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Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse
interstitial lung disease of unknown cause, characterized by
progressive fibrosis of lung
parenchyma[1,2]. A major process in fibrosis development is the differentiation of
fibroblastic cells into myofibroblasts which express
α-smooth muscle actin (α-SMA). Since myofibroblasts are virtually
absent in normal lungs, their differentiation is one of the
decisive events in the pathogenesis of progressive lung
fibrosis[3_5] and are largely responsible for the accumulation
of extracellular matrix (ECM). Therefore, it is very important
to identify the signaling pathway responsible for
myofibro-blasts differentiation and accumulation in the lung tissue.
Transforming growth factor
(TGF)-β1 has been widely recognized as a key fibrogenic cytokine and has been
demonstrated to activate fibroblasts differentiation into
myofibroblasts in vitro and in
vivo[6,7], and stimulate ECM
production[8]. The major signaling pathway of
TGF-β1 is through its transmembrane receptor serine/threonine kinases
and activate the cytoplasmic Smad
proteins[9,10]. Among those, Smad2 and Smad3 are receptor-activated Smad
proteins (R-Smad) phosphorylated by TGF-β receptor
(TbR)type I, and subsequently form a heteromeric
complex with Co-Smad (Smad4),
respectively[9,10]. Such complexes
subsequently translocate into the nuclear to regulate the
expression of target genes. In addition to these positively acting
Smad, Smad7 antagonizes TGF-β1 signal by interacting with
the receptor complex, and preventing phosphorylation of
R-Smad[9,10]. All Smad proteins have conserved amino-terminal
MH1 and carboxy-terminal MH2 domains. The MH1 domain
is important for mediating the DNA-binding activity of Smad.
The MH2 has a Serine-Serine-X-Serine (SSXS )conserved
domain which is phosphorylated by
TbR-I[11]. Although Smad2 is structurally highly similar to Smad3, and both Smad2
and Smad3 are phosphorylated directly by the TβR-I, they
do not share similar DNA-binding activity. This notable
difference in transcriptional activation by Smad2 and Smad3
involves an insertion of 30 amino acids in the N-terminal
MH1 domain of Smad2[12]. Studies have found that the
targeted deletion of Smad2 or Smad3 genes in mice has
revealed distinct developmental roles for these closely
related Smad[13,14]. Studies about fibroblasts derived from
embryos null for either Smad3 or Smad2 also revealed that
TGF-β-regulated genes depend on either Smad2 or Smad3 or
both[15]. The role of individual Smad signaling in
myofibro-blasts differentiation is still unclear.
TGF-β1 plays important roles in a variety of
developmental and pathological
processes[11]. It is obvious that any attempt to reverse fibrosis by using neutralizing antibodies
to block the whole function of TGF-β1 will result in disaster,
therefore, it will be more applicable that we select a
down-stream point of this pathway to more specifically block the
process of fibrosis.
Materials and methods
Cell culture Human fetal lung fibroblasts were purchased
from Cell Center, Chinese Academy of Medical Sciences
(Beijing, China) and Peking Union Medical College (China)
The cells were grown in Dulbecco's modified Eagle's
medium, supplemented with 10% fetal bovine serum (FBS),
antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin)
and 25 mmol/L HEPES under conditions of humidified 5%
CO2/95% air at 37 °C.
Antibodies and reagents Active human recombinant
TGF-β1 was purchased from Peprotech (London, UK); mouse
monoclonal anti-α-SMA antibody from DAKO (Glostrup,
Denmark); mouse monoclonal anti-flag M2 antibody from
Sigma (St Louis, Missouri, USA).
Plasmids: The human α-SMA promoter gene was cloned
by PCR from human genomic DNA with primers
5'-GAATT-CGAGACGAGATTTGGG-3'(_895/_875) and
5'-GTGGTGTT-CAGGGAAGCTGA-3' (+9/_11). It was inserted into vector
pGal3-basic (Promega,Madison,WI,USA) at the
MluI/XhoI site to form the α-SMA-luciferase(Luc) fusion plasmid
p895-Luc. pFlag-Smad7, pFlagSmad3,
pFlagSmad3mut and pFlagSmad2 were expression vectors for Smad7, Smad3,
Smad3mut and Smad2, respectively. These vectors were kindly
supplied by Dr Dijke-Peter TEN (Ludwig Institute for Cancer
Research, Uppsala, Sweden), Dr Harvey F LODISH
(Depart-ment of Chemistry and Biochemistry, University of Boulder,
Colorado, USA) and Dr Joan MASSAGUE (Sloan Kettering
Memorial Cancer Center, New York, NY, USA).
Smad3mut is a dominant negative mutant with 3 C-terminal serine
phosphorylation sites changed to
alanines[16]. pFlag-Smad2 was used as the template for obtaining
Smad2mut by PCR and also has 3 C-terminal serine phosphorylation sites changed to
alanines. Smad2mut was subcloned in vector pFlag-Smad2 to
form the pFlagSmad2mut. pSV-β-galactosidase vector was
used as the standard for transfection efficiency.
Transfection of cells and reporter gene assay
Transient cotransfections were performed using the
FuGENETM 6.0 reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA)
according to the manufacturer's instructions. Supercoiled
DNA was isolated with an endotoxin-free Qiagen column kit
(Qiagen, Inc Valencia, CA). Unless otherwise indicated, cells
were seeded in 24-well plates at a density of
2.5×104 cells per well in medium containing 10% FBS. Twenty-four hours later,
the indicated DNA plasmids were transfected in medium
containing 0.1% FBS. The total amount of plasmid DNA within
each experiment was kept constant by the addition of
appropriate empty vectors. For reporter gene assay,
pSV-β-galactosidase control vector was cotransfected for normalization.
Eight hours following transfection, cells were incubated with
TGF-β1. After 24 h the cells were harvested, and luciferase
activity and β-galactosidase activity was measured by
using the luciferase assay system and β-galactosidase assay
kit from Promega, respectively. There were 3 independent
experiments and triplicates every time for each plasmid. The
results are expressed as relative luciferase activity (RLA).
The RLA of the control group is 1.
Western blot analysis Transient cotransfections were
performed as above. Eight hours following transfection, the
cells were incubated with TGF-β1 for 3 d, then whole cell
lysates were prepared with lysis buffer containing protease
inhibitor. In the animal experiment, lung tissues were
homogenized and proteins were extracted in lysis buffer. The
protein concentrations were measured by using protein
assay reagent kit (Pierce, Rockford, IL, USA). Fifty
mg cell protein and 50 mg tissue protein were electrophoresed on
12% SDS-polyacrylamide gel, and wet transferred onto
polyvinylidene difluoride membrane, respectively. The
membranes containing the cell proteins were incubated
overnight at 4oC with anti-α-SMA antibody, anti-Smad4 antibody
and anti-Flag M2 antibody, respectively. The membranes
containing tissue proteins were incubated with
anti-α-SMA antibody. Then the membranes were incubated with
horseradish peroxidase-conjugated secondary antibody for 1 h at
room temperature. Signals were visualized with
chemiluminescence reagents (Pierce, Rockford, IL,USA).
Quantification of the bands was performed by using densitometric
analysis software-Quantity One (Bio-Rad, Hercules, CA, USA)
Animal model To further confirm the role of Smad3 on
myofibroblasts differentiation in vivo, we used Smad3
knockout mice, which were bred from heterozygous mice of a
targeted disruption of exon8 of the Smad3 gene, kindly
supplied by Dr Chu-xia DENG (NIH, Washington, USA). The
genotype of the mice was determined by PCR analysis of tail
DNA. The mice were maintained on standard chow and
allowed access to food and water ad libitum. The mice (8_10
weeks of age) were randomly selected for either bleomycin
or saline vehicle control treatment. The average body weight
for wild-type mice was ~27 g and for the Smad3 knockout
mice, ~17 g. Administration of bleomycin (Nippon Kayaku
Co Ltd, Tokyo, Japan) or saline vehicle was performed by a
constant subcutaneous infusion through a micro-osmotic
pump (model 1007D; Alza, Palo Alto, CA, USA) from d 0_7.
In the mice anesthetized with pentobarbital sodium (~50
mg/g, ip), the mini pump loaded with bleomycin (0.15 mg/g
mouse body weight dissolved in saline) was implanted
subcutaneously on the back of the mice, slightly posterior to the
scapulae[17]. The mice were killed after 4 weeks, and the
pumps were examined to ensure that they had delivered the
entire dosage in each mouse. No significant changes of the
body weight were observed after 4 weeks' treatment. The
lungs were removed for protein extraction, measurements of
hydroxyproline content, or fixation for histological analysis.
Histology and immunohistochemistry The mouse lungs
were fixed by perfusion with 10% formaldehyde before
routine processing and paraffin embedding. Serial sections, 4
µm thick, were prepared and stained with hematoxylin and
eosin (H&E) for histological examination. Alternatively, lung
sections were processed for Masson's trichrome stain for
collagen distribution. Lung tissue sections were
deparaf-finized, and endogenous peroxidase was blocked. Sections
were treated with blocking goat serum for 15 min and
incubated overnight with anti-α-SMA antibody (1:100), then with
biotinylated link secondary antibody and peroxidase-labeled
streptavidin followed by a diaminobenzidine revelation, and
a counterstaining with Mayer's hematoxylin.
Hydroxyproline quantification of lung tissue
The total hydroxyproline content of 50 mg lung tissue was measured
as an assessment of lung collagen content. The lung
tissues were homogenized in ice-cold PBS, hydrolyzed for 3 h
at 130 oC in 4 mL 6 mol/L HCl. After NaOH neutralization, and
adjusted solution, the pH level was between 6 and 8.
Hydroxyproline content was determined with a color-based
reaction as described by Stegemann and
Stalde[18]. Concentrations of unknown hydroxyproline were quantified
according to the standard curve for hydroxyproline.
Statistical analysis Data were expressed as mean±SD.
The differences in mean values were analyzed by one-way
ANOVA test and post hoc analysis with the Bonferroni
method with SPSS 11.0 software (SPSS Inc, Chicago, Illinois,
USA). P<0.05 was considered statistically significant.
Results
Analysis of human α-SMA promoter-driven Luc activity
in human lung fibroblasts To study α-SMA expression at
the transcriptional level, a ~904 bp human α-SMA promoter
was fused with the luciferase reporter gene to form an
α-SMA-Luc fusion plasmid called p895-Luc. We first
measured α-SMA promoter-driven Luc activity in human lung
fibroblasts. As shown in Figure 1, 5 ng/mL
TGF-β1 can induce α-SMA promoter-driven Luc activity in a time-
dependent manner. Compared to the cells without
TGF-β1 treatment, α-SMA promoter-driven Luc activity increased
about 2.44-fold (P=0.01) in the
TGF-β1-treated 24 h cells and 3.91-fold
(P=0.000) in the TGF-β1-treated 48 h cells. This
indicates that the construction of the plasmid p895-Luc is
fully functional and can be used in our further studies.
Effect of Smad protein on α-SMA expression in human
lung fibroblasts We then examined the possible role of
individual Smad protein on α-SMA expression at the
transcriptional level and at protein translational level in human lung
fibroblasts. To confirm the function of each Smad in
TGF-β1-induced human α-SMA expression, we employed
loss-of-function mutations Smad2mut or
Smad3mut, in which the replacement of 3 C-terminal serine residues with alanine
interferes with Smad2 or Smad3 phosphorylation by the activated
TGF-β1 receptor. If the Smad cascade is responsible for the
upregulation of α-SMA gene expression by
TGF-β1 receptor activation, then adding plasmids containing a
dominant-negative Smad2mut, dominant-negative
Smad3mut, or inhibitory Smad7 should reverse
α-SMA gene expression induced by TGF-β1.
Smad plasmid transfection did not affect Smad4
expression in human lung fibroblasts Smad4 is essential
for the Smad3/Smad2 signaling pathway of
TGF-β1. First, we confirmed that the overexpression of the Smad3,
Smad3mut, Smad2 or Smad2mut does not affect Smad4 basic expression.
As shown in Figure 2, Smad4 protein was still present after
the cells were transfected with Smad3,
Smad3mut, Smad2, or Smad2mut expression plasmids.
Smad2 did not affect α-SMA expression in human lung
fibroblasts As shown in Figure 3A, when p895-Luc
was cotransfected with either the Smad2-expressing plasmid
or Smad2mut-expressing plasmid into the fibroblasts, both
Smad2 and Smad2mut overexpression
did not affect basal and TGF-β1-induced
α-SMA promoter activity. Western blot analysis also showed that transient overexpression of Smad2
and Smad2mut had no effect on
TGF-β1-induced human α-SMA protein expression (Figure 3B).
Smad3 upregulated α-SMA expression in human lung
fibroblasts When p895-Luc was cotransfected with either
the Smad3-expressing plasmid or
Smad3mut-expressing plasmid into fibroblasts, as shown in Figure 4A, although Smad3
and Smad3mut by themselves had no effect on basal
α-SMA promoter activity, overexpression Smad3 was able to
stimulate TGF-β1-induced promoter activity in a dose-dependent
manner, Smad3-expressing plasmid 1.0 µg could significantly
increased TGF-β1-induced α-SMA promoter activity
(P<0.01). Overexpression of Smad3mut was able to inhibit
TGF-β1-induced α-SMA promoter activity in a dose-dependent
manner. Smad3mut expressing plasmid 1.0 µg could
significantly inhibit TGF-β1-induced α-SMA promoter activity
(P<0.05). Western blot analysis also showed that the
transient overexpression of Smad3 increased
TGF-β1-induced human α-SMA protein expression, and overexpression of
Smad3mut inhibited TGF-β1-induced human
α-SMA protein expression (Figure 4B).
Smad7 downregulated α-SMA expression in human lung
fibroblasts Smad7 forms stable interaction with the
activated TGF-β receptor and thus blocks ligand-induced
Smad3 phosphorylation. As shown in Figure 5A, transient
overexpression of Smad7 in fibroblasts abrogated the
TGF-β1-induced α-SMA promoter activity in a dose-dependent
manner. 1.0 µg Smad7 expressing plasmid could significantly
inhibit TGF-β1-induced α-SMA promoter activity
(P<0.05). Western blot analysis also showed that the transient
overexpression of Smad7 prevented the
TGF-β1-induced
α-SMA protein expression (Figure 5B). This indicated that
the Smad7 signaling pathway was responsible for the
inhibitory response elicited by TGF-β1.
Differentiated myofibroblasts are significantly reduced
in the lung tissue of Smad3 knockout mice treated by
bleomycin To further ascertain the effect of the Smad3
pathway in vivo, we examined myofibroblast differentiation in
bleomycin-induced fibrotic lesions in wild-type mouse and
Smad3 knockout mouse.
First, histological examination demonstrated that both
wild-type and Smad3 knockout mice treated with saline
vehicle demonstrated a well-alveolized normal histology
(Figure 6A, 6C). Fibrotic lesions induced by bleomycin in
wild-type mouse primarily distributed subpleural regions with
thickened interalveolar septa (Figure 6B). However, when
Smad3 knockout mice were given bleomycin treatment,
fibrotic lesions were much less severe in the subpleural
regions, and only a slight degree of interstitial fibrogenesis
was detected (Figure 6D). The hydroxyproline content of
lung tissue was determined to quantify the amount of
collagen. As shown in Figure 7, hydroxyproline content
significantly increased in the bleomycin-treated wild-type
mice vs saline control mice (1.37±0.12
vs 0.38±0.08 µg/mg lung tissue;
P<0.05). Hydroxyproline content was much less
in the bleomycin-treated Smad3 knockout
mice than in the bleomycin-treated wild-type mice (0.68±0.16
vs 1.37±0.12 µg/mg lung tissue;
P<0.05). These results show that loss of
Smad3 can attenuate bleomycin-induced fibrotic lesions in
mice.
Second, we examined the distribution of collagen
deposition by Masson's trichrome staining and distribution of
myofibroblasts by immunohistological stain using α-SMA
antibodies. As shown in Figure 8A, collagen deposition
was primarily distributed in the subpleural regions. There
were many differentiated myofibroblasts (Figure 8C) in
collagen deposition regions in the bleomycin-treated wild-type
mice. However, in the bleomycin-treated Smad3 knockout
mice, collagen deposition (Figure 8B) and the number of
myofibroblasts (Figure 8D) were reduced.
Next we quantified the expression of α-SMA in lung
tissue by Western blotting. As shown in Figure 9, the
expression of α-SMA was lower in bleomycin-treated Smad3
knockout mice than that in bleomycin-treated wild-type
mice (P<0.05). However, this decrease did not drop down to the basic level,
compared with the saline-treated wild-type mice.
Discussion
Interstitial myofibroblasts are identified as a key
participant in abnormal remodeling and progressive lung fibrosis
in IPF[3]. However, relatively little is known about the
underlying mechanisms that regulate myofibroblasts
differen-tiation. TGF-β1 has been widely recognized as a key
fibro-genic cytokine. Extracellular blockade of
TGF-β1 signaling, involving interference at the level of the ligand or of its
receptor, could ameliorate the fibrotic process in animal model
of lung fibrosis[19,20,21], but this is a non-specific blockade
and will result in many unacceptable side
effects. The targeting of individual intracellular mediators could permit
selective blockade of pathological TGF-β1responses such as fibrosis, without affecting other physiologically important
TGF-β1responses. Therefore, the aim of the current study
was to determine the role of individual
TGF-β1/Smad signal proteins in mediating
α-SMA gene expression, which is the well-known key marker of myofibroblasts differentiation.
It is also known that Smad signal transduction pathways
are crucial in mediating several TGF-β1 response in
fibro-blasts, such as collagen[22,23], tissue inhibitor of
metallopro-teinase-1(TIMP-1)[24], and plasminogen activator
inhibitor-1( PAI-1)[25,26]. These transcriptional responses appear to be
mediated predominantly through Smad3. In the present study,
we demonstrated that it is Smad3, not Smad2, that functions
as the major mediator for TGF-β1 signaling in activating
human α-SMA gene expression in vitro. This result was
also confirmed by a bleomycin-treated Smad3 knockout mice
fibrosis model. Hu et al[27] found that the regulation of both
basal and TGF-β1-induced rat α-SMA promoter activity is
involved in Smad3, but our study demonstrated Smad3 and
Smad3mut fail to affect the basal activities of the human SMA
promoter. Some investigators also have shown that Smad 2
is capable of activating a TGF-β1-responsive mouse
vascular smooth muscle a-actin promoter just as well as Smad 3.
The reason for the different results is that the transcriptional
regulation of the α-SMA gene is complex and likely to be
tissue- and cell-specific[28,29]. There are different nuclear
transcription factors which take part in the regulation of
α-SMA gene expression in different strain cells and different
binding sites for Smad transcriptional complex binding within the
α-SMA promoter gene[30]. So the human promoter behaves
differently from the mouse promoter in this regard. These
need further investigation. This is so far the first report that
shows the unique role of Smad3 in the regulation of human
α-SMA promoter activity.
It is very clear that both Smad2 and Smad3 are involved
in the TGF-β1 signaling pathway, even though they share
92% homology in their amino acid sequence; extensive study
has shown that Smad2 and Smad3 have different functions.
Smad2 knockout mice are embryonic lethal because of a
failure to develop mesoderm[14]. On the other hand, Smad3
knockout mice are viable, although with limb malformations
and a defect in immune functions[13]. The studies using
Smad2 and Smad3-deficient fibroblasts showed the
expressions of both α2 (I) procollagen and matrix
metalloproteinase-1(MMP-1 )are dependent on
Smad3[23,31], and the gelatinase expression is dependent on
Smad2[15]. In some instances, such distinct roles can be explained by the lower affinity of
Smad2 in DNA binding due to an insertion of 30 amino acids
in the N-terminal MH1 domain[12]. Whether there is a factor
to differentiate the function of Smad2 from Smad3 in
α-SMA gene regulation remains to be determined.
Our study also showed that TGF-β1-induced
α-SMA gene expression did not drop down to the basic level when
exogenous Smad7 and Smad3mut were added into the
fibroblasts. Furthermore, Smad3-deficient mice treated with
bleomycin displayed an attenuated pulmonary fibrosis, but
the α-SMA level was higher than that of the saline control
group. There are 3 explanations for this result. First, a
multiplicity of downstream pathways have been shown to be
activated by TGF-β1[32], and they form a complex signaling
network with extensive cross-talk, such as
c-jun-NH2-terminal kinase-dependent pathway which mediates phenotypic
modulation of human lung fibroblasts to myofibroblasts
induced by TGF-β1[6]. Other signaling pathways may
potentially contribute to induce α-SMA gene expression when
Smad3 signaling is disrupted. The crosstalk between
Smad-dependent and Smad-independent pathways is yet unknown.
Second, at whole body level, in addition to
TGF-β1, multiple growth factors, cytokines and chemokines have also been
implicated in the pathogenesis of
fibrosis[33]. Recently, a novel molecule, FIZZ1 (found in inflammatory
zones;also known as RELM-α or resistin-like
molecule-α) was found to be highly expressed in the bleomycin-induced lung fibrosis
model and could activate fibroblast and myofibroblast
differentiation independent of
TGF-β1[34]. Third, because the
origin of myofibroblasts is unclear, cells other than lung
interstitial fibroblast, such as circulation fibrocytes and bone
marrow-derived multipotent cells could differentiate into
myofibroblasts[35,36]. Since the mechanisms of myofibroblasts
which appear in damaged lung tissue is complex, we can not
exclude the possibility that the decreased number of
myofibroblasts in bleomycin-treated Smad3 knockout mice
was either a result of the inhibition of these precursors of
myofibroblast migration to the lung or the inhibition of the
fibroblast-myofibroblast differentiation process.
Taken together, TGF-β1/Smad3 is a major pathway that
regulates myofibroblast differentiation. This result indicates
a potential significance for future attempts for attenuating
the progression of human lung fibrosis by the inhibition of
the Smad3 cascade.
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