Adachi And Koji Suzuki Shino Shimizu, Esteban C. Gabazza, Tatsuya ...
Shino Shimizu, Esteban C. Gabazza, Tatsuya Hayashi, Masaru Ido, Yukihiko
Adachi and Koji Suzuki
279:503-510, 2000.
Am J Physiol Lung Cell Mol Physiol
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Am J Physiol Lung Cell Mol Physiol
279: L503–L510, 2000.
Thrombin stimulates the expression of PDGF
in lung epithelial cells
SHINO SHIMIZU,1 ESTEBAN C. GABAZZA,1,2 TATSUYA HAYASHI,1
MASARU IDO,1 YUKIHIKO ADACHI,2 AND KOJI SUZUKI1
1Department of Molecular Pathobiology and 2Third Department of Internal Medicine,
Mie University School of Medicine, Tsu-city, Mie 514-8507, Japan
Received 8 January 2000; accepted in final form 28 April 2000
Shimizu, Shino, Esteban C. Gabazza, Tatsuya Ha-
of the bronchial wall is responsible for the underlying
yashi, Masaru Ido, Yukihiko Adachi, and Koji Suzuki.
irreversible airway hyperreactivity observed in pa-
Thrombin stimulates the expression of PDGF in lung epithe-
tients with chronic bronchial asthma (23). The morpho-
lial cells. Am J Physiol Lung Cell Mol Physiol 279:
logical changes of airway remodeling include epithelial
L503–L510, 2000.—Several growth factors, including plate-
sloughing, thickening of the epithelial basement mem-
let-derived growth factor (PDGF), have been implicated in
brane, edema, inflammatory cell infiltration of the sub-
Downloaded from
the mechanism of lung and airway remodeling. In the
present study, we evaluated whether thrombin may promote
mucosa, hyperplasia of mucus glands, accumulation of
lung and airway remodeling by increasing PDGF production
myofibroblasts, and thickening of medial smooth mus-
from lung and airway epithelial cells. Conditioned medium
cle layers (2, 7, 8). Several growth factors such as
(CM) was prepared by treating epithelial cells with increas-
platelet-derived growth factor (PDGF), basic fibroblast
ing concentrations of thrombin; before use in the assays, CM
growth factor (bFGF), transforming growth factor- ,
was treated with hirudin until complete inhibition of throm-
epidermal growth factor (EGF), and insulin-like
ajplung.physiology.org
bin activity. CM from epithelial cells stimulated the prolifer-
growth factor, secreted by inflammatory or resident
ation of lung fibroblasts and bronchial smooth muscle cells.
structural cells, have been implicated in the mecha-
Anti-PDGF antibody significantly inhibited this CM prolifer-
nism of the structural changes associated with lung
ative activity, implicating PDGF in this effect. Enzyme im-
and airway remodeling (15, 16).
munoassay and RT-PCR demonstrated that thrombin in-
Thrombin is a serine protease that, besides its criti-
duced the secretion and expression of PDGF from bronchial
cal role in thrombosis and hemostasis, has also been
and alveolar epithelial cells. RT-PCR showed that epithelial
cells express the thrombin receptors protease-activated re-
described as inducing cellular and molecular events
ceptor (PAR)-1, PAR-3, and PAR-4. The PAR-1 agonist pep-
relevant to tissue remodeling. For example, thrombin
on March 17, 2010
tide was also found to induce PDGF secretion from epithelial
may induce platelet aggregation and platelet release of
cells, suggesting that the cellular effect of thrombin occurs
thromboxane A , serotonin, procoagulant, and growth
2
via a PAR-1-mediated mechanism. Overall, this study
factors. Thrombin may also stimulate chemotaxis of
showed for the first time that thrombin may play an impor-
monocytes and lymphocytes, the motility and prolifer-
tant role in the process of lung and airway remodeling by
ation of fibroblasts and smooth muscle cells, and the
stimulating the expression of PDGF via its cellular receptor,
secretion of growth factors and proteases from the
PAR-1.
vascular endothelium (9, 11, 22). These observations
coagulation; lung fibrosis; bronchial asthma; platelet-derived
together with previous studies (10, 18, 19) showing
growth factor receptor
increased generation of thrombin in pulmonary dis-
eases suggest that thrombin may also play an impor-
tant role in the pathogenesis of lung and airway re-
STRUCTURAL REMODELING of the lung tissue is believed to
modeling. We hypothesized that thrombin may also
be the consequence of repeated cycles of epithelial
indirectly promote lung and airway remodeling by in-
injury and repair process associated with chronic pul-
creasing the production of growth factors from alveolar
monary disease (3). Remodeling of the alveolar com-
and bronchial epithelial cells. To demonstrate this pos-
partment of the lung, such as that occurring in pulmo-
sibility, in the present study, we evaluated the effect of
nary fibrosis, is characterized by interstitial and intra-
thrombin on the secretion of PDGF from alveolar and
alveolar deposition of connective tissue and collapse
bronchial epithelial cells and the effect of conditioned
and distortion of air spaces by scar contraction (5).
medium (CM) of these cells stimulated with thrombin
Tissue remodeling is also a severe complication of air-
on the proliferation of lung fibroblasts and bronchial
way disorders, particularly of bronchial asthma. It is
smooth muscle cells. It is known that the cellular
believed that airway remodeling leading to thickening
effects of thrombin depend on stimulation of the pro-
Address for reprint requests and other correspondence: E. C.
The costs of publication of this article were defrayed in part by the
Gabazza, Third Dept. of Internal Medicine, Mie Univ. School of
payment of page charges. The article must therefore be hereby
Medicine, Edobashi 2-174, Tsu-city, Mie 514-8507, Japan (E-mail:
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
gabazza@clin.medic.mie-u.ac.jp).
solely to indicate this fact.
http://www.ajplung.org
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society
L503
L504
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
tease-activated receptor (PAR)-1, PAR-3, or PAR-4 (6).
were positive for smooth muscle cell -actin but negative for
In the present study, we also evaluated the expression
von Willebrand factor. The cells were cultured with a culture
of these receptors in alveolar and bronchial epithelial
kit (Clonetics) containing a modified MCDB131 medium sup-
cells.
plemented with 5% FBS, 50 g/ml of gentamicin, 50 ng/ml of
amphotericin B, 0.5 ng/ml of human EGF, 5 g/ml of insulin,
and 2 ng/ml of human FGF. All cells were cultured in 10-cm
MATERIALS AND METHODS
dishes in an atmosphere composed of 5% CO and 95% air.
2
Reagents. Dulbecco’s modified Eagle’s medium (DMEM),
Confluent cells were harvested by a brief exposure to 0.025%
L-glutamine, vitamin solution, sodium pyruvate, nonessen-
trypsin-0.02% EDTA in HEPES-buffered saline (50 mM
tial amino acids, transferrin, bovine pituitary extract, TRIzol
HEPES and 150 mM NaCl, pH 7.4) and passaged after 5 7
reagent, and Superscript preamplification system were
days.
purchased from GIBCO BRL (Life Technologies, Grand Is-
Preparation of CM. A549, NHBE, and BEAS-2B cells were
land, NY). Fetal bovine serum (FBS) was from BioWhit-
grown to 80 90% confluence in 10-cm dishes, and human
taker (Walkersville, MD); and penicillin, streptomycin, and
nasal epithelial cells were grown in 12-well tissue culture
dexamethasone were from Nacalai Tesque (Kyoto, Japan).
plates. After incubation for 24 h in basal medium without
BSA, Ham’s F-12 medium, epinephrine, gentamicin, am-
supplements, the cells were washed and incubated for 48 h in
photericin B, insulin, triiodothyronine, cholera toxin, endo-
the presence of varying concentrations of thrombin. The CM
thelial growth supplement, L-leucine, L-lysine, L-methionine,
was harvested, centrifuged at 1,200 g for 15 min, and stored
HEPES, and nonspecific protease from Streptomyces griseus
at
80°C until used. To evaluate the participation of the
were from Sigma (St. Louis, MO). Human EGF was from
thrombin receptor (PAR-1), after preincubation in FBS- and
Higeta Shouyu (Tokyo, Japan). Hydrocortisone and retinoic
supplement-free medium for 24 h, NHBE cells were treated
acid were from Wako (Osaka, Japan). bFGF, cDNA probes of
with the PAR-1 agonist peptide (SFLLRNP) or its negative
Downloaded from
PDGF-A and PDGF-B, goat anti-human PDGF antibody, and
control (YFLLRNP) for 48 h, and then the CM was collected
Quantikine enzyme immunoassay kits for EGF, bFGF, and
and used in the assays.
PDGF-AB were from R&D Systems (Minneapolis, MN). All
Proliferation assay. CM was used in the proliferation as-
other chemicals and reagents used were of the best quality
says after it was treated with hirudin (250 antithrombin
commercially available.
units) at 37°C for 30 min and after confirmation of the
Cell culture. The human lung fibroblast cell line WI-38 was
absence of thrombin activity in CM with the chromogenic
obtained from RIKEN Cell Bank (Ibaraki, Japan). The hu-
substrate S2238 for thrombin (Chromogenix, Mo¨lndal, Swe-
ajplung.physiology.org
man lung carcinoma-derived alveolar epithelial cell line
den) by amidolytic assay. The proliferative effect of CM on
A549 and the human bronchial epithelial cell line BEAS-2B
fibroblasts and bronchial smooth muscle cells was assessed
were obtained from the American Type Culture Collection
by a colorimetric method as previously described (21).
(Manassas, VA). Normal human bronchial epithelial (NHBE)
Briefly, after the cells were cultured in 96-well plates up to
cells and human bronchial smooth muscle cells were pur-
70 80% of confluence, their growth was arrested by cultur-
chased from Clonetics (Walkersville, MD) as primary culture
ing overnight in medium without FBS and growth factors.
cells. Both lung fibroblasts and A549 cells were cultured in
After the cells were washed, 100
l of hirudin-treated CM
DMEM containing 10% heat-inactivated FBS, 50
g/ml of
were applied to each well and incubated for 48 h under an
penicillin, 50
g/ml of streptomycin, 2 mM L-glutamine, 2%
atmosphere of 5% CO and 95% air. After being appropri-
on March 17, 2010
2
vitamin solution, 110
g/ml of sodium pyruvate, and 0.1 mM
ately washed, the cells were fixed by treatment with 10%
nonessential amino acids. NHBE cells were cultured in
Formalin for 30 min; the wells were then washed, and 1%
CCMD161 medium (Clonetics) supplemented with 30
g/ml
(wt/vol) methylene blue in 0.01 M borate buffer was added.
of bovine pituitary extract, 0.5
g/ml of BSA, 0.5
g/ml of
After the wells were washed with borate buffer, a 1:1 dilution
epinephrine, 50
g/ml of gentamicin, 50 ng/ml of amphoter-
of ethanol in 0.1 M HCl was added, and the absorbance at 650
icin B, 0.5 ng/ml of human EGF, 0.5 g/ml of hydrocortisone,
nm was measured with a microplate reader. This method has
5
g/ml of insulin, 7 ng/ml of triiodothyronine, 10
g/ml of
been previously validated with a number of characterized
transferrin, and 0.1 ng/ml of retinoic acid. BEAS-2B cells
growth factors (21). In addition, proliferative assays were
were cultured in serum-free Ham’s F-12 medium supple-
also carried out with a cell-counting kit purchased from
mented with 5
g/ml of insulin, 5
g/ml of transferrin, 20
Dojindo (Kumamoto, Japan); this kit contains a tetrazolium
ng/ml of human EGF, 0.1
M dexamethasone, 20 ng/ml of
compound, WST-1, a sodium salt of 4-[3-(4-iodophenyl)-2-(4-
cholera toxin, 30
g/ml of bovine pituitary extract, and 1
M
nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, which
retinoic acid. Nasal epithelial cells were isolated from nasal
is used for the colorimetric determination of cell number.
polyps of patients with chronic sinusitis according to the
In separate experiments to evaluate whether the prolifer-
method previously described (27). Briefly, stroma from
ative effect of CM depends on the presence of PDGF, the CM
polyploid lesions was removed, and the epithelial cells were
prepared from cells stimulated with the same concentration
dissociated by incubation with 0.1% nonspecific protease
of thrombin (250 nM) was treated with varying concentra-
from Streptomyces griseus in Ham’s F-12 medium at 4°C for
tions of anti-human PDGF antibody at 37°C for 30 min. The
20 h. The cell suspension was then filtered through a 60- m
anti-PDGF antibody-treated CM was then used in prolifera-
Nitex mesh, centrifuged at 500 g for 10 min, washed, and
tion assays performed as described above.
resuspended in DMEM-Ham’s F-12 medium containing 0.5
Enzyme immunoassay. Enzyme immunoassays were car-
mM L-leucine, 0.5 mM L-lysine, 0.1 mM L-methionine, 0.3
ried out to assess the concentration of growth factors in CM.
mM MgCl , 0.4 mM MgSO , 1 mM CaCl , 9 mg/l of phenol
Commercial immunoassay kits (described in Reagents) were
2
4
2
red, 14 mM NaHCO , 7 mM
used to measure the concentrations of PDGF-AB, bFGF, and
3
L-glutamine, 50
g/ml of peni-
cillin, 50 g/ml of streptomycin, 10 g/ml of insulin, 0.1 g/ml
EGF.
of hydrocortisone, 0.1
g/ml of cholera toxin, 5
g/ml of
RNA isolation and RT-PCR. RT-PCR was carried out to
transferrin, 25 ng/ml of human EGF, 8
g/ml of endothelial
evaluate the expression of thrombin receptors (PAR-1,
growth supplement, 50 g/ml of bovine pituitary extract, and
PAR-3, and PAR-4) and PDGF receptors ( and ) in alveolar
30 mM HEPES. The human bronchial smooth muscle cells
and airway epithelial cells, lung fibroblasts, and bronchial
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
L505
Table 1. Primer sequences for RT-PCR
Annealing
mRNA
Temperature,
Species
5 Primer
Nucleotides
3 Primer
Nucleotides
°C
PDGF-A
5 -CCTGCCCATTCGGAGGAAGAG-3
624–664
5 -TTGGCCACCTTGACGCTGCG-3
829–848
59
PDGF-B
5 -CCATTCCCGAGGAGCTTTATGA-3
1308–132
5 -GTCTTGTCATGCGTGTGCTTGA-3
1914–1935
59
PDGFR-
5 -CTGGAAGAAATCAAAGTCCCATCC-3
909–932
5 -TGAGCCATGGTGATCATCGACC-3
1388–1409
65
PDGFR-
5 -GTGGTGAGCACACTGCGTCTG-3
1827–1847
5 -GTAACGTGGCTTCTTCTGCCA-3
2022–2042
66
GAPDH
5 -CCACCCATGGCAAATTCCATGGCA-3
146–169
5 -TCTAGACGGCAGGTCAGGTCCACC-3
720–743
59
PAR-1
5 -CAGTTTGGGTCTGAATTGTGTCG-3
505–527
5 -TGCACGAGCTTATGCTGCTGAC-3
1075–1096
55
PAR-3
5 -TCCCCTTTTCTGCCTTGGAAG-3
152–172
5 -AAACTGTTGCCCACACCAGTCCAC-3
641–664
55
PAR-4
5 -AACCTCTATGGTGCCTACGTGC-3
949–970
5 -CCAAGCCCAGCTAATTTTTG-3
1471–1490
55
PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAR, protease-
activated receptor.
smooth muscle cells. Total RNA was extracted from confluent
Statistical analysis. All data are expressed as means
SE
cells by the guanidine isothiocyanate procedure with the
unless otherwise specified. The difference between three or
TRIzol Reagent. Two micrograms of total RNA were reverse
more variables was calculated by analysis of variance. Sta-
transcribed with oligo(dT) primer and the Superscript pre-
tistical analyses were carried out with the StatView 4.1
amplification system kit following the manufacturer’s in-
package software for the Macintosh (Abacus Concepts,
structions. The primers used in the PCR are listed in Table 1.
Berkeley, CA).
Downloaded from
The
PCR
mixture
contained
PCR
buffer
(100
mM
Tris HCl, pH 8.3, and 200 mM KCl), 25 mM MgCl , 10
M
2
RESULTS
deoxyribonucleosides (dATP, dGTP, dCTP, and dTTP), 10
M each forward and reverse primer, an appropriate amount
Effect of CM on proliferation of lung fibroblasts and
of diethyl pyrocarbonate-treated distilled water, and 1 U of
bronchial smooth muscle cells. To evaluate the role of
Taq polymerase (Boehringer Mannheim, Mannheim, Germa-
thrombin in the secretion of PDGF from bronchial and
ny). The mixture was then divided into aliquots in PCR tubes
ajplung.physiology.org
alveolar epithelial cells, the proliferative activity of CM
containing 2
l of cDNA sample, and the amplification was
performed with a PC-800 Programmable Temp control sys-
from these cells stimulated with varying concentra-
tem cycler (Astec, Fukuoka, Japan). The PCR products were
tions of thrombin on lung fibroblasts and bronchial
then electrophoresed on a 2% agarose gel and stained with
smooth muscle cells was assessed after complete inhi-
0.5
g/ml of ethidium bromide. PCR of samples treated
bition of residual thrombin activity in the CM with
without RT was also carried out to ensure the absence of
hirudin. In the methylene blue-based proliferation as-
contaminating DNA in total RNA prepared from epithelial
says, CM from the primary bronchial epithelial cells
cells.
(NHBE cells) significantly stimulated the proliferation
Southern hybridization. Southern hybridization was car-
of fibroblasts and bronchial smooth muscle cells in a
on March 17, 2010
ried out to assess the effect of thrombin on gene expression of
dose-dependent manner (Fig. 1A). Similarly, CM from
PDGF in airway epithelial cells. Preparation of total RNA
the alveolar epithelial cell line A549 significantly stim-
and PCR products was performed as described in RNA iso-
lation and RT-PCR. After the PCR products were resolved on
ulated the proliferation of fibroblasts and bronchial
a 2% agarose gel, the gel was incubated in a solution con-
smooth muscle cells in a dose-dependent fashion (Fig.
taining 1.5 M NaCl and 500 mM NaOH for 30 min at room
1B). These findings were in good agreement with the
temperature and then incubated again in a solution contain-
results obtained with the cell-counting kit containing
ing 1 M ammonium acetate and 20 mM NaOH at room
the tetrazolium salt WST-1. For preparation of the
temperature for 30 min. The cDNA was transferred for 24 h
control sample, aliquots of culture medium were incu-
by capillary action onto a Hybond-N
membrane (Amersham
bated in the presence of the same concentrations of
Pharmacia Biotech, Tokyo, Japan) and fixed to the mem-
thrombin but in the absence of epithelial cells; after
brane by ultraviolet irradiation with a Stratalinker (Strat-
treatment with hirudin, the control medium was also
agene, La Jolla, CA). The membrane was then prehybridized
used in the proliferative assays. These control media
in a solution containing 20 mM Tris HCl, pH 7.5, 750 mM
NaCl, 2.5 mM EDTA, 1% SDS, 5
Denhardt’s solution, 0.5
did not show proliferative activity on lung fibroblasts
g/ml of denatured salmon sperm DNA, and 50% formamide
(data not shown), further ensuring that hirudin com-
and then hybridized with cDNA probes of human PDGF-A,
pletely inhibited residual thrombin activity in our as-
PDGF-B, or human glyceraldehyde-3-phosphate dehydroge-
says.
nase (Cayman Chemical) that were labeled with [ -32P]dCTP
Effect of anti-PDGF antibody on proliferative activity
with a DNA labeling kit (Takara Shuzo, Kyoto, Japan).
of CM from airway epithelial cells. To assess whether
Northern hybridization. Northern hybridization was car-
the proliferative activity of CM depends on the secre-
ried out with a digoxigenin Northern starter kit (Boehringer
tion of PDGF from epithelial (NHBE) cells, the inhib-
Mannheim). Ten micrograms of total RNA were electro-
itory effect of anti-PDGF antibody that recognizes
phoretically separated on 1.2% agarose gels and transferred
PDGF-AA, PDGF-BB, and PDGF-AB was evaluated.
to positively charged Boehringer Mannheim nylon mem-
branes by capillary elution. The membranes were hybridized
Compared with control samples (0 g/ml of anti-PDGF
with digoxigenin-11-UTP-labeled RNA probes. Immunologic
antibody), the anti-PDGF antibody significantly inhib-
detection was then performed following the manufacturer’s
ited the proliferative activity of CM prepared with
instructions.
NHBE cells stimulated with 250 nM thrombin in a
L506
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
Fig. 2. Effect of anti-platelet-derived growth factor (PDGF) antibody
on proliferative activity of CM. CM prepared from NHBE cells
stimulated with the same concentration of thrombin (250 nM) was
treated with indicated concentrations of anti-human PDGF antibody
at 37°C for 30 min before the assay. Fibroblasts were incubated in
each medium for 48 h. Values are means
SE expressed as percent-
Downloaded from
age over basal medium that was taken as 100% of fibroblast prolif-
eration. Assays were done in triplicate in 5 separate experiments.
* P
0.05. ** P
0.005 [both compared with control sample (0 g/ml
of anti-PDGF antibody)].
Thrombin also significantly increased the concentra-
tion of PDGF-AB in CM prepared with primary cells
ajplung.physiology.org
from bronchial or nasal epithelium; thrombin in-
creased PDGF-AB concentration from a concentration
of 62.5 nM for NHBE cells (Fig. 5A) and 125 nM for
nasal epithelial cells (Fig. 5B). The secretion of
PDGF-AB from thrombin-treated BEAS-2B cells was
Fig. 1. Effects of conditioned medium (CM) from normal human
also time dependent (Fig. 6).
bronchial epithelial (NHBE; A) and alveolar epithelial (A549; B) cells
Effect of thrombin on PDGF gene expression in epi-
treated with indicated concentrations of thrombin for 48 h on prolif-
eration of lung fibroblasts (open bars) and bronchial smooth muscle
thelial cells. The effect of thrombin on the gene expres-
on March 17, 2010
cells (solid bars). Cell supernatant was used in this assay as CM.
sion of PDGF-A and PDGF-B in NHBE cells was eval-
Residual thrombin activity in CM was inhibited by hirudin before
uated by RT-PCR and Southern blot analysis. As
the assay. Lung fibroblasts and bronchial smooth muscle cells were
shown in Fig. 7, thrombin significantly increased the
incubated in each CM for 48 h. CM from NHBE and A549 cells
stimulated the proliferation of fibroblasts and bronchial smooth
expression of the PDGF-B gene but not that of the
muscle cells. Values are means
SE, with 100% being the no. of cells
that proliferated in the presence of CM from epithelial cells prepared
with 0 nM thrombin. Assays were done in triplicate in 5 separate
experiments. * P
0.05. ** P
0.01. # P
0.05. ## P
0.03 [all
compared with control samples (0 nM thrombin)].
dose-dependent manner (Fig. 2). This inhibition of fi-
broblast proliferation was significant above 10 g/ml of
the anti-PDGF antibody.
Secretion of PDGF and other growth factors by epi-
thelial cells treated with thrombin. To assess whether
thrombin stimulation increases the secretion of PDGF
and other growth factors from epithelial cells, the con-
centration of these growth factors in CM was mea-
sured. As shown in Fig. 3, thrombin significantly and
specifically stimulated the secretion of PDGF-AB but
not that of bFGF or EGF from NHBE cells. The in-
crease in the concentration of PDGF-AB in CM stimu-
Fig. 3. Concentrations of epidermal growth factor (EGF), basic fibro-
lated with thrombin was significant from a thrombin
blast growth factor (bFGF), and PDGF-AB in CM from NHBE cells
concentration of 62.5 nM for BEAS-2B cells (Fig. 4A);
treated with 125 nM thrombin. The cells were grown in 12-well
in A549 cells (Fig. 4B), thrombin stimulated the secre-
tissue culture plates. They were incubated in the presence (125 nM;
solid bars;
) and absence (open bars;
) of thrombin for 48 h, and
tion of PDGF-AB at a thrombin concentration of 62.5
then the concentration of each growth factor in CM was determined
nM and reached a plateau with 125 nM thrombin.
with commercial enzyme immunoassay kits.
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
L507
(A549) and airway (NHBE) epithelial cells expressed
not only PAR-1 but also PAR-3 and PAR-4; however,
the bronchial epithelial cell line BEAS-2B did not ex-
press PAR-3 (Fig. 9).
Expression of PDGF receptors in epithelial cells, lung
fibroblasts, and bronchial smooth muscle cells. Because
it is known that the effect of PDGF depends on the
presence of its receptors,
and
, on target cells, the
expression of PDGF receptors was evaluated. RT-PCR
showed that both PDGF receptors (
and
) are ex-
pressed by epithelial cells, lung fibroblasts, and bron-
chial smooth muscle cells (Fig. 10).
DISCUSSION
This study showed that CM from alveolar and bron-
chial epithelial cells treated with thrombin signifi-
cantly stimulates the proliferation of lung fibroblasts
and bronchial smooth muscle cells compared with that
in the medium of untreated cells. Immunoassays and
Downloaded from
inhibition experiments revealed that the increased
ajplung.physiology.org
Fig. 4. Effect of various concentrations of thrombin on PDGF-AB
secretion from established epithelial cell lines. BEAS-2B (A) and
A549 (B) cells were grown in 10-cm-diameter dishes. The cells were
incubated with indicated concentrations of thrombin for 48 h, and
on March 17, 2010
then the concentration of PDGF-AB in CM was measured. Values are
means
SE. Assays were done in triplicate in 2 separate experi-
ments. * P
0.01. ** P
0.001. # P
0.0001 [all compared with
control samples (0 nM thrombin)].
PDGF-A gene in NHBE cells. Similarly, Northern blot
analysis also showed that thrombin stimulates the
expression of the PDGF-B gene at a transcriptional
level (data not shown).
Effect of PAR-1 agonist peptide on PDGF secretion in
epithelial cells. To evaluate the participation of the
thrombin receptor on PDGF secretion induced by
thrombin, the effect of the PAR-1 agonist peptide on
the concentration of PDGF in CM from NHBE cells was
assessed. Like thrombin, the PAR-1 agonist peptide
also induced PDGF-AB secretion from NHBE cells in a
dose-dependent manner (Fig. 8). This effect was signif-
icant from a peptide concentration of 25
M. The neg-
ative control peptide did not induce PDGF-AB secre-
Fig. 5. Effect of indicated concentrations of thrombin on PDGF
tion (data not shown). These results suggest that
secretion from primary bronchial (NHBE; A) and nasal epithelial (B)
thrombin stimulation of PDGF-AB secretion from air-
cells. NHBE cells were grown in 10-cm-diameter dishes, and human
way epithelial cells is mediated by PAR-1.
nasal epithelial cells were grown in 12-well tissue culture plates. The
Expression of PARs in epithelial cells. To evaluate
cells were incubated with for 48 h, and then the concentration of
the type of thrombin receptors expressed by epithelial
PDGF-AB in CM was determined. Values are means
SE. Assays
were done in triplicate in 2 separate experiments. * P
0.001; ** P
cells, mRNAs of PAR-1, PAR-3, and PAR-4 were ana-
0.001. # P
0.005 [both compared with control samples (0 nM
lyzed by RT-PCR. RT-PCR showed that both alveolar
thrombin)].
L508
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
dent manners; RT-PCR analysis showed that this in-
creased secretion of PDGF-AB depends on increased
mRNA expression of PDGF-B. It is worth noting that
upregulation of PDGF-AB was induced by thrombin
not only in transformed epithelial cell lines but also in
primary bronchial and nasal epithelial cells, suggest-
ing that this effect is probably also observed in vivo at
sites of inflammation. Inhibition assays carried out
with anti-PDGF antibody confirmed that PDGF was
responsible for the proliferative activity of CM from
epithelial cells. Overall, these findings suggest that
stimulation of PDGF secretion from epithelial cells
may constitute another mechanism of thrombin partic-
ipation in lung and airway remodeling. The biological
relevance of PDGF in the mechanism of tissue remod-
Fig. 6. Time course of PDGF-AB secretion from thrombin-treated
eling is illustrated by the important role it plays in the
epithelial cells (BEAS-2B). BEAS-2B cells were grown in 12-well
tissue culture plates and incubated in the presence (solid bars) and
pathogenesis of fibroproliferative processes such as
absence (open bars) of thrombin (125 nM) for indicated times, and
wound healing, pulmonary fibrosis, atherosclerosis,
then the concentration of PDGF-AB in CM was measured. Values are
myelofibrosis, scleroderma, and inflammatory joint
means
SE. Assays were done in triplicate in 2 separate experi-
diseases. Properties that implicate PDGF in the mech-
Downloaded from
ments.
anism of tissue remodeling are its strong mitogenic and
chemotactic activities on fibroblasts and smooth mus-
growth of these cells depends on the enhanced produc-
cle cells and its ability to stimulate angiogenesis and
tion and secretion of PDGF-AB induced by thrombin in
the secretion of cytokines (e.g., transforming growth
epithelial cells.
factor- ) that, in turn, may upregulate the expression
It is well known that increased thrombin generation
ajplung.physiology.org
occurs in the lungs of most patients with inflammatory
diseases of the pulmonary parenchyma and airways
and that it plays a critical role in tissue remodeling (10,
18, 19). After injury, a tissue factor-activated factor VII
complex is formed on lung and airway epithelial and/or
endothelial cells, leading to activation of the extrinsic
pathway of coagulation, thrombin generation, fibrin
formation, and fibrosis (4, 12, 14). In the lung and
airways, generated thrombin may modulate the tissue
on March 17, 2010
repair response by altering vascular permeability, by
stimulating the secretion of proteases and the produc-
tion or degradation of components of the connective
tissue, and by promoting adhesion, spreading, and
proliferation of fibroblasts and smooth muscle cells (1).
In addition to its direct effects, thrombin may also play
a role in lung tissue remodeling by stimulating the
secretion of growth factors from several cells. For ex-
ample, it has been reported that thrombin may induce
increased expression of PDGF from lung fibroblasts,
vascular smooth muscle cells, endothelial cells, and
alveolar macrophages (13, 19, 20, 22, 26). In the
present study, we hypothesized that thrombin may
also participate in tissue remodeling by stimulating
the secretion of growth factors from alveolar and bron-
chial epithelial cells.
The CM prepared by treating alveolar and bronchial
epithelial cells with different concentrations of throm-
bin was found to stimulate the proliferation of lung
Fig. 7. Effect of thrombin on gene expression of PDGF in epithelial
fibroblasts and bronchial smooth muscle cells. This CM
(NHBE) cells. Total RNA was prepared with NHBE cells treated
effect did not depend on the direct proliferative effect of
with and without thrombin (125 nM) for 48 h, and RT-PCR and
thrombin because its activity in CM was blocked com-
Southern hybridization (top) were performed as described in MATE-
pletely by hirudin before use in the proliferative as-
RIALS AND METHODS. The intensities of the bands were densitometri-
says. Measurement of growth factors in CM showed
cally quantified (bottom), and the results were normalized to the
internal standard band glyceraldehyde-3-phosphate dehydrogenase
that thrombin upregulated the secretion of PDGF-AB
(GAPDH). Thrombin increased the expression of PDGF-B gene but
from bronchial epithelial cells in dose- and time-depen-
not that of PDGF-A gene in NHBE cells.
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
L509
mediates the effect of thrombin in primary airway
epithelial cells (NHBE cells), the effect of the PAR-1
agonist peptide on PDGF-AB secretion was assessed.
The PAR-1 agonist peptide induced PDGF-AB secre-
tion from NHBE cells in a similar fashion as thrombin.
RT-PCR analysis showed that besides the PAR-1 gene,
PAR-3 and PAR-4 genes are also expressed by bron-
chial (NHBE and BEAS-2B) and alveolar (A549) epi-
thelial cells. These findings suggest that PARs may
play an important role in the mechanism of lung and
airway remodeling by mediating the mitogenic and
secretory activity of thrombin in epithelial cells.
PDGF is known to occur in the form of three dimers
(PDGF-AA, PDGF-BB, and PDGF-AB) and to bind to
Fig. 8. Concentration of PDGF-AB in CM from protease-activated
specific high-affinity receptor subunits (
,
, and
)
receptor (PAR)-1 agonist peptide-treated epithelial cells. NHBE cells
on the surface of responsive cells (fibroblasts and
were grown in 12-well tissue culture plates and incubated with
smooth muscle cells). The effect of PDGF depends on
indicated concentrations of PAR-1 agonist peptide for 48 h, and
the type of PDGF receptor subunit available on the cell
then the concentration of PDGF-AB in CM was determined. Values
are means
SE. Assays were done in triplicate in 2 separate
surface. PDGF-BB can bind to any one of these recep-
experiments. Control peptide did not affect PDGF secretion (data
tor subunits; PDGF-AA can bind only to an
-recep-
Downloaded from
not shown). * P
0.05 compared with control sample (0
M PAR-1
tor; and PDGF-AB can bind either to an
-receptor or
agonist).
to an
-receptor (24). In the present study, RT-PCR
analysis showed that both the - and -subunits were
of extracellular matrix components (17, 24, 25). A pre-
equally expressed by lung fibroblasts and bronchial
vious study (24) has shown that the PDGF-B chain is
smooth muscle cells. These findings explain the prolif-
the polypeptide that is mainly expressed in lung tis-
erative activity of PDGF-rich CM on fibroblasts and
ajplung.physiology.org
sues from patients with lung fibrosis (24); the demon-
bronchial smooth muscle cells observed in the present
stration in the present study that thrombin upregu-
study. In addition, airway epithelial cells express both
lates the expression of PDGF-B but not that of PDGF-A
subunit receptors, suggesting that they may also play
reinforces the important role that thrombin plays in
an important role in epithelial regeneration in the lung
the process of lung fibrosis.
and airways.
The cellular effects of thrombin have been found to
In conclusion, the results of this study showed for the
depend on stimulation of the thrombin receptors
first time that 1) thrombin regulates PDGF expression
PAR-1, PAR-3, and PAR-4 (6). After cellular activation
and secretion from nasal, bronchial, and alveolar epi-
on March 17, 2010
by thrombin, a new NH -terminal peptide, which func-
thelial cells; 2) the cellular effect of thrombin on PDGF
2
tions as a tethered ligand for the receptor itself, is
secretion by epithelial cells is mediated by its receptor
unmasked on PAR-1, PAR-3, or PAR-4. The tethered
PAR-1; 3) airway epithelial cells express the thrombin
ligand of PAR-1 has been shown to promote prolifera-
receptors PAR-1, PAR-3, and PAR-4; and 4) the throm-
tion of fibroblasts and vascular smooth muscle cells,
bin-induced secretion of PDGF is sufficient for stimu-
cytokine secretion, and deposition of collagen in the
lating the proliferation of fibroblast and bronchial
extracellular matrix. To evaluate whether PAR-1 also
smooth muscle cells. Overall, these findings suggest
that thrombin may play an important role in the pro-
cess of lung and airway remodeling by stimulating the
secretion of PDGF via its PAR-1 receptor from alveolar
and bronchial epithelial cells.
Fig. 9. RT-PCR analysis of PAR-1, PAR-3, and PAR-4 in epithelial
Fig. 10. RT-PCR of PDGF receptors in NHBE cells, lung fibroblasts,
cells. Total RNA was prepared with cells incubated in basal medium
and bronchial smooth muscle cells (BSMC). Total RNA was prepared
without supplements for 24 h, and RT-PCR was performed as de-
with the respective cells incubated in basal medium without supple-
scribed in MATERIALS AND METHODS. Both alveolar (A549) and bron-
ments for 24 h, and RT-PCR was performed as described in MATERI-
chial (NHBE) epithelial cells expressed not only PAR-1 (592 bp) but
ALS AND METHODS. Both PDGF-
(501 bp) and PDGF-
(216 bp)
also PAR-3 (513 bp) and PAR-4 (542 bp) thrombin receptors, but
receptors were expressed by NHBE cells, lung fibroblasts, and
BEAS-2B cells did not express PAR-3.
BSMC.
L510
THROMBIN INDUCES LUNG REMODELING THROUGH PDGF
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