autophosphorylation, in cells
stimulated with TGF-
1 was not observed at early time points (15 and
30 min), and delayed autophosphorylation of Src-Y416 did not reveal
significant and consistent responses. However, TGF-
1 consistently
induced phosphorylation of FAK on Tyr-397, its major
autophosphorylation site, in a time-dependent manner (Fig.
3A). These results were further confirmed by immunoprecipitation of cell lysates obtained 16 h following TGF-
1 (2 ng/ml) and immunoblotting with
anti-phosphotyrosine antibody (PY20) and vice versa. In addition to
demonstrating tyrosine phosphorylation of FAK, these studies indicated
that this effect of TGF-
1 is inhibitable by PP2 but not by PP3 (Fig.
3B). The delayed induction of FAK autophosphorylation by
TGF-
1 suggested that new protein synthesis might be required to
mediate this effect. Fig. 3C demonstrates that when cells
are co-incubated with cycloheximide (1 µM; a protein
synthesis inhibitor) or actinomycin-D (0.1 µM; an
inhibitor of gene transcription), TGF-
1-induced FAK
autophosphorylation is blocked. The time course of FAK
autophosphorylation was compared with the up-regulation of
-SMA
induced by TGF-
1. FAK autophosphorylation was increased almost
2-fold by 6 h while significant
-SMA induction required about
12 h and steady increases thereafter (Fig. 3, C and
D). These results demonstrate that TGF-
1 induces FAK
autophosphorylation by a mechanism that requires new protein synthesis,
and this effect precedes the induction of
-SMA by TGF-
1.
View larger version (46K):
[in a new window]
Fig. 3.
A, TGF- 1 induces
time-dependent induction of FAK autophosphorylation in
human lung fibroblasts. Quiescent, adherent IMR-90 cells were
stimulated with TGF-
1 (2 ng/ml) in serum-free conditions for the
times indicated. Cell lysates were then obtained and subjected to
SDS-PAGE followed by immunoblotting with a phosphorylation
site-specific (Tyr-397) antibody to FAK. The blot was then stripped and
re-probed with an antibody against FAK. B, delayed
autophosphorylation of FAK by TGF-
1 is sensitive to inhibition by
PP2 but not PP3. Quiescent, adherent IMR-90 cells were stimulated with
TGF-
1 (2 ng/ml for 16 h) in the absence and presence of the
FAK/Src kinases inhibitor, PP2 (10 µM), or its inactive
analog, PP3 (10 µM). Cell lysates were immunoprecipitated
(IP) with anti-FAK antibody followed by SDS-PAGE and
immunoblotting (IB) with anti-phosphotyrosine
(PY-20) antibody and vice versa as indicated (upper
panels). Non-immunoprecipitated cell lysates were subjected to
SDS-PAGE, and immunoblotting was performed with antibodies against
397Y-phospho-FAK and total FAK
protein (bottom panels). C, effect of
cycloheximide and actinomycin-D on TGF-
1-induced FAK
autophosphorylation. Adherent, quiescent IMR-90 cells were stimulated
with/without TGF-
1 (2 ng/ml) in the presence/absence of
cycloheximide (1 µM, CHX) or actinomycin-D
(0.1 µM, Act-D) for 12 h. Cell lysates
were then obtained and subjected to SDS-PAGE followed by immunoblotting
with antibodies to 397Y-phospho-FAK
and total FAK protein. D, time course of TGF-
1-induced
FAK autophosphorylation. Densitometric analyses of the ratio of
phospho-specific FAK (Tyr-397) to total FAK plotted against time (from
A). E, time course of TGF-
1-induced
-SMA
induction. Densitometric analyses of the ratio of
-SMA to
-actin
plotted against time (from Fig. 1A).
Receptor Signaling Does Not Require Cell
Adhesion--
TGF-
receptor(s) signaling is primarily mediated by
the rapid phosphorylation and activation of the Smad proteins (14). To
address the question directly as to whether early TGF-
1 signaling events require cellular adhesion, TGF-
1-induced Smad2
phosphorylation was examined in adherent and suspended cells. Rapid
phosphorylation of Smad2 was observed within 15 min of TGF-
1
stimulation of cells under both conditions (Fig.
4A). Interestingly, Smad2
phosphorylation in response to TGF-
1 was higher in
suspended cells (Fig. 4, A and B). In contrast,
delayed autophosphorylation of FAK was almost completely abrogated in
suspended cells (Fig. 4, A and C), consistent with the requirement for cell adhesion to mediate FAK
autophosphorylation/activation. To determine whether
Smad-dependent transcriptional events were altered under
non-adherent conditions, the effect of TGF-
1 to induce activation of
the plasminogen activator inhbibitor-1 (PAI-1) gene was assessed
utilizing the stably transfected PAI-1 gene promoter-reporter MLECs.
Similar to the effects on Smad2 phosphorylation, PAI-1 promoter
activity in response to TGF-
1 was higher in suspended versus adherent cells, 11.9- versus 7.5-fold,
respectively, relative to adherent control cells (Fig. 4D).
This suggests that loss of cell adhesion does not interfere
with the ability of TGF-
1 to bind/activate its receptor and mediate
early Smad signaling; furthermore, the transcriptional activation of a
well characterized TGF-
1-responsive gene (PAI-1) is not altered
under non-adherent conditions.
View larger version (34K):
[in a new window]
Fig. 4.
A, cell adhesion is required for delayed
FAK autophosphorylation but not for the rapid phosphorylation of Smad2
induced by TGF- 1 in human lung fibroblasts. Adherent and
non-adherent (suspended) human lung fibroblasts (IMR-90)
were incubated in serum-free medium in the absence (control, lane
C) or presence of 2 ng/ml TGF-
1 (TGF). Cell lysates
obtained at early (15 min) and delayed (16 h) time points were
subjected to SDS-PAGE. Immunoblotting was performed with antibodies
against phospho-Smad2,
397Y-phospho-FAK, phospho-tyrosine
(PY-20), and FAK. B, rapid Smad2 phosphorylation
by TGF-
1 is enhanced in suspended cells. Densitometric analyses of
the ratio of phospho-Smad2 to Smad2 in adherent and suspended cells
stimulated with TGF-
1 (2 ng/ml) for 15 min (represented in
A, top panel). C, delayed FAK
autophosphorylation by TGF-
1 is inhibited in suspended cells.
Densitometric analyses of the ratio of phospho-FAK to total FAK in
adherent and suspended cells stimulated with TGF-
1 (2 ng/ml) for
16 h (represented in A, bottom panels).
D, TGF-
1-induced activation of the PAI-1 promoter is
enhanced in suspended cells. Adherent and non-adherent
(suspended) MLECs stably transfected with the PAI-1 promoter
gene linked to a luciferase reporter were stimulated in the absence
(control) or presence of 2 ng/ml TGF-
1.
Promoter/luciferase activities on cell lysates were determined 6 h
following stimulation with TGF-
1. Values are mean ± S.D.,
n = 3.
1-induced
Myofibroblast Differentiation--
Because protein kinase inhibitors
have the potential to target multiple protein kinases, the specific
role of FAK autophosphorylation in TGF-
1-induced myofibroblast
differentiation was examined by expressing kinase-deficient FAK in
these cells. Kinase-deficient FAK (Y397F-FAK, substitution of Phe for
Tyr-397) was co-expressed with green fluorescent protein (GFP;
transfected in 1:10 ratio by weight) by transient transfections. Cells
expressing mutant FAK (GFP-encoding "green" cells) were incapable
of expressing
-SMA (red fluorescence), whereas untransfected cells
(GFP-negative) in the same field expressed high levels of
-SMA in
response to 2 ng/ml TGF-
1 (Fig.
5A). To confirm these findings
at the level of protein expression, cells were transfected with
Y397F-FAK or "mock"-transfected, and the effect of TGF-
1 on
-SMA expression was determined by Western blotting. In cells
transfected with mutant FAK (Y397F-FAK), protein expression of
-SMA
in response to TGF-
1 was markedly diminished (Fig. 5B).
Together, these results indicate that autophosphorylation of FAK on
Tyr-397 is required for induction of myofibroblast differentiation by
TGF-
1.
View larger version (52K):
[in a new window]
Fig. 5.
A, expression of kinase-deficient
FAK inhibits the formation of actin stress fibers by TGF- 1 in human
lung fibroblasts. IMR-90 cells were transfected with either
kinase-deficient FAK (substitution of Tyr-397 with Phe,
Y397F-FAK) or without plasmid (transfection reagents alone,
control transfection). To localize individual cells that had
been successfully transfected, a plasmid encoding green fluorescent
protein (GFP) was cotransfected in a 10:1 (w/w) ratio. Cells
were stimulated with TGF-
1 (2 ng/ml) in serum-free medium for
24 h, and immunofluorescent cell staining was performed with a
monoclonal antibody against
-SMA. The same fields were photographed
under green and red fluorescent light for GFP and
-SMA, respectively
(inner panels); the merged image is also shown (right
panels). B, expression of kinase-deficient FAK inhibits
the TGF-
1-induced increase in
-SMA. Cells were transiently
transfected with a kinase-deficient FAK mutant (Y397F-FAK) plasmid or
without plasmid (transfection reagents alone; control
transfection) prior to incubation in the absence (lane
C) and presence (TGF) of TGF-
1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to SDS-PAGE followed by
immunoblotting with antibodies against
-SMA and
-actin.
1 Induces Expression of Fibronectin and the Integrin
Receptor Subunits
4,
5, and
1 in Association with Enhanced FAK
Autophosphorylation--
The observation that
adhesion-dependent signals involving FAK
autophosphorylation are required for TGF-
1-induced
-SMA
expression and the relatively delayed nature of the response suggested
a secondary effect of this cytokine to modulate adhesion
signaling. Therefore, we examined the ability of TGF-
1 to alter the
protein expression of fibronectin and the integrin receptor subunits,
4,
5, and
1 in these
cells. All three integrin subunits as well as fibronectin were
up-regulated by TGF-
1 in a time-dependent manner (Fig.
6). These effects of TGF-
1 are
associated with adhesion-dependent activation of FAK (Figs.
3A and 4C). This provides further support for the
concept that alterations in cell adhesive capacity, by up-regulated
expression/activation of integrin receptors and fibronectin by
TGF-
1, may be important in FAK activation and myofibroblast differentiation.
View larger version (69K):
[in a new window]
Fig. 6.
TGF- 1 induces the
expression of integrin subunits
(
4,
5, and
1) and fibronectin in adherent human
lung fibroblasts. Quiescent, adherent human lung fibroblasts
(IMR-90) were treated with TGF-
1 (2 ng/ml) for varying times as
indicated. Cell lysates were then obtained and subjected to SDS-PAGE
followed by immunoblotting with specific antibodies against the
proteins indicated.
-SMA varies significantly depending on the ECM protein
substrate where cells are grown. Cells grown on fibronectin expressed
the highest level of
-SMA, whereas expression on laminin and
poly-D-lysine was decreased (Fig. 7A).
-SMA
expression also correlated with levels of both FAK autophosphorylation
and total FAK, which appeared to be enhanced on fibronectin (results not shown). These results further support a role for integrin signaling
in myofibroblast differentiation and suggest that specific integrin-ECM
interactions may differentially regulate this process. When adherent
cells on different ECMs are stimulated with TGF-
1, the ability to
differentiate into myofibroblasts is restored under all conditions
(Fig. 7B), supporting the concept that the ability of
TGF-
1 to induce expression of integrin/ECM proteins and activate FAK
may be critical for this differentiation response.
View larger version (44K):
[in a new window]
Fig. 7.
A, expression of -SMA in human
lung fibroblasts grown on different ECM substrates. IMR-90 cells
were plated at the same density on cell culture-treated dishes
(tissue culture plastic), dishes coated with various ECM
proteins (collagen I, fibronectin, laminin, and
collagen IV) and a non-integrin-binding adhesion polypeptide
(poly-D-lysine). At ~90%
confluency, cells were serum-deprived for 48 h and cell lysates
obtained. Cell lysates were then subjected to SDS-PAGE followed by
immunoblotting with antibodies against
-SMA and
-actin.
B, effect of TGF-
1 on induction of
-SMA on ECM
substrates. Quiescent cells were stimulated in the presence/absence of
TGF-
1 (2 ng/ml) for 24 h prior to cell lysis and Western blotting as
described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (12); however, molecular mechanisms for
this effect are unclear. An understanding of these mechanisms is
critical for the development of new therapeutic approaches for these
otherwise fatal and largely treatment-unresponsive disorders. In this
study, we demonstrate that cell adhesion/integrin-dependent
autophosphorylation and activation of FAK is required for the induction
of myofibroblast differentiation by TGF-
1.
1. However, in
contrast to the model for adhesion-dependent proliferative
responses, growth factor receptor(s) activation does not appear to
require simultaneous integrin activation. Several lines of evidence
support this notion. First, we found efficient and rapid activation of
TGF-
receptor(s) signaling of Smad phosphorylation even in the
absence of cell adhesion. In fact, the phosphorylation of
Smad2 by TGF-
1 was enhanced in non-adherent cells;
moreover, these changes were associated with preserved (and even
enhanced) PAI-1 promoter activity in non-adherent cells. Second,
adhesion signals provided several hours after early TGF-
receptor(s)-mediated signaling had been transduced was, at least
partially, sufficient to induce differentiation. Third, FAK activation
in response to TGF-
1 is adhesion-dependent but occurs in
a delayed manner relative to early post-receptor signaling. FAK
autophosphorylation, although appreciable at 3 h post-stimulation
with TGF-
1, is maximal at 16 h. We have observed previously
(15) a similar response to this cytokine in the induction of protein
tyrosine phosphorylation in lung fibroblasts but had not identified FAK
as a potential candidate molecule for this regulation. Finally, the
phosphorylation of FAK is temporally associated with the up-regulation
of integrin receptors and fibronectin, suggesting that these effects of
TGF-
1 may be related. Cumulatively, our data suggest a "serial"
regulatory pathway by which TGF-
1 modulates adhesion signaling that
is critical for the induction and, perhaps more importantly, for the
maintenance of a stably differentiated myofibroblast phenotype. It is
important to recognize that adhesion signal(s), although necessary, are
unlikely to be sufficient for full expression of this phenotype.
Plating cells on fibronectin (in the absence of TGF-
1) only
partially promoted
-SMA expression. Components of early TGF-
receptor(s) signaling may function in a "parallel" (or synergistic)
manner to transmit other signals for myofibroblast differentiation (see
Fig. 8).
View larger version (29K):
[in a new window]
Fig. 8.
Schematic of the proposed
adhesion-dependent and -independent regulatory pathways
involved in myofibroblast differentiation by
TGF- 1 in human lung fibroblasts. TGF-
receptor(s) signaling is primarily mediated by rapid
phosphorylation/activation of Smad proteins, which occurs by an
adhesion-independent mechanism. Other unidentified non-Smad pathways
may also be activated early post-TGF-
receptor(s) activation. FAK
autophosphorylation/activation is delayed relative to Smad signaling
and is associated with TGF-
1-induced expression of both integrin
subunits and fibronectin/collagens. Integrin signaling via FAK
autophosphorylation/activation is essential for induction/maintenance
of the stably differentiated myofibroblast phenotype.
The findings of this study also support the concept that specific
integrin-ECM interactions may differentially regulate this cell
adhesion-dependent pathway. The basal expression of -SMA was lower in cells grown on laminin, relative to other integrin-binding ECM surfaces, and was highest on fibronectin. The observation that
fibronectin promotes this differentiation response is consistent with
reports that the ED-A spliced variant of fibronectin is essential for
TGF-
1-induced myofibroblast differentiation (16). Interestingly, this variant of fibronectin is preferentially produced by fibroblasts grown on rigid planar surfaces and in response to TGF-
1 (34, 35).
This may explain the relatively strong induction of
-SMA on
"rigid" tissue culture dishes even in the absence of ECM coating of
dishes. Furthermore, the ability of TGF-
1 to up-regulate fibronectin and integrin receptors may serve to augment and sustain this fibroblast phenotype by promoting cell adhesive interactions and altering the
biochemical/biomechanical microenvironment of the cell. We have shown
recently (36) that biochemical, and potentially biophysical, alterations of the ECM can be induced in an inflammatory milieu by
activated myofibroblasts via the generation of extracellular hydrogen
peroxide and oxidative cross-linking of ECM proteins. More recent data
from our laboratory also suggest that such oxidative modifications
occur preferentially on
fibronectin.2 Thus, complex
and dynamic changes in the cellular microenvironment influence the
ultimate phenotypic fate of cells and, eventually, the outcome of the
tissue injury and repair process.
The involvement of cell adhesion/integrin-FAK signaling in promoting
the myofibroblast phenotype has important implications for potential
new approaches to the therapy of fibrotic diseases. Although blocking
TGF- signaling is likely to be of benefit in abrogating the fibrotic
response (37), it could also prove detrimental because this pathway
functions in the control of epithelial cell proliferation and tumor
suppression (38). This is further complicated by the reported increased
incidence of epithelial cell malignancies in fibrotic diseases (39).
Thus, the identification of a non-tumor-suppressive pathway critical
for myofibroblast differentiation provides an ideal target for the
development of new therapies for fibrotic diseases characterized by
TGF-
1 and myofibroblast activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Seth Corey (Department of Pediatrics, University of Texas, MD Anderson Cancer Center, Houston, TX) for assistance with Src kinase assays. We also thank Dr. Hong-Chen Chen (Institute for Biochemistry, National Chung Hsing University, Taiwan) for providing the FAK-Y397F mutant plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant HL-67967 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: University of Michigan Medical School, Division of Pulmonary and Critical Care Medicine, 6301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 734-936-9371; Fax: 734-764-4556; E-mail: vjt@umich.edu.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M208544200
2 V. J. Thannickal and P. E. Thomas, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SMA, smooth muscle
actin;
TGF-1, transforming growth factor-
1;
FAK, focal adhesion
kinase;
ECM, extracellular matrix;
GFP, green fluorescent protein;
PIPES, 1,4-piperazinediethanesulfonic acid;
PAI-1, plasminogen
activator inhibitor-1;
MLECs, mink lung epithelial cells.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sappino, A. P., Schurch, W., and Gabbiani, G. (1990) Lab. Invest. 63, 144-161[Medline] [Order article via Infotrieve] |
2. | Skalli, O., Schurch, W., Seemayer, T., Lagace, R., Montandon, D., Pittet, B., and Gabbiani, G. (1989) Lab. Invest. 60, 275-285[Medline] [Order article via Infotrieve] |
3. | Gabbiani, G., Ryan, G. B., and Majne, G. (1971) Experientia (Basel) 27, 549-550 |
4. | Grinnell, F. (1994) J. Cell Biol. 124, 401-404[Medline] [Order article via Infotrieve] |
5. |
Ignotz, R. A.,
and Massague, J.
(1986)
J. Biol. Chem.
261,
4337-4345 |
6. | Zhang, K., Rekhter, M. D., Gordon, D., and Phan, S. H. (1994) Am. J. Pathol. 145, 114-125[Abstract] |
7. |
Finlay, G. A.,
Thannickal, V. J.,
Fanburg, B. L.,
and Paulson, K. E.
(2000)
J. Biol. Chem.
275,
27650-27656 |
8. | Thannickal, V. J., Aldweib, K. D., Rajan, T., and Fanburg, B. L. (1998) Biochem. Biophys. Res. Commun. 251, 437-441[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Heino, J.,
Ignotz, R. A.,
Hemler, M. E.,
Crouse, C.,
and Massague, J.
(1989)
J. Biol. Chem.
264,
380-388 |
10. |
Thannickal, V. J.,
and Fanburg, B. L.
(1995)
J. Biol. Chem.
270,
30334-30338 |
11. |
Border, W. A.,
and Noble, N. A.
(1994)
N. Engl. J. Med.
331,
1286-1292 |
12. | Desmouliere, A., Geinoz, A., Gabbiani, F., and Gabbiani, G. (1993) J. Cell Biol. 122, 103-111[Abstract] |
13. |
Sime, P. J.,
Xing, Z.,
Graham, F. L.,
Csaky, K. G.,
and Gauldie, J.
(1997)
J. Clin. Invest.
100,
768-776 |
14. |
Jayaraman, L.,
and Massague, J.
(2000)
J. Biol. Chem.
275,
40710-40717 |
15. |
Thannickal, V. J.,
Aldweib, K. D.,
and Fanburg, B. L.
(1998)
J. Biol. Chem.
273,
23611-23615 |
16. |
Serini, G.,
Bochaton-Piallat, M. L.,
Ropraz, P.,
Geinoz, A.,
Borsi, L.,
Zardi, L.,
and Gabbiani, G.
(1998)
J. Cell Biol.
142,
873-881 |
17. |
Arora, P. D.,
Narani, N.,
and McCulloch, C. A.
(1999)
Am. J. Pathol.
154,
871-882 |
18. | Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve] |
19. | Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5192-5196[Abstract] |
20. |
Giancotti, F. G.,
and Ruoslahti, E.
(1999)
Science
285,
1028-1032 |
21. | Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 1680-1688[Abstract] |
22. |
Chen, H. C.,
Appeddu, P. A.,
Isoda, H.,
and Guan, J. L.
(1996)
J. Biol. Chem.
271,
26329-26334 |
23. |
Salazar, E. P.,
and Rozengurt, E.
(2001)
J. Biol. Chem.
276,
17788-17795 |
24. |
Schaller, M. D.,
Hildebrand, J. D.,
and Parsons, J. T.
(1999)
Mol. Biol. Cell
10,
3489-3505 |
25. | Halevy, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D., and Lassar, A. B. (1995) Science 267, 1018-1021[Medline] [Order article via Infotrieve] |
26. | Skapek, S. X., Rhee, J., Spicer, D. B., and Lassar, A. B. (1995) Science 267, 1022-1024[Medline] [Order article via Infotrieve] |
27. |
Adams, J. C.,
and Watt, F. M.
(1993)
Development
117,
1183-1198 |
28. | Abe, M., Harpel, J. G., Metz, C. N., Nunes, I., Loskutoff, D. J., and Rifkin, D. B. (1994) Anal. Biochem. 216, 276-284[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Plow, E. F.,
Haas, T. A.,
Zhang, L.,
Loftus, J.,
and Smith, J. W.
(2000)
J. Biol. Chem.
275,
21785-21788 |
30. | Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C., and Brown, R. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 349-363[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Schwartz, M. A.
(1997)
J. Cell Biol.
139,
575-578 |
32. |
Renshaw, M. W.,
Ren, X. D.,
and Schwartz, M. A.
(1997)
EMBO J.
16,
5592-5599 |
33. | Roskelley, C. D., Srebrow, A., and Bissell, M. J. (1995) Curr. Opin. Cell Biol. 7, 736-747[CrossRef][Medline] [Order article via Infotrieve] |
34. | Dugina, V., Fontao, L., Chaponnier, C., Vasiliev, J., and Gabbiani, G. (2001) J. Cell Sci. 114, 3285-3296[Medline] [Order article via Infotrieve] |
35. | Borsi, L., Castellani, P., Risso, A. M., Leprini, A., and Zardi, L. (1990) FEBS Lett. 261, 175-178[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Larios, J. M.,
Budhiraja, R.,
Fanburg, B. L.,
and Thannickal, V. J.
(2001)
J. Biol. Chem.
276,
17437-17441 |
37. |
Wang, Q.,
Wang, Y.,
Hyde, D. M.,
Gotwals, P. J.,
Koteliansky, V. E.,
Ryan, S. T.,
and Giri, S. N.
(1999)
Thorax
54,
805-812 |
38. |
Like, B.,
and Massague, J.
(1986)
J. Biol. Chem.
261,
13426-13429 |
39. |
Hubbard, R.,
Venn, A.,
Lewis, S.,
and Britton, J.
(2000)
Am. J. Respir. Crit. Care Med.
161,
5-8 |